CN103740809A - miR-218 mimetic and application thereof, and method for screening drugs capable of inhibiting glioma cell migration and invasion - Google Patents

Abstract

A method for screening drugs capable of inhibiting glioma cell migration and invasion, the steps comprising: using quantitative PCR to detect the inhibitory effect of the drug to be screened on Bmi1 transcription level; transfecting the drug to be screened into glioma cells and using the Trizol method to extract Total RNA, after digesting DNA, was reverse-transcribed into cDNA by reverse transcription kit, and the transcription level of Bmi1 was detected by quantitative PCR; the migration of glioma cells was detected by scratch method; the migration of glioma cells was detected by transwell method Invasion is detected. The invention has simple process and low cost, and can effectively screen out the drugs with inhibitory effect on the migration and invasion activities of glioma cells. The invention also discloses an RNA molecule miR-218 mimic and its application in drugs for inhibiting the migration and invasion of glioma cells.

Description

miR-218 mimics, their applications and a method for screening drugs that can inhibit glioma cell migration and invasion

technical field

The invention belongs to the application field of biotechnology, and in particular relates to an application of miRNA. the

Background technique

Glioma is the most common malignant tumor of the central nervous system clinically, accounting for about 45% of all primary intracranial tumors. According to the degree of malignancy of glioma, it is divided into grades I-IV, the higher the grade, the greater the degree of malignancy. Among all glioma patients, 25% to 50% are grade II; 50% to 75% are grade III; 75% to 100% are grade IV. Patients with high degree of malignancy often have necrosis and hemorrhage in the tumor, rich blood vessels, and obvious hyperplasia of the outer layer of blood vessels and endothelial cells. Except for surgical resection, the efficacy of drugs and radiotherapy for gliomas are not ideal, so gliomas are It is one of the malignant tumors with the worst curative effect. According to statistics, the one-year overall survival rate of glioma patients is less than 30%, and the median survival period of high-grade patients is only about 15 months. One of the main reasons for the poor prognosis of gliomas is the high invasiveness of gliomas. Highly malignant gliomas show invasive growth, and the boundary between tumor components and normal brain tissue is blurred. Surgery often cannot completely remove the tumor tissue. The recurrence rate of posterior glioma is higher. More than 90% of recurrent gliomas originate from infiltrating tumor cells in the surgical field or from cancer cells at the edge of the surgical field. Understanding the migration mechanism of glioma, finding and discovering new genes and signaling pathways closely related to the occurrence, development and prognosis of glioma are of great significance for the molecular intervention of glioma. the

The proto-oncogene Bmi1 (B-cell-specific Moloney murine leukemia virus insertion site1) was first reported in 1991. It belongs to the Polycomb Group genes (PcG) member and is an important gene related to cell cycle transition and cell proliferation. Transcriptional repressor, directly involved in the regulation of cell growth and proliferation, Bmi1 gene is abundantly expressed in a variety of malignant tumors such as glioma, lung cancer, leukemia, prostate cancer, colon cancer, breast cancer, etc., and is associated with the invasion and metastasis of these tumors , Prognosis is closely related. INK4a/ARF is the target of Bmi1 gene to regulate cell proliferation and aging. In Bmi1-deficient mouse fibroblasts (MEFs) and lymphocytes, the expression level of INK4a/ARF was significantly increased. Conversely, in Bmi1-overexpressed MEF cells INK4a/ARF protein is down-regulated, and cells can be immortalized, so Bmi1 maintains the self-renewal of cells by inhibiting the expression of INK4a/ARF gene. Bmi1 is highly expressed in human glioma cells, and the tumorigenic ability of glioma cells knocked out in Bmi1 is weakened in vivo. the

