The Study Of LncRNA H19 Regulating Biological Behavior And EMT Process Via MiR-15b/CDC42 Signaling Axis In Hepatocellular Carcinoma

Backgroud:Hepatocellular carcinoma is the most commonly occurring refractory malignancy in adults due to the unique anatomical features of the liver.Hepatocellular carcinoma could undergo extensively introhepatic metastasis and extrohepatic metastasis at an early stage.EMT plays a crucial role in early steps of metastasis.Therefore,it is of paramount importance to explore the mechanism of the regulating biological behavior in HCC,especially to explore the mechanism of regulating EMT process.Our data have verified that IncRNA H19、miR-15b and CDC42 were abnormally expressed in the hepatic carcinoma.The bioinformatic analysis revealed that miR-15b could bind H19 and 3’UTR of CDC42 with a portion of complementary sequences.Therefore,we intend to explore the relationship between IncRNA H19r miR-15b,CDC42 expression and HCC clinic pathological factors in the HCC tissues and cell lines.Then perform a serial of cell function experiments to verify the interactive effect of IncRNA H19,miR-15b and CDC42.Furthermore,we wish to deeply explore the mechanisms of H19/miR-15b/CDC42 axis involved in HCC biological behavior and EMT process.Part Ⅰ:Expression of IncRNA H19,miR-15b and CDC42 in Hepatocellular CarcinomaObjective To investigate differential expression of IncRNA H19,miR-15b and CDC42 in human hepatocellular carcinoma(HCC)tissues and adjacent tissues,human HCC cells and normal hepatocytes.The associations between lncRNA H19,miR-15b,CDC42 expression and HCC clinicopathological factors were explored.Methods A total of 46 human HCC tissues and their adjacent paired tissues were collected.A variety of HCC cell strains and normal human hepatocytes were obtained in logarithmic growth phase.Total RNA was extracted by Trizol,and cDNA was prepared with specific reverse transcription primers.Based on the complete sequences of lncRNA H19 and miR-15b in Genebank,PCR detection primers targeting lncRNA H19 and miR-15b were designed for quantitative real-time PCR(qRT-PCR)detection of lncRNA H19 and miR-15b content.A variety of hepatoma cell strains and human normal liver cells in logarithmic growth phase were applied to extract total cellular protein and quantified by the BCA assay.The protein of CDC42 was detected by Western blot.The protein expression of CDC42 was detected by immunohistochemistry in the 46 human HCC tissues and adjacent paired tissues.Differential expression and correlation of IncRNA H19,miR-15b and CDC42 were analyzed over human HCC tissues and adjacent tissues,human HCC cells and normal hepatocytes.Results Compared with adjacent tissues and human normal hepatocytes,qRT-PCR revealed that lncRNA H19 was highly expressed in human HCC tissues and cells strains(P<0.05);while miR-15b was low expressed(P<0.05).Western blot exhibited that CDC42 protein was highly expressed in human HCC strains when compared with human normal hepatocytes(P<0.05).Immunohistochemistry results manifested that CDC42 was highly expressed in human HCC tissues when comparing to adjacent tissues(P<0.05).Moreover,we confirmed that the expression of lncRNA H19,miR-15b and CDC42 was up-regulated in 50.0%(23/46),47.8%(22/46)and 54.3%(25/46)of HCC tissues compared with adjacent benign tissues,repectively.Statistical analyses revealed that H19 expression levels were significantly associated with tumor number(χ2=9.698,p=0.002)and histological differentiation(χ2=4.934,p=0.026).However,we did not find any correlation between H19 expression levels and other clinicopathological features,including gender,age,tumor size,AFP level,tumor size,PVTT(portal vein tumor thrombi)and AJCC(American Joint Committee on Cancer)stage.miR-15b expression levels were significantly associated with tumor number(χ2=7.066,P=0.008).However,we did not find any correlation between miR-15b expression levels and other clinicopathological features,including gender,age,tumor size,AFP level,PVTT,AJCC stage and histological differentiation.Furthermore,CDC42 expression levels were significantly associated with AJCC stage(χ2=5.275,P=0.022)and histological differentiation(χ2=5.100,P=0.024).However,we did not find any correlation between