MiR-19 Promotes The EMT,Migration And Invasion In Lung Adenocarcinoma Cells And Its Mechanism

Background and objectiveLung cancer,with its fastest growing in incidence and mortality,is currently one of the malignancies most dangerous to human health.lt is the leading cause of cancer-related death.The annual number of deaths due to lung cancer is more than the sum of deaths caused by breast cancer,eolorectal cancer and prostate cancer.In China,lung cancer has replaced liver cancer became the first cause of cancer-elated death.There are mainly two types of lung cancer tissue,that is Small cell lung cancer and Non small cell lung cancer(NSCLC).Wherein NSCLC accounts for about 80%of all patients with lung cancer.While the majority of lung cancer patients is locally advanced(stage Ⅲ)or metastatic(IV period)at presentation.Only approximately 25%NSCLC patients are diagnosed with early stage NSCLC,which surgical cure is possible,but the consequences is wide.The most common treatment for NSCLC are Chemotherapy-radiotherapy(CRT)and triple therapy,which includes Chemotherapy-radiotherapy followed by surgical resection.Due to the extensive heterogeneity under different pathological conditions of advanced non-small cell lung cancer,the treatment of of it remains a difficult and controversial field.Overall,commonly used CRT parallel fashion treatment can achieve a median survival of 17-20 months,3 year survival rate of 23-27%.The prognosis of patients with stage IV NSCLC generally worse,with a median survival of only 6 months.Therefore metastasis of lung cancer is the most important reason leading to treatment failure and death in patients with lung cancer.Metastasis is one of the main features of malignancy as well as a leading cause of death in cancer patients.Metastasis is an important stage of cancer development,and most patients die of tumor metastasis.clarifying the critical mechanisms of invasion and metastasis in cancer,and thus to establish corresponding way to deter tumor metastasis is the hope to cure cancer.Invasion and metastasis of lung cancer is a multi-factor controlling,multi-step,multi-stage,continuous and complex biological processes.Epithelial-mesenchymal transition(EMT)is considered to be an inevitable critical event occurred early of the metastasis in malignancies of epithelial cell origin.The proposal of this conception make people understand the mechanisns of tumor invasion and metastasis more profoundly.EMT plays a very critical role in cancer in situ invasion and distant metastasis.To investigate the molecular mechanism of the startup,maintaining and reversing of cancer EMT process systematically and in-depth is helpful to elucidate the molecular mechanisms regulating cancer cell EMT processes and reveal its inducements,thus help to identify key molecules in the regulation of EMT.It will also provide strong evidence and reliable theoretical basis for clinical treatment in designing a new treatment program by curbing or reversing EMT in tumor cells thus blocking or reversing the process of tumor invasion and metastasis.Development of new treatment programs can achieve early intervention and treatment of malignant tumor metastasis.But presently,little is known about the molecular mechanism of EMT,invasion and metastasis in cancer cells(including lung cancer).Research on mechanisms of tumor EMT,invasion and metastasis will contribute to understand transfer process,and may screen meaningful therapeutic targets,laying the foundation for clinical diagnosis and treatment.miRNA is a class of small non-coding RNA.In a certain way,it is involved in a variety of physiological and pathological processes in human body.miRNA is also involved in the regulation of EMT,invasion and metastasis in tumor cells.miR-17-92 gene cluster is a typical multi-functional gene,which participates in many physiological and pathological processes.Abnormally high expression of the gene cluster is occurred in a variety of cancers(including lung cancer),and it can promote tumorigenesis,tumor cell proliferation,tumor angiogenesis and other functions,thus considered to be cancerous micro RNA(oncomiR).miR-17-92 gene cluster consists of six miRNAs.According to their seed sequences,they can be divided