The Expression,Function And Regulatory Mechanisms Of MiR-1296-5p In Gastric Cancer

Part one Expression of miR-1296-5p in gastric cancer tissues and cell lines and the corelation with the clinicopathological featuresObjective:The high morbidity and mortality of gastric cancer in China is a major health problem that threatens our citizen.Therefore,the study of biomarkers for the definitive diagnosis of gastric cancer and the targets of effective treatment have become hot spots to be solved.miRNAs are deregulated in the development and progression of gastric cancer,and the changes of their expression are related to the malignancy and prognosis of gastric cancer.miR-1296-5p is abnormally expressed in various tumorigenesis and has different expression patterns in different tumors of the digestive system.This study aimed to clarify the expression of miR-1296-5p in gastric cancer and its relationship with clinical features of gastric cancer patients.Methods:The gastric cancer and matched adjacent tissue samples were collected from the patients with gastric cancer.The gastric cancer cell lines and normal gastric epithelial cell strain were cultured.The expressions of miR-1296-5p in gastric cancer tissues and cells were detected by Real-time PCR.The clinical data of the patient was collected to analyze the corelation of miR-1296-5p expression and clinical pathological features of the patients.Results:The expression of miR-1296-5p in gastric cancer tissues was significantly lower than that in the matched adjacent tissues?P<0.01?.Compared with the normal gastric epithelial cell GES-1,the expressions of miR-1296-5p in gastric cancer cell lines BGC-823,NCI-N87,MGC-803,MKN-28,AGS,and SGC-7901 were all decreased?P=0.037,P=0.033,P=0.035,P=0.023,P=0.006,P=0.005?.The expression of miR-1296-5p was significantly negatively correlated with the depth of tumor invasion?P=0.007?and TNM stage?P=0.003?of gastric cancer.Conclusions:The expression of miR-1296-5p was down-regulated in gastric cancer tissues and cells,and the low expression of miR-1296-5p was associated with the depth of tumor invasion and TNM stage of gastric cancer.miR-1296-5p might be involved in the development of gastric cancer.Part two Effect of miR-1296-5p on biological behavior of gastric cancer cellsObjective:miR-1296-5p is abnormally expressed in various tumors,and it exerts different or even opposite effects in different types of tumors.It may act as a tumor promoter or a tumor suppressor.This study focused on the effect of miR-1296-5p on the biological behavior of gastric cancer cells.Methods:The effect of miR-1296-5p on the biological behavior of gastric cancer cells was determined by overexpressing and inhibiting the expression of miR-1296-5p by transfection with miR-1296-5p-mimics or miR-1296-5p-inhibitors in gastric cancer cell lines SGC-7901 and MGC-803.The expression of miR-1296-5p was verified by Real-time PCR.Cell cycle distribution was detected by flow cytometry,cell viability was measured by MTT assay,and cell migration and invasion ability were detected by scratch healing assay and Transwell migration and invasion assay.Results:The percentage of cells in S phase?P=0.009,P<0.001?and cell viability?P=0.012,P=0.032?in SGC-7901 and MGC-803 cell lines transfected of miR-1296-5p-mimics were significantly lower than those transfected with NC-mimics;conversely,the percentage of cells in the S phase?P=0.001,P=0.026?and the cell viability?P=0.002,P=0.006?after transfected with1296-5p-inhibitors,were significantly higher than those transfected with NC-inhibitors.The percentage of wound clousure?P=0.046,P<0.001?and the number of Transwell migratory?P=0.005,P=0.005?and invasive cells?P=0.002,P=0.047?in SGC-7901 and MGC-803 cells transfected with miR-1296-5p-mimics were significantly lower than those transfected with NC-mimics;conversely,the percentage of wound clousure?P=0.002,P=0.036?and the number of Transwell