CDC42 expression levels and other clinicopathological features,including gender,age,tumor number,AFP level,PVTT and tumor size.Conclusions IncRNA H19 and CDC42 were highly expressed in human HCC tissues and cells,and miR-15b was lower expressed in human HCC tissues and cells.LncRNA H19 expression levels and CDC42 were positively correlated,while miR-15b expression levels were negatively correlated with IncRNA H19 and CDC42.High H19,CDC42 and low miR-15b expression were correlated with the clinicopathological factors,such as tumor number,histological differentiation and AJCC stage in HCC patients.Part Ⅱ:Regulatory Relationship among lncRNA H19,miR-15b and CDC42Objective To explore the regulatory relationships of lncRNA H19 and miR-15b,miR-15b and CDC42.Methods Possible binding sites among lncRNA H19 and miR-15b,miR-15b and CDC42 were predicted by Bioinformatics software.miR-15b mimics and miR-15b inhibitors were synthesized respectively.A short hairpin RNA for lncRNA H19 was designed,synthesized and cloned into the pSicoR plasmid vector(shH19).HepG2 and Bel-7402 cell lines were transfected with the shH 19,miR-15b mimics,and miR-15b inhibitors,respectively,and the expression of lncRNA H19,miR-15b,and CDC42 mRNA were analysed by qRT-PCR after the transfection.The H19,CDC42 3’UTR region and their mutants were cloned into the luciferase vector psiCHECK-2 to construct wild-type and mutant plasmids of the H19,CDC42 3’ UTR region.The H19 wild-type and mutant plasmids were co-transfected with miR-15b mimics,miR-15b inhibitor,miR-15b mimics negative control or miR-15b inhibitor negative control in 293T cells,respectively.The luciferase activity of various groups was detected by dual luciferase reporter system after cells were collected,so as to verify the targeted regulatory relationship of IncRNA H19 and miR-15b,miR-15b and CDC42.Results Bioinformatics software found that there are potential binding sites between lncRNA H19 and miR-15b,and miR-15b and CDC42 3’UTR regions.After transfection with shH19,the expression levels of IncRNA H19 and CDC42 mRNA in HepG2 and Bel-7402 cells were significantly decreased compared with the control and negative control groups,while the expression level of miR-15b was significantly increased compared with the control and negative control groups(P<0.05).After transfected with miR-15b mimics,the expression level of CDC42 mRNA in HepG2 and Bel-7402 cells was significantly decreased compared with the control and negative control groups,and the expression level of miR-15b was significantly increased comparing to control and negative control groups(P<0.05);while transfected with HepG2 and Bel-7402 cells,the expression level of CDC42 mRNA of miR-15b inhibitor was significantly increased comparing to the control and negative control group,and miR-15b expression level was significantly decreased comparing to the control and negative control groups(P<0.05).Dual luciferase report revealed that the dual luciferase activity in the H19 wild-type group was significantly lower than that in the control group after co-transfected miR-15b mimics with H19 wild-type or mutant recombinant plasmids into 293T cells,respectively(P<0.05);furthermore,the dual luciferase activity in the H19 wild-type group was significantly increased comparing to the control group after co-transfected miR-15b inhibitor with H19 wild-type or mutant recombinant plasmids into 293T cells,respectively(P<0.05).However,the dual luciferase activity of CDC42 3’UTR wild-type group was significantly lower than that of the control group after co-transfected miR-15b mimics with CDC42 3’UTR wild-type or mutant recombinant plasmids into 293T cells,respectively;the dual luciferase activity of CDC42 3’ UTR wild-type group was significantly increased comparing to the control group after co-transfected miR-15b inhibitor with CDC42 3’UTR wild-type or mutant recombinant plasmids,respectively,(P<0.05).MiR-15b mimic,miR-15 binhibitor had no influence on luciferase activity of H19 and CDC-42 3’UTR mutant recombinant plasmids(P>0.05).Conclusions The IncRNA H19 can target and bind miR-15b,and CDC42 is a target gene of miR-15b.In HCC cells,H19 can up-regulate the expression of CDC42 via targeting miR-15b.Part Ⅲ:Effects of lncRNA H19/miR-15b/CDC42 