| Gastric cancer is one of the most common digestive tract tumors.The development of gastric cancer is a multi-step and multi-factor process involving a series of complex molecular mechanisms.To explore the new molecular mechanism in its occurrence and development is of great significance for the identification of biological targets,intervention in cancer,and early diagnosis.In recent years,studies have shown that non-coding RNA plays an important role in regulating biological functions such as cell proliferation,differentiation,invasion,and transfer.MiRNA is a class of non-encoded single-stranded small molecular RNA with a length of 20-24 nucleotides,with a 5 terminal phosphate group and a 3' terminal hydroxyl group.Through literature review and database analysis,the group found that miR-129-5p expression in gastric cancer tissue was significantly reduced compared to normal tissue expression.The miR-129-5p belongs to the miR-129 family and is a mature product of the 5 'end of the precursors miR-129-1 and miR-129-2,and is the main embodiment of the function.Previous studies have shown that miR-129-5p has abnormal expression in a variety of tumors and has effects on the proliferation,apoptosis,invasion,and metastasis of tumor cells.This study focuses on the role of miR-129-5p in the proliferation of gastric cancer and its mechanism.For this reason,in this study,we first examined the expression of miR-129-5p in the tissue of fresh gastric cancer collected by ourselves and in the tissue paired with cancer,and analyzed its correlation with the clinicopathological data of the patient.The expression level of miR-129-5p was detected in gastric cancer cell lines,and was specifically expressed in the microR-129-5p low-expression gastric cancer cell line through over-expression techniques.The effect on the proliferation ability of gastric cancer cells was observed.At the same time,bioinformatics technology was used to screen the potentially regulated target genes of miR-129-5p and verify and perform functional recovery experiments.According to the important target genes selected,further research on the mechanism is carried out.Material and methods:Part ?:1.Expression of miR-129-5p in gastric cancer tissue and its correlation with clinicopathological data? In 60 pairs of gastric cancer tissues and paired normal gastric tissues,the expression of miR-129-5p was detected by real-time quantitative PCR(qRT-PCR);? The correlation between the expression level of miR-129-5p and the patient data,tumor size and stage was analyzed with the clinicopathologic data of gastric cancer patients.? QRT-PCR was used to detect the expression of miR-129-5p in 4 gastric cancer cells and 1 normal gastric mucosal epithelial cell.2.Effects of miR-129-5p on the proliferation of gastric cancer in vitro? The effects of miR-129-5p on cell proliferation of MKN45 and BGC-823 were observed using Cell Counting Kit-8 kit(CCK-8);? Using EdU(5-ethynyl-2 '-deoxyuridine)experiment to detect the effect of miR-129-5p mimic on the percentage of cells in S phase in MKN45 and BGC-823 cells;? Cell cycle changes of MKN45 and BGC-823 after microR-129-5p mimic expression were detected by flow cytometry.3.Effects of miR-129-5p on the proliferation of gastric cancer in vivoBGC-823 cells stably transfected with miR129-5p plasmids or NC plasmids were injected into right posterior flank of nude mice.Once every 3 to 5 days,the growth of transplanted tumors was recorded and continued to be observed until 35 days.Part ?:1.miR-129-5p potential target gene screening and verificationUsing bioinformatics technology(TarigetScan,miRTar Base,miRDB,miRWalkk,etc.)to predict the downstream genes that miR-129-5p may regulate,in conjunction with The Caner Genome Atlas(TCGA)gastric cancer gene database,selected the highly expressed downstream gene HOXC10,The regulation of miR-129-5p and HOXC10 was verified by using qRT-PCR,Western Blot,and double luciferase reporting gene systems.2.HOXC10 expression in gastric cancer tissue and its correlation with miR-129-5p expression? In 60 pairs of gastric cancer tissues and paired normal gastric tissues,the expression difference of HOXC10 in and near cancer was detected by real-time quantitative PCR(qRT-PCR),and its correlation with miR-129-5p was analyzed;? QRT-PCR was used to detect the expression of HOXC10 