Study On The Role And Mechanism Of RPL32 Gene In The Tumorigenesis And Development Of Lung Cancer

Background and ObjectivesLung cancer is one of the most common malignant tumors.The tumorigenesis and development of many malignant tumors,including lung cancer,are related to the dysfunction of ribosomes.Relevant researches have confirmed that the expression of various ribosomal protein genes were up-regulated in lung malignant tumors,and the high expression of ribosomal protein genes were correlated with the poor prognosis of lung cancer patients.Moreover,ribosomal proteins have also been proven to have the function of regulating the biological behavior of tumor cells or tumor-related signaling pathways.RPL32 is one of the ribosomal proteins,and previous studies have confirmed that it could promote the progression of various malignant tumors.But so far,its correlation with the occurrence and development of lung malignant tumors as well as its mechanisms are still unclear.This thesis studies this topic from the following three aspects:A.To study the role and specific mechanism of RPL32 in lung cancer cells through in vitro experiments.B.To study the expression of key genes in the RPL32-p53 regulatory pathway in lung cancer,and the correlation between the expression of these genes and the prognosis of patients with lung cancer.We studied the expression of key genes in the RPL32-p53 regulatory pathway and its correlation with patients’ prognosis in lung adenocarcinoma and lung squamous cell carcinoma.Because the p53 mutation is one of the most common genetic variants in lung cancer,and the structure and function between mutant p53 protein and wild-type p53 protein are very different,we studied whether there are differences in the expression of key genes in the RPL32-p53 regulatory pathway and the correlation between these genes and the prognosis of patients.C.To study the correlation between the expression of key genes in the RPL32-p53 regulatory pathway and the immune infiltrations and immune functions of lung cancer.Previous studies have confirmed that three factors in lung cancer tissues were closely related to the occurrence and development of lung cancer and the prognosis of lung cancer patients: the infiltration levels of immune cells,the expressions of immune checkpoint genes,and the activities of important cytokine signaling pathways.Previous studies have also confirmed that ribosomal proteins and p53 were involved in the regulation of immune function in tumor tissues.So,we studied whether there are correlations between the expression of key genes in the RPL32-p53 regulatory pathway and the following three factors: the infiltration levels of immune cells,the expressions of immune checkpoint genes,and the activities of important cytokine signaling pathways.MethodsA.Study on the following by immunohistochemical staining:(a)The difference in the expression of RPL32 gene between lung cancer tissues and paratumor tissues.(b)The correlation between the expression of RPL32 gene and the prognosis of lung cancer patients.B.The expression of RPL32 gene in several lung cancer cell lines was knocked down by si RNA-based silencing strategy.Based on this,the role of RPL32 in lung cancer cells and its mechanisms were studied through Western blot analysis,cell cycle analysis,realtime quantitative PCR analysis,and immunoprecipitation analysis.C.The following characteristics of key genes in the RPL32-p53 regulatory pathway were analyzed by bioinformatics methods:(a)differential expressions.(b)co-expressions,(c)protein-protein interactions.(d)Study on the correlation between gene expression and patient prognosis.Moreover,the differences of these characteristics between lung adenocarcinoma and lung squamous cell carcinoma,and between p53 mutant lung cancer and p53 wild-type lung cancer were analyzed.D.The correlations between the expression of key genes involved in the regulation of RPL32-p53 and the following factors in lung adenocarcinoma tissues were analyzed through bioinformatics methods:(a)infiltration levels of immune cells.(b)expressions of immune checkpoint genes.(c)activities of important cytokine signaling pathways.ResultsA.By the immunohistochemical analysis and in vitro cell experiments,the following results are obtained:(a)The expression of RPL32 gene is up-regulated in lung cancer tissues,and the high expression of RPL32 gene is correlated with the poor prognosis of lung cancer patients(P = 0.0247).(b)The high expression of RPL32 gene promotes the proliferation of lung cancer cells in vitro and reduces the level of p53 in lung cancer cells.