| Background:Hepatocellular carcinoma(hepatic cancer)is one of the most prevalent cancers and the leading causes of human death worldwide.Aberrant gene expression and somatic mutations induced by any genotoxic agents that can cause DNA damage remain one key process leading to HCC.Most patients missed the best period for treatment due to late detection of disease,and systematic chemotherapy plays a crucial role in HCC treatment especially for patients with terminally staged tumors.Cisplatin is a widely used chemotherapeutic agent for the treatment of HCC.However,the high dose of cisplatin causes serious side effects that also kill normal cells and by the long-term side effects of cisplatin,which are carcinogenic and can cause secondary cancers.Thus,decreasing drug resistance of cisplatin has become a hot problem.MiRNAs belong to a class of small,endogenous,non-coding,single-strandedRNAs,which are approximately 22 nucleotides in length.Growing evidence indicates that miRNAs,functionally repress target proteins by pairing with the 3'-UTR of specific target mRNA,inducing mRNA degradation or translational repression..In recent years,large study shown that miRNAs are involved in many biological processes such as cell proliferation,cell differentiation,embryo development,apoptosis and autophagy.MiRNAs also play important roles in development of cancer,which can act as either oncogenic molecules or tumor suppressors by regulating their respective target genes.However,the role of miRNAs in cancer chemotherapy remains largely unknown.Mechanism research on the role of miR-193 b in the therapy of HCC,we can build a solid foundation for clinical study.MicroRNAs(miRNAs)belong to a class of small,endogenous,non-coding,single-strandedRNAs,which are approximately 22 nucleotides in length.Growing evidence indicates that miRNAs,functionally repress target proteins by pairing with the 3'-untranslated region(UTR)of specific target mRNAs,inducing mRNA degradation or translational repression.In recent years,a number of studies have shown that miRNAs are involved in many biological processes,such as cell proliferation,differentiation,development and apoptosis.MiRNAs also play important roles in development of almost all cancers in human beings,which can act as either oncogenic molecules or tumor suppressors by regulating their respective target genes.However,the effects of miR-193 on the onset and development of hepatocellular carcinoma remains largely unknown.Mechanism research on the role of miR-193 b in the therapy of hepatocellular carcinoma,we can build a solid foundation for further clinical study.Objective:MiR-193 b is a polyphonic miRNA,which possesses wide effects of anti-cancers.However,it remains unclear whether overexpression of miR-193 b is able to increase the sensitivity of cisplatin to hepatic cancer cells.The aim of this study was to analyze the influence of cell proliferation and apoptosis through the combine miR-193 b with the chemotherapy agent cisplatin dealing with the cells of Hep G2 cells(a classic cell line of hepatic cancer),and to provide a strategy for the therapy and prevetion of hepatocellular carcinoma.Methods:Forty hepatocellular carcinoma tissue samples and corresponding adjacent non-tumor tissue samples were collected from May 2011 to March 2014 in the first affiliated hospital of University of South China.In addition,the normal hepatic cell lines L-02 as well as hepatic cancer cell lines Huh7,Hep G2 and PLC were cultured.In the current study,we first detected the expression of miR-193 b in 40 paired HCC tumor and adjacent normal liver tissues by using the quantitative real-time polymerase chain reaction(qRT-PCR)method.At the same time,qRT-PCR was used to analyze the expression levels of miR-193 b in the L-02,Huh7,Hep G2 and PLC cells.Secondly,the relationship of miR-193 b with clinical pathological parameters including sex,age,tumor size,tumor differentiation,metastasis,vein offence,satellite focus,tumor number,AJCC stage and ?-fetoprotein(AFP)were analyzed in hepatocellular carcinoma patients.Finally,40 hepatocellular carcinoma patients were divided into miR-193 b low expression group and miR-193 b high expression group according to the expression levels of miR-193 b.The five-year survival rate was compared between miR-193 b low expression group and miR-193 b high expression group.