in 4 gastric cancer cells and 1 normal gastric mucosal epithelial cell.3.Effects of stable knockout HOXC10 on cell proliferation function in gastric carcinoma? The effect of the stable knocking of HOXC10 on the proliferation of MKN45 and BGC-823 cells was observed using Cell Counting Kit-8 kit(CCK-8);? Using EdU(5-ethynyl-2 '-deoxyuridine)experiment to detect the effect of stable knocking down of HOXC10 on the percentage of cells in S stage in MKN45 and BGC-823 cells;? Cell cycle changes of MKN45 and BGC-823 of stable knocking down of HOXC10 were detected by flow cytometry.4.Effect of co-transfection miR-129-5p and HOXC10 on the proliferation of gastric carcinoma cells? The effects of co-transfection of miR-129-5p mimic and HOXC10 vectors on the proliferation of MKN45 and BGC-823 cells were observed using Cell Counting Kit-8 kit(CCK-8);? Using EdU(5-ethynyl-2 '-deoxyuridine)experiment to detect the cell percentage influence in S stage in MKN45 and BGC-823 cells after transfection of miR-129-5p mimic and HOXC10 vector.Part ?:1.HOXC10 transcription factor downstream target gene screening and verificationChIP-seq was used to screen potential targets downstream of the HOXC10 transcription factor.According to pre-functional experiments and KEGG analysis,CCND1 was selected as an important target gene and the binding system was verified by qRT-PCR,Western Blot,ChIP-PCR,and double fluorescence enzyme reporting gene systems.2.Effect of CCND 1 on gastric cancer cell cycle in HOXC10 stable knockout cell lineThe influence of CCND 1 on gastric cancer cell cycle was observed by flow cytometry.3.The immunohistochemical analysis of HOXC10 and CCND1 on customized tissue chipHOXC10 and CCND 1 antibodies were used to staining tissue chips containing 90 pairs of gastric cancer and paraplastic tissue.The correlation between two proteins and prognostic analysis were studied further.ResultsPart I:1.The expression level of miR-129-5p decreased significantly in the tissue of gastric cancer.?The expression of miR-129-5p was reduced in fresh frozen gastric cancer compared with its paired normal tissue by qRT-PCR(p<0.001).? The level of miR-129-5p was related to the tumor size of gastric cancer patients.? The expression of miR-129-5p in MKN45 and BGC-823 gastric cancer cell lines was relatively low,so these two gastric cancer cells was selected to study further.2.miR-129-5p obviously inhibited the proliferation ability of gastric cancer cells in vitro.? The upregulation of miR-129-5p on MKN45,BGC-823 gastric cancer cell lines was verified by qRT-PCR.? Through CCK8 cell proliferation experiment,it was found that the expression of miR-129-5p can significantly inhibit the proliferation of gastric cancer cells compared to the control group(p<0.001).? EdU results showed that the cell percentage of S phase in MKN45 and BGC-823 of miR-129-5p upregulation group was significantly less than that of control group.? The cell cycle results show that the expression of miR-129-5p significantly affects the cell cycle compared to the control group.The main manifestation is G1/S phase block(MKN45:G1 phase increase,S phase decrease,p<0.01;BGC-823:Phase G1 increase,Phase S decrease,p<0.01);3.miR-129-5p obviously inhibited the tumor proliferation in vivo.After 35 days,the results indicated that overexpression of miR129-5p can significantly reduce tumor size and tumor volume compared with the control group(p<0.01).Besides,the tumor weight of miR-129-5p group showed much lighter than the control group(p<0.01).Part ?:1.HOXC10 is an important target gene downstream of miR-129-5p? Bioinformatics technology predicts 53 downstream target genes of miR-129-5p.? Combining the gene expression profile database of gastric cancer in The Cancer Genome Atlas(TCGA),HOXC10,a high-expression downstream target gene,was selected.The over-expression miR-129-5p mimic can reduce the expression level of HOXC10 at the mRNA and protein levels.Through the detection of biluciferase reporting genes,it is verified that miR-129-5p can bind to the 3 'UTR region of HOXC10 and degrade the HOXC10 modification after transcription.2.HOXC10 was upregulated in gastric cancer and the negative correlation was observed between the expression of HOXC10 and miR-129-5p? HOXC10 was upregulated in fresh frozen gastric cancer compared with its paired normal tissue