| Gastric cancer is a common malignant tumor of the digestive tract.It is formed by the deterioration of gastric epithelial cells and is the fourth largest malignant tumor in the world.In 2012,there were approximately950,000 new cases of gastric cancer worldwide,with approximately720,000 deaths.The occurrence of gastric cancer is related to Helicobacter pylori infection,obesity,eating habits and other factors.The early symptoms are not obvious.Most patients are in the middle and advanced stage of tumor metastasis at the time of diagnosis.The median survival time is only 5-12 months.The 5-year survival rate is less than 10%.Although early diagnosis methods such as endoscopic diagnosis and imaging diagnosis,as well as the continuous development of surgery,chemotherapy,radiotherapy and palliative care is developing,the overall survival rate of patients has improved.However,the treatment of patients with distant metastatic gastric cancer still faces great challenges.The mortality of advanced gastric cancer is related to peritoneal dissemination,hematogenous dissemination and lymph node metastasis.Therefore,it is necessary to explore the mechanism of gastric cancer progression and elucidate the proliferation,growth and migration of gastric cancer cells.Molecular mechanisms of invasion and apoptosisNumerous studies have shown that non-coding RNA plays an important role in the progression of gastric cancer.A non-coding RNA is anRNA molecule that does not encode a protein.Based on length,non-coding RNAs fall into two broad categories.Large RNAs(greater than 50nucleotides)include long non-coding RNAs(lncRNAs),small nucleolar RNAs,circular RNAs,tRNAs and rRNAs.Small RNAs(less than 50nucleotides)include microRNAs,siRNAs,and piRNAs.At present,many studies have found that miRNA plays an important role in the progression of gastric cancer.Recently,miR-338-3p was found to be down-regulated in malignant tumors such as breast cancer and hepatocellular carcinoma,and its up-regulation can inhibit the proliferation,migration and invasion of tumor cells,but the expression and role of miR-338-3p in gastric cancer is still unclear.For this reason,the clinical tissue samples of gastric cancer patients were collected,and gastric cancer cells were cultured in vitro.The expression and function of miR-338-3p in gastric cancer were analyzed.Combined with bioinformatics and dual luciferase reports,the downstream target genes of miR-338-3p is explored to reveal the molecular mechanisms involved in the proliferation,migration and invasion of gastric cancer cells,and to provide a theoretical foundation for miR-338-3p as a molecular marker for predicting gastric cancer metastasis and the development of targeted drugs for gastric cancer treatment.This research mainly includes:CHAPTER Ⅰ MIR-338-3P FUNCTIONAL VERIFICATIONOBJECTIVE: To investigate the expression of miR-338-3p in gastric cancer cells and tissues,and to analyze the effect of miR-338-3p on proliferation,Apoptosis,migration and invasion of gastric cancer cells.METHODS: Thirty-one patients with gastric cancer who underwent surgery at The First People’s Hospital Hospital of Yibin from February2016 to March 2018 were enrolled.The specimens of gastric cancer tissues and corresponding adjacent tissues(≥5 cm from tumor tissue)were collected.GES-1,MGC-803,HGC-27,MGC-803,SGC-7901,BGC-823 cell line was cultured in vitro.qRT-PCR was used to detect the expression of miR-338-3p in gastric cancer cells,tissues and normal gastric mucosa cells and tissues.The cells were transfected with con mimics,miR-338-3p mimics,cell proliferation was detected by MTT assay,cell Apoptosis was Flow cytometry,and cell migration was detected by Transwell assay.And invasion.RESULTS: 1.The expression of miR-338-3p in gastric cancer tissues was down-regulated compared with normal gastric mucosa(P <0.05),and in gastric cancer cell lines MGC-803,HGC-27,MGC-803,SGC-7901,BGC-823,the expression level of miR-338-3p was significantly lower than that of GES-1 gastric mucosal epithelial cells(P<0.05).2.The expression level of miR-338-3p was significantly increased after transfection of miR-338-3p mimics(P <0.05);3.Compared with miR-con group,the cell proliferation activity of miR-338-3p group was significantly decreased at48 h and 72h(P <0.05);4.Compared with miR-con group,the cell apoptosis of Mi-338-3p group was significantly promoted(P <0.05);5.Compared with miR-con group,the migration and invasion ability of HGC-27 cells in miR-338-3p group were significantly decreased(P <0.05).CONCLUSION: miR-338-3p is down-regulated in normal gastricmucosa cells and tissues.When miR-338-3p is up-regulated,apoptosis of gastric cancer cells is promoted and the proliferation,migration and invasion of gastric cancer cells is inhibited.CHAPTER Ⅱ BIOINFORMATICS ANALYSIS AND FUNCTIONAL VERIFICATION OF MIR-338-3P TARGET GENEOBJECTIVE: To search for downstream target genes of miR-338-3p and analyze its effects on proliferation,Apoptosis,migration and invasion of gastric cancer cells.METHODS: Thirty-one patients with gastric cancer who underwent surgery at The First People’s Hospital Hospital of Yibin from February2016 to March 2018 were enrolled.The specimens of gastric cancer tissues and corresponding adjacent tissues(≥5 cm from tumor tissue)were collected.GES-1,MGC-803,HGC-27,MGC-803,SGC-7901,BGC-823 cell line was cultured in vitro.Combined with bioinformatics prediction