MicroRNA (miR, also known as miRNA) is a kind of non-coding single-stranded RNA with a length of 20-25 nucleotides, which is a single-stranded RNA precursor with a hairpin structure of about 70-90 nucleotides. Dicer enzyme is produced after processing, and inhibits the expression of target genes at the post-transcriptional level by complementary binding with mRNA, and participates in the regulation of the occurrence and development of various diseases such as heart disease, vascular disease, nerves, and tumors. Tumor invasiveness is an important biological feature that distinguishes malignant tumors from benign tumors, and is also an important reason why malignant tumors are difficult to cure and eventually lead to death. Therefore, studying the regulatory mechanism of tumor invasion has always been the main direction of the tumor field. However, tumor invasion is a complex regulatory process involving multiple genes, so mircoRNA that can simultaneously regulate the expression of multiple target genes has become a research hotspot in the process of tumorigenesis and invasion since its discovery. the

Contents of the invention

The technical problem to be solved by the present invention is to provide a screening method for drugs that can inhibit the migration and invasion of glioma cells. The effect of tumor cell migration and invasion, so as to achieve the purpose of inhibiting glioma. the

The first aspect of the present invention relates to an RNA molecule miR-218 mimetic, the base sequence of which contains the hairpin structure of the miR-218 nucleic acid sequence "stem-loop" structure, and the main sequence is as follows: 5'-UUGUGCUUGAUCUAACCAUGU-3 ' (SEQ ID NO. 3). the

The second aspect of the present invention relates to the application of the RNA molecule miR-218 mimetic according to the first aspect of the present invention in drugs for inhibiting migration and invasion of glioma cells. the

The third aspect of the present invention relates to a method for screening drugs capable of inhibiting glioma cell migration and invasion, wherein the drug is miRNA or its mimics, the steps comprising:

(1) Use quantitative PCR to detect the inhibitory effect of the drug to be screened on the transcription level of Bmi1;

After the drug to be screened was transfected into glioma cells, the total RNA was extracted by the trizol method. After the DNA was digested, it was reverse-transcribed into cDNA by a reverse transcription kit, and the transcription level of Bmi1 was detected by quantitative PCR. the

The primers for amplifying Bmi1 are as follows:

Bmi1 gene upstream primer Bmi1-F: GCTTCAAGATGGCCGCTTG (SEQ ID NO.1),

Bmi1 gene downstream primer Bmi1-R: TTCTCGTTGTTCGATGCATTTC (SEQ ID NO.2);

(2) Detect the migration of glioma cells by scratch method;

(3) The invasion of glioma cells was detected by transwell method. the

Preferably, the quantitative PCR system in the step (1) is: 10 μl of Premix Ex Taq (SYBR qPCR) (2×), 10 μmol/L of Bmi1 upstream and downstream primers, 0.5 μl each, cDNA template to be tested or negative control 1 μl, add sterilized deionized water to make up the volume to 20 μl. the

Preferably, the PCR reaction conditions in step (1) are: pre-denaturation at 95°C for 30s; then 40 cycles of reaction at 95°C for 15s, 60°C for 20s, and 72°C for 20s. the

The step of detecting the migration of glioma cells by "scratch method" in step (2) is to transfect the glioma cells with the drug to be screened for 48 hours and then change the serum-free glioma cell culture medium to continue culturing The cells were starved for 12h. Use a sterilized pipette tip to scratch a single layer of glioma cells in one direction at a uniform speed and force, and scratch once in parallel in each well to form a cell-free scratch area. The cells were washed twice with PBS, and serum-free glioma cell culture medium was added. At 0 hour, 12 hours and 24 hours, observe the distance changes on both sides of the scratch under the microscope and calculate the distance on both sides of the scratch before and after stimulation. the

In step (3), the step of using the transwell method to detect the invasion of glioma cells is to transfect the glioma cells with the drug to be screened for 48 hours, and then dilute with serum-free glioma cell culture medium, and inoculate 5×105 Each cell was placed in a transwell chamber, and glioma cell culture medium was added to the outside. After 48 hours of culture, the upper layer of uninvaded cells was wiped off with a cotton swab, fixed with methanol for 10 minutes, dried in the air, stained with DAPI, and examined under a fluorescent microscope. Observe and take pictures, randomly count the number of cells in 10 fields of view, and judge whether the drug with screening can inhibit the invasion of glioma cells. the

