| BackgroundLung cancer,the disease incidence of which has a tendency to rise year by year,is one of the leading causes of cancer-related death worldwide,and the reason of 90%of deaths related with metastasis.Increasing number of studies has shown that deregulated miRNAs are involved in tumor formation and metastasis.The epithelial-emesenchymal transition(EMT)is the process of differentiated epithelial cells into dedifferentiated migratory mesenchymal phenotype.Several important signaling pathways that can initiate EMT can activate EMT transcription factors(EMT-TF)at the transcription and translation level.EMT-TFs include three families:the zinc finger SNAIL(Snaill and Snail2/Slug),ZEB(Zeb1and Zeb2/SIP1)and basic helix-loop-helix transcription factors(Twist1,Twist2 and E12/E47).ZEB2 is one of the important members of EMT-TF.It can promote the occurrence of EMT by inhibiting the transcription of cytokeratin,E-cadherin,mac-1,desmoglein and mucin.The miR-200 can directly target and repress ZEB1 or ZEB2 expression to reverse EMT.MiR-200c as one of the important members of miR-200 family has been widely studied,which found its expression and function are different in different tumors.The miR-200c is down-regulated in gastric cancer,hepatocellular carcinoma,and renal clear cell carcinoma and so on,and with the function of tumor suppressor.However,miR-200c is up-regulated in ovarian cancer,bladder cancer,endometrial cancer and other tumors,which as an oncogenous miRNA.At present,the expression level,regulation mechanism and the correlation with non-small cell lung cancer(NSCLC)clinical pathological features of miR-200c is controversial.Some studies found that the expression of miR-200c in NSCLC tissue specimens was higher than that of normal tissues and high expression of miR-200c in tumor tissue is associated with poor survival.Other studies found that miR-200c is down-regulated in NSCLC tissue and the expression of miR-200c was associated with tumor stage,differentiation,lymph node metastasis,E-cadherin expression and pathological types.We detected the expression of miR-200c in tissue samples and a variety of NSCLC cell lines,and investigated its biological function in vitro,which are looking forward to expound the expression and function of miR-200c in NSCLC.It is thought that miR-200c modulates EMT by two types of pathways:(1)miR-200c suppressed EMT by regulating ZEB1/2/E-cadherin axis.(2)miR-200c reduced EMT by other targets except ZEB1/2.This regulation pathway has been proved in gastric cancer,pancreatic cancer and breast cancer.But the role of miR-200c in NSCLC mechanism need to be further researched.ObjectivesThe aim of the present study was to detect the expression of miR-200c in NSCLC tissues and cell lines and its impact on the migration and invasion,which could provide theory basis on understanding the function and possible molecular mechanism of miR-200c in NSCLC cell and theoretical foundation for the research of diagnosis and effective treatment for NSCLC.Methods1.The expressions and correlation of miR-200c and ZEB2 in NSCLC tissue samples qPCR was used to analyze the expressions of miR-200c and ZEB2 in 39 samples of NSCLC tissue and its adjacent normal lung tissue.The correlation of miR-200c and ZEB2,and the relationships between the expression of miR-200c and age,sex,tumor pathological type,tumor cell differentiation degree,tumor size,lymph node metastasis,distant metastasis.T,N,M stage,tumor TNM staging,etc.were analyzed.2.The expression and impact on the invasion and metastasis of miR-200c in NSCLC cell linesqPCR was performed to detect the expression difference of miR-200c between NSCLC cell lines and the normal lung epithelial cells(BEAS-2 b),and the most obvious different cell line was used in the followed experiments.The analogues and competitive analogue of miR-200c was transfect to A549 cells respectively.And then qPCR was used to detect the expression of miR-200c in each experiment group,the scratch test and transwell experiment were used to detect the proliferation,migration and invasion of each experiment group.3.miR-200c inhibits the invasion and metastasis of A549 cell lines by target to ZEB2 Dual luciferase reporter gene system was used to verify whether the miR-200c could directly combine to 3'-UTR sequence of ZEB2 in the A549 cell line.The analogues and competitive analogue of miR-200c was transfect to A549 cells respectively,and the expression of ZEB2 was detected.Then the relationship between miR-200c and ZEB2 was analyzed.The expression of ZEB2 