results,SIRT2 was selected as a candidate target gene of miR-338-3p.qRT-PCR and Western blot were used to detect the difference of SIRT2 mRNA and protein expression in gastric cancer cells,tissues and normal gastric mucosa cells and tissues.Linear regression was used to analyze whether the expression of SIRT2 was correlated with the expression of miR-338-3p.The gastric cancer cells were transfected with con mimics,miR-338-3p mimics.The expression of SIRT2 mRNA and protein was detected by qRT-PCR and Western blot.The dual luciferase reporter assay was used to test miR-338-3p can directly regulate SIRT2.Artificially silence SIRT2 expression,MTT assay detects gastric cancer cell proliferation,Transwell assay detects cell migration and invasion.RESULTS:The expression of SIRT2 in gastric cancer tissues was significantly higher than that in normal gastric mucosa(P<0.05).Western blot analysis also confirmed that SIRT2 protein expression was significantly increased in gastric cancer tissues;2.SIRT2 expression leveland miR-338-3p was negatively correlated(r2=0.1017,P<0.05).3.The expression of SIRT2 mRNA in miR-338-3p group was significantly lower than that in the corresponding control group,and the expression of SIRT2 mRNA in anti-miR-338-3p group was significantly higher than that in the corresponding control group.<0.05),Western blot analysis of SIRT2 protein expression also showed similar results;4.In the dual luciferase reporter gene experiment,after wild-type SIRT2 3’-UTR and miR-338-3p mimics co-transfection the luciferase activity decreased(P <0.05),but the fluorescence intensity did not change significantly after co-transfection of SIRT2 3’-UTR mutant with miR-338-3pmimics(P > 0.05);5,compared with si-con group,cultured for 48 h At 72 h,the cell proliferation activity of SIRT2 group was significantly decreased(P <0.05).6.Compared with si-con group,the cell apoptosis of SIRT2 group was significantly promoted(P <0.05);7.Compared with si-con group,the migration and invasion ability of HGC-27 cells in si-SIRT2 group was significantly decreased(P<0.05).CONCLUSION: SIRT2 is a downstream target gene directly regulated by miR-338-3p.Its expression is up-regulated in gastric cancer cells and tissues compared with normal gastric mucosa cells and tissues.When SIRT2 is down-regulated,it can Promote the apoptosis of gastric cancer cells and it can inhibit the proliferation,migration and invasion of gastric cancer cells.CHAPTER Ⅲ MOLECULAR MECHANISM OF MIR-338-3P REGULATING MIGRATION AND INVASION OF GASTRIC CANCER CELLSOBJECTIVE: To further investigate the effects of miR-338-3p/SIRT2 functional axis on proliferation,Apoptosis,migration and invasion of gastric cancer,and to explore the response of Wnt/β-catenin signaling pathway to miR-338-3p/SIRT2 regulation.METHODS: mimics control,miR-338-3p mimics,inhibitor control,miR-338-3p inhibitor,control siRNA,SIRT2 siRNA,miR-338-3p mimics and pcDNA 3.1 empty vector,miR-338-3p mimics and pcDNA 3.1-SIRT2 overexpression vector was transfected into HGC-27 cells and labeled as miR-Con group,miR-338-3p group,anti-miR-con group,anti-miR-338-3p group,si-con group,si-SIRT2 group,miR-338-3p + Ctrl group and miR-338-3p + SIRT2 group,qRT-PCR was used to detect miRNA expression in each group,MTT assay was used to detect cell proliferation,Flow cytometry was used to detect cell Apoptosis was and Western blot was used to detect protein expression.RESULTS:After transfection of miR-338-3p mimics,the expression of SIRT2 in gastric cancer cells was significantly lower than that in miR-con group(P<0.05).The expression of SIRT2 in miR-338-3p-Ctrl group co-transfected with pcDNA 3.1 empty plasmid was not significantly different from that in miR-338-3p group(P>0.05).The expression of SIRT2 protein in the miR-338-3p+SIRT2 group co-transfected with pcDNA3.1-SIRT2 was significantly higher than that in the miR-338-3p-Ctrl group(P< 0.05),and the expression of SIRT2 protein in miR-338-3p+SIRT2group was not significantly different from that in miR-con group(P>0.05).2.The proliferation ability of gastric cancer cells in miR-338-3p group wassignificantly lower than that in miR-con group(P<0.05).The proliferation of gastric cancer cells in miR-338-3p+SIRT2 group was restored to some extent(P<0.05).3.Compared with the MIC-con group,the MIC-338-3p+SIRT2 group,the MIC-338-3p+SIRT2 group and the MIC-338-3p+Ctrl group were significantly promoted cell apoptosis(P <0.05);there was no significant difference between the MIC-338-3p group and the MIC-338-3p group(P > 0.05);the apoptotic ability of the MIC-338-3p+SIRT2 group and the MIC-338-3p group decreased significantly(P < 0.05).4.The migration and invasion ability of gastric cancer cells in miR-338-3p group were better than those in miR-con group.The migration and invasion ability of gastric cancer cells in miR-338-3p+SIRT2 group recovered to some extent(P< 0.05).5.The expression of Wnt and β-catenin protein in si-SIRT2 group was higher than that of si-con group(P < 0.05).CONCLUSION: Up-regulation of SIRT2 can partially reverse the proliferation,migration and invasion inhibition of gastric cancer cells induced by down-regulation of miR-338-3p.The Wnt/β-catenin signaling pathway is one of the downstream signaling pathways that miR-338-3p/SIRT2 regulates the proliferation,apoptosis,migration and invasion inhibition of gastric cancer cells. |
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