into four families,including miR-19 family(containing miR-19a and miR-19b-1).Overexpression of miR-17-92 gene cluster can promote c-Myc-induced lymphoma generation in transgenic mice.miR-19 has been shown to be a critical component of miR-17-92 gene cluster playing tumorigenic role.The expression level of miR-19a in advanced NSCLC tissue is much higher than in the early stage NSCLC tissue.In B cell lymphoma mouse model based on c一Myc transgenic mice,lymphoma of Em-myc/19b-1 experimental group shows highly aggressiveness(compared with the Em-myc group)with lymphoma cells invaded into thymus,bone marrow,liver and lungs and other organs.miR-19a expression level of highly metastatic human osteosarcoma cell lines was significantly higher than of low metastatic osteosarcoma cell lines.These evidence suggests,miR-19 may play a role in tumor invasion and metastasis,meanwhile,our previous studies also support this view.But it still needs sufficient in vivo and in vitro experiments to confirm.Other studies have shown that overexpression of miR-17-92 in lung cancer cells can promote cell proliferation.Then,what kind of role miR-19 is playing in miR-17-92 gene cluster’s promoting proliferation in lung cancer is worthy of further study.In view of our group members found miR-19 demonstrated a significant role in terms of tumor invasion,metastasis and proliferation in lung cancer,we carried out this study in depth.MethodsPart Ⅰ miR-19 regulates EMT,migration and invasion in lung cancer cells1.For miRNA and mRNA analyses,total RNA of lung cancer cells was extracted.Total RNA was reversely transcribed with The expression levels of mature miRNA determined by qRT-PCR.2.Plasmid extraction;Plasmid was purified with NaAC-ethanol precipitation method.3.Establishment of miR-19 stably expressing lung cancer cellsLentivirus was produced by cotransfection of expressing plasmid along with packaging plasmids psPAX2 and pMD2.G into 293T cells using Lipofectamine 2000 reagent.Using the produced lentivirus to infect lung cancer cell,by observing the EGFP expression efficient we determined the establishment of miR-19 stably expressing lung cancer cell line.4.qRT-PCR and Western blot were used to detect the proteic level of EMT-related genes.5.Human genome-wide expression profiling microarray analysis to analyze gene expression profiles of miR-19-expressing lung cancer cells and its vector control cells.6.Total RNA extraction,reverse transcription and qRT-PCR were carried out to verify microarray results.7.Transwell was used for migration assay.8.Cell adhesion experiments were carried out to verify the ability of cell adhesion.9.F-actin labeled phalloidin staining experiment were used to observe the cytoskeleton variation of lung cancer cells.10.Scanning electron microscopy was used to survey the morphological variation of lung cancer cells.11.MTT assay was used to draw cell growth curve;Colony formation assay was used to survey cell proliferation.12.Cell-cycle analysis was used to detect cell-cycle distribution of lung cancer cells.13.Animal experiment:cells were injected subcutaneously into nude mice to detect cell growth in vivo.14.Tumors formed subcutaneous in mice were used to detect EMT-related genes expression by Immunohistochemistry and Western blot.15.TNF-α induced apoptosis was used to detect apoptosis of cells and qRT-PCR was used to detect changes in apoptotic gene expression.16.Statistical analysis:Statistical analysis was performed using a SPSS 16.0 software package.Each experiment is repeated at least three times.The 2-△△Ct of qRT-PCR results for each lung cancer cell lines were compared by ANOVA(One-way ANOVA);Comparison between groups for qRT-PCR were performed by one sample t test;Cell cycle assay,plate cloning,transwell chamber assay and apoptosis detection experiment were performed by two independent samples t test;EDTA cell adhesion assay,MTT assay and growth curve in nude mice was analysed by analysis of variance of two repeated measurement factors.Each value was represented by the mean± standard deviation(X± S).P<0.05 was considered as statistically significant.Part Ⅱ Regulatory mechanisms of miR-19 triggered EMT,migration and invasion in lung cancer cells1.Western blot was used to