Signaling Axis on Biological Behavior of Hepatoma CellsObjective Establish cell models with different expression levels of lncRNA H19 and miR-15b,then explore its influence on the expression of CDC42 and the biological processes of hepatoma cells,such as cell proliferation,invasion,migration,and apoptosis.Methods Bel-7402 and HepG2 cells were transfected with shH19,miR-15b mimics,miR-15b inhibitor and shH19/miR-15b inhibitor complex liposomes,respectively,and the expression of CDC42 mRNA and protein in the cells were detected by qRT-PCR and Western blot.In addition,cell proliferation,invasion and migration,and apoptosis were observed by plate cloning test,scratch test,Transwell test and apoptosis test.Results After shH19 and miR-15b mimics transfected,cell proliferation,invasion and migration,and CDC42 expression were significantly decreased comparing to the control group,while the apoptosis was significantly increased(P<0.05)After miR-15b inhibitor transfection,cell proliferation,invasion and migration,and CDC42 expression were significantly increased,while the apoptosis in the tumor cells was significantly reduced(P<0.05).Additionally,after shH19/miR-15b inhibitor complexes were transfected into HCC cells,cell proliferation,invasion and migration ability,and CDC42 expression were significantly increased comparing to the shH19 single transfection group,while the apoptosis in the tumor cells was significantly reduced(P<0.05).Conclusions miR-15b inhibitor reversed shH19-mediated changes in the biological behaviors of cell proliferation,invasion,migration,and apoptosis in HCC cells.LncRNA-H19 could promote cell proliferation,migration and invasion via miR-15b/CDC42 signaling axis.Part Ⅳ:Preliminary Study of lncRNA H19/miR15b/CDC42 Signaling Axis in the EMT progression of HCCObjective:To explore the effect of lncRNA H19/miR-15b/CDC42 signaling axis in the epithelial-mesenchymal transition(EMT)progression of HCC.Method:Bel-7402 and HepG2 cells were transfected with shH19,miR-15b mimics,miR-15b inhibitor and shH19/miR-15b inhibitor complex liposomes,respectively.The mRNA expression of CDC42,PAK1 and EMT related genes in the cells were detected by qRT-PCR.The expression of CDC42,p-PAK1 and EMT related proteins were detected by Western blot.Moreover,the expression of EMT-related genes and proteins in 46 cases of HCC and its adjacent paired tissues were detected by qRT-PCR and Western blot.The correlation of lncRNA H19,miR-15b and CDC42 were analyzed over human HCC tissues and adjacent tissues.Results:After transfected with shH 19 and miR-15b mimics,the cells’ mRNA and protein expression levels of CDC42,PAK1(p-PAK1),N-cadherin,Vimentin were significantly lower than those in the control group,while the mRNA and protein expression levels of E-cadherin were significantly higher than those in the control group(P<0.05);after miR-15b inhibitor transfection,the cells’ mRNA and protein expression levels of CDC42,PAK1(p-PAK1),N-cadherin,Vimentin were significantly increased comparing to the control group,while the mRNA and protein expression levels of E-cadherin were significantly decreased comparing to the control group(P<0.05).In addition,after transfected with shH19/miR-15b inhibitor complex,the cells’ mRNA and protein expression levels of CDC42,PAK1(p-PAK1),N-cadherin,vimentin mRNA and CDC42,p-PAK1,N-cadherin,and Vimentin were significantly increased comparing to the shH19 single transfection group,while the mRNA and protein expression levels of E-cadherin were significantly decreased comparing to the shH19 single transfection group(P<0.05).Compared to adjacent tissues,the mRNA and protein expression levels of E-cadherin were significantly lower in HCC tissues whereas N-cadherin and Vimentin expression levels were significantly increased(P<0.05).LncRNA H19 expression levels were positively correlated with N-cadherin and Vimentin,while IncRNA H19 levels were negatively correlated with E-cadherin.Conclusions:miR-15b inhibitor reversed the regulation of shH19-mediated EMT-related proteins in HCC cells.lncRNA H19 could activates CDC42/PAK1 pathway to promote EMT progression via targeting miR-15b in HCC cells,LncRNA H19 involved in the EMT progression of HCC through the miR-15b/CDC42 signaling axis.