in the A549 cell lines was down-regulated by transfect with ZEB2 siRNA.The scratch test and transwell experiment were used to detect the proliferation,migration and invasion in each experiment group.Western blot was used to detect the expressions of N-cadherin,E-cadherin and vimentin in the A549 cell lines that was over-expression or suppressed expression of miR-200c.Results1.In NSCLC specimens,low expression of miR-200c and high expression of ZEB2 were negatively correlated(P<0.05).The expression of miR-200c was negatively correlated with tumor size,lymph node metastasis and TNM stage(P<0.05),and had no significant correlation with age,sex,pathological type,tumor differentiation,T stage and distant metastasis.2.The qRT-PCR was used to detect the expression of miR-200c in BEAS-2B and NSCLC cell lines.The expression of miR-200c in BEAS-2B cell line was defined as 1,and the expressions of that were 0.24,0.51,0.76,and 0.39 in A549,H358,H460 and H1229,respectively.The results showed that the expressions of miR-200c were all down-regulated in NSCLC cell lines(P<0.05),and the most significant down-regulated expression was found in the A549 cell lines.3.The qRT-PCR was used to detect the effect of miR-200c analogue to the expression of miR-200c in A549 cell lines.Compared with the control group,the expression of miR-200c in which was defined as 1,the expression of miR-200c was 7.30 and 0.95 in miR-200c analogue transfect group and NC group,respectively.The results showed that the expression of miR-200c was significant increased in the analogue transfect group(P<0.05).The first scratch width was defined as 1,and the scratch widths were 0.66,0.61,0.89 in the normal control group,negative control group,miR-200c analogue transfect group,respectively,after being cultured for 48 hours,which indicated that miR-200c analogue could decrease the ability of proliferation and migration of A549 cell(P<0.05).The results of transwell test showed that the penetrated cells were 100/vision,95/vision and 35/vision,respectively,in the normal control group,negative control group and miR-200c analogue transfect group,which indicated that miR-200c analogue could decrease the ability of migration and invasion of A549 cell(P<0.05).4.The wild type and mutant expression plasmids of ZEB2 3'-UTR,pMIR-ZEB2 and pMIR-Mut ZEB2,were successfully constructed.The luciferase activity in A549 cells with co-transfection of pMIR-ZEB2 and miR-200c analogues were 1.25.And the luciferase activity in A549 cells which co-transfection of pMIR-Mut ZEB2 and miR-200c analogues,as well as the control and negative control groups were all between 2.70 to 2.80(P<0.01).The present results confirmed that miR-200c could directly combine to ZEB2 in NSCLC A549 cells.5.The qRT-PCR was used to detect the expression of miR-200c in the miR-200c analogue transfect group and inhibitor transfect group.The expression of miR-200c in control group was defined as 1,and the expressions of that were 7.30 and 0.30 in A549 cells of analogue transfect group and inhibitor transfect group,respectively.The results indicated that the expression of miR-200c was significant increased in analogue transfect group,and that was significant decreased in the inhibitor transfect group(P<0.05).Western blot was used to detect the expression of ZEB2.The expression of ZEB2 in control group was defined as 1,and the expressions of that were 0.23 and 1.6 in the miR-200c analogue transfect group and inhibitor transfect group,respectively,which indicated that the over-expression of miR-200c could inhibit the expression of ZEB2 and inhibit expression of miR-200c could increase the expression of ZEB2 significantly(P<0.05).Our results indicated that ZEB2 was the target gene of miR-200c,and miR-200c negatively regulated the expression of ZEB2.6.Western blot was used to detect the expression of ZEB2 in the A549 cell which was transfect with ZEB2 siRNA.The expression of ZEB2 in control group was defined as 1.and the expressions of that were 0.95 and 0.14 in A549 cells of NC group and ZEB2 siRNA transfect group,respectively.The results indicated that ZEB2 protein was significant decreased in the ZEB2 siRNA transfect group(P<0.05).Cell migration and invasion experiments were used to analyze the effect of ZEB2 on cell invasion and metastasis ability.The first scratch width was defined as 1,and the scratch widths were 0.61,0.63 and 0.82 in the normal control group,NC group and ZEB2 siRNA transfect group,respectively,after being cultured for 48 hours,which indicated that down-regulated