The Effect And Mechanism Of Enhancement And Inhibition Of SMP30 Gene Expression On The Biological Behavior Of Hepatocellular Carcinoma Cells

Objective: Liver cancer is a common cancer.It has a high incidence throughout the world and ranks second in cancer deaths among men worldwide.Globally,70%–90% of primary liver cancers are diagnosed as hepatocellular carcinoma(HCC).The highest incidence of liver cancer is in East and Southeast Asia.In China,HCC occupies 50% of the incidence and death of liver cancer in the world,and Guangxi is also a high-risk area of liver cancer in China,accounting for 18.8% of all cancer deaths.The occurrence and development of HCC involves a variety of molecular structures,tumor oncogenes,tumor suppressor genes,gene expression profiles,and intracellular signaling pathways.However,the precise molecular mechanisms affecting its occurrence and development and the synergy between changes are still not completely clear.Therefore,the study of new HCC gene functions and the molecular mechanisms that influence the occurrence and development of disease will provide important theoretical basis for the early diagnosis,prognosis and treatment of liver cancer,and it is of great significance for the prevention and treatment of liver cancer.SMP30 is a liver cancer-related antigen that we have been investigating,and a series of studies have shown that SMP30 protein and its antibodies are important in the diagnosis of liver cancer.However,the effect of SMP30 overexpression and downregulation on the malignant biological characteristics of hepatocellular carcinoma like proliferation,invasion and metastasis,and its molecular mechanism in vitro is unclear.The aim of this study was to elucidate the influence of SMP30 gene on the biological behavior of hepatocellular carcinoma and its regulatory mechanism.Method: 1.17 cases of hepatocellular carcinoma(HCC)of patients with hepatocellular carcinoma who underwent surgical treatment were collected from the First Affiliated Hospital of Guangxi Medical University.The expression level of SMP30 was detected by QPCR;WB and QPCR were used to detect the expression of SMP30 mRNA and protein in SK-hep-1,MHCC97-L and Hep3 B cells;2.The lentivirus with SMP30 CDS and the empty lentivirus LV5 were transfected into SK-hep-1 hepatoma cells respectively.Flow cytometry was used to establish a stable transfected cell model of overexpressing SMP30.QPCR and Western-blot were used to detect the expression of SMP30 at the transcriptional and translational levels before and after transfection.Transwell assay and Scratch test were performed to detect cell migration ability,and Transwell assay was used to detect cell invasive ability;CCK8 assay was used to detect the cell proliferation ability,and the difference of cell proliferation ability between transfected cell line and control group was observed.The expression of mRNAs in SK-hep-1-SMP30 and SK-hep-1-NC groups was detected by microarray,Bioinformatic Analysis SMP30 related genes,and verified by PCR;3.Lentiviral vector encoding small interfering RNA targeting SMP30 and the empty lentivirus LV3 were transfected into Hep3 B hepatoma cells respectively.Flow cytometry was used to establish a stable transfected cell model of suppressing SMP30.QPCR and Western-blot were used to detect the expression of SMP30 at the transcriptional and translational levels before and after transfection.Transwell assay and Scratch test were performed to detect cell migration ability,and Transwell assay was used to detect cell invasive ability;CCK8 assay was used to detect the cell proliferation ability,and the difference of cell proliferation ability between transfected cell line and control group was observed.PCR was used to detect the expression of wnt pathway related genes;4.Use Targetscan and other software tools to predict microRNAs that may regulate SMP30.Expression of miR-382 in Hepatocellular Carcinoma Cells after Transfection with miR-382 mimic by QPCR.QPCR and Western blot(WB)were used to detect the difference of SMP30 expression before and after miR-382 transfection.Construction of SMP30-3'UTR luciferase reporter plasmid,miR-382 mimics,mutation and control group of liver cancer cells were co-transfected SMP30-3 'UTR luciferase reporter plasmid,detection of luciferase activity,to determine the regulation of miR-382 on SMP30-3'UTR.QPCR was used to detect the expression of miR-382 in hepatocellular