Further, the above method for screening drugs capable of inhibiting glioma cell migration and invasion also includes the step of constructing pmirGLO-Bmi13'-UTR and pmirGLO-Bmi13'-UTR-mut recombinant plasmids before step (1), two kinds of plasmids The 293T cell line was co-transfected with the drug to be screened respectively. the

The construction method of the recombinant plasmid pmirGLO-Bmi13'-UTR is as follows: according to the sequence of Bmi1 on NCBI (BC011652.2), a 3'-UTR sequence with a length of 390 bp containing the miR-218 binding site was artificially synthesized, and the enzyme contained at both ends The cleavage site is Sac I and XbaI, and these two sites are used to connect into the pmirGLODual-Luciferase miRNA Target Expression Vector vector (promega). The insertion sequence is as follows:

>Bmi1-wt

ACATTTCATTGTCCCCAGTCTGCAAAAG AAGCACAA TTCTATTGCTTTGTCTTGCTTATAGTCATTAAATCATTACTTTTACATATATTGCTGTTACTTCTGCTTTCTTTAAAAATATAGTAAAGGATGTTTTATGAAGTCACAAGATACATATATTTTTATTTTGACCTAAATTTGTACAGTCCCATTGTAAGTGTTGTTTCTAATTATAGATGTAAAATGAAATTTCATTTGTAATTGGAAAAAATCCAATAAAAAGGATATTCATTTAGAAAATAGCTAAGATCTTTAATAAAAATTTGATATGAA AAGCACAA TGTGCAGAAGTTATGGAAAACCTATAGAGGATTACAACAGGTAAACGTTAAAGAGAATACATTGCTGACTTAT（SEQ ID NO.4）

The construction method of the recombinant plasmid pmirGLO-Bmi13'-UTR-mut is as follows: According to the sequence of Bmi1 on NCBI (BC011652.2), a 3'-UTR sequence with a length of 390 bp containing a mutation of the miR-218 binding site was artificially synthesized. Two 8-bp sequences in pmirGLO-Bmi13'-UTR: AAGCACAA are replaced by TTTGTGTT . The enzyme cutting sites at both ends are Sac I and XbaI, and these two sites are used to connect into the pmirGLO Dual-Luciferase miRNA Target Expression Vector vector (promega). The insertion sequence is as follows:

>Bmi1-mut

ACATTTCATTGTCCCCAGTCTGCAAAAG TTTGTGTT TTCTATTGCTTTGTCTTGCTTATAGTCATTAAATCATTACTTTTACATATATTGCTGTTACTTCTGCTTTCTTTAAAAATATAGTAAAGGATGTTTTATGAAGTCACAAGATACATATATTTTTATTTTGACCTAAATTTGTACAGTCCCATTGTAAGTGTTGTTTCTAATTATAGATGTAAAATGAAATTTCATTTGTAATTGGAAAAAATCCAATAAAAAGGATATTCATTTAGAAAATAGCTAAGATCTTTAATAAAAATTTGATATGAA TTTGTGTT TGTGCAGAAGTTATGGAAAACCTATAGAGGATTACAACAGGTAAACGTTAAAGAGAATACATTGCTGACTTAT（SEQ ID NO.5）

The recombinant plasmid map is shown in Figure 1. the

Further, the above method for screening drugs capable of inhibiting glioma cell migration and invasion, after step (1), further includes the step of: detecting the inhibitory effect of the drug to be screened on Bmi1 protein expression by Western blot method. The specific steps are:

Load an equal amount of 50 μg protein per well. The film was transferred at a constant pressure of 400mA for 100min. Put the membrane into 1% casein/TBS and shake at room temperature for 1h. Add Bmi1 antibody (Cell Signaling Technology) diluted in TBST or control α-tubulin (Santa Cruz Biotechnology) antibody primary antibody (Bmi1 antibody is diluted at 2:1000, anti-α-tubulin antibody is diluted at 1:2000), and incubated at 4°C with shaking overnight. Aspirate the primary antibody, rinse the membrane with TBST for 10min×3; add the diluted secondary antibody (both diluted 1:4000), and incubate at room temperature for 1h on a horizontal shaker. Absorb the secondary antibody, rinse the membrane with TBST, 10min×4, mix ECL (A:B=100:1 solution), fully cover the surface of the wet NC membrane, and let it stand for 1min; seal the NC membrane with plastic wrap in the dark room, and put The X-ray film was laid flat on the NC film to develop and record the data. the

