Expression Of PDX1 In Xuanwei Female Lung Adenocarcinoma And Its Effect On Biological Behavior Of Cells

Backgrounds and objectives:Lung adenocarcinoma in non-smoking women in Xuanwei area is a major regional disease that seriously endangers people’s health,and its occurrence and development mechanism is not clear.Although a large number of studies have shown that the high incidence of Xuanwei lung cancer is closely related to the use of indoor coal combustion,the mortality and morbidity of Xuanwei lung cancer have not decreased significantly after the improvement of national stoves.Some studies have found that Xuanwei female lung adenocarcinoma has obvious family aggregation,and its gene mutation map is different from that of ordinary lung adenocarcinoma.In order to study the unique genetic basis of lung adenocarcinoma in Xuanwei women,we conducted transcriptional sequencing and differential expression analysis of lung adenocarcinoma in Xuanwei women.It was found that the expression of pancreatic duodenal homeobox 1(PDX1)was significantly increased in Xuanwei non-smoking women,and negatively correlated with disease-free survival((DFS)),suggesting that PDX1 may play a cancer-promoting role in lung adenocarcinoma in Xuanwei non-smoking women.It is a potential specific target for Xuanwei regional high incidence lung cancer with unique genetic basis.However,at present,the mechanism of PDX1 in malignant tumors is not clear,especially the research on PDX1 in lung cancer is even less.The purpose of this study was to explore the expression of PDX1 in lung adenocarcinoma tissues of Xuanwei non-smoking women and paired normal lung tissues of the same patient,and to analyze and compare the effects of PDX1 expression on proliferation,invasion,migration,apoptosis and cell cycle of lung adenocarcinoma cells.Methods:1.35 pairs of lung adenocarcinoma tissues and normal lung tissues from Xuanwei non-smoking female patients were collected,and the expression level of PDX1 protein in the two tissues was analyzed and compared by immunohistochemical technique.2.Using immortalized human bronchial epithelial cell line Beas-2b as control,the expression level of PDX1 protein in human lung adenocarcinoma cell lines A549,XWLC-05 and H838 was compared by WesternBlot technique.3.The cell line with the highest expression level of PDX1 was knocked out of PDX1 gene by CRISPR-Cas9 technique.Two sgRNA,were designed on each of the two exons of PDX1 gene to construct PDX1-sgRNA vector,and U6 sequencing was used to detect whether the knockout plasmid was constructed successfully.After the constructed knockout plasmids were extracted from plasmids,the three plasmid packaging systems(lentiCRISPRv2,psPAX2,PMD2G)were used to package the lentivirus through 239T cells.The virus was collected and infected with lung adenocarcinoma cells.48 hours later,the successfully transfected cells were screened with puromycin.Finally,the knockout efficiency was verified by WesternBlot experiment.The two lung adenocarcinoma cell lines with the highest knockout efficiency were selected from 4 sgRNA-guided CRISPR-Cas9 knockout lung adenocarcinoma cell lines for follow-up phenotypic study.4.The lung adenocarcinoma cell line with the lowest level of PDX1 expression was selected for PDX1 overexpression.After plasmid transformation,plasmid extraction and lentivirus packaging,the purchased PDX1 overexpression plasmid psin-ef1-puro-PDX 1 was collected to infect lung adenocarcinoma cells.After screening by puromycin,the success of overexpression was verified by WesternBlot experiment,and the functional experiments were carried out on the cells with successful overexpression.5.The effects of PDX1 knockout and overexpression on the proliferation of lung adenocarcinoma cells were observed by CCK8 assay,the effects of PDX1 knockout and overexpression on the invasion/migration ability of lung adenocarcinoma cells were evaluated by Transwell test and scratch test,and the effects of PDX1 knockout and overexpression on the growth cycle and apoptosis of lung adenocarcinoma cells were detected by flow cytometry.Results:1.The expression of PDX1 in lung adenocarcinoma tissues of Xuanwei non-smoking women was significantly higher than that in matched normal lung tissues.2.The expression level of PDX1 protein in lung adenocarcinoma cell lines A549,XWLC-05 and H838 was higher than that in Beas-2b,in which A549 was the highest,H838 was the second,and XWLC-05 was the lowest,so A549 cell line was selected for CRISPR-Cas9 knockout of PDX1 gene and XWLC-05 cells were selected for overexpression of PDX1.3.Four PDX1-sgRNA knockout plasmids were constructed successfully.After lentivirus infection,compared with the control group A549-NC,the expression level of PDX1 protein in A549-sg1,A549-sg2,A549-sg3 and A549-sg4 cells in knockout group was significantly decreased,and A549-sg2 and A549-sg4 were the lowest.A549-sg2 and A549-sg4 were used as knockout cell lines for subsequent phenotypic experiments.4.Compared with XWLC-05-NC cells,the level of PDX1 protein in XWLC-05-PDX1 cells in overexpression group was significantly higher.5.The proliferation rate of A549-sg2 and A549-sg4 in the PDX1 knockout group was significantly lower than that in the control group,and the cell proliferation ability was decreased,while the proliferation ability of XWLC-05-PDX1 in PDX1 overexpression cells was not significantly different from that in the control group(P>0.05).After PDX1 gene knockout,the number of A549 cells passing through Transwell chamber decreased significantly,the migration rate of A549 cells decreased significantly during scratching,and the invasion and migration ability of A549 cells decreased significantly after PDX1 gene knockout.After overexpression of PDX1 gene,there was no significant change in the number of XWLC-05 cells penetrating to the outside of Transwell chamber and the rate of cell migration during scratching.After knockout PDX1 gene in A549 cells,the proportion of cells in G1 phase decreased and the proportion of cells in S phase increased.Combined with the results of CCK8 proliferation test,it was suggested that knockout of PDX1 gene could block the growth cycle of A549 cells in S phase,while the apoptosis rate of A549 cells with PDX1 gene knockout increased significantly.After overexpression of PDX1 gene in XWLC-05 cells,there was no significant change in cell cycle proportion and apoptosis rate.Conclusions:1.The expression of PDX1 in lung adenocarcinoma tissue of Xuanwei non-smoking women was higher than that in normal lung tissue.The expression level of PDX1 protein in different lung adenocarcinoma cell lines A549,XWLC-05 and H838 was different,indicating that the expression level of PDX1 in lung adenocarcinoma cells from different sources was different.2.Knockout of PDX1 gene can inhibit the proliferation,invasion and migration of lung adenocarcinoma cells,lead to S-phase arrest and promote apoptosis of lung adenocarcinoma cells.Overexpression of PDX1 gene can not change the biological behavior of XWLC-05 cells,indicating that PDX1 may be a potential target for the treatment of lung adenocarcinoma.range.