CN108315349B - Application method of circular RNA circRNF13 - Google Patents

Abstract

The invention discloses an application method of circular RNA circRNF13. Specifically, it is the application of a preparation that overexpresses circRNF13 in the preparation of a preparation that reduces the resistance of tongue squamous cell carcinoma cells to cisplatin drugs. Studies have confirmed that the overexpression of circRNF13 in tongue squamous cell carcinoma drug-resistant cells can significantly reverse the drug resistance of tongue squamous cell carcinoma cells. Therefore, the use of circRNF13 overexpression preparations to reduce the drug resistance of tongue squamous cell carcinoma cells has far-reaching clinical significance and important prospects for promotion and application.

Description

Application method of circular RNA circRNF13

technical field

The invention belongs to the field of tumor molecular biology, and in particular relates to the application of a reagent for promoting the expression of circRNF13 in the preparation of a preparation for reducing the drug resistance of tongue squamous cell carcinoma cells to cisplatin drugs.

Background technique

Human Genome Project and its follow-up DNA Element Encyclopedia Project (The Encyclopedia of DNAElements Project, ENCODE) research results show that protein-coding gene sequences only account for 1-3% of the human genome sequence, while most of the human genome can be transcribed The sequence is non-coding RNA (non-coding RNA, ncRNA). Although non-coding RNA does not encode protein, it has been attracting much attention in the field of biomedical research due to its extensive involvement in the regulation of gene expression in cells. The most studied are linear ncRNA molecules, which can be divided into microRNA (microRNA, miRNA, 19-23nt) and long non-coding RNA (long non-coding RNA, lncRNAs, >200nt) according to the length of the RNA sequence. It plays an important role in the occurrence and development of many common human diseases, including malignant tumors. Recently, a new class of ncRNA molecules, circular RNA (circRNA), has become a new frontier and hotspot in the field of biomedicine due to its unique configuration and increasingly important biological functions.

circRNA was once regarded as an error product in the post-transcriptional processing of genes. Similar to the processing process of linear RNA such as mRNA and lncRNA, the formation of circRNA is also generated when the splicing complex splices and processes the RNA precursor, but in this process, the flanking looping sequences are first combined to form a loop, The splicing complex then performs splicing, and finally the flanking sequence is excised to form circRNA. In recent years, with the development of sequencing technology, especially next-generation sequencing technology such as RNA-seq, more and more circRNAs have been discovered, and studies have revealed that some circRNAs play an important role in many life activities. In addition, due to its unique structure, circRNA is not sensitive to nucleases, and circRNA also has obvious advantages in the development and application of tumor markers, so it is very likely to be an important molecular marker and a target for emerging drug development. However, due to the emerging of this field, most circRNAs have not been discovered or studied yet.

Tongue squamous cell carcinoma (TSCC) is the most common oral cancer with the highest incidence rate. Tongue squamous cell carcinoma is mostly squamous cell carcinoma, which mostly occurs on the edge of the tongue, followed by the tip of the tongue, the back of the tongue and the root of the tongue, etc., often ulcers type or infiltration type. Generally, the degree of malignancy is high, the growth is fast, and the infiltration is strong. It often affects the tongue muscle, resulting in restricted tongue movement and difficulties in speaking, eating, and swallowing. Surgical treatment combined with cisplatin-based chemotherapy is the main clinical treatment for tongue squamous cell carcinoma. However, clinically, it is found that the treatment effect of patients with squamous cell carcinoma of the tongue is often unsatisfactory, and the 5-year survival rate is only 55-65%. The ubiquitous drug resistance of circulating tumor cells leads to tumor recurrence and metastasis, which leads to treatment failure. Therefore, finding out the resistance mechanism of tongue squamous cell carcinoma cells to chemotherapy drugs, screening new drug targets, and ultimately improving the survival rate and quality of life of patients are important topics worthy of research by clinical researchers.

In order to study the role and mechanism of circRNA in the resistance of tongue squamous cell carcinoma cells to cisplatin drugs, we used Agilent's circRNA gene chip Arraystar Human Circular RNA Microarray V2.0 to detect two pairs of tongue squamous cell carcinoma cell lines before and after cisplatin treatment The expression of circRNAs in (Tca8113 and Cal27) was analyzed. We found that the expression of circRNF13 changed most significantly, suggesting that circRNF13 may be related to cisplatin resistance in tongue squamous cell carcinoma, but so far, the biological function and mechanism of circRNF13 have not been reported.

Since gene chips can only design probes based on the upstream and downstream sequences of circRNA circular splicing sites, these probes can specifically capture circRNAs to detect the expression abundance of circRNAs in cells or tissues; specifically for circRNF13 that we found through circRNA chips Said, through the technical information provided by the gene chip, we can find the corresponding probes with a total of 60bp. The previous section is the 30bp sequence at the 3' end of exon 8 of the RNF13 gene, and the latter section is the 5' of exon 2 of the RNF13 gene. The 30 bp at the end indicates that it is processed end-to-end by exons 2 and 8 of the precursor RNA transcribed by the RNF13 gene, but which exons of RNF13 are processed (is it all exons 2 to 8? Included? Does it also include intron sequences? Because some circRNAs include intron sequences) is not clear. Subsequently, we designed primers and successfully cloned the full-length sequence of circRNF13 by two nested PCRs, confirming that circRNF13 is indeed derived from the RNF13 gene (ring finger protein 13, NM_183381.2) located on chromosome 3q25.1. Exons 2-8 were formed by circular splicing, with a full length of 716bp.

In order to further study the biological function of circRNF13, we designed a sequence-specific small interfering RNA (siRNA) targeting circRNF13 to knock down the expression of circRNF13, and successfully constructed a circRNF13 overexpression vector to overexpress circRNF13 in tongue squamous cell carcinoma cells. MTT and flow cytometry assays confirmed that upregulation of circRNF13 can significantly inhibit the proliferation of tongue squamous cell carcinoma cells by arresting the cell cycle and inducing apoptosis, while knocking down circRNF13 has the opposite result. Furthermore, we successfully obtained chemotherapy-resistant cell lines of tongue squamous cell carcinoma through induction culture. Real-time quantitative PCR detected the expression level of circRNF13 in the cells, and found that the expression of circRNF13 in drug-resistant cell lines was significantly higher than that in the original cell line. Down-regulated; while we overexpressed circRNF13 in drug-resistant cells, it can significantly reverse the drug-resistant phenotype of tongue squamous cell carcinoma cells, and knocking down the expression of circRNF13 in original cell lines can significantly increase the resistance of tongue squamous cell carcinoma cells to cisplatin. tolerance.

Finally, we detected the expression of circRNF13 in clinical samples of tongue squamous cell carcinoma by real-time fluorescent quantitative PCR and in situ hybridization, and found that the expression of circRNF13 was significantly down-regulated in tongue squamous cell carcinoma, and the survival time of patients with low circRNF13 expression was shorter than Patients with high expression of circRNF13, therefore, detection preparations targeting this lncRNA can also be used for auxiliary diagnosis and prognosis of tongue squamous cell carcinoma.

Contents of the invention

The purpose of the present invention is to provide the application method of circular RNA circRNF13. Specifically, it is the application of a reagent that promotes the expression of circRNF13 in the preparation of a preparation for reducing the resistance of tongue squamous cell carcinoma cells to cisplatin drugs, and the sequence of the circular RNA circRNF13 is shown in SEQ NO:1.