Beneficial effect

The invention has simple process and low cost, can effectively screen out the drugs that can inhibit the migration and invasion activity of glioma cells, and simultaneously provides effective intervention targets for the prevention and treatment of gliomas. the

Description of drawings

Fig. 1 is recombinant plasmid map;

Figure 2 shows that computer software analysis shows that there are two targets of miR-218 in the 3'untranslated region of Bmi1;

Figure 3 shows that miR-218 mimics can inhibit the relative activity of luciferase carrying the Bmi1 target sequence;

Figure 4 shows that miR-218 mimics can inhibit the expression of Bmi1 at the transcriptional level;

Figure 5 shows that miR-218 mimics can inhibit the expression of Bmi1 at the protein level;

Figure 6 shows that miR-218 mimics effectively reduce the migration of glioma cells;

Figure 7 shows that miR-218 mimics are effective in reducing the invasion of glioma cells. the

The biochemical reagents and biological materials described in the present invention can be obtained from commercially available channels. For example, the tool cell 293T cell line can be purchased from Shanghai Baili Biotechnology Co., Ltd., and glioma cells (U251) were purchased from the Chinese Academy of Sciences Cell Bank/ Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Bmi1 antibody was purchased from Cell Signaling Technology). the

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. the

Example 1

1. Screening of miR-218 target genes

Using PicTar, TargetScan and other software to search for possible target genes of miR-218, it was found that the 3' untranslated region (3'UTR) of the Bmi1 gene contained two target sites of miR-218 (Fig. 2). Therefore, similar sequences of miR-218 may have the function of inhibiting the expression of proto-oncogene Bmi1. the

2. Reporter gene verification that Bmi1 is the target gene of miR-218

Construction of pmirGLO-Bmi13'-UTR and pmirGLO-Bmi13'-UTR-mut recombinant plasmids

Construction method: Artificially synthesized the Bmi13'-UTR sequence with a length of 390bp containing the miR-218 binding site, and ligated it into the pmirGLO Dual-Luciferase miRNA Target Expression Vector vector with the same restriction enzymes Sac I and XbaI The pmirGLO-Bmi13'-UTR vector was obtained in the middle. The pmirGLO-Bmi13'-UTR two sequences with a length of 8bp: AAGCACAA was replaced by TTTGTGTT to construct the pmirGLO-Bmi13'-UTR-mut vector. the

In the pmirGLO-Bmi13'-UTR vector, the Bmi13'-UTR sequence containing the site that can bind to miR-218 is fused with the firefly luciferase gene, so that the transcribed firefly luciferase 3'-UTR contains miR-218 binding site, so that when co-transfected with miR-218, miR-218 will inhibit the expression of firefly luciferase at the post-transcriptional level by binding to the binding site. The miR-218 binding site was mutated in the pmirGLO-Bmi13’-UTR–mut vector, so co-transfection with miR-218 did not affect the expression of firefly luciferase. Sea cucumber luciferase in the carrier is used as an internal reference, and the content of firefly luciferase in the system can be determined by measuring the activity ratio of firefly luciferase to sea cucumber luciferase, and then it can be judged whether the predicted binding site of miR-218 is really related to miR -218 combined. Specific method: pmirGLO empty vector, pmirGLO-Bmi13'-UTR vector, and pmirGLO-Bmi13'-UTR–mut were co-transfected with miR-218 mimics into tool cell 293T, and 24 hours after transfection, the cells were lysed with a special lysate. Hole 100μl, beat evenly, fully lyse the sample for later use. Using the luciferase detection kit from Promega, add 50 μl of LARII and 5 μl of lysate into a 1.5ml centrifuge tube and mix well, and detect firefly luciferase with a microplate reader; add 50 μl Stop&Glo solution to inactivate firefly luciferase, and detect with a microplate reader Sea cucumber luciferase, calculate the ratio of firefly luciferase to sea cucumber luciferase. The results showed that the expression of luciferin decreased by about 50% after transfection of the mimic of miR-218, and the expression of firefly luciferase was restored after the miR-218 mimic binding site was mutated (see Figure 3). Bmi1 was proved to be the direct target gene of miR-218. the