detect changes of critical protein expression within EMT-related signaling pathway in miR-19 stably overexpressed lung cancer cells.2.Dual luciferase reporter assay was performed to verify whether MST4 is a direct target of miR-19 in lung cancer cells after cotransfection of MST4 wt 3’UTR and miR-19 mimics or inhibitor.3.Lentiviral vectors was constructed,lentivirus and then produced and MST4 or dnMST4 stablely expressing lung cancer cell lines were establishished.4.Western blot was used to analysis EMT-related gene protein expression levels.5.Immunofluorescence experiments was used to observe EMT-related genes expression locations and levels in stablely expressing MST4 or dnMST4 lung cancer cell lines.6.Transwell and Boyden chamber assay were performed to analyze migration and invasion ability of lung cancer cells.7.Reexpression of MST4 in lung cancer cells undergone EMT induced by overexpression of miR-19.Western blot was used to detect protein expression of EMT-related genes;Transwell and Boyden chamber assay were used to analyze migration and invasion ability of lung cancer cells.8.F-actin labeled phalloidin staining test was used to detect cytoskeleton variation of lung cancer cells.Western blot were used to detect expression of cytoskeleton-associated protein.9.Xenograft mouse model:Nude mice were subcutaneously inoculated with A549 cells stablely expressing dnMST4 or MST4 to examine lung cancer cell grovrth in vivo and BrdU staining to analyze cell proliferation.10.Statistical analysis:Statistical analysis was performed using a SPSS 16.0 software package.Each experiment was performed at least three times.Comparison between groups for qRT-PCR were performed by one sample t test;Dual luciferase reporter assay,transwel and Boyden chamber assay and BrdU staining were compared by two independent samples t-test.Growth curve in nude mice was analysed by analysis of variance of two repeated measurement factors.Each value was represented by the mean ± standard deviation(X±S).P<0.05 was considered as statistically significant.Part Ⅲ MST4,a target gene of miR-19,modulates proliferation,EMT,migration and invasion in hepatocellular carcinoma cells1.Total RNA of hepatocellular carcinoma cells was extracted and reversely transcribed;qRT-PCR were carried out to detect mRNA expression level of MST4 in these cell lines.2.Establishment of MST4 or dnMST4 stablely expressing HCC cell lines.3.Western blot was used to detect changes in protein expression level.4.Trans well and Boyden chamber assay were carried out to detect the migration and invasion ability of liver cancer cells stably expressing MST4 or dnMST4 genes.5.Xenograft mouse model:Nude mice were subcutaneously inoculated with HCC cells stablely expressing dnMST4 or MST4 to examine liver cancer cell growth in vivo and BrdU staining to analyze cell proliferation.6.F-actin labeled phalloidin staining test was performed to survey the cytoskeleton variation of liver cancer cells.7.Statistical analysis:Statistical analysis was performed using a SPSS 16.0 software package.Each experiment is repeated at least three times.The 2-△△Ct of qRT-PCR results for each HCC cell line were compared by ANOVA(One-way ANOVA);Comparison between groups for transwell and Boyden chamber assay and BrdU staining were performed by two independent samples t-test;Growth curve in nude mice was analysed by analysis of variance of two repeated measurement factors.Each value was represented by the mean±standard deviation(X±S).P<0.05 was considered as statistically significant.ResultsPart Ⅰ miR-19 regulates EMT,migration and invasion in lung cancer cells1.Expression of miR-19 in lung cancer cell linesqRT-PCR data showed that miR-19 had a certain degree of expression level in each lung cancer cell line.lt was significantly high expressed in H23 cell line,but low expressed in A549-Luc cell line.2.Overexpression of miR-19 in A549 and HCC827 cells induced EMT-like cellular marker alterationsTo address the potential contribution of miR-19 to lung tumorigenesis,miR-19 family(miR-19a and miR-19b-1)was ectopically expressed in lung cancer A549 and HCC827 cells.The ectopic expression of miR-19 in A549 and HCC827 cells led to the emergence of a spindle-like,fibroblastic