MiR-338 Targeted NFATc1 Inhibited The Proliferation Of NSCLC Cells And The EMT Process

Part Ⅰ: Expression and correlation analysis of miR-338 and NFATc1 in NSCLC tissues and adjacent tissues Objective: Differential expression of miR-338 and NFATc1 in NSCLC tumor tissues and NSCLC tumor systems was analyzed to determine their cancer-promoting and cancer-inhibiting properties and their correlation in the pathological development of NSCLC.Materials and Methods: From January 2016 to December 2018,20 human non-small cell lung cancer tissues and adjacent non-small cell lung cancer tissues were collected in Shengjing Hospital affiliated to China Medical University for research and analysis.RT-PCR was used to detect the m RNA expression analysis of miRNAs series(miR-143,miR-124,miR-338,miR-137 and miR-218)in NSCLC tissues and adjacent normal tissues,as well as the m RNA expression analysis of NFATc family NFATc1,NFATc2,NFATc3 and NFATc4,so as to determine the role of miR-338 and NFATc1 in the pathological tissues of NSCLC.Analyze the correlation between the two,analyze the data,and draw conclusions.Results: Differential expression of miRNA series in NSCLC tissues and adjacent tissues: miRNAs series.The expressions of miR-143,miR-124,miR-137 and miR-218 in NSCLC tissues were all lower than those in adjacent tissues,indicating tumor inhibition characteristics.The expression difference of miR-338 was the largest,and the tumor inhibition was the most obvious.Differential expression of miR-338 in NSCLC tissues and adjacent tissues: the expression level of miR-338 in NSCLC cancer tissues was significantly lower than that in adjacent tissues.Comparison of the two groups showed that the difference was statistically significant.Differential expression of NFATc family in NSCLC tissues and adjacent tissues: NFATc1,NFATc2,NFATc3 and NFATc4 are highly expressed in NSCLC tissues,with obvious cancer-promoting characteristics.Among them,the highest expression of NFATc1 is found in NSCLC tissues.Correlation analysis of the expression of miR-338 and NFATc1 in NSCLC tissues: the expression of miR-338 in NSCLC tissues was negatively correlated with the expression of NFATc1.With the increase of the expression of miR-338,the expression of NFATc1 decreased gradually(r=-0.8232,P=0.0007),The difference was statistically significant.Conclusions: In the pathological development of NSCLC,the expression of miR-338 was more sensitive than that of other miRNAs,showing tumor suppressive characteristics.Compared with other members of the NFAT family,NFATc1 is more sensitive to cancer promotion.miR-338 and NFATc1 show negative correlation in the pathological development of NSCLC,and the regulatory mechanism of the above two molecules in the pathological process of NSCLC will be discussed in the following studies.Part Ⅱ: Studies on the regulation of proliferation and EMT of NSCLC cells by miR-338 and NFATc1Objective: To explore the role of miR-338 and NFATc1 in regulating the proliferation and EMT of NSCLC cells,so as to provide a basis for subsequent studies.Materials and Methods: Lung cancer cells A549,H1650,spca-1,H460,SW900,H226,H1299 and bronchial epithelial cell line beas-2b were selected as the research objects.The primary culture of cell lines was carried out,and the third layer of monolayer cells was selected as the research object.m RNA expression of miR-338 and NFATc1 in the above cell lines was detected by RT-PCR.The sensitive cell line A549 was selected as the follow-up research object.A549 cells were cultured in primary culture,and the third-generation monolayer cells were selected as the research object.Lipofectamine 2000 reagent was used to transfect miR-338 mimics(50 n M),miR-338 inhibitor(100 n M)and si-nfatc1(100n M)for the synthesis of miR-338 mimics,miR-338 inhibitor,miR-nc,miR-anti-miR-nc,si RNA(si-NFATc1)and si-nc of NFATc1.Total RNA and protein were collected 48 hours after transfection of A549 cells,and relevant items were analyzed: miR-338 and NFATc1 were overexpressed and silenced by RT-PCR.The proliferation trend of cells in each group was detected by Brd U.The expression of proliferation-related protein genes PCNA,CDK4,cyclin D1 and p27 in A549 cells was detected by western-blot analysis.Expression analysis of E-cadherin,N-cadherin and Vimentin of EMT process-related proteins;Analyze the data and make a summary Results: The expression of miR-338 and NFATc1 in different NSCLC cell lines: the A549 cell line had the lowest expression of miR-338 and the highest expression of NFATc1.Compared with the control group of NSCLC cell lines,the difference was statistically significant.In the subsequent research,we chose A549 as the research object.Differential expression of miRNAs in A549 cell line: Among the differential expressions of miR-124,miR-338,miR-137 and miR-218 in A549,miR-338 showed the lowest expression content and obvious tumor inhibition.Compared with the control group,all the miRNAs series showed,indicating a statistically significant difference.Study on the regulation of NFATc1 on NSCLC cell proliferation and EMT process: NFATc1 was silenced in A549 cells;When the expression of NFATc1 was reduced,the proliferation of A549 cells could be significantly inhibited,which reflected the characteristics of cancer promoting.The comparison between the two groups showed that the difference was statistically significant.When NFATc1 was silenced in A549 cells,the expression levels of oncogenic genes PCNA,CDK4 and cyclin D1 were decreased,and the expression levels of tumor suppressor genes p27 were increased.Compared with the control group,P<0.05 showed a statistically significant difference.The silencing expression of NFATc1 can increase the expression level of e-cadherin protein,reduce the expression level of n-cadherin and Vimentin protein,decrease the EMT process