The reagent for promoting the expression of circular RNA circRNF13 is a circRNF13 overexpression vector.

The construction process of the circRNF13 overexpression vector is as follows:

(1) According to the formation mechanism of circRNA and the basic rules of eukaryotic RNA splicing, design a circular sequence suitable for circRNA expression; design a suitable multiple cloning site according to the sequence information of the commercial vector pcDNA3.1, so as to obtain The complete DNA sequence to achieve circRNA overexpression is as follows:

GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTT CTTAAGCTTGGTACCGAGCTCGG ATCCACATCGATTGGTGGAATTCTGCAGATATCCACCGCGGTGGCGGCCGCTCGAGTCTAGA GAAAAGAATTAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCA , the underlined part in the middle is the multiple cloning site, and the two ends are the upstream and downstream looping sequences respectively;

(2) Add the NheI restriction site sequence to one end of the DNA sequence synthesized to realize the overexpression of the circRNA, and add the ApaI restriction site sequence to the other end, and construct it into the pcDNA3.1 vector after digestion with NheI and ApaI to obtain pcCirc Blank plasmid, confirmed by sequencing that the plasmid is correct, and the sequence is shown in SEQ NO:13;

(3) The Cla I and Sac II restriction sites were selected for restriction digestion of the pcCirc blank plasmid, and the circRNF13 sequence was inserted between the upstream and downstream looping sequences of the vector.

The specific process of step (3) is as follows:

1) Using the Tca8113 cDNA of tongue squamous cell carcinoma cells as a template, using TaKaRa LA Enzyme PCR amplification of the full-length circRNF13 sequence; circRNF13 full-length sequence amplification primers are as follows:

Upstream primer: 5'-GTGATTTTACAACGAGAT-3';

Downstream primer: 5'-CTTTCTTGAATTTATGTA-3';

After adding restriction endonuclease Cla I and Sac II recognition sites and protective bases to the 5' ends of the upstream and downstream primers, the primer sequences are as follows:

Upstream primer: 5'-AGGA ATCGAT GTGATTTTACAACGAGAT-3', the underlined part is the Cla I recognition site;

Downstream primer: 5'-ATGC CCGCGG CTTTCTTGAATTTATGTA-3', the underlined part is the Sac II recognition site;

2) PCR amplification, the full-length sequence of circRNF13, the PCR reaction conditions are as follows:

PCR reaction steps

3) Electrophoresis of the PCR product, gel recovery of the target fragment, electrophoresis after Cla I and Sac II double enzyme digestion, and gel recovery again;

4) The pcCirc blank plasmid was double digested with Cla I and Sac II, and the target fragment was recovered by electrophoresis gel;

5) Ligate the gel recovery product of step 3) and step 4) with T4 DNA ligase to obtain a vector plasmid overexpressing circRNF13;

6) The eukaryotic expression plasmid containing the full-length sequence of circRNF13 obtained in step 5) was transformed into competent Escherichia coli to amplify the plasmid.

After we knocked down the expression of circRNF13 (si-circRNF13) in primitive tongue squamous cell carcinoma cells (NC) or overexpressed circRNF13 (OE-circRNF13) in drug-resistant cells (CR), the cells were treated with the same concentration of cisplatin, and then Cell apoptosis was detected by flow cytometry, and it was found that knocking down the expression of circRNF13 could increase the resistance of cells to cisplatin (decreased apoptosis), while overexpressing circRNF13 could significantly reduce the drug resistance of drug-resistant cells. Therefore, the use of circRNF13 overexpression preparations to reduce the drug resistance of tongue squamous cell carcinoma cells has far-reaching clinical significance and important prospects for promotion and application.

Description of drawings

Figure 1: It was verified by qRT-PCR that the expression of circRNF13 was significantly up-regulated after cisplatin treatment (left), and the sequencing results confirmed that circRNF13 was indeed detected by qRT-PCR (right). The arrow represents the splicing site, the left side of the arrow is the 3' end of exon 8 of RNF13, and the right side of the arrow is the sequence of the 5' end of exon 2 of RNF13 gene.

Figure 2: Through two nested PCR-sequencing, the full-length sequence of the circRNF13 molecule was successfully cloned for the first time, proving that the circRNA is composed of exons 2-8 of the RNF13 gene transcript NM_183381.2 located on the chromosome 3q25.1 region Formed by circular splicing, the full length is 716bp.

Figure 3: Designed and successfully constructed the circRNF13 overexpression vector, using the pcDNA3.1 vector as the basic backbone, by adding two circular sequences and restriction enzyme sites downstream of the CMV promoter, exon 2-8 of RNF13 CircRNF13 was successfully overexpressed in tongue squamous cell carcinoma cells (right) by PCR, enzyme digestion, ligation into the vector (left), and then transfection into tongue squamous cell carcinoma cells.

Figure 4: A siRNA sequence (left) specifically targeting the circular RNA was designed for the circular splicing site of circRNF13 (the junction of exon 8 and exon 2), and then transfected into tongue squamous cell carcinoma cells, The expression of circRNF13 was successfully knocked down in tongue squamous cell carcinoma cells (right).

Figure 5: MTT proliferation assay shows that overexpression of circRNF13 (OE-circRNF13) can inhibit the proliferation of tongue squamous cell carcinoma cells, while knockdown of circRNF13 expression (si-circRNF13) can promote the proliferation of tongue squamous cell carcinoma cells compared with the control group (NC). proliferation.

Figure 6: Flow cytometry analysis found that compared with the control group (NC), the distribution of tongue squamous cell carcinoma cells in G2/M phase was significantly increased after overexpressing circRNF13 (OE-circRNF13), indicating that the cell cycle was arrested in G2/M phase , and after knocking down the expression of circRNF13 (si-circRNF13), the S phase distribution was significantly increased, indicating that the cell cycle progression was accelerated.

Figure 7: Analysis of cell apoptosis by flow cytometry, it was found that compared with the control group (NC), the apoptosis rate of tongue squamous cell carcinoma cells after overexpression of circRNF13 (OE-circRNF13) was significantly increased. Under normal culture conditions, tumor cell apoptosis Therefore, knocking down the expression of circRNF13 slightly reduces the proportion of apoptotic cells.

Figure 8: circRNF13 expression was significantly reduced in chemotherapy-resistant cells (CR) compared with controls (NC).

Figure 9: After knockdown of circRNF13 expression (si-circRNF13) in primitive tongue squamous cell carcinoma cells (NC) or overexpression of circRNF13 (OE-circRNF13) in drug-resistant cells (CR), cells were treated with the same concentration of cisplatin , and then flow cytometry to detect cell apoptosis, and found that knocking down the expression of circRNF13 can increase the resistance of cells to cisplatin (decreased apoptosis), while overexpressing circRNF13 can significantly reduce the drug resistance of drug-resistant cells.

Figure 10: The expression level of circRNF13 was detected by qRT-PCR in 28 pairs of fresh tongue squamous cell carcinoma biopsy tissues (T) and paracancerous control tissues (N), and it was found that the expression of circRNF13 was significantly down-regulated in tongue squamous cell carcinoma biopsy tissues.

