| ObjectivesTo observe the effect of altered expression of miRNA-21 on the growth and metastasis of human lung cancer cell in vitro and explore its potential Molecular mechanism.Methods Part OneThe miRNA-21 overexpression vector(named p-miR-21)was constructed by molecular cloning technique;After the vector was constructed,human lung cancer 95 D were transiently transfected with p-mi R-21-ASOs and p-miR-21 respectively in vitro.Then,the expression level of miR-21,cell growth cycle-associated factors(CDK2,CDK3,CDK4 and CDK6)and metastasis-associated factors(MMP-2,MMP-9,E-cadherin and CXCR4)were detected by real-time PCR assay.The proliferation and migration of cells were analyzed by CCK-8 assay and Scratch assay respectively;Further,The metastasis and invasion of cells were detected by Transwell assay.Finally,the transduction of related signal pathway,including Akt/mTOR and Erk,was detected by Western blot assay.Part TwoThe candidate target genes of miR-21 were screened by bioinformatics.The expression level of target genes were detected by Real-time PCR in transfected 95 D cells.Then,the putative target genes of miR-21 was verified by Western blot assay.RNAi technology were used to synthesize small interfering LEMD3(named si-LEMD3);And human lung cancer 95 D cells were transiently transfected with si-LEMD3 in vitro.The expression level of LEMD3 was detected by real-time PCR and Western blot assay.The proliferation and migration of cells were analyzed by CCK-8 assay and Scratch assay respectively.The clone formation ability of cells also was observed respectively.And the expression level of cell growth cycle-associated factors(CDK2,CDK3,CDK4 and CDK6)and metastasis-associated factors(MMP-2,MMP-9,E-cadherin and CXCR4)were detected by real-time PCR assay.Finally,the transduction of related signal pathway,including Akt/mTOR and Erk,were detected by Western blot assay.Then,human lung cancer 95 D cells were transiently co-transfection with 50 nM si-LEMD3 and 4 ?g p-miR-21-ASOs(named p-miR-21-ASOs+si-LEMD3)in vitro.And the proliferation and migration of cells were analyzed by CCK-8 assay and Scratch assay respectively.Further,the expression of cell growth cycle-associated factors and metastasis-associated molecules by Real-time PCR assay.Finally,the level of phosphorylation of Akt,phospho-Erk,total Akt and Erk protein and LEMD3 protein were analyzed by Western blot assay.Results Part OneThe miRNA-21 overexpression vector was successfully constructed.And the expression level of miRNA-21 in 95 D cells was significantly decreased in p-miR-21-ASOs transfected group,compared with those in control group(P<0.05).However,the expression level of mi RNA-21 in p-miR-21 transfected group was significantly up-regulated(P<0.05).And the number of tumor cells in the p-miR-21-ASOs transfected group was significantly decreased when compared with that in control group(P<0.05).However,the number of tumor cells in p-miR-21 transfected group was significantly increased(P<0.05).Compared with those in control group,the proliferation and migration of human lung cancer cells in p-miR-21-ASOs transfected group decreased significantly,however,increased significantly in the p-miR-21 transfected group(P<0.05).Compared with that in control group,metastasis and invasion of cells in p-miR-21-ASOs transfected group decreased significantly,however,incrased obviously in p-miR-21 transfected group(P<0.05).Compared with those in control group,the relative expression level of lung cancer cell growth cycle-associated factors such as CDK2,CDK3,CDK4 and CDK6 in p-miR-21-ASOs transfected group were significantly decreased(P<0.05),however,increased remarkedly in the p-miR-21 transfected group(P<0.05).Further analysis showed that the expression level of metastasis-associated molecules of E-cadherin in p-miR-21-ASOs transfected group was significantly increased(P<0.05),conversely,decreased significantly in p-miR-21 transfected group(P<0.05).Moreover,the expression level of metastasis-associated molecules MMP-2?MMP-9 ? CXCR4 