| Objective:By studying the relationship between retinoic acid-inducible gene I(RIG1)and the occurrence and development of hepatocellular carcinoma,and analyzing the binding relationship between miR-362-5p and RIG1,we studied the effects of miR-362-5p combined with RIG1 on invasion and metastasis of hepatocellular carcinoma cells by regulating PI3K/AKT signaling pathway and cellular immune response in microenvironment.Methods:(1)Immunohistochemical method was used to detect the expression of RIG1 in hepatocellular carcinoma and adjacent tissues,and the relationship between RIG1 and clinicopathological features of hepatocellular carcinoma was analyzed;(2)Predicting the Interaction of miRNA Molecule with RIG1 Gene by bioinformatics: miR-362-5p;(3)The expression of miR-362-5p was detected by q RT-PCR and RIG1 was detected by Western blot in hepatocellular carcinoma and adjacent tissues respectively.The relationship between miR-362-5p and RIG1 was analyzed;(4)The expression of RIG1 and PI3K/AKT related proteins was detected by q RT-PCR and Western blot in hepatocellular carcinoma cell lines with different metastatic potential.The relationship between the expression of miR-362-5p and RIG1 and PI3K/Akt related proteins was analyzed;(5)Double luciferase assay confirmed that RIG1 is a target gene for downstream regulation of miR-362-5p;(6)To study the effects of miR-362-5p combined with RIG1 on PI3K/AKT signaling pathway and cellular immunity: miR-362-5p mimic,inhibitor and blank control group(NC)were synthesized and transfected into hepatocellular carcinoma cell Hep G2 respectively.The expression of miR-362-5p in recombinant cell lines was detected by q RT-PCR.The expression levels of RIG1,PI3 K,AKT,IL-2,IL-4 and TNF-a were detected by Western blot,and the miR-362-5p table was analyzed.The correlation between the expression of RIG1,PI3K/AKT,IL-2,IL-4 and TNF-a protein;(7)Transwell assay was used to detect the change of invasion and metastasis ability of hepatocellular carcinoma cells after combination of miR-362-5p and RIG1.Results:(1).The positive rate of RIG1 protein was 20.0%(10/50)in hepatocellular carcinoma tissues and 85.0%(17/20)in adjacent tissues.There was significant difference between them(P< 0.05).The expression of RIG1 protein was not related to sex,age,maximum diameter of tumors,distant metastasis(P > 0.05),but was related to clinical stage and lymph node metastasis(P < 0.05).It was found that the binding stability and specificity of miR-362-5p to RIG1 were the best.When micro RNA-362-5p was overexpressed in hepatocellular carcinoma,RIG1 was underexpressed;when miR-362-5p was underexpressed in adjacent tissues,RIG1 was overexpressed(P < 0.05).(2)In hepatocellular carcinoma cell lines,the high expression of miR-362-5p and the low expression of RIG1 lead to the over-expression of PI3 K and Akt,and the low expression of miR-362-5p and RIG1 lead to the low expression of PI3 K and Akt,and the low metastasis ability of hepatocellular carcinoma cells.miR-362-5p can bind to the 3'-UTR site of RIG1 and inhibit its expression.RIG1 is a potential target gene downstream of miR-362-5p.Increasing the expression of miR-362-5p can inhibit the expression of RIG1 and enhance the invasion and metastasis ability of hepatocellular carcinoma cells,while the low expression of miR-362-5p can increase the expression of RIG1 and decrease the invasion and metastasis ability of hepatocellular carcinoma cells.Conclusion:1.The expression of RIG1 is related to the occurrence and development of hepatocellular carcinoma,and hepatocellular carcinoma cells with low expression of RIG1 have strong metastasis ability.2.Mi R-362-5p can bind to the 3'-UTR site of RIG1 and regulate the expression of RIG1.3.RIG1 can inhibit PI3K/AKT signaling pathway and restore cellular immune function in microenvironment.4.Mi R-362-5p regulates PI3K/AKT pathway through RIG1,inhibits cellular immune function and promotes invasion and metastasis of hepatocellular carcinoma cells. |
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