MiR-139-5p,miR-940 And Mir-193a-5p Inhibited HCC Development Through Targeting SPOCK1

BackgroundHepatocellular carcinoma(HCC)is characterized with high mobidity and high mortality in China because of the great population of hepatitis B infection.Due to most of the patients were diagnosed as advanced disease,the prognosis were poor although the treatments of HCC have been developed rapidly.Since the molecular biology played a significant role in the development of HCC,it is urgent to study the molecular mechanism of HCC in order to improve the prognosis through early diagnosis and treatment.MicroRNAs could inhibit the translation and promote the decompositon of the target genes by binding to the 3'UTR region of target gene mRNA,thus down-regulating the expression level of target genes.Studys have confirmed that miRNAs play an important role in the occurence,invasion and metastasis processes of malignant tumors.Among them,mir-139-5p,mir-940 and mir-193a-5p have been proved to play a negative regulatory role in the occurrence and development of hepatocellular carcinoma,but the mechanism is not clear yet.SPOCK1,which encodes the protein core of a seminal plasm proteoglycan,is a member of the Calcium Binding Proteoglycan family.It may be involved in the interaction between cells and extracellular matrix,and is closely related to cell proliferation,apoptosis and metastasis.Studies have shown that SPOCK1 is up-regulated in a variety of tumors,which plays an important role in promoting the proliferation,invasion,metastasis and inhibiting the apoptosis of tumor cells,but there are few studies in hepatocellular carcinoma.Object iveTo clarify the expression of miR-139-5p,miR-940 and miR-193a-5p in hepatocellular carcinoma(HCC),and their relationship with proliferation,invasion and apoptosis of HCC cells.To determine whether there are common target gene of miR-139-5p,miR-940 and miR-193a-5p,and to study the expression of the common target gene in hepatocellular carcinoma and the relationship of the common target gene and the proliferation,invasion,apoptosis of hepatocellular carcinoma cells.Methods1.Forty-six samples of hepatocellular carcinoma tissues and their adjacent normal tissues were collected.HepG2,Hep3b and HL-7702 hepatocellular carcinoma cell lines were cultured.The expressions of miR-139-5p,miR-940 and miR-193a-5p in hepatocellular carcinoma cells and adjacent normal hepatocytes,as well as in different cell lines,were detected by qRT-PCR.2.Three miRNAs(miR-139-5p,miR-940 and miR-193a-5p)mimics were used to transfect hepatocellular carcinoma cell lines HepG2 and Hep3b,respectively.The expression of three miRNAs was up-regulated in the two cell lines.The transfection effect was confirmed by qRT-PCR.The proliferation activity of cells before and after transfection was determined and compared by MTT assay,the number of invasive cells before and after transfection was determined and compared by Transwell assay,and flow cytometry assay was used to determined the cell apoptosis,in order to evaluate the effects of three miRNAs on proliferation,invasion and apoptosis of hepatocellular carcinoma cells.3.Bioinformatics databases were used to predict the common target genes of mir-139-5p,mir-940 and mir-193a-5p.The most probable target gene SPOCK1 was selected by RNA pull down method for further validation.The expression of three miRNAs,SPOCK1 mRNA and protein were detected by qRT-PCR and Western Blot respectively in hepatocellular carcinoma tissues and adjacent normal tissues.Three miRNA mimics were transfected into hepatocellular carcinoma cell lines HepG2 and Hep3b respectively.The expression of SPOCK1 mRNA and protein was detected by qRT-PCR and Western Blot respectively before and after transfection.The dual luciferase reporter assay was used to confirm whether SPOCK1 is a common target gene of miR-139-5p,miR-940 and miR-193a-5p.4.HepG2 and Hep3b cells were transfected with full-length SPOCK1 and si-SPOCKl pcDNA3.1 vectors respectively to construct SPOCK1 overexpressing and low-expressing cells.Three miRNAs mimics were transfect to SPOCK1 overexpressing cells.The expression of SPOCK1 mRNA and protein of each group was determined by qRT-PCR and Western blot respectively.Cell viability and invasive ability of each group were determined by MTT assay and Transwell assay.Cell apoptosis of each group was detected by flow cytometry assay..5.The miR-139-5p,miR-940 and miR-193a-5p mature sequences were first cloned after the cytomegalovirus promoter into the pcDNA3.1 vector.HepG2 cells were transfected with these vectors to up-regulate the expressions of miR-139-5p,miR-940 and miR-193a-5p respectively.MiR-139-5p,micR-940 and miR-193a-5p overexpressed HepG2 cells and ordinary HepG2 cells were injected into the back of 