of A549 cells,and inhibit the cell proliferation and migration.Compared with the data of the control group,the difference was statistically significant.Studies on the regulation of miR-338 on the proliferation and EMT of NSCLC cells: after transfection with different plasmids,miR-338 was successfully overexpressed and partially silenced in A549 cells.Overexpression of miR-338 could inhibit the proliferation activity of A549 cells.When miR-338 was partially silenced,the proliferation activity of A549 cells was significantly increased,and the difference was statistically significant.After the overexpression of miR-338,the expression levels of cancer-promoting genes PCNA,CDK4 and cyclin D1 were decreased,and the expression levels of tumor suppressor genes p27 were increased,and the cell proliferation activity was decreased.P<0.05,the difference was statistically significant.After the overexpression of miR-338,the expression of e-cadherin protein concentration increased,and the expression of n-cadherin and Vimentin protein concentration decreased,the difference was statistically significant.After the over-expression of miR-338,the expression concentrations of EMT gene protein Snail,Slug and ZEB1 all decreased to a certain extent,and the differences were statistically significant.Conclusions:(1)In many NSCLC tissue cells,A549 is the most sensitive to the expression of miR-338 and NFATc1,showing the most obvious anti-cancer or pro-cancer effect.In this study,A549 was selected as the cell line for subsequent study.(2)After NFATc1 was silenced in A549 cells,the proliferation activity of the cells decreased,the expression of oncogenic genes PCNA,CDK4 and cyclin D1 decreased,the expression of tumor suppressor gene p27 increased,and the EMT process of the cells decreased,showing obvious cancer-promoting characteristics.After verexpression of miR-338 in A549 cells,the proliferation activity of the cells decreased,the expression of oncogenic genes PCNA,CDK4 and cyclin D1 decreased,the expression of tumor suppressor gene p27 increased,and the EMT process of the cells decreased,which showed obvious anticancer characteristics.It was consistent with the conclusion of the first part of the study and provided a basis for the following study.Part Ⅲ: Study and analysis of the regulation of NSCLC cell proliferation and EMT by miR-338 targeting NFATc1Objective: To investigate the role of miR-338 targeting NFATc1 in the regulation of NSCLC cell proliferation and EMT.Materials and Methods:A549 human lung cancer cells were selected for primary culture,and the third-generation monolayer cells were selected as the research object.miR-338 mimics+pc DNA3.1 and miR-338 mimic+ pc DNA-NFATc1 were synthesized by transfection of miR-338 mimics(50 n M)and pc DNA-NFATc1 with Lipofectamine 2000 reagent and pc DNA-NFATc1.Total RNA and protein were collected 48 hours after transfection of A549 cells,and relevant analysis was carried out.Primary hybridization was performed to determine the targeted regulatory effect of miR-338 on NFATc1.The over-expression of miR-338 and NFATc1 were detected by western-blot.Cell proliferation trend was detected by Brd U.The expression of proliferation-related protein genes PCNA,CDK4,cyclin D1 and p27 in A549 cells was detected by western-blot analysis.Expression analysis of e-cadherin,n-cadherin and Vimentin of EMT process-related proteins;Analyze the data and make a summary.Results:Validation and analysis of mir-338 targeted NFATc1: NFATc1 was the least expressed in A549 cells with overexpression of mir-338,and NFATc1 was most significantly affected by changes in mir-338 content.Compared with the control group,miRNAs in each group were P<0.05,indicating a statistically significant difference.When mir-338 was overexpressed,the expression level of NFATc1 was significantly reduced.The comparison between the two groups showed that the difference was statistically significant.The hybridization experiment of mir-338 targeting NFATc1 verified that the increased expression of mir-338 could significantly inhibit the luciferase gene activity of the wild type of 3’utr NFATc1,with little influence on the mutant type.When the expression of mir-338 was effectively reduced,the luciferase gene activity of the wild type of 3’UTR NFATc1 was significantly increased.The comparison between the two groups showed that the difference was statistically significant.Studies on the regulation of proliferation and EMT of NSCLC cells by mir-338 targeting NFATc1: after overexpression of mir-338 alone,the expression levels of PCNA,CDK4,cyclin D1,etc.in the cells decreased,while the expression levels of p27 increased,and the cell proliferation trend decreased.However,after transfection with NFATc1 plasmid,the expression levels of PCNA,CDK4 and cyclin D1 in the cells increased,and the expression levels of p27 decreased,and the cell proliferation trend increased.P<0.05,the difference was statistically significant.Analysis of genes involved in the regulation of EMT process by miR-338 targeting NFATc1 in NSCLC cells: after the overexpression of miR-338 alone,the expression levels of N-cadherin,Vimentin,Snail,Slug and ZEB1 in the cells decreased,and the expression levels of E-cadherin increased,thus the EMT process of the cells was blocked.However,after transfection with NFATc1 plasmid,the expression levels of N-cadherin,Vimentin,Snail,Slug and ZEB1 in the cells increased,the expression levels of E-cadherin decreased,and the EMT process of the cells was enhanced.The difference was statistically significant.Conclusions:miR-338 negatively targets NFATc1 for physiological regulation of NSCLC cells.The overexpression of mir-338 can significantly reduce the proliferation trend and EMT process of A549 cells,while the transfection of NFATc1 plasmid can significantly slow down the above phenomenon.