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide(MTT)was used to examine the effect of miR-193 b on cisplatin(1?m,2?m,3?m,4?m,5?m,and 6?m)treatment in Hep G2 cells,and apoptotic rate of Hep G2 cells was determined using Annexin V/PI staining after treatment of cisplatin(2?M)plus miR-193 b mimic or NCO(50 pmol/ml).In the next moment,in order to explore the molecular mechanisms responsible for the sensitization of miR-193 b to cisplatin treatment,we used Target Scan database and found Mcl-1 may be the putative target of miR-193 b,meanwhile,myeloid cell leukemia(Mcl-1)expression levels were determined after miR-193 b mimic transfection by qRT-PCR analysis and western blot analysis.To validate whether Mcl-1 is an actual target of miR-193 b,a Mcl-1 3'-UTR fragment containing either a wild-type or mutant miR-193 b binding sequence was cloned into p MIR reporter plasmid,and Hep G2 cells were cotransfected with p MIR reporter and miR-193 b,incubating for 24 h,firefly luciferase activity was measured,and normalized to Renilla luciferase.Moreover,Hep G2 cells were treated with cisplatin(2?M)plus NCO,miR-193 b or Mcl-1 small Interfering(siRNA,50pmol/ml)for 48 h,cell viability was determined by MTT assay and the expression of Mcl-1that pc DNA3.1-Mcl-1(2 ?g/ml)abolished the suppression of Mcl-1 caused by miR-193 b mimic transfection was determined by q PCR and western blot analysis.Finally,Hep G2 cells were cotransfected with pc DNA3.1-Mcl-1(2 ?g/ml)and miR-193 b mimic for 24 h,and then,cisplatin(2 ?M)was added,incubating for another 48 h,the viability and apoptotic rate of Hep G2 cells were determined by MTT assay and Annexin V/PI staining on flow cytometry;meanwhile,Hep G2 cells were treated with cisplatin(2 ?M)plus miR-193 b or NCO for 24 h in the absence or presence of z VAD-fmk(10 ?M),western blot analysis was performed to measure the cleavage of caspase-3 and its substrate PARP and the cell viability was determined by MTT assay.Result:Our results showed that miR-193 b expression was significantly decreased in hepatocellular carcinoma tumor tissues when compared with the adjacent normal liver tissues(P<0.05).At the same time,the expression levels of miR-193 b were lower in L-02 cells than those in HCC cell lines than in Huh7,Hep G2 and PLC cells(P<0.05).Moreover,the expression levels of miR-193 b were the lowest in Hep G2 cells.We thus selected Hep G2 cells as the subsequent investigation objective.The expression levels of miR-193 b were associated with tumor differentiation,metastasis,vein offence,and AJCC stage of hepatocellular carcinoma patients(P<0.05).However,the expression levels of miR-193 b were not associated with sex,age,tumor size,satellite focus,tumor number,and AFP of hepatocellular carcinoma patients(P>0.05).In addition,five-year survival rate in miR-193 b low expression group was lower than that in miR-193 b high expression group(?2=25.38,P<0.01).MTT cell proliferation assay showed that the enforced expression of miR-193 b plus cisplatin led to a significant decrease in the viability of Hep G2 cells compared to the group of cisplatin combined with NCO(P<0.05).Furthermore,we selected a moderate concentration of cisplatin(2 ?M)for combination treatment with miR-193 b mimic to detect the apoptosis induction.Flow cytometry analysis was performed that more apoptotic cells were observed in the group that treated with the combination of cisplatin and miR-193 b mimic than the single treatment group(P<0.05).Then,Target Scan database found Mcl-1 maybe the putative target of miR-193b(P<0.05),and the transfection of miR-193 b mimic significantly suppressed the expression of Mcl-1 in Hep G2 cells by western blot and qRT-PCR analysis(P<0.05).Firefly luciferase activity showed that a reduction in luciferase activity was observed for the wild-type construct-containing Hep G2 cells(P<0.05);More importantly,transfection of Mcl-1 siRNA plays a similar function with miR-193 b mimic on sensitizing Hep G2 cells to cisplatin inducing cytotoxicity(P<0.05).In addition,transfection with pc DNA3.1-Mcl-1 which contains no 3?-UTR sequences totally overcomes the suppression of Mcl-1 caused by miR-193 b mimic(P<0.05).Meanwhile,MTT cell proliferation assay and flow cytometry analysis shown that the forced expression of Mcl-1 significantly decreased the inhibition effect(P<0.05)and apoptotic rate in combined treatment of cisplatin plus miR-193b(P<0.05).Finally,MTT cell proliferation assay and western blot showed that single cisplatin treatment showed a weak activation of caspase-3 in Hep G2 cells and its substrate poly-ADP-Ribose polymerase(PARP)(P<0.05).More importantly,cell death of Hep G2 cells induced by cisplatin in combination with miR-193 b mimic treatment was inhibited in the presence of z VAD-fmk,suggesting the sensitization of miR-193 b mimic to cisplatin-mediated cell death was caspase-dependant.Conclusion:1)The expression levels of miR-193 b are significantly decreased in hepatic cancer tissues and cell lines.Moreover,its expression levels are associated with the prognosis of hepatic cancer patients.2)Overexpression of miR-193 b can inhibit the growth and induce apoptosis of Hep G2 cells by targeting Mcl-1,which leads to a marked increase in the sensitivity of cisplatin to Hep G2 cells.3)MiR-193 b increased cisplatin-induced apoptosis of Hep G2 cells via activating the caspase-3-dependent signaling pathway. |
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