Figure 11: The expression of circRNF13 was detected in archived tongue squamous cell carcinoma (right) and paracancerous control tissues (left) by in situ hybridization, which further confirmed that circRNF13 was less or not expressed in tongue squamous cell carcinoma biopsy tissues.

Figure 12: We collected 88 cases of tongue squamous cell carcinoma tissue specimens with clinical follow-up data. The expression of circRNF13 was detected by in situ hybridization, and the expression of circRNF13 was not detected in 21 cases (Negative). The survival time of these patients was longer than that of other patients (Although the expression level of circRNF13 is not high, it can be detected, Positive) is shorter and the prognosis is worse.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, rather than limiting the present invention.

Example 1, PCR sequencing determined the full-length sequence of circRNF13 in tongue squamous cell carcinoma cells

1. Materials and methods

1.1 Cell lines

Tongue squamous cell carcinoma cells Tca8113 and Cal27 were purchased from the Cell Center of Central South University and cultured under conventional conditions.

1.2 Reagents and kits

TRIZOL ^TM Reagent (Invitrogen); Gel Recovery Kit (OMEGA); Reverse Transcription Kit (Promega); Proteinase K, DNase I, RNAsin, RNase A (GBICOL Company); Enzyme (Takara).

1.3 Real-time quantitative PCR detection of circRNF13 expression in tongue squamous cell carcinoma cells

Total RNA was extracted, and 1 μg of RNA was reverse-transcribed into cDNA, followed by real-time fluorescent quantitative PCR. The forward primer of circRNF13 is 5-GTCCAGGATAGACATAGAGC-3, as shown in SEQ NO:2, and the reverse primer is 5-GTGTAGACTTGTGTGGCTGA-3. As shown in SEQ NO:3.

The GAPDH forward primer used for the control is 5'-ACCACAGTCCATGCCATCAC-3' as shown in SEQ NO:4, and the reverse primer 5'-TCCACCACCCTGTTGCTGTA-3' as shown in SEQ NO:5.

Real-time fluorescence quantitative PCR reaction system

Real-time fluorescent quantitative PCR reaction steps

After the reaction, the amplification curve and melting curve of real-time fluorescent quantitative PCR were confirmed, and the expression intensity of each gene was standardized according to the CT value (threshold cycle values) and the internal reference gene (GAPDH), and the P value was calculated by group t-test test.

Finally, we sequenced the fragments amplified by real-time fluorescent quantitative PCR, compared the sequences of the amplified fragments, confirmed that what we amplified was circRNF13, and obtained the specific site of circRNF13 circular splicing.

1.4 Reverse transcription PCR to obtain the full length of circRNF13

1) Using tongue squamous cell carcinoma cell Tca8113 cDNA as a template, two pairs of primers were designed to amplify the full-length circRNF13 sequence by overlapping and nesting PCR twice (primer design and PCR amplification sequence are shown in Figure 2). The primers for amplification of the full-length sequence of circRNF13 are as follows:

The first amplification, upstream primer: 5-GCTAGAAGAAACAGACTTCGTAAA-3; as shown in SEQ NO:6;

Downstream primer: 5-CTAATGAGGTCATCAGAATCAACA-3, as shown in SEQ NO:, 7;

Second amplification, upstream primer: 5-AATGTTGATTCTGATGACCT-3, as shown in SEQ NO:8,

Downstream primer: 5-AGATTGTGTAGACTTGTGTGG-3, as shown in SEQ NO:9.

2) PCR amplification, the full-length sequence of circRNF13, the PCR reaction conditions are as follows:

PCR reaction steps

3) The PCR product was gelled to recover the target fragment and then sequenced and analyzed.

2. Results

2.1 The expression of circRNF13 was significantly increased in tongue squamous cell carcinoma cells treated with cisplatin

After tongue squamous cell carcinoma cells were treated with cisplatin (P), we detected the expression of circRNF13 in the cells by real-time fluorescent quantitative PCR, and found that the expression level of circRNF13 was significantly increased compared with the negative control (NC) (Fig. 1, left); we In order to confirm that the real-time fluorescent quantitative PCR detection is indeed circRNF13, we performed a sequencing analysis on the real-time fluorescent quantitative PCR product and found that the amplified fragment was indeed the splicing of the 3' end of exon 8 and the 5' end of exon 2 of RNF13 , is indeed a new circRNA molecule.

2.2 We amplified and sequenced to confirm the full-length sequence of circRNF13

Because circRNF13 is a new circular RNA molecule, its specific sequence, especially the transcript sequence expressed in tongue squamous cell carcinoma needs further confirmation. We designed two pairs of primers and successfully cloned the full-length sequence of circRNF13 by two nested PCRs, confirming that circRNF13 is indeed derived from the RNF13 gene (ring finger protein 13, NM_183381.2) located on chromosome 3q25.1. Exons 2-8 were formed by circular splicing ( FIG. 2 ), with a full length of 716 bp (as shown in SEQ NO: 1).

Example 2, circRNF13 inhibits cell cycle arrest and induces apoptosis in tongue squamous cell carcinoma cells

1. Materials and methods

1.1 Reagents and kits

Restriction enzymes Cla I and Sac II and T4 DNA ligase were purchased from TakaRa Company; TRIZOL ^TM Reagent (Invitrogen); Plasmid Extraction Kit, Gel Recovery Kit (OMEGA); Reverse Transcription Kit (Promega); Protease K, DNase I, RNAsin, RNase A (GBICOL); tetramethylazolazolium blue (MTT, Sigma); antibiotic G418 (Ameresc); cell cycle detection kit (Invitrogen), cell apoptosis detection kit (Invitrogene ).

1.2 Induction and culture of tongue squamous cell carcinoma drug-resistant cells

In the cell culture medium, a low dose of cisplatin was added, and the concentration of cisplatin was gradually increased. After a long period of induction culture, a drug-resistant cell line resistant to cisplatin was finally obtained.

1.3 Construction of circRNF13 eukaryotic expression vector

We first inserted the upstream and downstream looping sequences at the multiple cloning site in the pcDNA3.1 vector (derived from Invitrogen), namely GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTT as shown in SEQ NO: 10, GAAAAGAATTAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCA as shown in SEQ NO: 11) to help insert The fragment is transcribed and processed into circular RNA; this is the circular RNA eukaryotic expression blank expression vector; the specific construction process is as follows:

According to the formation mechanism of circRNA and the basic rules of eukaryotic RNA splicing, the circular sequence suitable for circRNA expression is designed; according to the sequence information of the commercial vector pcDNA3.1, an appropriate multiple cloning site is designed to achieve a complete realization DNA sequence of circRNA overexpression:

GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGGCCCACTTTTT CTTAAGCTTGGTACCGAGCTCGG ATCCACATCGATTGGTGGAATTCTGCAGATATCCACCGCGGTGGCGGCCGCTCGAGTCTAGA GAAAAGAATTAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCA , as shown in SEQ NO: 12;

The underlined part in the middle is the multiple cloning site, and the two ends are the upstream and downstream looping sequences respectively;

Add the NheI restriction site sequence to one end of the DNA sequence synthesized above to realize the overexpression of circRNA, and add the ApaI restriction site sequence to the other end, and construct it into the pcDNA3.1 vector after digestion with NheI and ApaI to obtain the pcCirc blank plasmid ( The sequence is shown in SEQ NO:13), and sequencing confirmed that the plasmid was correct.

