CN103131710B - ShRNA (short hairpin ribonucleic acid) for inhibiting tumor cell invasion - Google Patents

Abstract

The invention discloses a shRNA for inhibiting tumor cell invasion, its sense strand sequence is shown in SEQ NO.1 in the sequence listing, and its antisense strand sequence is shown in the sequence listing SEQ NO.2. The shRNA described in the present invention is designed, synthesized and constructed through the nucleotide sequence of the CD14 molecule gene on the surface of human cells, and the shRNA expression vector for inhibiting tumor cell migration is obtained through functional experiment verification. The invention also discloses the application of the shRNA.

Description

A shRNA that inhibits tumor cell invasion

technical field

The invention belongs to the field of molecular biology and relates to the construction and application of a novel shRNA. Specifically, the present invention designs and synthesizes shRNA aiming at the nucleotide sequence of human tumor cell surface molecule CD14 gene, and said shRNA can inhibit the migration of said tumor cells after being transferred into gastric cancer cells.

Background technique

RNA interference is a double-stranded RNA-mediated sequence-specific gene silencing phenomenon that widely exists in the body [Fire A, Xu SQ, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature1998; 391:806-811], because of its advantages over traditional gene knockout methods such as high silencing efficiency, strong specificity, and easy operation, it has been rapidly developed and has become an important research tool in the field of life sciences [Arziman Z, Horn T, Boutros M. E-RNAi: a web application to design optimized RNAi constructs. Nucleic AcidsRes2005; 33: W582-W588].

shRNA is a small hairpin RNA or short hairpin RNA (a small hairpin RNA or short hairpinRNA, shRNA), which is an exogenous RNA sequence with a stem-loop structure, which can be processed into siRNA in the cell, and siRNA is then combined with protein to form RNA Induced silencing complex (RNA-inducedsilencing complex, RISC). This complex is able to bind to cognate mRNA and induce its degradation. The role of shRNA has strict targeting, and the selection of specific target sites has a position effect. Different target sites have great differences in the interference efficiency of the same gene [Reynolds A, Leake D, Boese Q, et al.Rational siRNA design for RNA interference. Nat Biotechnol 2004;22:326-330].

Although CD14 is a high-affinity receptor for LPS, it does not have a transmembrane region. It needs the cooperation of TLR4 to transmit the signal into the nucleus and activate NF-kB [Gioannini TL, Weiss JP. Regulation of interactions of Gram-negative bacterial endotoxins with mammalian cells.Immunol Res 2007; 39:249-260], some scholars also believe that CD14 is the main signaling molecule that receives LPS stimulation and triggers the transcriptional activation of nuclear factors, which can mediate cell apoptosis and regulate cell cycle [Zanoni I, Ostuni R, Capuano G, et al. CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation 2009;460:264-8]. CD14 Gene Promoter Polymorphisms and Gastric Cancer, Colorectal Cancer, Prostate Cancer [Dan Zhao, Tong Sun, Xuemei Zhang. ] and other susceptibility have been reported, and its direct connection with cell proliferation and apoptosis has gradually attracted people's attention. Kevyn et al [Kevyn L, Comstock, Kevin A, et al. LPS-Induced TNF-a Release from and Apoptosis in Rat Cardiomyocytes: Obligatory Role for CD14 in Mediating the LPS Response. J Mol Cell Cardiol1998; 30: 2761-2775] think CD1 Can respond to LPS signal, induce the release of TNF-α and further promote cardiomyocyte apoptosis. CD14 neutralizing antibody can delay neutrophil apoptosis induced by BLP, and its anti-apoptotic effect is enhanced after adding TLR2 neutralizing antibody. Waldemar et al [WaldemarKanczkowski, Piotr Tymoszuk, et al.Abrogation of TLR4 and CD14 Expression and Signaling in Human Adrenocortical Tumors.J Clin EndocrinolMetab2010; 95(12):421-429] found that CD14 mRNA was expressed in adrenocortical carcinoma, adenoma and The expression in cancer cell lines was significantly reduced or even lost, and the activation of NF-kB was detected in the adrenocortical cancer cell lines co-transfected with CD14 and TLR4. At the same time, the cell activity was inhibited and the number of apoptotic cells increased, indicating that CD14 may mediate NF-kB The -kB pathway is involved in the regulation of cancer cell proliferation and apoptosis.