3. Using quantitative PCR and Western blot to detect the inhibitory effect of miR-218 mimics on the transcription level and protein expression of Bmi1

72 hours after miR-218 mimic or miR-control transfected glioma cells, total RNA was extracted by trizol method, after DNA was digested, it was reverse transcribed into cDNA by reverse transcription kit; the quantitative PCR system was: 10 μl of Premix Ex Taq (SYBR qPCR) (2×), 0.5 μl of 10 μmol/L upstream and downstream primers of Bmi1, 1 μl of cDNA template to be tested or negative control, add sterilized deionized water to make up the volume to 20 μl. The PCR reaction conditions were: pre-denaturation at 95°C for 30s; then 40 cycles of 95°C for 15s, 60°C for 20s, and 72°C for 20s. It was found that after increasing the expression of miR-218 mimics in glioma cells, the expression of Bmi1 transcript level decreased significantly. the

Seventy-two hours after miR-218 mimic or miR-control transfected glioma cells, cell protein samples were collected for electrophoresis. During electrophoresis, load an equal amount of 50 μg protein per well. The film was transferred at a constant pressure of 400mA for 100min. Put the membrane into 1% casein/TBS and shake at room temperature for 1h. Add Bmi1 antibody (Cell Signaling Technology) diluted in TBST or control α-tubulin (Santa Cruz Biotechnology) antibody primary antibody (Bmi1 antibody is diluted at 2:1000, anti-α-tubulin antibody is diluted at 1:2000), and incubated at 4°C with shaking overnight. Aspirate the primary antibody, rinse the membrane with TBST for 10min×3; add the diluted secondary antibody (both diluted 1:4000), and incubate at room temperature for 1h on a horizontal shaker. Absorb the secondary antibody, rinse the membrane with TBST, 10min×4, mix ECL (A:B=100:1 solution), fully cover the surface of the wet NC membrane, and let it stand for 1min; seal the NC membrane with plastic wrap in the dark room, and put The X-ray film was laid flat on the NC film to develop and record the data. The results showed that after increasing the expression level of miR-218 mimics in glioma cells, the expression level of Bmi1 protein decreased significantly. (See Attachment 5)

4. Migration assay of glioma cells

The migration activity of glioma cells was studied by "scratch method". MiR-218 mimics or miR-control transfected glioma cells for 48 hours, then changed to serum-free glioma cell culture medium and continued to culture cells for 12 hours to starve them. At this time, the cells should cover the bottom of the culture plate, forming a single layer of cells. Use a 200 μl sterilized pipette tip to scratch a single layer of glioma cells in one direction at a constant speed and force, and scratch once in parallel in each well to form a cell-free scratch area. The cells were washed twice with PBS, and then added to serum-free glioma cells for culture. Mark the scratched edge of the cells on the bottom of the culture plate with a marker pen and take a picture under the microscope. At 0 hour, 12 hours and 24 hours, observe the distance change on both sides of the scratch and take pictures. Image-pro plus6.0 software was used to calculate the distance on both sides of the scratch before and after stimulation. It was found that the migration activity of glioma cells transfected with miR-control was greatly increased, and the scratch healing rate was 25.1% at 12 hours and 43.2% at 24 hours. However, using miR-218 mimics to increase the expression of miR-218 in glioma cells can significantly inhibit the migration of glioma cells to the scratch area, and the scratch healing rate is 19.2% at 12 hours and 34.2% at 24 hours (see appendix Figure 6). the

5. Invasion assay of glioma cells

The invasive activity of glioma was studied by transwell method. MiR-218 mimic or miR-control transfected glioma cells for 48 hours, then diluted with serum-free glioma cell culture medium, planted 5× ¹⁰⁵ cells in a transwell chamber, and added glioma cell culture medium outside After culturing for 48 hours, wipe off the uninvaded cells in the upper layer with a cotton swab, fix with 100% methanol for 10 minutes, air-dry, add DAPI to stain the nucleus, observe and take pictures under a fluorescent microscope, and randomly count the number of cells in 10 fields of view , found that using miR-218 mimics to increase the expression of miR-218 in glioma cells can significantly inhibit the invasion of glioma cells (see Figure 7).

