Objective To investigate miR-183-5p targeting to forkhead box protein O1(FOXO1)and its corresponding effect on the proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT)of non-small cell lung canc...Objective To investigate miR-183-5p targeting to forkhead box protein O1(FOXO1)and its corresponding effect on the proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT)of non-small cell lung cancer(NSCLC)cells.Methods NSCLC tissues and adjacent normal tissues from 60 patients with NSCLC adenocarcinoma were obtained via pathological biopsy or intraoperative resection.Several cell lines were cultured in vitro,including the human normal lung epithelial cell line BEAS-2B and human NSCLC cell lines A549,SPCA-1,PC-9,and 95-D.miR-183-5p and FOXO1 mRNA expression in tissues and cells were detected by qRT-PCR;the corresponding correlations in NSCLC tissues were analyzed using the Pearson test,and the relationship between miR-183-5p expression and clinicopathological parameters was analyzed.The miR-183-5p-mediated regulation of FOXO1 was verified by bioinformatics prediction alongside double luciferase,RNA-binding protein immunoprecipitation(RIP)assay,and pull-down experiments.A549 cells were divided into control,anti-miR-NC,anti-miR-183-5p,miR-NC,miR-183-5p,miR-183-5p+pcDNA3.1,and miR-183-5p+pcDNA3.1-FOXO1 groups.Cell proliferation,invasion,migration,apoptosis,and cell cycle distribution were detected using an MTT assay,clone formation assay,Transwell assay,scratch test,and flow cytometry,respectively.The expression of EMT-related proteins in the cells was analyzed by western blotting.The effect of miR-185-3p silencing on the development of transplanted tumors was detected by analyzing tumor formation in nude mice.Results miR-183-5p expression was significantly higher in NSCLC tissues and cells than in adjacent normal tissues,whereas FOXO1 mRNA expression was significantly down-regulated.There was a significant negative correlation between miR-183-5p and FOXO1 mRNA in NSCLC tissues(P<0.05).Additionally,the expression of miR-183-5p was significantly correlated with tumor size,tumor differentiation,and tumor-node-metastasis stage in patients with NSCLC(P<0.05).miR-183-5p targeted and inhibited FOXO1 expression.Compared to the anti-miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the anti-miR-183-5p group,whereas the protein expression of E-cadherin andα-catenin and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion were significantly lower in the anti-miR-183-5p group(P<0.05).Compared to the miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells in the miR-183-5p group were significantly higher,whereas the E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly lower;furthermore,the frequency of colony formation and invasion were significantly higher in the miR-183-5p group(P<0.05).Compared with the miR-183-5p+pcDNA3.1 group,the OD value,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group,whereas E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion was significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group(P<0.05).Overall,silencing miR-185-3p inhibited the growth of transplanted tumors and promoted FOXO1 expression.Conclusion Overexpression of miR-183-5p can inhibit apoptosis and promote the proliferation,migration,invasion,and EMT,of NSCLC cells by down-regulating FOXO1 expression.展开更多
文摘Objective To investigate miR-183-5p targeting to forkhead box protein O1(FOXO1)and its corresponding effect on the proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT)of non-small cell lung cancer(NSCLC)cells.Methods NSCLC tissues and adjacent normal tissues from 60 patients with NSCLC adenocarcinoma were obtained via pathological biopsy or intraoperative resection.Several cell lines were cultured in vitro,including the human normal lung epithelial cell line BEAS-2B and human NSCLC cell lines A549,SPCA-1,PC-9,and 95-D.miR-183-5p and FOXO1 mRNA expression in tissues and cells were detected by qRT-PCR;the corresponding correlations in NSCLC tissues were analyzed using the Pearson test,and the relationship between miR-183-5p expression and clinicopathological parameters was analyzed.The miR-183-5p-mediated regulation of FOXO1 was verified by bioinformatics prediction alongside double luciferase,RNA-binding protein immunoprecipitation(RIP)assay,and pull-down experiments.A549 cells were divided into control,anti-miR-NC,anti-miR-183-5p,miR-NC,miR-183-5p,miR-183-5p+pcDNA3.1,and miR-183-5p+pcDNA3.1-FOXO1 groups.Cell proliferation,invasion,migration,apoptosis,and cell cycle distribution were detected using an MTT assay,clone formation assay,Transwell assay,scratch test,and flow cytometry,respectively.The expression of EMT-related proteins in the cells was analyzed by western blotting.The effect of miR-185-3p silencing on the development of transplanted tumors was detected by analyzing tumor formation in nude mice.Results miR-183-5p expression was significantly higher in NSCLC tissues and cells than in adjacent normal tissues,whereas FOXO1 mRNA expression was significantly down-regulated.There was a significant negative correlation between miR-183-5p and FOXO1 mRNA in NSCLC tissues(P<0.05).Additionally,the expression of miR-183-5p was significantly correlated with tumor size,tumor differentiation,and tumor-node-metastasis stage in patients with NSCLC(P<0.05).miR-183-5p targeted and inhibited FOXO1 expression.Compared to the anti-miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the anti-miR-183-5p group,whereas the protein expression of E-cadherin andα-catenin and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion were significantly lower in the anti-miR-183-5p group(P<0.05).Compared to the miR-NC group,the cell proliferation,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells in the miR-183-5p group were significantly higher,whereas the E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly lower;furthermore,the frequency of colony formation and invasion were significantly higher in the miR-183-5p group(P<0.05).Compared with the miR-183-5p+pcDNA3.1 group,the OD value,scratch healing rate,N-cadherin and vimentin protein expression,and the proportion of S phase cells were significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group,whereas E-cadherin andα-catenin protein expression and the proportion of G0/G1 phase cells were significantly higher;additionally,the frequency of colony formation and invasion was significantly lower in the miR-183-5p+pcDNA3.1-FOXO1 group(P<0.05).Overall,silencing miR-185-3p inhibited the growth of transplanted tumors and promoted FOXO1 expression.Conclusion Overexpression of miR-183-5p can inhibit apoptosis and promote the proliferation,migration,invasion,and EMT,of NSCLC cells by down-regulating FOXO1 expression.