(c)The high expression of RPL32 gene facilitates MDM2-mediated p53 ubiquitination in lung cancer cells,thereby promoting the degradation of p53.(d)Knockdown of RPL32 gene expression in lung cancer cells triggers nucleolar stress,which causes the ribosomal proteins RPL5 and RPL11 to translocate from the nucleus to the nucleoplasm.RPL5 and RPL11 can bind to MDM2,thereby blocking the interaction between MDM2 and p53,allowing p53 to accumulate in cells,and finally leading to inhibition of lung cancer cell proliferation.B.By the differential expression analysis,co-expression analysis,protein-protein interaction analysis,and survival analysis,the following results are obtained:(a)7 genes are involved in the regulation of p53 by RPL32: RPL32,RPL5,RPL11,RPL23,MDM2,MDM4,and TP53.(b)The expressions of RPL32,RPL5,RPL23,MDM4,and TP53 in lung adenocarcinoma tissue is higher than that in normal lung tissue(P < 0.01).The expressions of RPL5,RPL23,MDM4,and TP53 in lung squamous cell carcinoma tissue is higher than that in normal lung tissue(P < 0.05),while the expression of RPL11 in lung squamous cell carcinoma tissue is lower than that in normal lung tissue(P < 0.01).(c)The expressions of RPL32,RPL11,RPL23,MDM2,MDM4,and TP53 in p53wild-type lung adenocarcinoma is higher than that in p53 mutant lung adenocarcinoma(P < 0.05).The expressions of RPL32 in p53 wild-type lung squamous cell carcinoma is higher than that in p53 mutant lung squamous cell carcinoma(P < 0.01).(d)The protein levels of RPL32,RPL5,RPL11,and p53 in lung adenocarcinoma and lung squamous cell carcinoma tissue are higher than that in normal lung tissue.The protein level of RPL23 in lung adenocarcinoma tissue is higher than that in normal lung tissue.The protein level of MDM4 in lung squamous cell carcinoma tissue is higher than that in normal lung tissue.(e)The expression correlation of key genes in the RPL32-p53 regulatory pathway in lung adenocarcinoma and lung squamous cell carcinoma is different.The expression correlation of key genes in the RPL32-p53 regulatory pathway in p53 wild-type lung adenocarcinoma and p53 mutant lung adenocarcinoma is different.The expression of p53 is positively correlated with the expression of RPL32,RPL5,RPL23 and MDM4 in p53wild-type lung adenocarcinoma(P < 0.05).(f)p53 protein interacts with other proteins in the RPL32-p53 regulatory pathway.(g)The high expressions of RPL32,RPL11,RPL23,and TP53 are significantly correlated with the poor overall survival of patients with lung adenocarcinoma(P < 0.05).The expressions of all key genes in the RPL32-p53 regulatory pathway are not significantly correlated with overall survival of patients with lung squamous cell carcinoma.In lung adenocarcinoma patients with low expression of RPL32,the prognosis of p53 wild-type patients is better than that of p53 mutant patients(P = 0.033).The prognosis of lung adenocarcinoma patients with high expression of RPL32 is not correlated with p53 mutation status.C.By correlation analysis between the expression of key genes in the RPL32-p53 regulatory pathway and the immune infiltrations and immune functions in lung cancer:the following results are obtained:(a)In lung adenocarcinoma,the expression of key genes in the RPL32-p53 regulatory pathway is correlated with the infiltration levels of a variety of immune cells(P < 0.05).(b)RPL32 affects immune checkpoint function by regulating p53.In lung adenocarcinoma,the expressions of immune checkpoint genes is correlated with the expression of key genes in the RPL32-p53 regulatory pathway and p53 mutation status(P < 0.05).(c)The expression levels of key genes in the RPL32-p53 regulatory pathway in lung adenocarcinoma are correlated with the activities of the following cytokine signaling pathways: IL-4,IL-13,TGF-β,and core serum response(P < 0.05).ConclusionsA.RPL32 promotes lung cancer progression by facilitating MDM2-mediated p53 ubiquitination and degradation.B.7 genes involved in the regulation of p53 by RPL32: RPL32,RPL5,RPL11,RPL23,MDM2,MDM4,and TP53.C.The activation of the RPL32-p53 regulatory pathway promotes the development of lung adenocarcinoma.D.The activity of RPL32-p53 regulation in p53 wild-type lung cancer stronger than that in p53 mutant lung cancer.E.The expressions of key genes in the RPL32-p53 regulatory pathway in lung adenocarcinoma is correlated with the infiltration levels of immune cells,the expressions of immune checkpoint genes,and the activities of important cytokine signaling pathways.