Expression Of Centromere Protein K In Lung Adenocarcinoma And Its Effects On The Biological Function Of Lung Adenocarcinoma Cells

Chapter 1 Introduction Lung cancer is not only one of the most common tumors in the world,but also the leading cause of death among cancer patients worldwide,with morbidity and mortality ranking first among all cancers.Lung adenocarcinoma is the most common subtype,accounting for the largest proportion of lung cancer.For the treatment of lung cancer,in addition to surgery,radiotherapy,and chemotherapy,targeted therapy and immunotherapy provide new directions.However,due to the problems of drug resistance,efficiency,and adverse reactions,its curative effects is still unsatisfactory.At present,the long-term survival rate of lung cancer patients is still very low,with a 5-year survival rate of only 4%~17%.Therefore,the study of abnormally expressed genes in lung cancer,especially in lung adenocarcinoma,is of great importance to further elucidate the mechanism of its occurrence and development,and to identify prognostic markers and therapy-related targets.Centromere protein K(CENPK),also known as P33,is a member of the centromere protein family.It plays a regulatory role in maintaining normal kinetochore function and is one of the necessary conditions in the process of mitosis.Currently,there are few reports on the effects of CENPK in tumors and its specific mechanism.Limited studies have shown that CENPK is an oncogene,which can promote the proliferation and metastasis ability of cancer cells in liver cancer and ovarian cancer.However,there are few reports on the biological function and prognostic value of CENPK in lung adenocarcinoma,and further study is still needed.Chapter 2 The expression of CENPK in lung adenocarcinoma and its clinical correlation analysisObjective: The study was conducted to investigate the expression level of CENPK in lung adenocarcinoma,to explore the relationship between the differential expression of CENPK and various clinicopathological parameters of patients with lung adenocarcinoma and its clinical prognostic significance.Methods: In this study,the expression level of CENPK in lung adenocarcinoma and normal samples were performed by using bioinformatics analysis,immunohistochemistry,Western blotting,and q RT-PCR.Secondly,Kaplan-Meier,univariate and multivariate Cox survival analysis were used to further explore the relationship between the differential expression of CENPK and the clinicopathological parameters of patients with lung adenocarcinoma and its clinical prognostic significance,according to the expression data of CENPK and the clinical data of patients with lung adenocarcinoma,Results: The results of bioinformatics analysis and q RT-PCR showed that the expression level of CENPK m RNA increased in lung adenocarcinoma.The results of Western blotting and immunohistochemical staining experiments showed that the expression level of CENPK protein increased in lung adenocarcinoma cell lines and tissues.The expression of CENPK was positively correlated with lymph node metastasis and pathological stage(P < 0.05).The high expression of CENPK was significantly correlated with the poor prognosis of patients with lung adenocarcinoma(P < 0.05).Multivariate Cox regression analysis showed that CENPK could be used as an independent predictor of poor prognosis of patients with lung adenocarcinoma.Conclusion: The high expression level of CENPK in lung adenocarcinoma is positively related to lymph node metastasis and pathological stage in patients with lung adenocarcinoma.Moreover,the high expression level of CENPK is related to poor prognosis in patients with lung adenocarcinoma and can be used as an independent predictor of prognosis in patients with lung adenocarcinoma.Chapter 3 The effects of CENPK on the biological behavior of lung adenocarcinoma cell line and its mechanism in vitro studyObjective: Based on the results of chapter 2,the effects of CENPK on the biological behavior of lung adenocarcinoma and its possible regulatory pathway was further explored.Methods: First,si RNA was used to interfere with the expression of CENPK in lung adenocarcinoma cell line A549,and si RNA-NC was transfected into the A549 cell line as a negative control group.The proliferation ability of A549 cells was detected by CCK-8 and cell clone formation assay,the migration ability of A549 cells was detected by cell scratch assay and transwell migration assay,and the invasion ability of A549 cells was detected by transwell invasion assay.Western blotting was used to detect the effects of reducing the expression of CENPK on epithelial-mesenchymal transition(EMT)of A549 cells.Finally,through bioinformatics analysis methods,based on the expression data of CENPK in lung adenocarcinoma in the TCGA database,gene set enrichment analysis(GSEA)was performed to explore the potential regulatory pathways involved in the high expression of CENPK in lung adenocarcinoma.Results: After interfering with the expression of CENPK in the lung adenocarcinoma cell line A549,the proliferation and migration ability of A549 cells was significantly reduced compared with the control group.Western blotting detection revealed that after knocking down CENPK,the protein expression level of epithelial marker(E-cadherin)increased significantly,and the level of interstitial markers(N-cadherin,Vimentin)and transcription factor(Snail)decreased significantly,proving CENPK decreased expression can inhibit EMT progress.Finally,GSEA results show that the high expression of CENPK in lung adenocarcinoma is mainly related to DNA repair,E2 F TARGETS,cell cycle(G2M CHECKPOINT),MTORC1 signaling pathway,and PI3K/AKT/MTOR signaling pathway.Conclusion: After interfering with the expression of CENPK,the proliferation and migration ability of lung cancer cell line A549 decreased,and the EMT progress in A549 cells is inhibited.The potential regulatory pathways that CENPK may participate in are DNA repair,E2 F TARGETS,cell cycle(G2M CHECKPOINT),MTORC1 Signal pathway,PI3K/AKT/MTOR signal pathway.Chapter 4 Effects of CENPK on tumor formation in nude mice of lung adenocarcinoma cell lineObjective: The effects of interference with CENPK expression on the proliferation of lung adenocarcinoma cells was further verified by in vivo experiments.Methods: After obtaining the interference sequence of the CENPK gene,the recombinant plasmid construction,packaging,and titer detection of the lentivirus were carried out.Then the lung adenocarcinoma cell line A549 was transfected with lentivirus,which could stably interfere with the expression of CENPK in the A549 cell line.The tumor formation experiment in nude mice was carried out:Ten 5-week-old Balb/C-nu nude mice were randomly divided into two groups,each with 5 mice.The experimental group was injected with the A549 stable transgenic strain of si-CENPK,and the control group was injected with the A549 stable transgenic strain of si RNA-NC,then the volume and weight of the transplanted tumor were observed and analyzed statistically.Results: Compared with the control group,the mean volume and mean weight of subcutaneous xenograft in nude mice in CENPK knockout group were significantly decreased,with statistical differences(P < 0.05).Conclusion: In vivo tumor formation experiments in nude mice further confirmed that silencing the expression of CENPK could inhibit the proliferation ability of lung adenocarcinoma cell line A549.