in p-miR-21-ASOs transfected group were significantly decreased(P<0.05),and increased obviously in p-miR-21 transfected group(P<0.05).Finally,Compared with those in control group,the level of phosphorylated Akt and Erk in 95 D cells were decreased in p-miR21-ASOs transfected group(P<0.05),however,increased in the p-miR-21 group significantly(P<0.05).Part TwoA total of 10 candidate target molecules of miR-21 were screened by bioinformatics analysis.The expression levels of tumor suppressor gene SOCS6,HBP1,PTEN,PDCD4,RECK,SPRY1,TIMP3 in 95 D cells were significantly increased in p-miR-21-ASOs transfected group when compared with these in control group(P<0.05).However,the expression of these genes in p-miR-21 transfected group was significantly decreased(P<0.05).And,the expression level of oncogene TIAM1,PIK3R1 and HIF-1? in 95 D cells was significantly decreased in p-miR-21-ASOs transfected group when compared with those in control group(P<0.05).However,the expression of these genes in p-miR-21 transfected group was significantly increased(P<0.05).Combined with the relevant literature,LEMD3 was selected as a new candidate target molecule of miR-21.Further analysis showed that the expression level of mRNA and protein of LEMD3 in 95 D cells significantly increased in p-miR-21-ASOs transfected group when compared with those in control group(P<0.05),however,increased significantly in p-miR-21 transfected group(P<0.05),which indicated that LEMD3 was a novel target molecule of miR-21.The expression level of mRNA and protein of LEMD3 in 95 D cells were significantly decreased in si-LEMD3 transfected group when compared with those in control group(P<0.05).And the total cell number of lung cancer cells in the si-LEMD3 transfected group was significantly increased(P<0.05).Moreover,the proliferation and migration of human lung cancer cells in si-LEMD3 transfected group were enhanced significantly compared with those in control group(P<0.05).Compared with those in control group,the relative expression level of lung cancer cell growth cycle-associated factors such as CDK2,CDK3,CDK4 and CDK6,as well as the relative expression level of metastasis-associated molecules MMP-2,MMP-9 and CXCR4,in si-LEMD3 treated group significantly increased(P<0.05).Conversely,to the relative expression of E-cadherin the in si-LEMD3 transfected group was decreased.Finally,that the level of phosphorylated Akt and Erk in 95 D cells were increased in si-LEMD3 transfected group,compared with those in control group(P<0.05).The protein level of LEMD3 was significantly decreased in p-miR-21-ASOs+si-LEMD3 co-transfected group compared with those in p-miR-21-ASOs+si-control group(P<0.05).Moreover,The proliferation and migration of human lung cancer cells in p-miR-21-ASOs+si-LEMD3 co-transfected group were enhanced significantly compared with those in p-miR-21-ASOs+si-control group(P<0.05).Compared with those in control group,the relative expression level of lung cancer cell growth cycle-associated factors such as CDK2,CDK3,CDK4 and CDK6,as well as the relative expression level of metastasis-associated molecules MMP-2,MMP-9 and CXCR4,in p-miR-21-ASOs+si-LEMD3 co-transfected group was significantly increased(P<0.05),Conversely,to the relative expression of E-cadherin the in p-miR-21-ASOs+si-LEMD3 co-transfected group(P<0.05).Finally,The level of phosphorylated Akt and Erk in 95 D cells were increased in p-miR-21-ASOs+si-LEMD3 co-transfected group,compared with those in that in p-miR-21-ASOs+si-control group(P<0.05).Conclusions Part One The altered expression of miR-21 could significantly affect the growth and metastasis of human lung cancer cells in vitro,suggesting that miR-21 might be one of the potential new targets for lung cancer gene therapy.Part Two The effect of miR-21 on the growth and metastasis of human lung cancer cells was closely related to changed expression of LEMD3,accompanied by altred transduction of related signaling including Akt and Erk pathway. |
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