The Mechanism Of MiR-218-5p In Gastric Cancer Biological Function Via Targeting HOXA10

ObjectiveTo explore the expression of miR-218-5p in gastric cancer(GC)cells and gastric cancer tissues,and to analyze the expression of miR-218-5p in gastric cancer,then to predict their target genes,and to explore the molecular mechanism of mir-218-5p regulating the occurrence and development of gastric cancer through target genes,so as to provide a new targeted therapeutic scheme for the treatment of gastric cancerclinically.MethodsReal-time fluorescence quantitative PCR was used to detect the expression of miR-218-5p between gastric cancer cell lines with different degrees of differentiation and normal gastric cell line GES-1,as well as in gastric cancer tissues and adjacent tissues,and the clinical significance was finally analyzed.The target genes of miR-218-5p were predicted using online database,such as miRecords,TargetScan,miRwalk,miRDB and Pictar websites for selecting target genes from the intersection part supported by the three softwares at least.The DAVID6.7 database was used to enrich and analyze the signal pathways involved in its target genes,and the OncomiR database was used to analyze the expression of miR-218-5p in gastric cancer.Human gastric cancer cells SGC-7901 and HGC-27 were cultured for next gene knocking in and knocking out operation experiments.The lentivirus vector containing miR-218-5p was designed to infect GC cells and to increase the expression of miR-218-5p in gastric cancer cells.The experiment was divided into LV-miR-218-5p and LV-NC group.An inverted fluorescence microscope was used to observe the green fluorescent cells in the dark field,and the Image J software was used to measure the proportion of fluorescent cells for estimating the efficiency of infection of LV-miR-218-5p roughly,next,the method of RT-qPCR was used to detect the upregulation of miR-218-5p to determine the transduction efficiency.After miR-218-5p was overexpressed,a series of cell function experiments including MTT,cell clone formation experiments were performed to detect cell proliferation,cell scratch healing,transwell chamber test was performed to detect cell migration and invasion ability,flow cytometry was conducted to detect changes in cell cycle distribution and apoptosis,RT-qPCR and Western blot were used to detect the gene expressions related to cell cycle and apoptosis.The bioinformatics software was used to predict the target genes of miR-218-5pto select target genes supported by more than three softwares,and the method of RT-qPCR was used to detect the expression of candidate target genes after miR-218-5p was overexpressed.Since then,the candidate target genes were screened and identified among the intersection part from bioinformatics prediction and acedemic reports,finally,the HOXA10 gene was choosed for more verification.When the HOXA10-3'UTR-WT and HOXA10-3'UTR-mut plasmids were designed and constructed,the dual luciferase reporter gene experiment was performed to detect the binding site of 3'UTR region of HOXA10 by miR-218-5p specifically.Furthmore,the lentiviral shRNA was used to silence HOXA10 in gastric cancer cells.The experiment was divided into shRNA-HOXA10 and shRNA-NC group.An inverted fluorescence microscope and RT-qPCR were used to observe the degree of infection and to detect the down-regulation of HOXA10 respectively.After lentivirus shRNA was successfully infected with GC cells,a series of cell function experiments were conducted,as well as the miR-218-5p overexpression operation performed in GC cells.In addition,the effects of HOXA10 silencing on the biological phenotypes such as proliferation,invasion,migration,cycle and apoptosis in GC cells were observed.Results1.Compared with GES-1 cells,the expression of miR-218-5p is reduced in gastric cancer cells with different degrees of differentiation.Compared with paracancerous tissues,miR-218-5p also showed a significant downward trend in gastric cancer tissues.The low expression of miR-218-5p was correlated with gastric cancer lymph node metastasis(P=0.000)and TNM stage(P=0.000).Bioinformatics analysis showed that miR-218-5p was down-regulated in gastric cancer,and the target gene of miR-218-5p was enriched in multiple tumor-related signaling pathways.2.After the expression of miR-218-5p was overexpressed in GC cells,it was found that about 80% GC cells emitted green fluorescence,and the expression of miR-218-5p was significantly higher in LV-miR-218-5p group than that of the LV-NC group(P<0.01),MTT experiments and cell clone formation experiments showed that the proliferation ability of gastric cancer cells was significantly reduced after miR-218-5p was overexpressed(P <0.01),cell scratch healing and transwell chamber experiments showed that miR-218-5p could inhibit the migration and invasion ability of GC cells significantly(P <0.01),flow cytometry results showed that miR-218-5p could block GC cells at S phase,and induce apoptosis.RT-qPCR and Western blot results showed that the expression levels of p21,p27,p53,Bax,C-Caspase3,and C-Caspase9 were up-regulated,CDK6 and Bcl-2 were down-regulated after miR-218-5p was overexpressed3.The bioinformatical prediction results showed that HOXA10,COL1A1,LASP1,TPD52,ClDN2,CDH2,HMGB1,LMNB,SLC6A1,MBNL1 were choosed as miR-218-5p candidate target genes,RT-qPCR results showed that the expression levels of these 10 target genes were significantly reduced(P <0.01)following miR-218-5p was overexpressed.Finally,HOXA10 was selected as a potentile target gene of miR-218-5p.RT-qPCR and Western blot results showed that HOXA10 was in a high expression status in gastric cancer cells when comparing with GES-1 cells(P <0.01),and the expression of HOXA10 was decreased following miR-218-5p was overexpressed GC cells indicating that HOXA10 was negatively regulated by miR-218-5p(P <0.01).The results of dual luciferase reporter gene experiments showed that miR-218-5p can bind to the 3'UTR region of HOXA10 specifically to regulate gene expression.After HOXA10 was successfully silenced,the mRNA and protein expression level of HOXA10 in the shRNA-HOXA10 group was significantly lower than that in the shRNA-NC group(P<0.01).MTT experiment and cell clone formation experiment showed that silencing HOXA10 significantly reduced the activity of gastric cancer cells,and the proliferation capacity was also reduced(P <0.01),cell scratch healing and transwell experiments showed that silencing HOXA10 can reduce the GC cells' migration and invasion ability,flow cytometry results showed that silencing HOXA10 could block GC cells at S phase and induce apoptosis.RT-qPCR and Western blot results showed that the expression levels of p21,p27,p53,Bax,C-Caspase3,and C-Caspase-9 were increased,while the expression levels of CDK6,and Bcl-2 were decreased after HOXA10 was silenced.Conclusions1.The expression of miR-218-5p is down-regulated in GC showing its potential anti-cancer capacity and becoming a new therapeutic target and prognostic marker.2.MiR-218-5p could inhibit the proliferation,invasion and migration of GC cells,block GC cells at S phase and induce GC cells apoptosis.3.HOXA10 is the target gene of mir-218-5p.Silencing HOXA10 can inhibit the proliferation,invasion and migration of GC cells,block GC cells at S phase and induce their apoptosis,which is consistent with the results of overexpression of mir-218-5p.Mir-218-5p can inhibit the occurrence and development of gastric cancer by regulating the expression of HOXA10 negatively.