| ObjectiveTo investigate the expression and mechanism of microRNA in hepatocellular carcinoma from serum and cell levels.On the serum level,the expression of miR-486-5p and miR-122-5p in hepatocellular carcinoma and the diagnostic value of combined diagnosis with AFP in hepatocellular carcinoma were investigated.At the cellular level,explore the potential target of miR-486-5p and its mechanism on the proliferation and migration of HCC cells.Methods1.Collected from June 2016 to March 2017 Guangzhou General Hospital of PLA,60 cases of HCC serum samples,including 57 males and 3 females,mean age 53.9 ± 11.2 years,at the same time collecting 30 cases of hepatitis patients,30 cases of cirrhosis patients,30 cases of gastric cancer,30 cases of breast cancer,30 cases of healthy patients as a control group.The relative expression of miR-486-5p and miR-122-5p in each experimental group and control group were detected by RT-PCR.Combined with AFP,ROC curve was used to demonstrate the value of serological diagnosis.2.The relative expression of miR-486-5p was detected with HCC cell line SMMC-7721,and a stable HCC cell line SMMC7721 overexpressing miR-486-5p was constructed..CCK-8,cell wound scratch assay and Transwell assay were performed to study the function of miR-486-5p on cell proliferation and migration in hepatocellular carcinoma.3.Through the four databases of TargetScan,miRanda,miRWalk and RNA22,22 potential targets of miR-486-5p were predicted.CBL was confirmed as the target of this study by qPCR and Western blot.The expression of CBL in miR-486-5p overexpressing HCC cells was detected by Western blot.The recombinant plasmid was transfected into miR-486-5p overexpressing HCC cells.The effect of CBL on cell proliferation and migration were performed by CCK-8,cell scratches and Transwell experiments.Results1.The expression of serum miR-486-5p and miR-122-5p in HCC were lower than those in the healthy control group,and the difference was statistically significant(P<0.05).Compared with the gastric cancer group and the breast cancer group,the expression of the HCC group was significantly decreased,and the difference was statistically significant(P<0.05).2.There was a negative correlation between miR-486-5p and miR-122-5p with AFP serum expression levels.The correlation analysis of miR-486-5p and miR-122-5p with AFP serum expression levels showed a negative correlation with AFP expression.3.The combination of miR-486-5p,miR-122-5p and AFP showed that using one serum marker to diagnosis HCC,miR-122-5p had the highest specificity(95%),and AFP had the highest sensitivity(73.7%).While combined two serum markers,AFP + miR-486-5p had the highest sensitivity(86%),AFP + miR-122-5p had the highest specificity(90%).The sensitivity,and specificity of the three markers were 83.5% and 90% respectively,The area under the ROC curve was 0.891(95% CI: 0.818-0.964).The combined detection of the three markers were higher than that of the single test.4.Tissue expression: a total of 422 samples were analyzed using the TCGA database,of which 372 were HCC specimens and 50 were adjacent specimens.The study showed that the expression of miR-486-5p in tumor samples was significantly lower than that in adjacent non-cancerous samples,with a statistically significant difference(P <0.0001).The analysis of tumor size,invasiveness and clinical stage showed no significant difference(P>0.05).GSEA analysis showed that 14 pathways were positively correlated with miR-486-5p levels and 4 pathways were negatively correlated with miR-486-5p levels.According to the database,the collected samples were divided into 2 and 3 subtypes by cluster analysis,and the miR-486-5p expression is detected in each subtype.5.miR-486-5p was lowexpressed in SMMC-7721 cells.The expression of miR-486-5p was significantly increased after miR-486-5p overexpressed in SMMC-7721 cells,The difference was statistically significant(P<0.001).Compared with the vector,miR-486-5p overexpressed group inhibit the proliferation and migration of hepatocellular carcinoma cells,the difference was statistical significance(P<0.05).6.With qPCR and Western blot verification,miR-486-5p overexpression inhibited the expression of CBL,overexpression of miR-486-5p HCC cells co-expressed CBL recombinant plasmid,the proliferation and migration of HCC cells increased,with statistical significance(P<0.05).Conclusion1.Serum miR-486-5p and miR-122-5p can be used as a potential biomarker for the diagnosis of liver cancer,and the combination of the two with AFP can improve the diagnosis of liver cancer.2.The low expression of miR-486-5p in HCC can inhibit the proliferation and migration of hepatocellular carcinoma cells.CBL may be an important target in the development and progression of HCC. |
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