The Study On The Mechanism Of ITGB1 Regulate The Radioresistance Of Human Non-small Cell Lung Cancer Cells By Modulating The Dna Damage Response And YAP1-induced Epithelial-mesenchymal Transition

Objectives: Lung cancer is the second most common cancer and the main cause of cancer deaths.In 2020,the number of deaths from lung cancer worldwide is as high as 1.8 million,which is much higher than other cancers.Lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),of which NSCLC accounts for 80-85% of the total incidence of lung cancer.At the time of clinical diagnosis,34% are in stage I and II,and 28% are in stage III.In the treatment of NSCLC patients in various clinical stages,radiotherapy is an important local treatment method,and about(64.3±4.7%)of patients need to receive radiotherapy at different periods of treatment.However,NSCLC still has a high local regional recurrence rate and distant metastasis rate after radiotherapy.It is currently believed that the resistance of NSCLC to radiotherapy is the main reason for this result.The mechanism of tumor cell resistance to radiotherapy is very complicated.Current studies have shown that multiple factors such as related gene changes,tumor microenvironment,hypoxia,metabolic changes,tumor stem cells,tumor heterogeneity,micro RNA,cell cycle,and DNA damage and repair lead to tumor radioresistance.However,the specific molecular mechanism of NSCLC radioresistance is still not fully understood.Therefore,studying the mechanism of lung cancer radiotherapy resistance and finding effective measures to reduce radiotherapy resistance and improve radiotherapy sensitivity is an urgent problem in lung cancer radiotherapy.Methods: Part 1: Based on the results of sequencing the NSCLC radioresistant cell line and its parental cell line in the early stage of the research group,further bioinformatics analysis of differentially expressed m RNA,combined with KEGG(Kyoto Encylopedia of Genes and Genomes)pathway enrichment analysis,and literature review.It was found that cell adhesion plays an important role in radioresistance.Therefore,differential analysis of genes enriched in adhesion and related pathways was carried out.STRING was used to construct protein-protein interaction network diagrams for differential adhesion-related genes.Cytoscape software screened hub genes.In NSCLC,the expression and prognosis of the top ten hub genes are analyzed.Part 2: Using flow cytometry apoptosis and cell clone formation experiments,the resistance of resistant cell lines was re-verified.PCR and Western Blot were used to verify the m RNA and protein expression of ITGB1.The R language was used to perform the expression of ITGB1 in NSCLC tissues from TCGA and analyze its expression with clinicopathological characteristics of NSCLC patients.PCR and Western Blot techniques were used to detect the expression of ITGB1 m RNA and protein levels in 1 human bronchial epithelial cell and 5 non-small cell lung cancer cell lines,and the flow cytometry apoptosis and cell clone formation experiment was conducted to explore the radiosensitivity of A549 and H522 cells.Using lentiviral technology to construct stable overexpression cell line of H522-ITGB1,A549 and H460R-sh ITGB1 stable knockout cell lines.CCK8 and cell clone formation experiments were used to explore the effects of ITGB1 on the proliferation and sensitivity of various cell lines after radiation.Cell apoptosis and cell cycle experiments were performed with flow cytometry to explore the effects of ITGB1 on cell apoptosis and cell cycle of various cell lines after radiation.Using immunofluorescence technology and Western Blot to detect DNA damage markers ?H2AX,and to explore the effects of ITGB1 on DNA damage in various cell lines after radiation.The STRING tool was used to conduct PPI network analysis on ITGB1 and DNA damage repair pathways to explore potential interactions,and Western blot technology was used to detect DNA damage repair pathways.Western blot was used to detect EMT related indicators after overexpression or knockout of ITGB1.Use Western Blot experiments to detect changes in the expression of E-cadherin,N-cadherin,Snail,Vimentin,and Zeb1 after overexpression or knockout of ITGB1.Using the "correlation analysis" in GEPIA2 to explore the Spearman correlation coefficient of ITGB1 and YAP1.Use the "survival analysis" in the GEPAI2 database to explore the impact of ITGB1 and YAP1 gene co-expression on the overall survival and disease-free survival of patients with non-small cell lung cancer.Use Western Blot experiment to explore the influence of ITGB1 on YAP1 protein expression in cytoplasm and nucleus,and its total protein expression.Results: Part 1: 1.The previous research team successfully established the NSCLC radiation resistant cell line H460 R,and the next-generation sequencing analyzed the differential expression of m RNA.Compared with the parental cells,the resistant cell line H460R cells had 508 genes up-regulated and 330 genes down-regulated.Enrichment analysis of KEGG pathway found that it was mainly enriched in cell adhesion related