At present, studies have shown that the high expression of CD14 is positively correlated with the invasion ability of cancer cells, Mina et al [MinaSong, Sayeon Cho. CD14 acts as an angiogenic factor by inducing basic fibroblast growth factor (bFGF). Bull. ): 1613-1614] intervened human umbilical vein endothelial cells with CD14 overexpression cell culture supernatant as a chemokine, and found that the migration ability of endothelial cells was significantly enhanced, Dong[Dong H, Liu MY, Cheng ZD, et al.Small interferingRNA-directed targeting of Toll-like receptor 4 inhibits human prostate cancer cellinvasion, survival, and tumorigenicity. Molecular Immunology 2009; 46: 2876-2884] found TLR4 and CD14, MD in the prostate cancer cell line PC3 with strong invasion ability The expression of other proteins increased, suggesting that the CD14/TLR4 pathway was positively correlated with the invasion ability of cancer cells.

Invasion and metastasis are the most common biological characteristics of malignant tumors, and they are also the key factors affecting the survival and prognosis of patients. Gastric cancer not only has a high incidence rate but also easily metastasizes in the middle and late stages, which has become the main reason for treatment failure. In order to inhibit the invasion and metastasis of gastric cancer cells, the present invention constructs shRNA targeting CD14 molecules, and after said shRNA is transferred into gastric cancer cells, it effectively inhibits the invasion of gastric cancer cells, providing a way for the treatment of malignant tumors.

Contents of the invention

In order to solve the problem of treatment failure caused by tumor cell metastasis, the present invention constructs a shRNA whose sense strand sequence is shown in sequence table SEQ NO.1, and whose antisense strand sequence is shown in sequence table SEQ NO. 2. The shRNA can reduce the content of CD14 protein in tumor cells, and can inhibit the migration of tumor cells.

According to the mRNA sequence of human CD14 in the GenBank database and the primer design principle of shRNA, the present invention designed a number of CD14-shRNAs targeting CD14 molecules, and also designed a negative control and a positive control shRNA that interferes with GAPDH, and the design will be completed The sequence of shRNA was sent to Sangon Biology (Shanghai) Co., Ltd. for synthesis. Among them, the CD14-shRNA targeting the 898th position of CD14 mRNA (NM_000591) (sense strand sequence is shown in sequence table SEQ NO.1, and its antisense strand sequence is shown in sequence table SEQ NO.2), can effectively reduce tumor The expression of CD14 in cells can inhibit the migration of tumor cells.

In the present invention, the oligonucleotides of the above-designed CD14-shRNA, negative control and positive control shRNA are routinely annealed to synthesize double strands, and after double enzyme digestion, they are connected to the pGCsi-H1/Neo expression vector, and the ligated product is transformed into Escherichia coli . A single colony was picked for PCR and sequencing identification, and positive clones and plasmids were obtained.

The present invention detects the expression of CD14 in four gastric cancer cell lines, SGC-7901, MGC-803, BGC-823, and MKN-28, through RT-PCR and Western blot techniques, and the results show that the MGC-803 cell line highly expresses CD14.

In the present invention, the CD14-shRNA expression vector is transfected into the MGC-803 cell line, the transfection efficiency is observed under a fluorescence microscope, the positive cells are selected for subculture, and G418 is used for screening to obtain a CD14-shRNA stably transfected cell line. Among them, tumor cells transfected with shRNA whose sense strand is SEQ NO.1 and antisense strand is SEQ NO.2 have CD14 mRNA and protein silencing efficiencies of 71.7% and 63.4%, respectively.

