Effect Of MCF2 On Biological Functions Of Lung Adenocarcinoma Cells

Background and Objectives: A previous study found that there is a unique gene mutation profile of lung adenocarcinoma in non-smoking women in Xuanwei,and MCF2 is one of the significant mutated genes.MCF2 expression level is negatively correlated with the prognosis of lung adenocarcinoma,but the role of MCF2 in lung adenocarcinoma is not clear.The aim of this study was to investigate the expression of MCF2 in lung adenocarcinoma tissues from Xuanwei non-smoking female patients,to explore the effect of MCF2 on the biological function of lung adenocarcinoma cells at the cellular level,and to provide a contribution to the exploration of novel molecular markers for lung adenocarcinoma.Methods: 1 Methods to detect the expression of MCF2 protein in Xuanwei female nonsmoking lung adenocarcinoma tissues From January 1,2017 to December 31,2019,30 cases of lung adenocarcinoma tissues and paired adjacent tissues of female non-smoking patients in Xuanwei area who underwent surgical treatment and were confirmed by pathology in the Third Affiliated Hospital of Kunming Medical University were selected.The expression level of MCF2 protein was detected by immunohistochemistry.The expression of MCF2 protein in lung adenocarcinoma tissues and paired adjacent tissues was compared by artificial semi-quantitative scoring analysis.2 Methods to investigate the effect of MCF2 on the biological function of lung adenocarcinoma cells 2.1 Tool cell lines were selected by Western Blot Using human immortalized bronchial epithelial cell Base-2b as a control,the cells were screened in Base-2b and 11 lung adenocarcinoma cell lines including PC-9,A549,H292 and H838 by Western Blot.The cells with the highest expression level of MCF2 protein were selected to knock out MCF2 by CRISPER-Cas9 technology,and the cells with the lowest expression level of MCF2 protein were selected to over-express MCF2.2.2 Stable transfected cell lines with MCF2 knockout and overexpression were constructed(1)Three sgrnas were designed for MCF2 knockout,and U6 sequencing was performed after the construction of the knockout plasmid to evaluate whether the construction was successful.After 72 hours of lentivirus transfection,the stable strains were screened by puromycin.The expression of fluorescent protein was observed under a fluorescence microscope and the knockout efficiency was verified by Western Blot.(2)The MCF2 overexpression lentivirus was transfected into lung adenocarcinoma cells for 72 hours,and the stable cell lines were screened by puromycin.The fluorescent protein expression was observed under a fluorescence microscope,and the overexpression efficiency was verified by RT-q PCR.2.3 To investigate the effect of MCF2 knockout and overexpression on the biological function of lung adenocarcinoma cells CCK8 assay was used to detect the effect of MCF2 knockout and overexpression on the proliferation of lung adenocarcinoma cells.Transwell assay was used to detect the effect of MCF2 knockout and overexpression on the invasion and longitudinal migration of lung adenocarcinoma cells.Scratch test was used to detect the effect of MCF2 knockout and overexpression on the lateral migration of lung adenocarcinoma cells.The effects of MCF2 knockout and overexpression on cell cycle and apoptosis of lung adenocarcinoma cells were detected by flow cytometry.Results:1 Expression of MCF2 protein in Xuanwei female non-smoking patients with lung adenocarcinoma The positive expression rate of MCF2 protein in lung adenocarcinoma tissues of non-smoking female patients in Xuanwei was 26.6%(8/30),and that in paired adjacent tissues was 3.33%(1/30),and the difference was statistically significant(χ2=6.405,P<0.05).MCF2 protein was expressed in the cytoplasm of Xuanwei non-smoking female lung adenocarcinoma cells,but not in the nucleus.2 Effects of MCF2 on the biological functions of lung adenocarcinoma cells 2.1 Expression of MCF2 protein in Xuanwei female non-smoking patients with lung adenocarcinoma Compared with Base-2b cells,the expression level of MCF2 protein was the highest in PC-9 cells and the lowest in H1734 cells among the 11 lung adenocarcinoma cell lines.Therefore,we used PC-9 cells to knock out MCF2 by CRISPER-Cas9 technology,and H1734 cells were transfected with lentivirus to overexpressed MCF2.2.2 MCF2 knockout and overexpression stably transfected cell lines were successfully constructed(1)U6 sequencing results showed that the three sgrnas were successfully transferred into the vector plasmid.The stable strains were screened by puromycin and observed under a fluorescence microscope,and the fluorescent protein expression was more than 90%.After Western Blot verification of knockdown efficiency,the cells with the highest knockdown efficiency were selected as tool cells for subsequent experiments.(2)The over-expression stable strain was screened by puromycin and observed under a fluorescence microscope.The expression of fluorescent protein was > 80%,and RTq PCR showed that the over-expression vector was successfully constructed.2.3 Effects of knockout and overexpression of MCF2 on biological functions of lung adenocarcinoma cells MCF2 knockout inhibited the proliferation of lung adenocarcinoma cells(P< 0.05),while MCF2 overexpression promoted the proliferation of lung adenocarcinoma cells(P<0.05).MCF2 knockdown inhibited the longitudinal migration ability of the cells (P<0.05),while MCF2 overexpression had no significant change(P>0.05).Knockout of MCF2 significantly inhibited the invasion ability of lung adenocarcinoma cells(P<0.01),while overexpression of MCF2 had no significant change(P>0.05).Knockout of MCF2 significantly decreased G0/G1 phase and increased S and G2/M phase in lung adenocarcinoma cells(P<0.01).Overexpression of MCF2 significantly increased G0/G1 phase and decreased S and G2/M phase in lung adenocarcinoma cells(P<0.01).Knockout of MCF2 significantly increased the apoptosis rate of lung adenocarcinoma cells,and the proportion of early apoptotic cells(Q2)and late apoptotic cells(Q3)increased,and the difference was statistically significant(P<0.01).Overexpression of MCF2 significantly decreased the apoptosis rate of lung adenocarcinoma cells,and the proportion of early apoptotic cells(Q2)and late apoptotic cells(Q3)decreased.And the difference was statistically significant(P<0.01).Conclusions: MCF2 protein is abnormally expressed in lung adenocarcinoma of non-smoking women in Xuanwei.MCF2 gene can promote the proliferation and growth cycle of lung adenocarcinoma cells and inhibit cell apoptosis,which may be a potential target for inhibiting the malignant biological behavior of lung adenocarcinoma.