MicroRNA-449a Suppresses Hepatocellular Carcinoma Cell Growth And Metastasis By Targeting C-Met Pathway

Primary liver cancer(PLC)is one kind of extremely malignant tumors.The histopathologic type of PLC can be categorized into hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC)and mix form carcinoma.It is known that HCC takes great majority of PLC,which is the second leading cause of all cancer deaths in China[1].Under most circumstances,HCC develops from long-term and chronic liver disease(accounts for about 70%?90%of etiology)[2].Hepatitis B virus(HBV)infection and aflatoxin B1 contaction are major etiological factors in southeast Asia and Africa.However,hepatitis C virus(HCV)infection and alcoholic cirrhosis are most important factors in Europe,North America and Japan[3].According to incidence worldwide,almost 50%of HCC patients result from HBV infection[4].Hence,HBV infection is the most risk factor for developing HCC.To date,growing report indicates that more and more HBV/HCV carriers and clinical cases have been diagnosed in China.At present,Chinese HBV patients are up to 93,000 thousands,nearly account for 1/4 worldwide.Concequently,Chinese HCC patients account for about 55%of all cases in the whole world.The new data from the National Central Cancer Registry(NCCR)demonstrated that the estimates of new HCC cases and deaths were 366 thousands and 321 thousands in 2012,respectively.The incidenceand mortality of HCC were 19.44/106 and 16.84/106,which respectively accounted for the third of all cancer cases and the second of all cancer deaths[1].Obviously,HCC is an extremely serous issue in China,which threatens people's health as well as result in huge expenses and social burden.Nowdays,HCC can be resort to hepatectomy,liver transplantation,chemotherapy,radiotherapy,interventional therapy,targeted drug medication,and so on[5,6].Despite recently great advances in therapeutic strategies above,long-term survival rate of HCC patients remains unsatisfactory mainly due to its high recurrence and metastatic rate.Recent studies have demonstrated that oncogene and anti-oncogene aberrant expression,as well as signal pathway dysregulation may be important reasons for the molecular behavior change,and ultimately lead to HCC recurrence and metastasis[7,8].Therefore,revealing the mechanisms of HCC molecular behavior and investigating its roles on HCC development are of importance in HCC clinical treatment.MicroRNAs(miRNAs)are a class of small non-coding RNAs,approximately 19 to 25 nucleotides,which are gradually produced by a series RNase ? enzymes and transport proteins in nucleus and cytoplasm[9]In nucleus,biogenesis of miRNA begins with RNA polymerase ?-dependent or RNA polymerase ?-dependent transcription that generates a long primary transcript(pri-miRNA).DGCR8 recognizes pri-miRNA at the stem single-stranded RNA junction and positions RNase? endonuclease Drosha at the correct catalytic sites to cleave approximately 11 bp away from the junction,releasing a hairpin-shaped pre-miRNA.The secondary double-stranded RNA(dsRNA)stem is then recognized by exportin-5(XP05)in complex with Ran-GTP,subsequently shuttling to the cytoplasm via GTP hydrolysis.In the cytoplasm,the terminal loop of pre-miRNA is cleaved by another dsRNA-specific RNase ? Dicer in cooperation with dsRNA-binding protein TAR RNA-binding protein or protein activator of PKR.Both the 5'-phosphorylated end and 3 overhang of the pre-miRNA are anchored by the PAZ domain of Dicer.TRBP facilitates Dicer-mediated cleavage through direct binding with the pre-miRNA via its two dsRNA-binding domains(dsRBD)and enhances the stability of Dicer-RNA complex.TRBP and PACT participate in the recruitment of Argonaute2(Ago2)to Dicer,forming the RISC loading complex(RLC).There are several characteristics in miRNA,such as highly conserving,sequence homology,time series and tissue specificity.MiRNAs can regulate gene expression by binding directly to 3'-UTR of collresponding target messenger RNAs(mRNAs)in a sequence-specific manner,which play an important role in embryo development,cell proliferation,growth,differentiation,apoptosis and tumorigenesis.According to bioinformatics analysis,miRNAs can regulate almost 20%?30%genes in human,which related to human tumors and cardiovascular disease[10].Emerging evidence has demonstrated that the aberrant expression