The present invention uses the Transwell chamber model to detect the invasion ability of tumor cells transfected with shRNA whose sense strand is SEQ NO.1 and antisense strand is SEQ NO.2. The specific method is as follows: stabilize the CD14-shRNA in the logarithmic growth phase The transfected cells were inoculated in the upper chamber of the Matrigel-coated transwell chamber, and the lower chamber was filled with complete culture solution, cultured in a 5% CO2 incubator at 37°C, and the number of invasive cells was detected after 48 hours, using SPSS16.0 software Data analysis showed that CD14 silencing caused by CD14-shRNA (sense strand is SEQ NO.1 and antisense strand is SEQ NO.2) can significantly inhibit the invasion and migration of gastric cancer cells.

Description of drawings

Figure 1 The content of CD14 mRNA and CD14 protein in each gastric cancer cell line, M: DNA molecular marker;

Figure 2 The expression of GAPDH in different groups of tumor cells, where Control is the control group: no transfection, lip2000 group: only transfection reagent is added, NC is the negative control group: transfected with a vector containing irrelevant sequences, shRNA is the positive control of GAPDH Group: transfect the vector containing GAPDH interference sequence;

Figure 3 Detection of CD14-shRNA interference effect, where Control is the control group: no transfection, lip2000 group: only transfection reagent is added, NC is the negative control group: transfected with a vector containing irrelevant sequences, shRNA is: transfected with CD14 - interference carrier of shRNA sequence;

Fig. 4 Fluorescent images of MGC-803 transfected with CD14-shRNA (A picture shows shRNA transient transfection for 48 hours, B picture shows shRNA stable transfection);

The expression level of CD14 in Fig. 5 stable transfection cell line;

Figure 6 Detection of cell invasion ability, Control group: tumor cells not transfected with CD14-shRNA, CD14-shRNA group: tumor cells transfected with CD14-shRNA.

Screening of high expression cell lines of embodiment 1CD14

1. Materials and methods

1. Materials

Gastric cancer cell lines SGC-7901, MGC-803, BGC-823, and MKN-28 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI1640 and 0.05% Trypsin were purchased from Gibco; fetal bovine serum was purchased from hyclone; OptiMEM culture medium was purchased from Invitrogen.

Total RNA extraction kit, TIAN Script cDNA first strand synthesis kit, 2×Taq PCRMaster Mix were purchased from Beijing Tiangen; CD14 antibody was purchased from santa cruz; GAPDH antibody was purchased from abcam; Lip2000 and G418 were purchased from Invitrogen; BamHI, HindIII Endonuclease, T4 DNA ligase were purchased from TaKaRa.

Escherichia coli DH5α and pGCsi-H1/Neo vectors were all preserved by our laboratory; primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; other drugs were of domestic analytical grade.

2. Method

2.1. RT-PCR detection of CD14 mRNA expression in different cell lines

a) Gastric cancer cell lines SGC-7901, MGC-803, BGC-823, and MKN-28 in the logarithmic growth phase were inoculated in a six-well plate;

b) When the cells grow to 90%, the total RNA of each group of cells is extracted by the TRIzol method,

c) Reverse transcription into cDNA using cDNA First Strand Synthesis Kit.

d) Design PCR primers according to the primer design principles, see the table below for the sequences of the upstream and downstream primers and the length of the amplified fragments of CD14, GAPDH, and β-actin

e) Perform PCR using cDNA as a template, the reaction system is as follows

The PCR reaction conditions are

f) Using β-actin as an internal reference, adjust the loading ratio so that the loading amount of β-actin is the same, load the sample at this loading ratio to detect the mRNA expression of CD14, and the results of grayscale analysis and electrophoresis confirm that CD14 is highly expressed at the mRNA level cell line.

2.2. Western blot detection of CD14 protein expression in each cell line

a) Gastric cancer cell lines SGC-7901, MGC-803, BGC-823, and MKN-28 in the logarithmic growth phase were inoculated in a six-well plate;

b) When the cells grow to 90%, RIPA lysate is added to extract the total protein of cells, and the protein is quantified by BCA method.

c) SDS-PAGE electrophoresis. Take 4 μg protein for SDS-PAGE.