carcinoma and different cells.Transwell was used to detect the invasion and migration of liver cancer cells before and after miR-382 mimic transplantation.Results: 1.The expression of SMP30 in hepatocellular carcinoma was lower than that in para-carcinoma tissues(P <0.01),SMP30 expression levels were different in different hepatocarcinoma cells,and SMP30 mRNA and protein were the lowest in SK-hep-1;2.SK-hep-1-SMP30 and SK-hep-1-NC cells were observed under the fluorescence microscope after Flow cytometry screened.The transfection was confirmed successful if all cells with green fluorescence.A lentiviral cell line stably expressing SMP30 was successfully constructed.QPCR and Western-blot showed that the expression of SMP30 mRNA and protein in SK-hep-1-SMP30 cells was higher than that in SK-hep-1-NC cells(P<0.01).The SK-hep-1-SMP30 group and the SK-hep-1-NC group were compared,there was no significant difference in the proliferation of HCC cells(P> 0.05),overexpression of SMP30 inhibited cell invasion and migration(P <0.01).Chip detection,bioinformatics prediction,WB validation showed that SMP30 may inhibit cell invasion and migration through EMT and Wnt/?-catenin pathway,(P <0.05);3.Hep3B-siRNA and Hep3B-NC cells were observed under the fluorescence microscope after Flow cytometry screened.The transfection was confirmed successful if all cells with green fluorescence.A lentiviral cell line stably expressing SMP30 was successfully constructed.QPCR and Western-blot showed that Hep3B-siRNA-939 has the most significant inhibitory effect in the four Hep3B-siRNAs(P<0.01),Hep3B-siRNA-939 was used to perform followup behavioral experiments.there was no significant difference in the proliferation of HCC cells(P> 0.05),suppression of SMP30 promoted cell invasion and migration(P <0.01).SMP30 may promote cell invasion and migration through the EMT and Wnt/?-catenin pathway(P <0.05);4.We used TargetScan,miRanda and other online detection combined with previous research results,analysis shows that miR-382 may be the potential regulation of SMP30 microRNA.The relative expression of miR-382 in miR-382 mimic group was 21.06963 times higher than that in miR-382 negative control group after transient transfection with miR-382 mimic(p< 0.01).The expression of SMP30 mRNA and protein was decreased in the hepatocarcinoma cells transfected with miR-382 mimics(p <0.01).Compared with the control group,the fluorescence intensity of miR-382 mimic \ SMP30-3'UTR was significantly decreased(P <0.05),but the fluorescence intensity of miR-382-mut group did not change significantly.QPCR results showed that the expression level of miR-382 in hepatocellular carcinoma was significantly higher than that in para-carcinoma tissues.The expression of miR-382 was different in different cells,The expression of miR-382 in SK-hep-1 cells was the highest in hepatocellular carcinoma cells.MiR-382 mimic promoted the invasion and migration of hepatocellular carcinoma cell line MHCC97-L after transient transfection(P <0.01).Western-blot showed that miR-382 may inhibit the expression of SMP30,and then inhibit the invasion and migration of cells through EMT and Wnt/?-catenin pathway.Conclusion: 1.SMP30 expression in hepatocellular carcinoma was lower than that in para-carcinoma tissues,indicating that SMP30 had certain clinical significance;According to the expression of SMP30 in different hepatocellular carcinoma cells,SK-hep-1 cells could be used as the model of overexpression of SMP30,MHCC97-L and Hep3 B were used as SMP30 inhibition experiment model;2.SK-hep-1-SMP30 and SK-hep-1-NC cell lines were successfully constructed.Overexpression of SMP30 inhibits migration and invasion of SKhep-1 cells.SMP30 may inhibit cell invasion and migration through the EMT and Wnt/?-catenin pathway;3.Hep3B-siRNA-939 and Hep3B-siRNA-NC cell lines were successfully constructed.Suppression of SMP30 promote migration and invasion of SK-hep-1 cells.SMP30 may regulate cell invasion and migration through the EMT and Wnt/?-catenin pathway.4.MiR-382 is one of the microRNAs that regulate the SMP30 gene.MiR-382 down-regulates the expression of SMP30.The expression of miR-382 was negatively correlated with SMP30 in tissues and cells.MiR-382 promotes the invasion and migration of hepatocarcinoma cells.miR-382 may inhibit the expression of SMP30,and then inhibit the invasion and migration of cells through EMT and Wnt/?-catenin pathway.