The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art shall be within the scope of protection defined by the claims. the

Claims (9)

1. An RNA molecule miR-218 mimic whose base sequence comprises the following sequence: 5'-UUGUGCUUGAUCUAACCAUGU-3'. 2. The application of the RNA molecule miR-218 mimic as claimed in claim 1 in inhibiting glioma cell migration and invasion drugs. 3. A method for screening drugs that can inhibit glioma cell migration and invasion, wherein the drug is miRNA or an analog thereof, the steps comprising: (1) Use quantitative PCR to detect the inhibitory effect of the drug to be screened on the transcription level of Bmi1; After the drug to be screened was transfected into glioma cells, the total RNA was extracted by Trizol method, after DNA was digested, it was reverse transcribed into cDNA by a reverse transcription kit, and the transcription level of Bmi1 was detected by quantitative PCR. The primers for amplifying Bmi1 are as follows: Bmi1 gene upstream primer Bmi1-F: GCTTCAAGATGGCCGCTTG, Bmi1 gene downstream primer Bmi1-R: TTCTCGTTGTTCGATGCATTTC; (2) The migration of glioma cells was detected by the scratch method; (3) The invasion of glioma cells was detected by transwell method. 4. The method according to claim 3, wherein, the quantitative PCR system in the step (1) is: 10 μl of Premix Ex Taq (2×), 0.5 μl of 10 μmol/L of Bmi1 upstream and downstream primers, to be tested cDNA template or negative control 1μl, add sterilized deionized water to make up the volume to 20μl. 5. The method according to claim 4, wherein the PCR reaction conditions in the step (1) are: pre-denaturation at 95°C for 30s; then 40 cycles of reaction at 95°C for 15s, 60°C for 20s, and 72°C for 20s. 6. The method according to claim 3, wherein the step of using the "scratch method" in step (2) to detect the migration of glioma cells is to transfect the glioma cells with the drug to be screened for 48 hours Change the serum-free glioma cell culture medium and continue culturing the cells for 12 hours to starve them. Use a sterilized pipette tip to scratch a single layer of glioma cells in one direction at a uniform speed and force, and scratch once in parallel in each well to form a cell-free scratch area. The cells were washed twice with PBS, and serum-free glioma cell culture medium was added. At 0 hour, 12 hours and 24 hours, observe the change of the distance on both sides of the scratch under the microscope and calculate the distance on both sides of the scratch before and after stimulation. 7. The method according to claim 3, wherein, in step (3), the step of using the transwell method to detect the invasion of glioma cells is to transfect the glioma cells with the drug to be screened for 48 hours and then change to serum-free Dilute the glioma cell culture medium, plant 5×105 cells in the transwell chamber, add glioma cell culture medium outside, cultivate for 48 hours, wipe off the upper layer of uninvaded cells with a cotton swab, fix with methanol for 10 minutes, and air DAPI was added to stain the nucleus, observed and photographed under a fluorescent microscope, and the number of cells in 10 fields of view was randomly counted to determine whether the drug with screening could inhibit the invasion of glioma cells. 8. The method according to claim 3, wherein, before step (1), it also includes the step of constructing pmirGLO-Bmi13'-UTR and pmirGLO-Bmi13'-UTR-mut recombinant plasmids, and the two plasmids are respectively combined with the drug to be screened Co-transfection tool cells 293T cell line. 9. The method according to claim 3, wherein, after step (1), further comprising the step of: detecting the inhibitory effect of the drug to be screened on Bmi1 protein expression by Western blot.

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