The formula of 12% SDS-PAGE separating gel is: 3.3ml of water, 4.0ml of 30% acrylamide solution, 2.5ml of 1.5mol/L Tris (pH8.8), 0.1ml of 10% SDS, 0.1ml of 10% ammonium persulfate solution, TEMED0.004ml.

The formula of 5% SDS-PAGE stacking gel is: water 3.4ml, 30% acrylamide solution 0.83ml, 1.0mol/L Tris (pH6.8) 0.63ml, 10% SDS 0.05ml, 10% ammonium persulfate 0.05ml, TEMED0 .005ml.

c) Transfer film. After electrophoresis, electrotransfer transfers protein samples from SDS-PAGE to PVDF membrane.

d) Block with 5% skimmed milk powder for 5 minutes. Add diluted primary antibody (CD14 1:500 dilution, GAPDH 1:1000 dilution), and incubate overnight at 4°C.

e) Wash the membrane with PBS for 3 times, 5 min each time, add 1:4000 times diluted secondary antibody, and incubate at 37°C for 45 min.

f) After the incubation, wash off non-specifically bound antibodies with PBS, the method is the same as above. Exposure was performed by ECL substrate luminescence.

g) Wash the PVDF membrane, add 1:10000 internal reference antibody after peeling off, and incubate overnight at 4°C.

h) The membrane was washed with PBS in the same way as above, and the secondary antibody diluted 1:4000 was added, and incubated at 37° C. for 45 minutes. After washing, the membrane was exposed by ECL substrate luminescence method.

i) Perform grayscale analysis on the exposure photos.

two results

Data analysis: SPSS16.0 software was used for statistical analysis of the data, and the experimental data was expressed as ±S. The comparison between the two groups was performed by the t test method, and P<0.05 indicated that the difference was statistically significant.

The expression of CD14 in each gastric cancer cell line (Figure 1) shows that the expression of CD14 mRNA and protein is the highest in MGC-803, followed by MKN-28 and BGC-823, but there is no significant difference compared with MGC-803. The expression of CD14 mRNA and protein in SGC-7901 was the lowest, and the difference was statistically significant compared with MGC-803 (P<0.01, P<0.05). Based on the results, the MGC-803 cell line was selected for subsequent CD14 interference experiments.

Example 2 Construction of CD14-shRNA interference vector

1. Materials and methods

1. Materials

Gastric cancer cell lines SGC-7901, MGC-803, BGC-823, and MKN-28 were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. RPMI1640 and 0.05% Trypsin were purchased from Gibco; fetal bovine serum was purchased from hyclone; OptiMEM culture medium was purchased from Invitrogen.

Total RNA extraction kit, TIAN Script cDNA first strand synthesis kit, 2×Taq PCRMaster Mix were purchased from Beijing Tiangen; CD14 antibody was purchased from santa cruz; GAPDH antibody was purchased from abcam; Lip2000 and G418 were purchased from Invitrogen; BamHI, HindIII Endonuclease, T4 DNA ligase was purchased from TaKaRa.

Escherichia coli DH5α and pGCsi-H1/Neo vectors were all preserved by our laboratory; primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; other drugs were of domestic analytical grade.

2. Method

2.1. Design of shRNA sequence

Search the GenBank database (http://www.ncbi.nlm.nih.gov/nucleotide) to obtain the cDNA sequence of the human CD14 gene, and follow the principles of shRNA primer design [Reynolds A, Leake D, BoeseQ, et al.Rational siRNA design for RNA interference.Nat Biotechnol2004; 22(3):326-330], designed and synthesized CD14-shRNA sequence and an irrelevant interference sequence, and synthesized GAPDH interference sequence as a positive control. The selected target sequence is: 5'-GGTACTGAGCATTGCCCAA-3', located at position 898 of CD14 mRNA (NM_000591). The sequence of the synthetic positive and antisense oligonucleotides is as follows: the sequence of the sense strand SEQ NO.1 is 5'-GATCCCCGGTACTGAGCATTGCCCAATTCAAGAGATTGGGCAATGCTCCAGTACCTTTTT-3', the sequence of the antisense strand SEQ NO.2 is 5'-AGCTAAAAAGGTACTGAGCATTGCCCAATCTCTTGAATTGGGCAATGCTCAGTACCGGG-3', with There are BamHI and HindIII enzyme cutting sites.