expression of ZEB2 could significant decrease the ability of proliferation and migration of A549 cell(P<0.05).The results of transwell test showed that the penetrated cells were 150/vision in the normal control group and negative control group,and that of 40/vision in the ZEB2 siRNA transfect group,which indicated that down-regulated expression of ZEB2 could significant decrease the ability of proliferation,migration and invasion of A549 cell(P<0.05).These results were consistent with that of miR-200c over-expression.7.Western blot was used to detect the expressions of E-cadherin,N-cadherin and vimentin,which related to EMT.The expression of E-cadherin,N-cadherin and vimentin in the control group was defined as 1,and the expression of the three proteins were 2.10.0.28 and 0.22 in the miR-200c analogue transfect group,which indicated that the over-expression of miR-200c could up-regulate the expression of E-cadherin,and down-regulate the expression of N-cadherin and vimentin(P<0.05).In contrast,the expression of the three proteins were 0.53,1.56 and 1.5 in the miR-200c inhibitors transfect group,which indicated that inhibit expression of miR-200c could down-regulate the expression of E-cadherin,and up-regulate the expression of N-cadherin and vimentin(P<0.05).The present results indicated that miR-200c inhibit tumor invasion and metastasis through ZEB2/E-cadherin axis.Conclusions1.In NSCLC tissue samples,low expression of miR-200c and high expression of ZEB2 were negatively correlated.The miR-200c expression level was negatively correlated with TNM stage,lymph node metastasis and tumor size,and there was no significant different in age,sex,pathological type,tumor differentiation,T stage and distant metastasis.Therefore,we speculated that miR-200c could be a biomarker for the diagnosis and monitoring of NSCLC.2.The expressions of miR-200c in NSCLC cell lines,A549,H358,H460 and H1229,were all down-regulated,of which the most significant down-regulated expression was in the A549 cell lines.High expression of miR-200c could inhibit the proliferation,migration and invasion of A549 cells,which further verified the results of clinical sample.Confirmed that miR-200c was as a tumor inhibitor,which may be the treatment target of inhibiting invasion and metastasis in NSCLC.3.The miR-200c regulated proliferation,migration and invasion of A549 cells by targeting to ZEB2.And there was the negative regulate relationship between miR-200c and ZEB2.The expression of miR-200c could affect the expressions of E-cadherin,N-cadherin and vimentin in A549 cells.The results confirmed that miR-200c affected tumor proliferation,migration and invasion through ZEB2/E-cadherin axis in A549 cells,which is helpful to understand the mechanism of invasion and metastasis of NSCLC,and provide theoretical basis for the development of new antitumor drugs.Innovations1.For the first time,we confirmed that the decreased expression of miR-200c was negatively correlated with the increased expression of ZEB2 in NSCLC tissue samples.Further analysis found that the expression of miR-200c was reversely correlated with tumor size,lymph node metastasis and TNM stage.There was no significant correlation with age,sex,pathological type,degree of tumor differentiation,T stage and distant metastasis.2.We confirmed,for the first time,that miR-200c inhibited tumor proliferation,migration and invasion by directly targeting to ZEB2,and there was negative regulate relationship between miR-200c and ZEB2 in NSCLC.Significance1.The present study confirmed the low expression of miR-200c in tumor tissues and cell lines of NSCLC.The expression of miR-200c was reversely correlated with tumor size,lymph node metastasis and TNM stage,which indicated that miR-200c could be a biomarker for the diagnosis and monitoring of NSCLC.2.Low expression of miR-200c accompanied by a higher tendency of invasion and metastasis.Up-regulation of miR-200c in A549 cell lines could inhibit the invasion and metastasis of tumor,which indicated that miR-200c might be a tumor suppressor and new antitumor drug.3.In NSCLC tissue samples,the expression of miR-200c and ZEB2 were negatively correlated.The study confirmed that miR-200c could regulate the process of EMT and inhibits the invasion and metastasis ability of tumor through ZEB2/E-cadherin axis in A549 cells,which is helpful to understand the mechanism of invasion and metastasis of NSCLC,and provide theoretical basis for the development of new antitumor drugs. |
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