morphology,suggesting that miR-19-expressing A549 and HCC827 cells might have undergone EMT.The Western Blot results showed that miR-19-overexpressing A549 and HCC827 cells demonstrated the typical EMT phenotype,including downregulation of epithelial markers E-cadherin,and upregulation of mesenchymal markers FN1,N-cadherin and Vimentin.Therefore,miR-19 induces a mesenchymal-like phenotype and EMT-like cellular marker alterations in miR-19-expressing A549 and HCC827 cells.3.miR-19 overexpression in A549 cells induces global changes in geneexpression consistent with EMT,migration and metastasisWe determined the changes in the mRNA expression profile between miR-19-and vector-expressing A549 cells by using microarrays.Our results demonstrated that 352 genes were significantly down-regulated and 501 genes were significantly up-regulated in the miR-19-expressing A549 cells compared with controls.Among 853 significantly changed genes,the expression of 206 EMT-,migration-and metastasis-related genes changed significantly(up-regulated:134;down-regulated:72).To validate the gene expression level of specific miR-19 target genes related with EMT,migration and metastasis,qRT-PCR and Western blot analysis were employed.qRT-PCR of IGFBP3,ID2,PTEN,LPAR1,TGFB2,PIK3CD,TGFB1,PDGFA,TUBB2B,PDGFC,WNT5A,MMP10 and MMP1 confirmed the microarray results of these genes,while Western blot analysis of PTEN,MMP10 and ITGA5 also confirmed their changes in microarray.These genes’ expression changes were consistent with the results of microarray.4.Increased cell motility and decreased cell adhesion demonstrated inmiR-19-expressing cells undergoing EMTWe further defined the changes in motility and cell adhesion ability of miR-19-expressing lung cancer cells which have undergone EMT by in vitro migration assay using transwell chamber and EDTA cell adhesion assay,respectively.miR-19-expressing A549 and HCC827 cells with mesenchymal-like phenotype exhibited significantly enhanced mobility compared with vector control cells(t=-5.860、-7.330,P=0.002、000).In addition,miR-19-expressing A549 cells with EMT-like cellular marker alterations demonstrated significantly decreased cell adherent ability compared with control cells.Collectively,the enforced expression of miR-19 in lung cancer cells triggers EMT accompanied by the increased cell migration and reduced cell adhesion ability.5.Ectopic expression of miR-19 in lung cancer cells restores the disruptedcytoskeleton and promotes filopodia&lamellipodia formationUsing phalloidin staining,we found that the disrupted F-actin stress fiber(polymerised actin)networks were restored in both miR-19-expressing A549 and HCC827 cells.Under a scanning electron microscope,the formation of filopodia and lamellipodia was found on the cell surfaces of miR-19-expressing A549 cells,as strongly supported by a significantly enhanced cell motility in miR-19-expressing A549 cells compared with control cells.In sum,the ectopic expression of miR-19 in lung cancer cells promotes the formation of stress fibres,filopodia and lamellipodia.In summary,our results suggest that the upregulation of miR-19 is sufficient to induce EMT and enhance the mobility of lung cancer cells in vitro.6.miR-19 induced growth inhibition in lung cancer cells undergoing EMTThe results of MTT assay indicated that miR-19 inhibited cell growth in A549 cells which have undergone EMT(F=127.498,P=0.000).The cell-cycle experiment showed that A549 cells stably overexpressing miR-19 displayed an increased percentage of cells in G1 phase and fewer cells in S phase.Additionally,as shown in colony formation assay,miR-19-expressing A549 cells and HCC827 cells formed much fewer colonies compared with vector-expressing cells(t=4.267、4.134,P=0.013、0.014).These results demonstrated that the growth-inhibition effect of miR-19 was partly due to a G1-phase arrest.Finally,miR-19 suppressed tumor growth of A549 cells in nude mice.To reveal which genes were involved in the growth inhibition of miR-19,we checked the mRNA levels of some cell-cycle regulators and cell proliferation-associated molecules in A549 cells by microarray and qRT-PCR.Microarray analysis revealed the up-regulation of some growth-related genes(i.e.,GNG4,MXD1,BCAT1,E2F7 and