2.2. Construction of CD14-shRNA vector and detection of silencing effect

1) Construction of interference carrier

a) Take an equal amount of 100mmol/L DNA oligonucleotide single-strand fragments and mix, heat in annealing buffer (100mmol/L NaCl) at 95°C for 5min, and slowly cool down to room temperature.

b) DNA double strands were purified by ethanol precipitation and identified by 2.5% agarose electrophoresis.

c) Digest the double-stranded DNA obtained above and the expression vector pGCsi-H1/Neo with BamHI and HindIII, and connect the fragment and the vector with T4 ligase.

d) The ligation system was transformed into Escherichia coli DH5α.

e) Pick a number of positive clones for culture and extract plasmids for identification by enzyme digestion.

f) Sequencing to verify whether the DNA sequence in the recombinant clone is consistent with the requirements.

g) The clones with correct sequencing were selected and inoculated into 5 ml medium, cultured overnight, and plasmids were extracted. It is the expression vector of the interference fragment.

2) Detection of CD14-shRNA interference effect and establishment of CD14 stable silencing cell line

a) Inoculate MGC-803 cells into a 6-well plate containing RPMI1640 medium containing 10% FBS, and perform transfection when the cells grow to 80%-90% confluent.

b) Prepare solution 1 and solution 2 according to the instructions of lip2000 transfection reagent, add the mixed transfection reagent drop by drop to the cells and shake gently. The experiment was divided into GAPDH positive control group: transfected with vectors containing GAPDH interference sequences, negative control group (NC): transfected with vectors containing irrelevant sequences, control group (Control): no transfection, lip2000 group: only added transfection Reagent, experimental group: transfected with interference vector containing CD14-shRNA.

c) The cells were cultured in a 5% CO ₂ incubator at 37° C. for 5 hours, and the medium was replaced with RPMI1640 culture medium containing 20% FBS.

d) After culturing for 48 hours, the transfection efficiency of the interference vector was observed under a fluorescence microscope.

e) detecting the expression levels of CD14 mRNA and protein, as described in Method Example 1.

F) The positive cells were digested with trypsin and passaged, and the medium was replaced with a complete culture medium containing 800 μg/m1G418, and the culture medium was changed every 2-3 days.

g) After 2 weeks, the concentration of G418 was reduced to 400 μg/ml to maintain the screening, until the normal cell morphology and strong expression of green fluorescent protein were observed under the fluorescence microscope, and positive clones were isolated and expanded for culture.

g) detecting the expression levels of CD14 mRNA and protein in the cells. The method is as described in Example 1.

2.3. Data analysis

SPSS16.0 software was used for statistical analysis of the data, and the experimental data were expressed as ±S. The comparison between the two groups was performed by the t test method, and P<0.05 indicated that the difference was statistically significant.

two results

1. Construction of CD14-shRNA vector and verification of silencing effect

The interference effect of the GAPDH positive control group is shown in Figure 2. The expression of GAPDH in the shRNA group was significantly reduced, while the other groups did not change significantly, indicating that this experimental system can be used for shRNA interference. The interference effect of CD14 is shown in Figure 3. Compared with the control group, the expression of mRNA and protein in the shRNA group (CD14-shRNA) was significantly reduced, while there was no significant change in the irrelevant interference sequence and Lip2000 group, indicating that the designed shRNA sequence can be effective Interfering with the expression of CD14. Compared with the control group, the expression levels of CD14 mRNA and protein decreased by 51.0% and 47.5%, respectively, so shRNA was selected as the interference sequence of CD14 for subsequent experiments.

