The Mechanism Of MiR-103a-3p Regulating Proliferation And Invasion Of Hepatocellular Carcinoma Cells By Targeting EVA1A

Objective: Hepatocellular carcinoma(HCC)is a common malignancy,and molecular targeted drug therapy has been shown to significantly improve the prognosis of patients with advanced HCC.Recent studies on the pathogenesis of HCC have found that micro RNAs play an important regulatory role in the development of HCC and have become an important target for the diagnosis and treatment of HCC.Among them,miR-103a-3p plays a cancer-promting role in human bladder cancer,gastric cancer,colorectal cancer and other tumors,but its role and mechanism in the tumorigenesis and progression of HCC are still not completely clear.Endoplasmic reticulum transmembrane protein EVA1A(Eva-1Homolog A)is an autophagy and apoptosis regulatory protein newly identified from human kidney c DNA library in recent years.Recent studies have shown that this protein is significantly down-regulated in HCC,and overexpression of EVA1 A can inhibit cell proliferation,migration and invasion in HCC via upregulating TP53.Furthermore,studies have shown that EVA1 A can be down-regulated by miR-125 b in oxaliplatin-sensitive HCC cells,suggesting that the low expression of EVA1 A in HCC may be regulated by some micro RNAs.Previously,through bioinformatics prediction,it was found that EVA1 A might be the target molecule of miR-103a-3p.This study will explore the function of miR-103a-3p in HCC and relative mechanism from the perspective of downstream target molecules,so as to provide theoretical basis for further improving and clarifying the pathogenesis mechanism of HCC and for the diagnosis and treatment of HCC.Methods: In this study,firstly,the expression of miR-103a-3p in HCC tissues,paired adjacent tissues and HCC cell lines(QGY-7703、Hccl-M3、PLC-PRF5),human normal liver cell line L02 were detected by real-time quantitative PCR(RT-qPCR).The effects of miR-103a-3p on the migration,invasion and proliferation of HCC cells were assessed by cell scratch assay,transwell assay,CCK8 assay and plate cloning assay,respectively.Secondly,bioinformatics was used to predict the downstream target genes of miR-103a-3p.The binding effect between miR-103a-3p and the 3’UTR region of EVA1 A was verified by dual-luciferase reporter gene assay.The effect of miR-103a-3p on the expression of EVA1 A was tested by Western blot and RT-qPCR.The effect of co-expression of miR-103a-3p and EVA1 A on the cancer-promoting effect of miR-103a-3p in HCC was evaluated.Finally,the effects of miR-103a-3p on TP53 expression and apoptosis were detected by Western blot and flow cytometry.JC-1 staining,DCFH-DA staining and ATP detection kit were used to check the effects of miR-103a-3p on mitochondrial membrane potential,the production of reactive oxygen species(ROS)and the generation of ATP,to analyze the effects of miR-103a-3p on mitochondrial function.Results: RT-qPCR results showed that miR-103a-3p levels in HCC tissues and cell lines were significantly higher than those in paired adjacent tissues and normal liver cell L02(P<0.001).The overexpression of miR-103a-3p significantly down-regulated the expression levels of EVA1 A and TP53(P<0.05)and promoted HCC cell migration(P<0.05),invasion(P<0.01)and proliferation(P<0.05).Inhibition of miR-103a-3p expression significantly up-regulated the expression levels of EVA1 A and TP53(P<0.05),inhibited HCC cell migration(P<0.05),invasion(P<0.01)and proliferation(P<0.05)and caused mitochondrial dysfunction and HCC cell apoptosis.Bioinformatics predicted that EVA1 A was a potential downstream target gene of miR-103a-3p.Dual-luciferase reporter assay showed that luciferase activity was significantly decreased in the group of miR-103a-3p co-transfected with EVA1A-3’UTR-wt(P<0.001).Compared with the overexpression of miR-103a-3p alone,the cell abiltity of migration(P<0.01),invasion(P<0.01)and proliferation(P<0.01)were significantly reduced in the group co-expressing Myc-EVA1 A.Compared with the inhibition of miR-103a-3p alone,the knockdown of EVA1 A together alleviated the apoptosis and mitochondrial dysfunction caused by the inhibition of miR-103a-3p expression alone.Conclusion: Our research comprehensively analyzed the role of miR-103a-3p in HCC.miR-103a-3p can significantly promote the migration,invasion and proliferation of HCC cells,which plays a cancer-promoting role by targeting EVA1 A in HCC.Inhibition of miR-103a-3p expression in HCC cells plays an anti-cancer role by inducing mitochondrial dysfunction andtriggering apoptosis.The up-regulation of miR-103a-3p expression in HCC may be a mechanism for the tumorigenesis of HCC.miR-103a-3p may be a potential target for the diagnosis and treatment of HCC.