In order to construct the circRNF13 overexpression vector. We selected Cla I and Sac II restriction sites for digestion of the pcCirc blank plasmid (that is, the pcDNA3.1 vector constructed above with the upstream and downstream circular sequences inserted), and inserted the circRNF13 sequence into the upstream and downstream components of the vector. between loop sequences (Fig. 3, left).

The steps to construct the circRNF13 overexpression vector (i.e. eukaryotic expression vector) are as follows:

1) Using the Tca8113 cDNA of tongue squamous cell carcinoma cells as a template, using TaKaRa LA Enzyme PCR was performed to amplify the full-length circRNF13 sequence. The primers for amplification of the full-length sequence of circRNF13 are as follows:

Upstream primer: 5'-GTGATTTTACAACGAGAT-3' as shown in SEQ NO:14;

Downstream primer: 5'-CTTTCTTGAATTTATGTA-3' as shown in SEQ NO:15;

After adding restriction endonuclease Cla I and Sac II recognition sites and protective bases to the 5' ends of the upstream and downstream primers, the primer sequences are as follows:

Upstream: 5'-AGGA ATCGAT GTGATTTTACAACGAGAT-3' (the underlined part is the Cla I recognition site) as shown in SEQ NO:16;

Downstream: 5'-ATGC CCGCGG CTTTCTTGAATTTATGTA-3' (the underlined part is the Sac II recognition site) as shown in SEQ NO:17;

2) PCR amplification, the full-length sequence of circRNF13, the PCR reaction conditions are as follows:

PCR reaction steps

3) Electrophoresis of the PCR product, gel recovery of the target fragment, electrophoresis after Cla I and Sac II double enzyme digestion, and gel recovery again;

4) The pcCirc blank plasmid was double digested with Cla I and Sac II, and the target fragment was recovered by electrophoresis gel;

5) Ligate the gel recovery product of steps 3) and 4) with T4 DNA ligase to obtain a vector plasmid that can be used for eukaryotic expression of circRNF13;

6) The eukaryotic expression plasmid containing the full-length sequence of circRNF13 obtained in step 5) was transformed into competent Escherichia coli to amplify the plasmid.

1.4 circRNF13 small RNA interference sequence (siRNA)

We designed a specific small interfering RNA (siRNA, Figure 4, the position of the siRNA is shown on the left) sequence for the splicing site of circRNF13 as follows:

5-AGAAAGGUGAUUUUACAACGA-3 is shown in SEQ NO:18,

At the same time, select the Scramble sequence that does not have a target in the human genome sequence as the control of siRNA:

The Scramble sequence is as follows:

5'-GACACGCGACUUGUACCAC-3'. As shown in SEQ NO:19,

The sequence was synthesized by Invitrogen Company.

1.5 Preparation of polylysine-coated silicon nanoparticles

Polylysine-coated silicon nanoparticles were synthesized by using OP-10/cyclohexane/ammonia microemulsion self-assembly technology to synthesize silicon nanoparticles (SiNPs), and using the surface energy of silicon nanoparticles and passing Electrostatic interaction of ions to prepare polylysine-modified silicon nanoparticles; the nanoparticles can be prepared by the following method:

1) Mix OP-10 (nonylphenol polyoxyethylene ether), cyclohexane and ammonia water, stir evenly at room temperature, add orthosilicate isoester (TEOS), continue stirring until the polymerization is complete, add an equal volume of acetone, and ultrasonically disperse , centrifuged, washed three times with double distilled water, centrifuged to collect the precipitate, dried at 80°C, and ground to obtain silicon nanoparticles (SiNP, particle size range 10-50nm). The molar ratio of H ₂ O to OP-10 and H ₂ O to TEOS is 2-10, the concentration of ammonia water is 1.6-28%, and the molar concentration of TEOS in cyclohexane is 0.1-3 mol/L.

2) Resuspend SiNP in 0.1-10 mg/ml in 0.6M NaCO ₃ solution, ultrasonically disperse, centrifuge, discard the supernatant, then re-suspend the precipitate in PBS (pH 7.4) at 0.1-10 mg/ml, ultrasonically disperse Disperse, add poly-lysine (final concentration: 4-15nmol/mL), mix well, and shake at room temperature; centrifuge, discard the supernatant, and resuspend the precipitate in double-distilled water at 0.1-10 mg/ml to obtain poly-lysine Lysine-modified silicon nanoparticles.

3) Ultrasonic disperse the modified silicon nanoparticles, add 10-50ug circRNF13 overexpression vector or 10-100pmol siRNA per milliliter nanoparticle suspension, mix, and let stand at room temperature to combine.

1.6 Cell culture and transfection

The tongue squamous cell carcinoma cells Tca8113 and Cal27 in good growth state or drug-resistant cells were inoculated in 6-well plates at ² ×105 cells/well, and the 6-well plates were placed in a 37°C, 5% _CO2 incubator until The cultured cells grow to 50-70% density to start the transfection of circRNF13 overexpression vector or siRNA; the transfection process is as follows:

Add 100 μl of prepared polylysine-modified silicon nanoparticle suspension carrying circRNF13 eukaryotic expression plasmid or siRNA to a sterile EP tube, mix gently with 100 μl serum-free medium; wash cells with D-Hank's solution 3 times; add 800 μl of serum-free medium (without antibiotics) to the above mixture, mix gently and add to 1 well of a 6-well plate; place the 6-well plate in a CO ₂ incubator, and incubate at 37°C for 6 hours, Then discard the supernatant and add complete medium to continue culturing overnight. Silicon nanoparticles modified with empty vector or poly-lysine of Scramble sequence were used as experimental control. 1.7 Real-time fluorescent quantitative PCR to detect the expression level of circRNF13 in cells

After the cells were treated, the cells were collected at an appropriate time point, and the total RNA was extracted. After 1 μg of RNA was reverse-transcribed into cDNA, real-time fluorescent quantitative PCR was performed. The forward primer of circRNF13 is 5-GTCCAGGATAGACATAGAGC-3, as shown in SEQ NO:2, and the reverse primer is 5-GTGTAGACTTGTGTGGCTGA-3. As shown in SEQ NO:3.

The GAPDH forward primer used for the control is 5'-ACCACAGTCCATGCCATCAC-3' as shown in SEQ NO:4, and the reverse primer 5'-TCCACCACCCTGTTGCTGTA-3' as shown in SEQ NO:5.

Real-time fluorescence quantitative PCR reaction system

Real-time fluorescent quantitative PCR reaction steps

After the reaction, the amplification curve and melting curve of real-time fluorescent quantitative PCR were confirmed, and the expression intensity of each gene was standardized according to the CT value (threshold cycle values) and the internal reference gene (GAPDH), and the P value was calculated by group t-test test.

1.8 MTT cell proliferation assay

1) Digest the cells obtained in the previous step, count the cells with a cell counter, inoculate cells transfected with circRNF13 and pcDNA3.1 empty vectors in a 96-well plate, inoculate 1000 cells per well, and inoculate 5 wells for each cell , and the result takes its mean value.

2) Incubate in a 5% CO ₂ incubator at 37° C. for 6 hours. After the cells adhere to the wall, add 20 μl of MTT solution (5 mg/ml) to each well. Continue to incubate for 4 hours, terminate the culture, and discard the culture medium. Add 150 μl DMSO to each well and shake for 10 minutes to dissolve the crystals.