by qRT-PCR(p<0.001)and the negative correlation was observed between the HOXC10 and miR-129-5p(r=-0.378,p<0.05)? The expression of HOXC10 in MKN45 and BGC-823 gastric cancer cell lines was relatively high,so these two gastric cancer cells was selected to study further.3.Steady knock down of HOXC10 obviously inhibits the proliferation ability of gastric cancer cells? Stable knock down of HOXC10 on the MKN45,BGC-823 gastric cancer cell lines was confirmed by Western blot.?Through CCK8 cell proliferation experiments,it was found that stable knocked down of HOXC10 could significantly inhibit the proliferation of gastric cancer cells compared to the control group(p<0.001).? EdU results showed that the cell percentage of S phase in the stable knocked down of HOXC10 in MKN45 and BGC-823 was significantly lower than that of the control group.? Cell cycle results showed that stable knocked down of HOXC10 significantly affected the cell cycle compared to the control group,mainly manifested as Gl/S phase block(MKN45:G1 phase increase,S phase decrease,p<0.01;BGC/823:Phase G1 increase,Phase S decrease,p<0.01)4.Co-transfection of miR-129-5p and HOXC10 can relieve the inhibition of miR-129-5p on the proliferation ability of gastric cancer cells.? In the MKN45 and BGC-823 gastric cancer cell lines,the miR-129-5p mimic and HOXC10 vectors were transfected together.The results of CCK-8 cell proliferation showed that compared with empty vector,up regulation of HOXC10,which can obviously relieve the inhibitory effect of miR-129-5p alone on the proliferation function of gastric cancer cells.?The EdU results show that the over-expression of HOXC10 can significantly increase the percentage of cells in the S-phase of gastric cancer cells compared with the co-transfection miR-129-5p mimics and empty carriersPart ?:1.CCND 1 is an important target gene downstream of HOXC10 and verificationAccording to the pre-functional experiment and the KEGG analysis of the results of the ChIP-seq,CCND1 was selected as an important target gene for the downstream of the HOXC10 transcription factor to promote cell proliferation.qRT-PCR,Western Blot results show that knocking down of HOXC10 can reduce the expression of CCND1 at mRNA and protein levels,while the ChIP-PCR results show that the HOXC10 protein antibody can specifically pull down the promoter region of CCND1.The double luciferase reporting gene experiment also further confirmed that HOXC10 can bind to the promoter region of CCND 1.2.CCND1 can relieve the G1/S phase block phenomenon in HOXC10 knocked down gastric cellsThe MKN45 and BGC-823 cell lines of the HOXC10 stable knocked down were successfully constructed.The cell flow results showed that the CCND1 can alleviate the G1/S block phenomenon in HOXC10 stably knocked down gastric cancer cells.3.The immunohistochemical results of tissue chips show that the expression of CCND1 and HOXC10 has a moderate correlation,and the expression of the two is related to the poor prognosis.The results of 90 customized gastric cancer tissue chips showed a moderate correlation between the expression of CCND1 and HOXC10(R=0.349,p<0.01).Prognostic survival analysis showed that patients with high expression of HOXC10 and CCND1 in gastric cancer showed relatively poor outcomes.Conclusions:1.The expression of miR-129-5p in human gastric cancer tissue was lower than normal tissue,and its expression was related to the tumor size of gastric cancer patients.Excessive expression of miR-129-5p can significantly inhibit the proliferation of gastric cancer cells,reduce the percentage of cells in S phase in gastric cancer cells,and lead to G1/S phase block.2.miR-129-5p can bind to the 3 'UTR region of HOXC10 and degrade the HOXC10 modification after transcription.The expression of the two has a moderate correlation in the tissue of gastric cancer.Hyperexpression of HOXC10 can relieve the inhibitory effect of miR-129-5p on cell proliferation in gastric cancer.3.CCND1 is an important downstream target gene that plays a role in promoting proliferation in gastric cancer.Excessive expression of CCND1 can relieve the effect of downregulation of HOXC10 on the cell cycle of gastric cancer cells.The prognosis of gastric cancer patients with high expression of CCND1 and HOXC10 was poor. |
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