DAB2)and the down-regulation of several growth-related genes[i.e.,Betacellulin(BTC),Ets homologous factor(EHF),epiregulin(EREG),E2F transcription factor 8(E2F8),cell division cycle associated 7-like(CDCA7L)].qRT-PCR data demonstrated that the levels of E2F1,E2F2,CCNA2 and CCNB1 were significantly decreased,whereas the level of tumor suppressor p21CIP1 was increased at least 3.5-fold after miR-19 overexpression,and the expression of CDK6 was increased in mRNA level.In summary,miR-19 limited the proliferation ability of lung cancer cells undergoing EMT.However,the underlying mechanisms remain unclear.7.miR-19 promotes the survival of lung cancer cells undergoing EMTMicroarray data revealed the up-regulation of anti-apoptosis genes and down-regulation of pro-apoptosis genes.Furthermore,the results of qRT-PCR analysis for all selected genes(including BCL2A1,XIAP,IL1RAP,IRAK2,TNFAIP3,IGFBP3 and TNFRSF11A)were basically in agreement with the microarray data.Next,we checked whether miR-19-expressing cells might also be resistant to apoptosis induced by proapoptotic signals such as TNF-α.Indeed,An approximately three fold decrease in the activity of caspase-8 was observed in miR-19-expressing A549 cells after 16 h of treatment compared with that of vector-expressing A549 cells.This result substantiates that the death observed in vector-expressing A549 cells was mediated by the activation of this pathway.As expected,the activity of the initiator,caspase-8,correlated with that of the effector,caspase-3,verified that the expression of miR-19 protects the A549 cells(undergoing EMT)from TNF-α-induced cell death.Therefore,miR-19 enhances the anti-apoptotic ability of lung cancer cells undergoing EMT.8.A diagram of miR-19 signaling pathways for the induced EMT,reduced proliferation and enhanced cell survival in lung cancer cells is indicatedBased on the above experimental results,we postulated the signaling pathway of miR-19 in lung cancer cells,that is miR-19 gene over-expression induces the loss of epithelial markers and the gain of mesenchymal markers,as well as inducing changes in cell shape,and changes related to morphology and to the acquisition of motility and invasive properties,while the miR-19 gene also regulates cell proliferation and cell death.Part Ⅱ Regulatory mechanisms of miR-19 triggered EMT,migration and invasion in lung cancer cells1.Overexpression of miR-19 induced the occurrence of lung cancer cell EMT by activating Akt/GSK-3β and JAK-STAT signaling pathway.To clarify the miR-19 mediated signaling pathway in lung cancer cells undergoing EMT,we used Western blot method to detect critical components of common EMT-related signaling pathways.We found that miR-19 overexpressing A549 and HCC827 cells promoted the phosphorylation of Akt and thus increased phosphorylation of GSK-3β,a downstream target of Akt,indicating that upregulation of miR-19 activated PI3K/Akt/GSK-3p pathway in A549 and HCC827 cells.;Overexpression of miR-19 also promoted phosphorylation of Stat3 in A549 and HCC827 cells,indicated that overexpression of miR-19 in lung cancer cells can also activate the JAK-STAT signaling pathway.2.MST4 is a direct target gene of miR-19Through online target gene prediction software,we found that miR-19 is capable of specifically binding to the 3’UTR of MST4.Western blot analysis found that miR-19-overexpression downregulated expression of MST4 in lung cancer cells.These above results suggest that MST4 is a target gene of miR-19.To prove a direct or indirect relationship of MST4 expression regulated by miR-19,we constructed a dual luciferase reporter gene vector carrying the 3’UTR of wild-type MST4,and verified whether MST4 was a direct target gene of miR-19 by dual luciferase reporter gene experiment.The results showed that miR-19a mimics significantly inhibited luciferase reporter gene activity by binding to 3’UTR region of MST4(t=4.471,P=0.043);On the contrary,miR-19a inhibitor can significantly enhance the dual luciferase reporter gene activity(t=-6.163,P=0.004).That is to say,MST4 is a direct target of miR-19.3.To establish lung cancer cell lines stablely carrying MST4 or dnMST4(Dominent-negative MST4)transgeneTo make it clear whether miR-19 mediates EMT process in lung