| Objective:According to the results of differential gene expression of 8 pairs of cancers and adjacent tissues in the database GSE89039 obtained by the research group using bioinformatics analysis in the early stage,it was found that there was a meaningful differential expression of the cell cycle checkpoint kinase 1(chk1)gene located upstream of the oncogene p53: the expression of chk1 in cancer tissues was higher than that in adjacent tissues.This project takes the results of this bioinformatics analysis as the starting point to explore the clinical significance of chk1 expression in Xuan-Wei lung cancer and its influence and mechanism on the malignant biological behavior of lung adenocarcinoma cells.High-throughput sequencing and bioinformatics analysis are combined to explore potential targets and related signaling pathways.Methods:1.Expression and clinical significance of chk1 in Xuan-Wei lung cancer1.1.Download the expression profile information of Xuan-Wei lung adenocarcinoma samples in GSE89039 database,use the independent sample T test to statistically analyze the chk1 expression of 8 pairs of Xuan-Wei lung cancer and adjacent tissues in GSE89039 database,and use the paired sample T test to statistically analyze the chk1 expression of 58 paired cancers and adjacent tissues.1.2.Data of 85 patients with lung adenocarcinoma in Xuan-Wei area who underwent surgery in the Department of Thoracic Surgery of Yunnan Cancer Hospital from January 2016 to May 2021 were collected,and the collected lung adenocarcinoma tissue samples were immunohistochemically stained for chk1 gene and p53 gene and statistically analyzed.Chi-square test was used to analyze the relationship between the expression of chk1 gene and p53 gene and the clinicopathological characteristics of lung adenocarcinoma patients.2.Effect of chk1 on in vitro biological behavior of lung adenocarcinoma cells2.1.Lentiviral transfection of A549,H1299 cell lines,puromycin screening,and construction of stable transfected cell lines.2.2.Cell function experiments: use CCK8 to detect cell proliferation;Scratch assay to detect cell migration ability;Transwell detects cell invasion;Flow cytometry to detect cell cycle and apoptosis.3.Effect of chk1 on in vivo biological behavior of lung adenocarcinoma cells Nude mice were randomly divided into A549 cell lines: control group,experimental group;H1299 cell line control group and experimental group,the experimental cell line was cultured in large quantities to prepare cell suspension.Nude mice were injected subcutaneously with suspension,and after 21 days,nude mice were sacrificed,tumors were peeled off,tumor volumes were measured and the tumor was weighed,and photographs were taken.4.Mechanism exploration of the effect of chk1 on the biological behavior of lung adenocarcinoma cells(sequencing and analysis)High-throughput sequencing was used to analyze the expression changes of differential genes and potentially closely related signaling pathways after knocking down chk1.Results:1.In lung adenocarcinoma samples in TCGA database,the expression of chk1 in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues(P<0.05).The immunohistochemical results showed that the positive expression rate of chk1 in lung adenocarcinoma tissues was higher than that in adjacent tissues(P<0.05),and there were no significant statistical differences between CHK-1expression and age,sex,pathological subtype,TNM stage and other factors(P>0.05).2.The expression of knockdown chk1 inhibits the proliferation,migration and invasion of lung adenocarcinoma A549 and H1299 cell lines.3.The results of tumorigenesis experiments in nude mice suggest that knocking down chk1 can inhibit the proliferation of lung adenocarcinoma cells in vivo,but there is a lack of convincing data support.4.The results of high-throughput sequencing suggested that chk1 may have potential regulatory factors such as invasion and metastasis,positive regulation of cell proliferation,and angiogenesis,and KEGG analysis suggested that chk1 may be related to PI3 K,MAPK and other signaling pathways.Conclusion:1.High expression of oncogene chk1 in Xuan-Wei lung cancer tissues;chk1 after knockdown can inhibit the proliferation,invasion and metastasis of lung adenocarcinoma cells and other malignant biological behaviors in vitro.But the role in vivo is not supported by data.2.High-throughput sequencing results showed that chk1 affected the biological behavior of tumor cells by regulating p53 signaling pathway and PI3 K in p53 wildtype cells(A549),while regulating PI3 K,MAPK and other signaling pathways in p53-deletion cells(H1299)affected tumor cell biological behavior.That is,chk1 may not only affect the malignant biological behavior of lung adenocarcinoma through the p53 pathway. |
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