3) Select a wavelength of 490nm, set the zero-adjustment well at the same time, measure the absorbance value of each well on the enzyme-linked immunosorbent assay instrument and record the result.

4) Repeat the steps above every 24 hours for a total of 6 days. The MTT curve is drawn with the absorbance value as the ordinate and the interval time as the abscissa.

5) The experiment was repeated three times. Taking each time point as the abscissa and the absorbance value as the ordinate, draw the cell growth curve.

1.9 Analysis of cell cycle by flow cytometry

1) When the cultured cells reach 85% confluency, digest the cells with trypsin, centrifuge at 1200rpm/min for 5min, and collect the cell pellet.

2) Resuspend the cells in 1xPBS, centrifuge at 1200rpm/min for 5min, and collect the cells. Repeat this step 2 times.

3) Resuspend the cells in 1xPBS, add pre-cooled 70% ethanol to fix the cells overnight.

4) Centrifuge at 1000 rcf/min for 5 min to collect the cell pellet.

5) Wash the cells once with pH 7.4 PBS, add PBS to resuspend the cells after centrifugation, and adjust the cell concentration to 1x 10 ⁶ /ml.

6) Add propidium iodide (PI) staining solution (containing 50mg/L PI, 1g/L Triton X-100, 100g/L RNase) to mix well, and incubate at 4°C in the dark for 30min.

7) Detection by flow cytometer FACStar (BD Company, USA). The received signal was processed by Cellquest software, and the fluorescence intensity of the detected cells was analyzed. The experiment was repeated three times.

1.10 Analysis of cell apoptosis rate by flow cytometry

1) When the cultured cells reached 85% confluency, the cells of each group were digested with trypsin and collected in a centrifuge tube, and the supernatant suspended cells of each group were collected at the same time. Take care to gently pipette the cells to avoid overdigestion with trypsin.

2) Merge the cells of each group and transfer them to centrifuge tubes, centrifuge at 1000rpm for 5min, discard the supernatant to collect the cells, resuspend gently in PBS, and count the cells.

3) Take 50,000 to 100,000 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and add 195ul Annexin V-FITC conjugate solution to gently resuspend the cells.

4) Add 5ul Annexin V and mix gently. Incubate at room temperature for 10 min in the dark.

5) Centrifuge at 1000rpm for 5min, discard the supernatant, and add 190ul Annexin V-FITC binding solution to gently resuspend the cells.

6) Add 10ul PI staining solution, mix gently, and keep in an ice bath to avoid light.

7) Immediately perform flow cytometry detection, Annexin V-FITC is green fluorescence, PI is red fluorescence.

2. Results

2.1 circRNF13 inhibits the growth of tongue squamous cell carcinoma cells

After transfection of the circRNF13 eukaryotic expression vector, we detected the expression of circRNF13 in the cells by real-time fluorescent quantitative PCR, and found that the expression level of circRNF13 was significantly increased (Figure 3); after transfection of small interfering RNA (siRNA) targeting circRNF13, The expression level of circRNF13 in cells was significantly down-regulated (Figure 4);

Compared with cells transfected with empty vectors, the growth rate of tongue squamous cell carcinoma cells Tca8113 and Cal27 overexpressing circRNF13 was significantly slowed down, while the growth rate of cells was accelerated after knocking down the expression of circRNF13 with siRNA interference sequence (Figure 5).

2.2 circRNF13 inhibits the growth of tongue squamous cell carcinoma cells by blocking the cell cycle and inducing apoptosis.

Flow cytometry analysis showed that after transfection of circRNF13 in tongue squamous cell carcinoma cells, the proportion of cells in G2/M phase increased significantly, while the proportion of cells in S phase decreased, indicating that circRNF13 can block tongue squamous cell carcinoma cells in G2/M phase, It slows down the speed of cell division (Figure 6). At the same time, the proportion of apoptotic cells increased significantly after transfection of circRNF13 in tongue squamous cell carcinoma cells (Figure 7), which is one of the reasons why circRNF13 inhibits the growth of tongue squamous cell carcinoma cells. Knocking down circRNF13 gave the opposite result.

2.3 Down-regulation of circRNF13 in tongue squamous cell carcinoma drug-resistant cells

We gradually increased the concentration of cisplatin in the culture medium and successfully induced Tca8113 and Cal27 cells resistant to cisplatin after several months of culture. Real-time quantitative PCR detected the expression level of circRNF13 in the cells and found that circRNF13 was in The expression in the drug-resistant cell line was significantly down-regulated compared with the original cell line (Figure 8).

2.4 Overexpression of circRNF13 can reverse the drug resistance phenotype of tongue squamous cell carcinoma cells

After knocking down the expression of circRNF13 (si-circRNF13) in primitive tongue squamous cell carcinoma cells (NC) or overexpressing circRNF13 (OE-circRNF13) in drug-resistant cells (CR), the cells were treated with the same concentration of cisplatin, and then flowed Cytometry was used to detect cell apoptosis, and it was found that knocking down the expression of circRNF13 could increase the resistance of cells to cisplatin (decreased apoptosis), while overexpressing circRNF13 could significantly reduce the drug resistance of drug-resistant cells (Figure 9).

Example 3, real-time fluorescent quantitative PCR method detection confirmed the down-regulation of circRNF13 in tongue squamous cell carcinoma

1. Materials and methods:

28 adjacent tongue squamous cell carcinoma tissues and 28 tongue squamous cell carcinoma tissues were collected, total RNA was extracted, 1 μg RNA was reverse transcribed into cDNA, and real-time fluorescent quantitative PCR was performed. The forward primer of circRNF13 is 5-GTCCAGGATAGACATAGAGC-3, as shown in SEQ NO:2, and the reverse primer is 5-GTGTAGACTTGTGTGGCTGA-3. As shown in SEQ NO:3.

The GAPDH forward primer used for the control is 5'-ACCACAGTCCATGCCATCAC-3' as shown in SEQ NO:4, and the reverse primer 5'-TCCACCACCCTGTTGCTGTA-3' as shown in SEQ NO:5.

Real-time fluorescence quantitative PCR reaction system

Real-time fluorescent quantitative PCR reaction steps

After the reaction, the amplification curve and melting curve of real-time fluorescent quantitative PCR were confirmed, and the expression intensity of each gene was standardized according to the CT value (threshold cycle values) and the internal reference gene (GAPDH), and the P value was calculated by group t-test test.

2. Results

The expression of circRNF13 was higher in paracancerous control tissues, while the expression in tongue squamous cell carcinoma tissues was significantly reduced P<0.01 (Figure 10).

Example 4, in situ hybridization detection found that the low expression of circRNF13 in tongue squamous cell carcinoma was associated with poor prognosis of patients

1. Material method

1.1 Design and synthesis of hybridization probes

In order to detect the expression of circRNF13 by in situ hybridization, we designed an oligonucleotide probe for the circular splicing site of circRNF13 (that is, the splicing site of exon 2 and exon 8 of the RNF13 gene), and used Three in situ hybridization oligonucleotide probes were used as positive controls.