cancer cells by down-regulation of MST4,we established lung cancer cell lines stablely carrying MST4 or dnMST4.We found that exogenous increase of MST4 expression in A549 cells led to reduction of the volume of A549 cells compared with control cells;whereas exogenous expression of dnMST4 increased the s volume of A549,and the cells became more scattered.While in HCC827 cells with exogenous expression of dnMST4,we observed a more obvious morphological change,i.e.,HCC827 cells of closely arranged epithelial-like morphology cells became relatively loosely arranged mesenchymal-like morphology cells.This suggested that MST4 loss of function may lead to EMT in the A549 cells and HCC827 cells.4.MST4 negatively regulats the EMT,migration and invasion in the lung cancer cellsWe used the Western Blot test and immunofluorescence assay to detect protein expression of EMT-related genes in A549 and HCC827 cells with MST4 dysfunction.Our results showed that dysfunction of MST4 in lung cancer cells led to downregulation of epithelial markers,ZO-1 and E-cadherin,and upregulation of mesenchymal markers,FN1、N-cadherinand and Vimentin at protein level.So far,our study have proved that MST4 dysfunction caused EMT in lung cancer cell line A549 and HCC827.In vitro migration and fast invasion experiments showed that MST4 dysfunctioned A549 and HCC827 displayed enhanced migration and invasion ability,P<0.05;On the contrary,exogenous expression of wild-type MST4 in A549 and HCC827 cells significantly decreased the migrated and invaded number of cells P<0.05.The above experiment showed that MST4 negatively regulated the process of EMT、migration and metastasis in lung cancer cells.5.Exogenous expression of MST4 is able to reverse the EMT,invasion and migration process induced by miR-19 overexpression in lung cancer cellsTo further illustrate whether miR-19 induced EMT process and enhanced migration ability and invasiveness was mediated through downregulation of MST4,we established the MST4 reoverexpressing lung cancer cell lines on the basis of the miR-19 overexpression cell lines which had undergone EMT,and the protein level of its EMT-related marker genes were detected and functional experiments of migration and invasion were done in vitro.The results showed that reoverexpressing of MST4 in lung cancer cells which had undergone EMT induced by miR-19 could partially reverse the EMT marker genes expression(vimentin Downregulation,i.e.)and the mesenchymal-like morphology changes;Meanwhile,MST4 reoverexpression also reversed the accelerated migration and invasion ability in lung cancer cells caused by miR-19.Namely,MST4 inhibited the acceleration of migration and invasion in lung cancer cells induced by miR-19.The above experiments fully demenstrated that MST4 is a direct target gene of miR-19 and mediates EMT,migration and invasion caused by miR-19.6.MST4 can negatively regulate the proliferation ability of lung cancer cells in vivoTo clarify the function of MST4 gene on the proliferation of lung cancer cells,we inoculated A549 cells stably expressing dnMST4 or MST4 and their control cells subcutaneously into nude mice,and the results showed that the growth rate of MST4 functional deficient A549 cells was significantly faster than the control cells,P<0.05;Whereas the growth rate of wild-type MST4 overexpressed A549 cells was significantly slower than the control cells.BrdU proliferation detection tests of the subcutaneous tumor tissue,also support this result.These data suggested that,MST4 negatively regulated the proliferation of A549 cells in vivo.7.Loss of function of MST4 enables the reorganization of cytoskeleton in lung cancer cells and enhances the activity of cytoskeleton-related proteinTo elucidate the mechanism of MST4 involved in the EMT,migration and invasion in lung cancer cells,we conducted F-actin phalloidin staining experiments on A549 and HCC827 cells carrying dnMST4 gene and their control cells,and it showed that loss of function of MST4 led to a remodeling of the cytoskeleton,actin filaments within cells increased significantly.Western Blot analysis showed that,loss of function of MST4 led to enhanced activiation of cytoskeleton-related protein,Rho,Rac and Cdc42 in A549 cells.So far,we