| Gastric cancer is one of the most common digestive system tumors worldwide,and its mortality rate has remained high.Due to the complicated pathogenesis and the extent of malignant lesions pose a serious threat to the patient's health,there is currently no clinically specific early diagnosis and effective treatment for it.Therefore,the development of new early diagnosis and treatment methods is the current research focus.In cancer research,the application of bioinformatics databases to screen genes related to tumorigenesis and development can provide new scientific evidence for clinical diagnosis of diseases and development of new drugs.As a small non-coding RNA,microRNA is involved in the occurrence and development of tumors,and plays an important role in many processes,including participating in regulating tumor cell proliferation,apoptosis,migration,differentiation,and so on.With the in-depth study of miRNA in recent years,more and more research results show that some miRNAs in tumors are dysregulated and can further participate in the regulation of tumor development by playing the role of proto-oncogene or tumor suppressor gene.Mitotic arrest defective protein 2(MAD2L1)is an important component of the mitotic checkpoint complex protein.The MAD2L1 gene is located on chromosome 4 in humans and on chromosome 6 in mice.In this study,using bioinformatics database analysis,we found that compared with normal tissues,MAD2L1 was significantly overexpressed in gastric cancer tissue;MAD2L1 was significantly negatively correlated with miR-30a-3p expression;miR-30a-3p could be 3'-UTR with MAD2L1 It is speculated that MAD2L1 and miR-30a-3p may play a very important role in the pathogenesis of gastric cancer.miR-30a-3p is a member of the miR-30 family,22 bp in length,and has been reported to be involved in regulating proliferation,apoptosis,invasion and migration in many tumors.In this study,bioinformatics methods were used to screen gastric cancer-related key genes from a public database,and the cycle target gene MAD2L1 was selected from it.Then,miR-30a-3p,which was significantly negatively correlated with its expression,was selected through analysis.The dual luciferase reporter gene Experiments,cell biology and molecular biology testing methods such as real-time fluorescence quantitative PCR(qRT-PCR),western blot analysis,cell proliferation ability test(MTT),and flow cytometry to detect cell cycle,explored the role of MAD2L1 and miR-30a-3p in gastric cancer and their interrelationships.Methods:1.The genes differentially expressed in normal gastric cancer samples and tumor samples were analyzed through the TCGA and GEO databases.The differentially expressed genes obtained from the two databases were intersected,and the differentially expressed genes were clustered through the String website analysis.Gastric cancer-related key genes were selected from GOD cluster analysis cycle-related target genes MAD2L1,and miR-30a-3p was further selected through linkedomics website combined with TCGA database analysis.2.The miR-30a-3p mature mimics were synthesized,and the transfection efficiency of miR-30a-3p-mimics in gastric cancer cells AGS and BGC-823 was examined by qRT-PCR experiments.Western blot was used to detect the effect of miR-30a-3p on MAD2L1 at the protein level.The effects of miR-30a-3p on the proliferation and cycle of gastric cancer cells AGS and BGC-823 were detected by MTT experiments,clone formation experiments and flow cytometry.3.Wild-type and mutant dual-fluorescent reporter vectors for MAD2L1 were synthesized,and dual-luciferase reporter gene experiments were used in HEK-293 cellsto determine the targeting relationship between miR-30a-3p and MAD2L1.4.The overexpression vector of MAD2L1 was constructed,and the changes of cell proliferation and cycle after co-transfection of miR-30a-3p mmics and ov-MAD2L1 were detected in gastric cancer cells AGS and BGC-823 cells by MTT and cell cycle experiments.Results:1.Combining the TCGA database and the GEO database and analyzing the results of STRING and cluster analysis to obtain the highest scoring group of genes KIF11,NCAPD2,TOP2 A,MCM3,MCM2,KIF23 and MAD2L1 are key genes for gastric cancer;MAD2L1 is highly expressed in tumors It may be a new marker gene for gastric cancer.Compared with normal tissues,miR-30a-3p expression was significantly reduced in gastric cancer tissues.MAD2L1 was significantly negatively correlated with miR-30a-3p expression.2.MiR-30a-3p mature mimics were synthesized,and qRT-PCR was used to detect the overexpression of miR-30a-3p in gastric cancer cell AGS and BGC-823 cells.The results showed that: miR The expression level of-30a-3p was significantly higher than that of the control group.The results of Western blot showed that compared with the control group,the expression level of MAD2L1 protein was significantly reduced after miR-30a-3p overexpression.The results of MTT and clone formation experiments showed that: Compared with the control group,overexpression of miR-30a-3p can inhibit the proliferation of AGS and BGC-823 cells;cell cycle experiments showed that compared with the control group,overexpression of miR-30a-3p can block the cell cycle at G0 / G1.3.The wild-type and mutant dual-fluorescent reporters of MAD2L1 were synthesized,and the dual-luciferase reporter gene experiment was used in HEK-293.The results showed that miR-30a-3p has a good targeting relationship with MAD2L1.4.MTT and cell cycle experiments were performed to simultaneously overexpressmiR-30a-3p and MAD2L1.The results showed that overexpression of MAD2L1 can rescue the inhibitory effect of cell overexpression of miR-30a-3p alone on AGS and BGC-823 cell proliferation and cell cycle Blocking effect.Conclusion:1.KIF11,NCAPD2,TOP2 A,MCM3,MCM2,KIF23 and MAD2L1 can be used as key genes for gastric cancer.2.The expression of miR-30a-3p is significantly reduced in gastric cancer tissues.Overexpression of miR-30a-3p can inhibit the expression of MAD2L1 at the protein level,at the same time can inhibit the proliferation of gastric cancer cells,and block cells in the G0 / G1 phase.3.MiR-30a-p can target MAD2L1 expression in model cells HEK-293.4.Simultaneous expression of miR-30a-3p and MAD2L1 can rescue the effect of miR-30a-3p overexpression alone on the proliferation and cycle of gastric cancer cells,further proving that there is a certain targeting relationship between miR-30a-3p and MAD2L1,thus Affects the proliferation of gastric cancer cells. |
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