The oligonucleotide probe used to detect the expression of circRNF13 by in situ hybridization: TCGTTGTAAAATCACCTTTCTTGAATTTAT, as shown in SEQ NO:20,

Positive control probe (to detect the housekeeping gene GAPDH):

GAPDH probe 1: 5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3', as shown in SEQ NO:21,

GAPDH probe 2: 5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3', as shown in SEQ NO:22,

GAPDH probe 3: 5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3', as shown in SEQ NO:23.

The gene-specific oligonucleotide probe sequences designed above were synthesized by chemical synthesis method.

1.2 Oligonucleotide probe labeling kit and in situ hybridization detection reagent

Digoxigenin oligonucleotide tailing reagent (Dig Oligonucleotide Tailing Kit 2 ^nd Generation, Roche Company), anti-digoxigenin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fabfragments, Roche Company ), the TSA signal amplification system (TSA ^TM BiotinSystem, NEL700 kit, PerkinElmer Company) to enhance the detection signal of in situ expression, DAB staining kit (Beijing Zhongshan Company), 20x sodium citrate buffer solution (saline sodium citrate, SSC), Dextran sulfate, Deionized Formamide, polyadenylic acid (Poly A), polydeoxyadenylic acid (Poly dA), denatured sheared frog Sperm DNA (denatured and sheared salmon sperm DNA, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), dithiothreitol (DTT), 50x Dunham’s buffer (Denhardts’ solution), phosphate buffer (PBS buffer ), pepsin K, bovine serum albumin (BSA), triethanolamine (TEA), TNB Buffer (0.1M Tris-HCl, pH7.5, 0.15M NaCL, 0.5% BlockingReagent), TNT Buffer (0.1M Tris-HCl , pH7.5, 0.15M NaCL, 0.05% Tween 20), acetic anhydride, blocking reagent (Blocking reagent agent, Roche Company).

1.3 Other main reagents and materials

Absolute ethanol, 90% alcohol, 70% alcohol, 50% alcohol, turpentine, double distilled water, PBS buffer (pH7.2～7.4, NaCl 137mmol/L, KCl 2.7mmol/L, Na ₂ HPO ₄ 4.3mmol/ L, KH ₂ PO ₄ 1.4mmol/L); 3% methanol-hydrogen peroxide solution (80% methanol and 30% hydrogen peroxide solution); 0.01mol/L citrate buffer (citrate buffer, CB, pH6.0±0.1, Add 9ml 0.1M citric acid solution and 41ml 0.1M sodium citrate solution to 450ml distilled water for temporary preparation and then correct the pH value of the working solution); 0.1% trypsin; hematoxylin; 1% hydrochloric acid alcohol (1ml concentrated hydrochloric acid + 99ml70% alcohol preparation) ; Mounting glue (PTS Cure Mount II); special cover glass (480×240mm ² ) was custom-made in Zhengzhou Glass Instrument Factory. Leica low melting point (58°C) paraffin, domestic beeswax, absolute alcohol, xylene, 10% neutral paraformaldehyde (0.01mol/L, pH7.4, prepared with DEPC double distilled water and PBS buffer), hematoxylin, i Red, neutral mounting gum, coverslip, glass slide.

1.4 Labeled probes

The 3-tailing DIG Oligucleutide Kit is used for oligonucleotide probe labeling, and the reaction system is as follows.

100pmol oligonucleotide+ddH ₂ O＝9μl (control: control oligonucleotide 5μl+ddH ₂ O 4μl)

Mix well and centrifuge slightly. React in a water bath at 37°C for 30 min, then add 2 μl of EDTA (0.2M, pH 8.0) to terminate the reaction.

1.5 Purification after oligonucleotide probe labeling

In order to increase the purity of labeled probes, the labeled probes need to be purified as follows:

1) Probe reaction mixture (22μl)+2.5μl 4M LiCL+75μl 100% cold ethanol (-20℃).

2) Precipitate at -70°C for 60 minutes, or at -20°C for 2 hours.

3) Centrifuge at 13.000×g at 4°C for 15 minutes.

4) Discard the supernatant and wash with 50 μl of ice-cold 70% (V/V) ethanol.

5) Centrifuge at 13.000xg at 4°C for 5 minutes.

6) Discard the supernatant and dry it under vacuum at 4°C.

7) Redissolve the probe with sterile double distilled water.

1.6 In situ hybridization detection of circRNF13 expression in archived paraffin sections

Pretreatment of paraffin sections for hybridization

1) Paraffin sections stored at 4°C were placed on a 58°C baking sheet for 30 minutes to melt the paraffin wax on the surface.

2) Xylene was dewaxed sequentially for 3×5min.

3) Stepwise alcohol washing, 100% alcohol 2×2min→95% alcohol 1×5min→70% alcohol 1×5min→50% alcohol 1×5min→DEPC water washing 2×3min→DEPC-PBS washing 2×5min.

4) Add 300 μl of pepsin K (10 μg/ml) dropwise on the slice, and digest at 37° C. for 20 minutes.

5) Wash the slices in PBS (0.1M PBS+2 mg/ml glutamic acid) for 1 min, and stop the reaction.

6) Slice into 0.2N HCL and react at 37°C for 20-30min to increase tissue permeability.

7) The slices were fixed with 4% paraformaldehyde (dissolved in 0.1M PBS) for 10 min at room temperature.

8) In order to increase the positive hybridization intensity of the tissue, acetylate the slices. Slice into 0.25% acetic anhydride Buffer I (0.1M triethanolamine), room temperature for 10 min.

9) Wash with 1M PBS for 2×5 min.

prehybridization and hybridization

Pre-hybridization: The pre-hybridization solution stored at -20°C was first incubated at 37°C for 60 minutes, the amount of the pre-hybridization solution was 50 μl, paraffin film covered sections, and pre-hybridization in a 37°C humid box for 2 hours. (Prehybridization solution components include: 2XSSC, 10% Dextransulphate, 1X Denhardt's solution, 50mM Phosphate Buffer (PH 7.0), 50mM DTT, 250μl, 100μg/ml poly A, 5μg/ml poly dA, 250μg/ml yeast t-RNA, 500 μg/ml ssDNA, 47% Deionized formamide).

1) Remove the paraffin film, shake off the pre-hybridization solution, and place the slice in 2×SSC for 5 minutes.

2) Hybridization reaction: hybridize overnight at 37°C (18-20h). Add 250 μl of hybridization solution to each section and cover with parafilm. The corresponding probes are added to the pre-hybridization solution to become the hybridization solution. The hybridization solution was prepared during pre-hybridization, and incubated at 37°C to fully dissolve the probes in the hybridization solution. In this experiment, multiple oligonucleotide probes were mixed, and each probe was prepared at a concentration of 500ng/ml for probe hybridization. liquid. Digoxigenin Tail Labeling Kit Labeling Probe Concentration Calculation Basis: The concentration of each probe is compared with the color development of the detection reaction of the positive quantitative probe and the 100pmol 30-base naked probe labeling reaction theory Probe yield is 900ng two kinds of standards are combined to calculate the concentration of labeled probe.

3) Wash after hybridization, immerse the slice in 2×SSC for 10 min, and remove the parafilm. Shake and wash sequentially on a shaker, 2×SSC (0.5% SDS), 2×15 min→0.25×SSC (0.5% SDS), 2×15 min.