migratory?P=0.003,P=0.004?and invasive cells?P=0.002,P<0.001?after transfected with miR-1296-5p-inhibitors,were significantly higher than those of the control group transfected with NC-inhibitors.Conclusions:miR-1296-5p inhibit the proliferation,migration and invasion of gastric cancer cells.miR-1296-5p plays a tumor suppressor role in gastric cancer cell lines.Part three Screen and verification of miR-1296-5p targets in gastric cancer cellsObjective:The results from the previous section indicate that miR-1296-5p inhibits the proliferation,migration and invasion in gastric cancer,however the mechanism is still unclear.It is widely accepted that miRNA usually exerts its function by inhibiting the translation of a target gene by binding to the 3'UTR of the mRNA of the target gene.Therefore,we will further screen and verify the target gene of miR-1296-5p to elucidate the mechanism in the present study.Methods:miR-1296-5p was overexpressed and knocked down by transfection with miR-1296-5p-mimics or miR-1296-5p-inhibitors in gastric cancer cell lines SGC-7901 and MGC-803.The target genes of miR-1296-5p were predicted by Targetscan software?targetscan.test/ucsc.html?,miRBase software?http://www.mirbase.org?and PicTar?http://pictar.mdc-berlin.de?.The mRNA and protein levels of target genes were detected by Real-time PCR and Western blot assay.The proteins that negatively regulated by miR-1296-5p were screened.The correlation between miR-1296-5p and target gene expressions were further verified in 40 pairs of tumors and adjacent tissues in patients with gastric cancer.To confirm the bindings of miR-1296-5p to the 3'UTR region of the target mRNAs,a fluorescent reporter plasmid containing the 3'UTR region of the mRNA of the target genes were constructed and analyzed by luciferase activity assay.The role of the target genes in the anticancer effect of miR-1296-5p was confirmed by a rescue experiment.The plasmids overexpressing the target genes were transfected into the cells overexpressing miR-1296-5p to detect the proliferation and invasion of gastric cancer cells.Results:The mRNA?CDK6,P=0.001,P=0.001;EGFR,P=0.002,P=0.012?and protein levels?CDK6,P=0.040,P=0.048,EGFR,P=0.002,P=0.008?of CDK6 and EGFR were significantly decreased in gastric cancer cell lines SGC-7901 and MGC-803 transfected with miR-1296-5p-mimics.Conversely,the expressions of the two proteins were significantly increased after transfection with miR-1296-5p-inhibitors?CDK6,P<0.001,P<0.001;EGFR,P<0.001,P=0.002?,while the expression of NCL was not changed,which suggested that CDK6 and EGFR are the target genes of miR-1296-5p in gastric cancer cells.The levels of CDK6 and EGFR in the gastric cancer tissues were significantly higher than those in the matched adjacent tissues,and the expression levels of CDK6 and EGFR were negatively correlated with the expression of miR-1296-5p in gastric cancer tissues?P<0.001,r=0.859;P=0.010,r=0.507?.The luciferase activity assay showed that the luciferase activity of psiCHECK-2/CDK6-3'-UTR^wt?P=0.004,P=0.038?and psiCHECK-2/EGFR-3'-UTR^wt?P=0.027,P=0.006?were significantly inhibited in SGC-7901 and MGC-803 cells transfected with miR-1296-5p-mimics,compared with the control group transfected with NC-mimics.However,the fluorescence of psiCHECK-2/CDK6-3'-UTR^mut?P=0.204,P=0.173?and psiCHECK-2/EGFR-3'-UTR^mut?P=0.362,P=0.486?was not affected by transfection with miR-1296-5p-mimics.The cell viability?CDK6,P=0.002,P=0.004;EGFR,P=0.032,P=0.026?and the number of Transewell invasive cells?CDK6,P=0.022,P=0.037;EGFR,P=0.003,P<0.001?in SGC-7901 and MGC-803 cells transfected with miR-1296-5p-mimics were significantly decreased,which were rescued by transfection of plasmid overexpressing CDK6 or EGFR.Conclusions:Our study indicates that miR-1296-5p inhibits the expressions of the target genes CDK6 and EGFR by binding to the 3'UTR region of their mRNAs,and thereby to exert its anti-cancer effect in gastric cancer.