8-week-old nude BALB/c mice respectively to construct the transplanted tumor model.Animals were killed after 4 weeks,the tumour volumes and weights were measured.qRT-PCR and Western blot were used to detemine the expression of SPOCK1 mRNA and protein in each group,and to determine whether microRNA-139-5p,microRNA-940 and microRNA-193a-5p could inhibit the growth of tumors in vivo.Result1.Compared with normal liver tissues adjacent to cancer,the expression of miR-139-5p,miR-940 and miR-193a-5p in hepatocellular carcinoma was down-regulated significantly;compared with HL-7702 cell lines,the expression of miR-139-5p,miR-940 and miR-193a-5p in HepG2 and Hep3b cell lines was down-regulated significantly.These results suggest that the expression of miR-139-5p,miR-940 and miR-193a-5p is low in hepatocellular carcinoma cells.2.After transfection of HepG2 and Hep3b cell lines with microRNA mimics,the relative expression levels of miR-139-5p,miR-940 and miR-193a-5p were up-regulated.MTT assay was used to detect the proliferation activity of cells before and after transfection.It was found that the cell viability of HepG2 and Hep3b cells decreased significantly when the expression of three miRNAs was up-regulated.The number of invasive cells before and after transfection was detected by Transwell assay.It was found that the number of invasive cells of HepG2 and Hep3b cells was significantly decreased after the expression of three miRNAs was up-regulated.Flow cytometry was used to detect the cell apoptosis of HepG2 and Hep3b cells before and after transfection.It was found that the apoptosis of HepG2 and Hep3b cells increased significantly after the expression of three miRNAs was up-regulated.Among them,Hep2 and Hep3b cells co-transfected with three miRNA mimics at the same time showed the most significant decrease in proliferation activity,invasive cell number and increase of cell apoptosis.3.Target Scan,starBase and microRNA.org databases were used to screen and predict the common target genes of miR-139-5p,miR-940 and miR-193a-5p.Four possible target genes,SPOCK1,GED1,TRAF 3 and TAOK1,were selected.Then Four genes were identified by RNA pull down assay,and find out SPOCK1 was the most likely target gene for three miRNAs.qRT-PCR and Western blot analysis showed that the expression of SPOCK1 mRNA and protein in hepatocellular carcinoma tissues was significantly higher than that in adjacent normal tissues.The expression of SPOCK1 mRNA and proteinin in HepG2 and Hep3b cells was significantly higher than that in HL-7702 cells.The expression of SPOCK1 mRNA and protein in HepG2 and Hep3b cells was significantly decreased after three miRNAs were transfected which up-regulated the expression of three.miRNAs in HepG2 and Hep3b cells.Further dual luciferase reporter assay showed that SPOCK1 gene could match with miR-139-5p,miR-940 and miR-193a-5p,and was the common target gene of three microRNAs.4.Overexpression of SPOCK1 in HepG2 and Hep3b cells could promote proliferation and invasion and inhibit apoptosis.After knockout of SPOCK1,the proliferation and invasion were inhibited,and the apoptosis was promoted.However,simultaneous overexpression of SPOCK1 and three miRNAs retrieved the viability and invasive ability of HCC cells.cell apoptosis remained stable in SPOCK1+miRNAs groups.SPOCK1 could promote growth and invasion of HCC cells,and its promotiveeffect could be attenuated by miR-139-5p,miR-940 and miR-193a-5p.5.Over-expression of miR-139-5p,miR-940 and miR-193a-5p reduced the volume and weight of tumor in vivo.The SPOCK1 expression in these tumour tissues showed that overexpression of three miRNAs significantly decreased the expression of SPOCK1.The protein expression of SPOCK1 in tumour tissues confirmed the down-regulation of SPOCK1 resulted from miR-139-5p,miR-940 and miR-193a-5p.Conclusions1.The expression of miR-139-5p,miR-940 and miR-193a-5p was low in hepatocellular carcinoma cells,which was negat.ively correlated with the proliferation activity and invasion ability of hepatocellular carcinoma cells,and positively correlated with the apoptosis of hepatocellular carcinoma cells.2.SPOCK1 is highly expressed in hepatocellular carcinoma cells.The expression of SPOCK1 is positively correlated with the proliferation activity and invasion ability of hepatocellular carcinoma cells,and negatively correlated with the apoptosis of hepatocellular carcinoma cells.3.SPOCK1 is the common target gene of miR-139-5p,miR-940 and miR-193a-5p.MiR-139-5p,miR-940 and miR-193a-5p inhibit the proliferation and invasion of hepatocellular carcinoma cells and induce apoptosis of hepatocellular carcinoma cells by targeting SPOCK1.In conclusion,miR-139-5p,miR-940 and miR-193a-5p successfully restrained HCC deterioration through down-regulating SPOCK1.