Chromogenic detection reaction after hybridization

1) Anti-Digoxigenin-POD is used to detect the complex of digoxin probe and mRNA; TSA amplification system enhances the positive signal of in situ hybridization reaction color reaction, and DAB color development.

2) The slices were transferred to TNT buffer, 3×5 min.

3) Add TNB blocking buffer dropwise, 300 μl/TMAs, room temperature, 30 min.

4) Suck off excess blocking agent, dilute Anti-Digoxigenin-POD (TBS+0.1% Triton X-100+1% blocking agent) at 1:100, and keep at room temperature for 4 hours.

5) Washing with TNT Buffer (0.1M Tris-CL, pH7.5, 0.15M NaCL, 0.05% Tween 20), 3x5min.

6) Add signal amplification reagent Biotinyl Tyramid, 300 μl/TMAs, (Biotinyl Tyramid stock solution: Biotinyl Tyramid dissolved in 0.2ml DMSO, Biotinyl Tyramid working solution: 1× dilution, 1:50 dilution of Biotinyl Tyramid stock solution) on the slice, 10 minutes at room temperature.

7) Wash with TNT, 3×5min.

8) SA-HRP (streptavidin-horseradish peroxidase) was added dropwise to the slices, 300 μl/TMAs, at room temperature for 30 min.

9) Wash with TNT, 3×5min.

10) Wash with distilled water, 1×1 min.

11) DAB color development, the color reaction is controlled under the microscope.

12) Hematoxylin counterstaining,

13) Alcohol cascade dehydration, slices are dried.

14) Add mounting glue dropwise, cover slips of corresponding specifications, and cross-link sections under ultraviolet light for 1 min.

1.7 Results Judgment and Standards

Use an optical microscope to observe under low magnification and high magnification respectively, and first observe the location of the positive expression signal of the target RNA in the observed target cell: located in the nucleus, cytoplasm or cell membrane.

Then, the intensity of the positive signal at the detected RNA expression site and the number of positively expressed cells were used for comprehensive scoring, and the judging criteria were: (1) Judgment based on the staining intensity of positive cells: a. No staining, 0 points; b. . Cells stained light brown as weak positive, score 1 point; c. Cells stained brown without background staining, or cells stained dark brown with light brown background, medium positive, scored 2 points; d. Cells stained dark brown Brown and no background coloring is strong positive, score 3 points. (2) Score based on positive cell expression: a. no positive cell expression, score 0; b. positive cell number ≤ 25%, score 1 point; c. 25% < positive cell number < 50%, score 2 Points; d. The number of positive expression cells ≥ 50%, score 3 points.

In order to reduce the subjective factors of the scoring results as much as possible, two pathologists judged and scored according to one of the above-mentioned standards, and then multiplied the two scores. ; ② 1 point and 2 points are finally counted as 1 point, which is considered as weak positive expression; ③ 3 and 4 points are finally counted as 2 points, which are considered as moderate positive expression; ④ 6 to 9 points are finally counted as 3 points, which are considered as strong positive expression Express.

1.8 Analytical and Statistical Software

SPSS13.0 statistical software was used to carry out statistical analysis on the experimental results, pairwise comparison was performed by χ ² test or Fisherexact test, and correlation analysis was performed by Spearmen correlation method; P<0.05 means the difference was statistically significant. Kaplan-Meier method and log-rank test were used for survival curve analysis; Cox'sproportional hazards model was used for multivariate analysis; P<0.05 means the difference was statistically significant.

2 results

2.1 The expression of circRNF13 in tongue squamous cell carcinoma was significantly higher than that in normal control tissues

circRNF13 was not expressed or expressed at a low level in tongue squamous cell carcinoma tissues, but it was expressed in normal control tissues adjacent to tongue squamous cell carcinoma (Figure 11), and there was a significant statistical difference between the two.

2.2 The prognosis of tongue squamous cell carcinoma patients with low expression of circRNF13 is worse

88 patients with tongue squamous cell carcinoma were followed up by telephone, and they were asked in detail about the time of onset, treatment, recurrence, other diseases, recurrence and death time, etc., and the survival time and status were registered. The survival analysis of the expression of circRNF13 in tongue squamous cell carcinoma tissue and the survival time and status of patients found that the average survival time of patients who did not express circRNF13 in tongue squamous cell carcinoma tissue was significantly shorter than that of patients with higher expression of circRNF13 (Figure 12). It shows that circRNF13 is a molecular marker related to the prognosis of tongue squamous cell carcinoma. The expression of this circRNA is low or not expressed, and the prognosis of patients is poor.

sequence listing

<110> Central South University

<120> Application method of circular RNAcircRNF13

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 716

<212> RNA

<213> Homo sapiens

<400> 1

gugauuuuac aacgagaugc ugcucuccau agggaugcuc augcugucag ccacacaagu 60

cuacaccauc uugacugucc agcucuuugc auucuuaaac cuacugccug uagaagcaga 120

cauuuuagca uauaacuuug aaaaugcauc ucagaacauuu gaugaccucc cugcaagauu 180

ugguauaga cuuccagcug aagguuuaaa ggguuuuuug auuaacucaa aaccagagaa 240

ugccugugaa cccauagugc cuccaccagu aaaagacaau ucaucuggca cuuucaucgu 300

guuaauuaga agacuugauu guaauuuuga uauaaagguu uuaaaugcac agagagcagg 360

auacaaggca gccaauaguuc acaauguuga uucugaugac cucauuagca ugggauccaa 420

cgacauugag guacuaaaga aaauugacau uccaucuguc uuuauuggug aaucaucagc 480

uaauucucug aaagaugaau ucacauauga aaaagggggc caccuuaucu uaguuccaga 540

auuuagucuu ccuuuggaau acuaccuaau ucccuuccuu aucauagugg gcaucugucu 600

caucuugaua gucauuuuca ugaucacaaa auuuguccag gauagacaua gagcuagaag 660

aaacagacuu cguaaagauc aacuuaagaa acuuccugua cauaaauuca agaaag 716

<210> 2

<211> 20

<212>DNA

<213> Unknown (Unknown)

<400> 2

gtccaggata gacatagagc 20

<210> 3

<211> 20

<212>DNA

<213> Unknown (Unknown)

<400> 3

gtgtagactt gtgtggctga 20

<210> 4

<211> 20

<212>DNA

<213> Unknown (Unknown)

<400> 4

accacagtcc atgccatcac 20

<210> 5

<211> 20

<212>DNA

<213> Unknown (Unknown)

<400> 5

tccaccaccc tgttgctgta 20

<210> 6

<211> 24

<212>DNA

<213> Unknown (Unknown)

<400> 6

gctagaagaa acagacttcg taaa 24

<210> 8

<211> 24

<212>DNA

<213> Unknown (Unknown)

<400> 8

ctaatgaggt catcagaatc aaca 24

<210> 8

<211> 20

<212>DNA

<213> Unknown (Unknown)

<400> 8

aatgttgatt ctgatgacct 20

<210> 9

<211> 21

<212>DNA

<213> Unknown (Unknown)

<400> 9

agattgtgta gacttgtgtg g 21

<210> 10

<211> 45

<212>DNA

<213> Unknown (Unknown)

<400> 10

gtgctgggat tacaggtgtg agctaccacc cccggcccac ttttt 45

<210> 11

<211> 47

<212>DNA

<213> Unknown (Unknown)

