The Inpacts Of MiR-122,Target Genes Citrate Synthase And Microtubule Binding Protein 1 On Proliferation,Migration And Apoptosis Of Hepatocarcinoma Cell Line

As a highly expressed miRNA in liver tissue,miR-122 plays a regulatory role in the physiological process of hepatocarcinomar cells,and when its expression level is dysregulated,it will lead to various liver diseases.Previous studies have reported that the expression of miR-122 in hepatocellular carcinoma cells is decreased,and the m RNA expression is affected by binding with downstream target genes,thus affecting the physiological processes such as cell proliferation and apoptosis.Therefore,it is important to study the function of downstream target genes of miR-122 for understanding its regulatory mechanism in hepatocarcinomar cells.At present,miRNA target genes are mainly obtained through various prediction software,but most of the predicted target genes are lack of experimental data confirmation.The citrate synthase(CS)and Microtubule-Associated Protein RP/EB Family Member1(MAPRE1)were found to be the predicted target genes of miR-122 by high-throughput sequencing and gene microarray technology.CS is the first key regulating enzyme of the tricarboxylic acid cycle(TCA),which plays an important role in the regulation of various metabolic pathways in vivo.MAPRE1 plays an important role in the production of microtubules and participates in the process of cell mitosis.However,the specific functional mechanism of them in hepatocarcinomar is still unclear.In view of the above problems,we detect the functions of miR-122 and target genes CS and MAPRE1 on a variety of physiological indicators by transfecting miR-122 mimic and siRNA to HepG2 cell,including growth,proliferation,cell migration,cell cycle apoptosis,ROS reactive oxygen species,mitochondrial membrane potential stability and ATP content.The main research results of this study are as follows:1.In this experiment,HepG2 hepatoma cells were transfected with mimics of miR-122 for several cell experiments.Compared with control cells,miR-122 overexpression would block cells from the G1 phase to the S phase,thus inhibiting the growth,proliferation and migration of HepG2 hepatoma cells.We detected the changes of mitochondrial membrane potential,the red/green fluorescence intensity ratio was 42.22% lower than the control group,the intracellular ATP content was 70% lower,but the ROS reactive oxygen level was 1.75 times higher than the control group.At the same time,the number of apoptosis was 39.36%higher than before.Mi R-122 can reduce the stability of mitochondrial membrane potential by activating intracellular ROS activity,and reduce the production of intracellular ATP,thus initiating the mechanism of apoptosis in HepG2 hepatoma cells.2.When the miR-122 was overexpressed,the m RNA levels of downstream target genes predicted by the software,such as CS,MAPRE1,DLAT and GNPDA1 were significantly down-regulated.CS and MAPRE1 were screened out by Dual luciferase assay,it was confirmed that they were target genes of miR-122.The protein levels of CS and MAPRE1 were also down-regulated.In this study,siRNA(si-CS and si-MAPRE1)were used to silence the expression of CS and MAPRE1,and the cell experiments found that: Compared with the control cells,si-CS and si-MAPRE1 blocked the cells in the G1 phase and reduced the DNA synthesis in the S phase;si-MAPRE1 has a better effect on inhibiting the cell cycle.The cell viability of si-CS and si-MAPRE1 treatment was significantly lower than that of the control group,and the cell migration rate was reduced by 68% and 84%,respectively.The apoptosis of si-CS and si-MAPRE1 cells was detected,and the percentage of apoptosis of si-CS cells increased by 42.25%,but si-MAPRE1 did not promote the apoptosis of cells.We detected the changes of mitochondrial membrane potential after si-CS treatment,indicating that the ratio of red/green fluorescence intensity was 39.43% lower than that of the control group,the intracellular ATP content was 50.71% lower,but the ROS reactive oxygen level was 2.19 times higher than that of the control group.CCNG1 m RNA levels were decreased after siCS and si-MAPRE1 treatment,while BCL-2 and caspase-9 m RNA levels were upregulated in si-CS,but there was no significant change in si-MAPRE1.Both si-CS and si-MAPRE1 inhibited the proliferation and migration of HepG2 hepatoma cells,si-MAPRE1 had a stronger effect on cell proliferation and migration.Si-CS can stimulate ROS activity and affect the stability of mitochondrial membrane potential,thus promoting cell apoptosis.This study has deepened the study on the biological functions of miR-122 in HepG2 hepatoma cells,and provides a theoretical basis for further exploring the target gene functions involved in the specific mechanism of miR-122 regulation on hepatoma cells,verifying the downstream predicted target gene CS and MAPRE1 of miR-122 by Target Scan,miRanda and Pic Tar databases was also completed.It has been confirmed that CS and MAPRE1 play an important role in the proliferation,migration,apoptosis and other biological processes of HepG2 hepatoma cells.Therefore,this study provides a new therapeutic idea for the future treatment of hepatoma.