consider MST4 involved in the EMT,migration and invasion in lung cancer cells by altering activity of cytoskeleton-related proteins in Rho/Rac signaling pathway leading to reorganization of cytoskeleton.However,whether MST4 is involved in EMT,migration and invasion of cancer cells through other mechnisms(such as common signaling pathways:PI3K/Akt signaling pathway,JAK/STAT3 signaling pathway)remains to be further studied.Part Ⅲ MST4,a target gene of miR-19,modulates proliferation,EMT,migration and invasion in hepatocellular carcinoma(HCC)cells1.The expression profiles of MST4 in HCC cell linesWe detected the expression of MST4 at the mRNA level.There was a certain expression of MST4 in immortalized human liver epithelial cells and several liver cancer cell lines,and compared with immortalized human liver epithelial cell line Chang liver and LO2,the expression level of MST4 is relatively much lower in HCC cell lines.Thus,we selected the Bel-7404 cell in which MST4 expression is not so low and Bel-7402 cell in which MST4 expression is much lower than other HCC cell lines for subsequent experiments.2.Establish stable transgenic carry MST4 or dnMST4 hepatoma cell linesBy production of lentivirus and infection of Bel-7402 and Bel-7404 cell with these lentiviruses,we established stablely expressing MST4 or dnMST4 HCC cell lines.qRT-PCR and Western blot were used to confirme that MST4 was significantly high expressed in MST4-or dnMST4-expressing Bel-7402 and Bel-7404 cell lines.3.MST4 negatively regulates EMT,migration and invasion in liver cancer cellsWestern blot analysis of EMT-related genes showed that,loss of function of MST4 upregulated protein expression of tipical mesenchymal genes,Fibronectin,N-cadherin,Vimentin in Bel-7402 and Bel-7404 cells,and downregulated expression of tipical epithelial genes,E-cadherin or a-catenin;While the protein expression of EMT-related genes in wild-type MST4-expressing Bel-7402 and Bel-7404 cells was just the opposite.It is suggested that functional deficiency of MST4 leads to EMT occurrence in Bel-7402 and Bel-7404 cells,whereas overexpression of wild-type MST4 leds to opposite changes of EMT in Bel-7402 and Bel-7404 cells.Transwell and Boyden chamber assay showed that overexpression of wild-type MST4 significantly weakened the in vitro migration and invasion ability of Bel-7402 and Bel-7404 cells(P<0.05);whereas overexpression of dnMST4 significantly increased the migration and invasion ability of Bel-7402 and Bel-7404 cells(P<0.05).4.Functional deficiency of MST4 promotes the proliferation of HCC cells in vivoTo clarify the function of MST4 gene on proliferation of HCC cells,we subcutaneously inoculated Bel-7402 cells and Bel-7404 cells stably expressing dnMST4 into nude mice,and the results showed that loss of function of MST4 contributed to proliferation of HCC cells in vivo(P<0.05).5.Impact on the cytoskeleton of MST4 geneTo demenstrate whether MST4 regulates EMT,migration and invasion of HCC cells by reorganization of the cytoskeleton,we conducted F-actin phalloidin staining experiments,and it showed that overexpression of wild-type MST4 or dnMST4 didn,t obviously affect the F-actin contents in Bel-7402 and Bel-7404 cells,but it is yet to study the effect of MST4 or dnMST4 on activity of proteins related to Rho/Rac pathway.Conclusions1.miR-19 promotes EMT,migration and invasion in lung cancer cells,accompanied by inhibition of cell growth;2.miR-19 promotes the EMT process by activating PI3K/Akt/GSK3βand JAK/Stat3 signaling pathway;3.MST4 is a miR-19 direct target gene.It can promote EMT,migration and invasion in lung cancer cells,and can negatively regulate proliferation of lung cancer cells;4.MST4 can negatively regulate the proliferation,EMT,migration and invasion process in HCC cells;5.5.By regulating the expression of target gene MST4 and by activating EMT-related pathways,miR-19 promots EMT,migration and invasion of lung cancer cells,while miR-19 mediated inhibition of lung cancer cell growth may be achieved by other means,suggesting that regulation of different biological processes of miR-19 is realized by different target genes or different approaches,which also reflects the complexity and diversity of gene regulatory networks.