of miRNAs may regulate biological behavior by targeting oncogenes,anti-oncogenes,cyclins key kinases in signal pathway,apoptosis factors,transcription factors and growth factors,which is one of important factors relating to therapeutic effectiveness and the medical prognosis[11,12].In recent years,some new miRNAs have been gradually found and further studied,whose important roles in HCC development and invasion have been increasingly understood[13].MiR-122 is the most abundant miRNA in hepatocyte,which can potentially regulate HCC invasion and metastasis by targeting ADAM 17[14]and miR-122/CUX1/RhoA pathway[15].MiR-199a/b-3p is the other abundant miRNA in hepatocyte,showing low expression in HCC tissues,and perfroming anti-cancer effect by inhibiting PAK4 and attenuating PAK4/Raf/MEK/ERK pathway.In last 5 years,our research team focused on studies of miRNA regulation and its molecular mechanisms in HCC development.Some miRNAs have been found to regulating HCC molecular behavior.For example,miR-34a repressed HCC cell proliferation and neutralized cell invasion by regulating p53 pathway and cell cycle[161.MiR-145 inhibited HCC cell growth by targeting IRS1 and regulating Akt/FOXO1 downstream pathway[17].At present,our team is in charge of some research projects about HCC etiology,such as "Histone acetylated modification induced ncRNA abnormal expression lead to metastasis and recurrence in HCC patients after hepatectomy","miR-106a regulate HCC cell apoptosis by targeting TIMP-2" and "c-MET regulate HCC cell growth and invasion via Ras-Raf-Erk/Wnt pathway".In our previous studies,some differentially expressed miRNAs in HCC tissues had been detected by the bead-based miRNA expression profiling method,some of which were related to HCC cell growth and invasion.MiR-449a,found in near recent research,is one of low expressed miRNAs in HCC tissues.Based on our research background previously,the present study aimed to explore the protential roles and underlying mechanisms of miR-449a in HCC development.Clinical trail was applied to detected the expression of miR-449a and c-Met in both HCC tissues and adjacent non-tumorous liver tissues(ANLT)using qRT-PCR technique.Experiments in vitro and in vivo were designed to study the effect of high or low expressing miR-449a on HCC cell growth and invasive capacity,and to investigate the molecular mechanisms relating to c-Met pathway.Chapter 1.The expression of miR-449a and c-Met in HCC tissuesObjective:To analyze correlation between the expression of miR-449a in HCC tissues and the clinicopathologic characteristics,as well as the prognosis of HCC patients.Methods:Thirty eight samples from both HCC tissues and adjacent non-tumorous liver tissues(ANLT)were obtained from May,2014 to April,2016 in Hepatobiliary Surgery Department of our hospital.Clinical data was recorded.All the patients were followed up.The expression of miR-449a and c-Met in HCC tissues and ANLT was detected by qRT-PCR.According to the mean of miR-449a expressing results in HCC tissues,38 cases were divided into low-expression group(n=28)and high-expression group(n=10).The correlations of miR-449a expression with clinicopathologic characteristics and prognosis of HCC patients were analyzed.Results:.The qRT-PCR results showed that the miR-449a expression in HCC tissues was remarkably lower than that in ANLT.Inversely,the expression of c-Met in HCC tissues was extremely higher than that in ANLT.The miR-449a expression was negatively correlated with the c-Met expression according to correlation analysis.The miR-449a expression in HCC tissues was found to be correlated with the tumor diameter,metastasis and recurrence.However,it had no significant correlation with gender,age,HBV infection,cirrhosis,AFP level,tumor number,tumor capsule and pathological grade.Comparing with high-expression group,low-expression group was significantly lower in over survival time and tumor-free survival time.Conclusion:Our study suggested that miR-449a expression in HCC tissues is remarkably lower than that in ANLT.It was negatively correlated with the c-Met expression level and might play a downregulated role in c-Met expressing.Low-expression miR-449a might induce invasive and metastatic potentiality of HCC,and would lead to poor prognosis of HCC patients.Chapter 2.The effect of miR-449a expression change on HCC cell growth