pathways: ECM receptor interaction pathway,focal adhesion and cell adhesion molecule signaling pathway.2.STRING and Cytoscape screened out Hub genes: ITGB1,ITGA1,ITGB4,ITGA3,ITGA2,LAMA5,COL4A5,LAMB1,LAMA1 and COL4A6.The first-ranked Hub gene ITGB1 is elevated in non-small cell lung cancer and is related to the prognosis.It will be studied later.Part 2: 1.The analysis of the NSCLC data set from TCGA(1,102 cases,including 103 normal tissues and 999 tumor tissues)shows that ITGB1 is highly expressed in NSCLC,and ITGB1 expression is related to clinical staging,and T classification and M classification.Through sensitivity verification,after 10 generations,the resistant cell line can still maintain its radioresistance.The results of q RT-PCR and Western Blot confirmed that ITGB1 is highly expressed in the resistant cell line compared with the parental cell line.2.ITGB1 is expressed to varying degrees in various non-small cell lung cancer cell lines.The ITGB1 high expression A549 and low expression H522 cell lines are selected for subsequent experiments.Compared with H522 cells,the apoptosis rate of A549 cells after irradiation is lower,and the colony forming ability and survival rate are higher.3.We used lentivirus to construct A549-sh ITGB1 and H460R-sh ITGB1 stable knockout cell lines,and constructed H522-ITGB1 stable overexpression cell line.After irradiation,the survival rate of A549-sh ITGB1 and H460R-sh ITGB1 was lower than that of the control group,and the survival rate of H522-ITGB1 cells was higher than that of in the control group.4.Apoptosis analysis by flow cytometry showed that the proportion of apoptotic cells in the A549-sh ITGB1 and H460R-sh ITGB1 groups was significantly higher than in the control group 48 h after irradiation.On the contrary,the incidence of radiation-induced apoptosis in H522-ITGB1 cells was significantly lower than that in negative control cells.The cell cycle distribution was evaluated by flow cytometry.Compared with the negative control,24 h after irradiation,A549-sh ITGB1 and H460R-sh ITGB1 cells have a higher proportion of cells in the G2/M phase,while the proportion of cells in the S phase is lower.The proportion of H522-ITGB1 cells in the G2/M phase is significantly lower than the negative control group,while the proportion of cells in the S phase is higher.5.We performed immunofluorescence detection to detect the expression of ?H2AX after or without irradiation.Compared with unirradiated cells,A549-sh ITGB1 and control cells had significantly more ?H2AX-positive nuclei at 30 minutes after irradiation.However,the number of positive nuclei in H522-ITGB1 cells was less than that of the control group.In both groups,the number of ?H2AX-positive nuclei decreased with the prolonged irradiation time,and returned to the basic level after 24 h.The protein level of ?H2AX was detected by Western Blot for verification,which was consistent with the results of the immunofluorescence experiments.Through the analysis of the protein interaction between ITGB1 and DNA damage repair pathway,ITGB1 may be related to the ATM / CHK2 pathway.after ITGB1 silence,the expression of ATM(p-ATM),CHK2(p-CHK2),CDC25 c and CDC25c(p-CDC25c)is reduced,and the overexpression of ITGB1 leads to higher expression of these proteins in H522 cells.6.After knocking out ITGB1,the expression of N-cadherin,Vimentin,Snail and Zeb1 was significantly down-regulated,and the expression of E-cadherin was up-regulated.On the contrary,the overexpression of ITGB1 down-regulated the expression of E-cadherin in H522 cells and up-regulated the expression of N-cadherin,Vimentin,Snail and Zeb1.7.We used GEPIA2 to study the correlation between the expression of ITGB1 and YAP1 in LUAD,LUSC and normal tissue samples.In LUAD(Cor = 0.37,P <0.01)and LUSC(Cor = 0.41,P <0.01)samples,the expression of ITGB1 was positively correlated with the expression of YAP1.In addition,the survival analysis of NSCLC patients in GEPIA2 showed that the overall survival and disease-free survival of patients with high expression of these two genes were significantly shortened.Western blot analysis showed that when ITGB1 was knocked out in A549 cells,YAP1 was down-regulated while inhibiting its nuclear entry.In the H522-ITGB1 group,YAP1 expression was up-regulated and YAP1 expression in the nucleus was significantly increased.Conclusions: 1.The second-generation sequencing method screened out the differentially expressed m RNAs between the resistant cell line and the parental cell line.Based on the KEGG pathway enrichment analysis,in-depth mining was carried out,which provided favorable clues for the subsequent research on the mechanism of radiation resistance.2.Adhesion-related pathways play an important role in the formation of radioresistance in NSCLC cells.The high expression of ITGB1,a key gene in adhesion-related pathways,is related to the clinicopathological characteristics,prognosis and formation of radioresistance.3.Silencing ITGB1 may inhibit the DNA repair ability of NSCLC cells,and regulate the EMT induced by YAP1,thereby reversing the radioresistance of NSCLC.