2. Establishment of CD14 stable silencing cell lines

After 48 hours of CD14-shRNA transfection, cells emitting green fluorescence can be seen (Fig. 4A). After 4 weeks of G418 selection, a large area of cells emit green fluorescence (Fig. 4B), and the expression rate is over 90%, indicating that CD14-shRNA has been obtained Stably transfected cell lines. The expression of CD14 in the stably transfected cell line was detected ( FIG. 5 ). Compared with the control group, the expression of mRNA and protein decreased by 72% and 63%, respectively, indicating that CD14 had been stably silenced. The cell line sh-CD14 was successfully constructed.

Example 3 CD14-shRNA inhibits tumor cell invasion

1. Materials and methods

1. Materials

Gastric cancer cell line MGC-803 was provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. CD14 stable silencing cell line sh-CD14 was constructed in this experiment. RPMI1640 and 0.05% Trypsin were purchased from Gibco; fetal bovine serum was purchased from hyclone.

Transwell plates were purchased from Corning; Matrigel gels were purchased from BD.

Total RNA Extraction Kit, TIAN Script cDNA First Strand Synthesis Kit, 2×Taq PCRMaster Mix were purchased from Beijing Tiangen; TNF-α was purchased from R&D; Lip2000 was purchased from Invitrogen; CD14, vimentin, TGF-β1 antibodies were purchased from Santa Cruz; TNF-α, E-cadherin, N-cadherin antibodies were purchased from abcam; other drugs were domestic analytically pure.

2. Method

a) Coating Matrigel, and pre-thawing Matrigel at 4°C. It was diluted 1:3 with serum-free RPMI1640 medium, mixed evenly, and 100 μl/well was added to the polycarbonate membrane in the upper chamber of the Transwell chamber. The whole operation was performed on ice and under sterile conditions. Incubate at 37°C for 30 minutes, at this time Matrigel has formed a gel.

b) Digest the cells, count the cells, inoculate the cells into the upper chamber of the Matrigel-coated Transwell chamber at a density of 2×10 ⁵ cells/well, and add RPMI1640 medium containing 10% fetal bovine serum to the lower chamber at 37°C , 5% CO ₂ , and saturated humidity conditions. The experiment was divided into control group and sh-CD14 group, the control group was gastric cancer cell line MGC-803, and the sh-CD14 group was gastric cancer cell line MGC-803 transfected with CD14-shRNA.

c) After culturing for 48 hours, the upper chamber was taken out and fixed in 40 ml/L formaldehyde for 15 minutes, dried and stained in hematoxylin staining solution for 20 minutes.

d) Wipe away the dye around the small chamber, observe and take pictures under an inverted microscope, take 5 fields of view from each well, and record the number of cells that invaded under the membrane of the small chamber.

two results

SPSS16.0 software was used for statistical analysis of the data, and the experimental data were expressed as ±S. The comparison between the two groups was performed by the t test method, and P<0.05 indicated that the difference was statistically significant.

The results of Transwell detection of cell invasion ability are shown in Figure 6. Under the inverted microscope (200×) the number of cells migrating in the sh-CD14 group was less than that in the control group, indicating that the CD14-shRNA designed in the present invention can effectively inhibit tumor cells invasion.

Claims (7)

1. a shRNA, comprises positive-sense strand and antisense strand, it is characterized in that, the nucleotide sequence of described positive-sense strand is as shown in sequence table SEQ NO.1, and the nucleotide sequence of described antisense strand is as shown in sequence table SEQ NO.2.

2. shRNA according to claim 1 reduces the application in cell CD14 mRNA reagent in preparation.

3. shRNA according to claim 1 is preparing the application in T suppression cell CD14 protein expression reagent.

4. shRNA according to claim 1 is preparing the application in tumor migration inhibition drug.

5. tumour according to claim 4, is characterized in that, described tumour is cancer of the stomach.

6. tumour according to claim 4, is characterized in that, described tumour is prostate cancer.

7. tumour according to claim 4, is characterized in that, described tumour is liver cancer.