and invasive capacityObjective:To investigate the effect of miR-449a expression change on HCC cell growth and invasive capacity.Methods:MiR-449a expression in hepatocyte L02 and hepatoma cell lines,such as HepG2,Hep3B,SMMC-7721 and Bel-7402,was respectively examined.Bel-7402 cells were transfected with either miR-449a mimic,miR-449a inhibitor,or their control vectors,by liposome transfection method,which were designated as miR-449a mimic cells,mimic-NC cells,miR-449a inhibitor cells and inhibitor-NC cells.Cell proliferation was detected by CCK-8 method.Cell cycle was analyzed by flow cytometer.Cell invasion was detected using Transwell assay with Matrigel.Bel-7402 cells with high expression miR-449a or control were subcutaneously transplanted into nude mice.Xenografts were detected in the nude mice 7 days after injection.At 28 days,xenografts in mice were successfully excised,of which weight and volume were measured.Results:The expression miR-449a in 4 hepatoma cell lines was significantly lower than that in L02 cells.Among the 4 hepatoma cell lines,Bel-7402 cells showed lowest expression in miR-449a and were chosen to be the experimental cells in following study.Comparing with mimic-NC cells,miR-449a mimic cells were extremely higher in miR-449a expression.Whereas,the miR-449a expression in miR-449a inhibitor cells was significantly lower than that in inhibitor-NC cells.Cell proliferation of miR-449a mimic cells in transfection 24h,48h and 72h was significantly lower than those of mimic-NC cells in corresponding time-point.Comparing with inhibitor-NC cells in transfection 24h,48h and 72h,miR-449a inhibitor cells was higher respectively in cell proliferation in corresponding time-point.The percentage of miR-449a mimic cells in G1 phase was obviously higher than that of mimic-NC cells.Whereas,the percentage of miR-449a inhibitor cells in G1 phase was significantly lower comparing with that of inhibitor-NC cells.Number of migrated cells per high-power field(HPF)in miR-449a mimic cells group was less than that in mimic-NC cells group.Inversely,miR-449a inhibitor cells group showed more migrated cells number per HPF comparing with inhibitor-NC cells group.Tumorigenesis experiment demonstrated that the weight and volume of xenografts in high-expression miR-449a group are much lower and smaller than those in control group.Conclusion:Bel-7402 cells proliferation inhibiting,G1/S phase arresting and invasive capacity attenuating were regulated by high-expression of miR-449a.Low-expression miR-449a may induce cell proliferation,promote Gl/S phase and enhance cell invasive capacity.High-expression miR-449a could suppress tumorigenesis in vivo.Chapter 3.miR-449a regulates HCC cell growth and invasive capacity by targeting c-MetObjective:To explore the regulation of miR-449a in HCC cell growth and invasive capacity by targeting c-Met,and analyze its molecular mechanism.Methods:The protein expression of Cdk2,Cdk4,Cdk6,Cyclin D1,Cyclin El,c-Met,pERK,Ras and Raf-1 was determined by Western blot.MiR-449a targets were predicted and verified by bioinformatics and Luciferase reporter assay.C-Met expression in Bel-7402 cell was silenced by siRNA technique.Cell proliferation was detected by CCK-8 method.Cell cycle was analyzed by flow cytometer.Cell invasion was detected using Transwell assay with Matrigel.Results:High-expression miR-449a may inhibit the protein expression of Cdk6,Cyclin D1,Ras,Raf-1 and pERK.C-Met is one of direct targets of miR-449a.MiR-449a suppressed c-Met expression by directly targeting 3'-UTR sequence of c-Met.Inhibition of c-Met in Bel-7402 cells resulted in cell proliferation suppressing,G1/S phase arresting and invasive capacity attenuating,which similar to those induced by high-expression miR-449a treatment.Conclusion:High-expressing miR-449a may induce G1/S phase arrest by inhibiting the protein expression of Cdk6 and Cyclin D1,and block Ras/Raf/ERK pathway by suppressing the protein expression of Ras,Raf-1 and pERK,which ultimately leads to cell growth and invasion neutralizing.MiR-449a regulates HCC molecular behavior by negative regulation on c-Met expression.Silencing c-Met expression may lead to inhibiting the protein expression of Cdk6,Cyclin D1 and pERK,Gl/S phase arresting and Ras/Raf/ERK pathway blocking,which eventually results in cell growth and invasive capacity attenuating.