<400> 11

gaaaagaatt aggctcggca cggtagctca cacctgtaat cccagca 47

<210> 12

<211> 177

<212>DNA

<213> Unknown (Unknown)

<400> 12

gtgctgggat tacaggtgtg agctaccacc cccggcccac tttttcttaa gcttggtacc 60

gagctcggat ccacatcgat tggtggaatt ctgcagatat ccaccgcggt ggcggccgct 120

cgagtctaga gaaaagaatt aggctcggca cggtagctca cacctgtaat cccagca 177

<210> 13

<211> 5515

<212>DNA

<213> Unknown (Unknown)

<400> 13

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tattacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtgctgggat tacaggtgtg agctaccacc cccggcccac ttttttttaa acttaagctt 960

ggtaccgagc tcggatccac atcgattggt ggaattctgc agatatccac cgcggtggcg 1020

gccgctcgag tctagagaaa agaattaggc tcggcacggt agctcacacc tgtaatccca 1080

gcagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 1140

gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 1200

tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 1260

ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 1320

gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 1380

ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 1440

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 1500

gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga 1560

tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 1620

gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 1680

agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 1740

ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 1800

tttaacgcga attaattctg tggaatgtgtgtcagttagg gtgtggaaag tccccaggct 1860

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 1920

agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 1980

ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 2040

ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct 2100

ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 2160

tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc 2220

gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat 2280

tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt 2340

cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac 2400

tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg 2460

tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc 2520

aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa 2580

tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc 2640

gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg 2700

aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg 2760

acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa 2820

atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg 2880

acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct 2940

tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc 3000

ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa 3060

cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat 3120

cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt 3180

cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 3240

aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 3300

caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg 3360

gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 3420

cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 3480

gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 3540

cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 3600

tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 3660

aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 3720

gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 3780

ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 3840

ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 3900

gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 3960

ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4020

cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4080

cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4140

gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 4200

aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 4260

tagctcttga tccggcaaac aaaccaccgc tggtagcggt ttttttgttt gcaagcagca 4320

gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 4380

cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 4440

cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 4500

gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 4560

tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 4620

gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 4680

agattatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 4740

tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 4800

agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 4860

gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 4920

catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 4980

ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 5040

atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 5100

tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 5160

cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 5220

cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 5280

atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 5340

aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 5400

ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5460

aaataaacaa atagggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc 5515

<210> 14

<211> 18

<212>DNA

<213> Unknown (Unknown)

<400> 14

gtgattttac aacgagat 18

<210> 15

<211> 18

<212>DNA

<213> Unknown (Unknown)

<400> 15

ctttcttgaa tttatgta 18

<210> 16

<211> 28

<212>DNA

<213> Unknown (Unknown)

<400> 16

aggaatcgat gtgattttac aacgagat 28

<210> 17

<211> 28

<212>DNA

<213> Unknown (Unknown)

<400> 17

atgcccgcgg ctttcttgaa tttatgta 28

<210> 18

<211> 21

<212> RNA

<213> Unknown (Unknown)

<400> 18

agaaagguga uuuuacaacg a 21

<210> 19

<211> 19

<212> RNA

<213> Unknown (Unknown)

<400> 19

gacacgcgac uuguacacac 19

<210> 20

<211> 30

<212>DNA

<213> Unknown (Unknown)

<400> 20

tcgttgtaaa atcacctttc ttgaatttat 30

<210> 21

<211> 30

<212>DNA

<213> Unknown (Unknown)

<400> 21

ccactttacc agagttaaaa gcagccctgg 30

<210> 22

<211> 30

<212>DNA

<213> Unknown (Unknown)

<400> 22

cagtagaggc agggatgatg ttctggagag 30

<210> 23

<211> 30

<212>DNA

<213> Unknown (Unknown)

<400> 23

gtcagaggag accacctggt gctcagtgta 30

Claims (3)

1. the application method of circular rna circRNF13, which is characterized in that the reagent for promoting circRNF13 expression is reduced in preparation Tca8113 cells are to the application on cis-platinum class drug resistance preparation, the sequence such as SEQ of the circular rna circRNF13 Shown in NO:1；The reagent for promoting circular rna circRNF13 expression is circRNF13 over-express vector.

2. application according to claim 1, which is characterized in that the building process of circRNF13 over-express vector is as follows:

(1) it according to the fundamentum of the Forming Mechanism of circRNA and eucaryote RNA montage, designs and is suitable for The loop-forming sequences of circRNA expression；According to the sequence information of commercial carrier pcDNA3.1, multiple cloning sites are designed, thus It is as follows to the complete DNA sequence for realizing that circRNA is overexpressed: GTGCTGGGATTACAGGTGTGAGCTACCACCCCCGG CCCACTTTTTCTTAAGCTTGGTACCGAGCTCGGATCCACATCGATTGGTGGAATTCTGCAGATATCCACCGCGGTG GCGGCCGCTCGAGTCTAGAGAAAAGAATTAGGCTCGGCACGGTAGCTCACACCTGTAATCCCAGCA, intermediate underscore Part is multiple cloning sites, and both ends are respectively upstream and downstream loop-forming sequences；

(2) above-mentioned synthesis is realized that NheI restriction enzyme site sequence is added in DNA sequence one end that circRNA is overexpressed, it is another End addition ApaI restriction enzyme site sequence obtains pcCirc by being built into pcDNA3.1 carrier after NheI and ApaI digestion Empty plasmid, sequencing confirmation plasmid is correct, and sequence is as shown in SEQ NO:13；

(3) selection Cla I and Sac II restriction enzyme site is used for digestion pcCirc empty plasmid, and circRNF13 sequence is inserted Enter between the upstream and downstream loop-forming sequences of carrier.

3. application according to claim 2, which is characterized in that detailed process is as follows for step (3):

1) using Tca8113 cells Tca8113 cDNA as template, PCR amplification overall length is carried out using TaKaRa LA Taq enzyme CircRNF13 sequence；CircRNF13 full length sequence amplimer is as follows:

Upstream primer: 5 '-GTGATTTTACAACGAGAT-3 '；

Downstream primer: 5 '-CTTTCTTGAATTTATGTA-3 '；

At 5 ' ends of upstream and downstream primer respectively plus restriction enzyme Cla I and Sac II recognition site and after protecting base, Primer sequence is as follows:

Upstream primer: 5 '-AGGAATCGAT GTGATTTTACAACGAGAT -3 ', underscore part are that Cla I identifies position Point；

Downstream primer: 5 '-ATGCCCGCGG CTTTCTTGAATTTATGTA -3 ', underscore part are that Sac II identifies position Point；

2) PCR amplification, circRNF13 full length sequence, PCR reaction condition are as follows:

PCR reaction step

3) by, through Cla I and Sac II double digestion rear electrophoresis, glue recycles again after PCR product electrophoresis, glue recycling target fragment；

4) pcCirc empty plasmid recycles target fragment through Cla I and Sac II double digestion rear electrophoresis glue；

5) vector plasmid of overexpression circRNF13 3) is arrived with 4) step glue recovery product with the connection of T4 DNA ligase；

6) by the eukaryon expression plasmid transformed competence colibacillus Escherichia coli comprising circRNF13 full length sequence that 5) step obtains, with Expand plasmid.