The Expression And Mechanism Of SUZ12 In Lung Adenocarcinoma

Chapter 1: IntroductionResearch background:Lung cancer still ranks first in cancer incidence and mortality in China,and lung adenocarcinoma accounts for the main part.The polycomb family is one of the key regulatory gene families in epigenetics,which is capable of post-transcriptional modification of histones and silencing gene expression.SUZ12 is one of the core members of the PRC2 complex in the polycomb family.PRC2 complex can trimethylate lysine at the 27 th position of histone H3,generate H3K27me3,initiate chromatin compression,and thus silence gene expression.Current studies have shown that SUZ12 is overexpressed in a variety of malignant tumor tissues and is closely associated with malignant clinicopathological features and poor prognosis.However,the expression of SUZ12 in lung adenocarcinoma and its relationship with clinicopathological features and prognosis remain unclear.In terms of molecular mechanism,previous studies have shown that SUZ12 can play a role in promoting cancer by silencing the expression of p21,p27 and other molecules,but its role in other molecular mechanisms related to tumor proliferation,invasion,metastasis,apoptosis and immunity are still unclear.In our previous work,immunohistochemical tests showed that SUZ12 protein was significantly overexpressed in paraffin-embedded tissues after operation of human lung adenocarcinoma,but was negatively expressed in its corresponding paracancer normal tissues.Research objectives:To explore the expression,biological function and downstream molecular mechanism of SUZ12 in lung adenocarcinoma tissues and cell lines,the inhibition function of tumor and the regulation of target genes in the tumorigenic model in nude mice,and further explore the binding effect of target gene promoter,the correlation between SUZ12 and target gene expression in human lung adenocarcinoma tissues was analyzed.Chapter 2: SUZ12 expression in lung adenocarcinoma and its clinical significanceMethods: The expression of SUZ12 in paraffin-embedded tissue of lung adenocarcinoma after operation in 100 cancer tissue and 38 paracancer normal tissue was detected by immunohistochemistry,and the relationship between SUZ12 expression and clinicopathological features and prognosis was analyzed.Results: The high expression rate of SUZ12 in lung adenocarcinoma tissues(58.00%)was significantly higher than that in adjacent normal tissues(15.79%)(P<0.001).The high expression rate of SUZ12 was significantly correlated with tumor size(P=0.006),p T stage(P=0.016),lymph node metastasis(P=0.004),p TNM stage(P=0.023),and recurrence(P=0.020).The disease-free survival(P=0.019),overall survival(P=0.017)and 5-year overall survival rate(P=0.023)in the high expression group of SUZ12 were significantly lower than those in the low expression group.Conclusion: SUZ12 is overexpressed in lung adenocarcinoma tissues,and is closely related to tumor proliferation,invasion,metastasis,recurrence and poor prognosis.Chapter 3: The expression and biological function of SUZ12 in lung adenocarcinoma cell linesMethods: The expression of SUZ12 in human lung adenocarcinoma cell lines was detected by q RT-PCR and western bolt.The effects of SUZ12 knockdown and overexpression on the proliferation,invasion,migration,apoptosis and cell cycle of lung adenocarcarcinoma cell lines were detected by CCK8,cloning formation assay,transwell invasion and migration assay,scar assay and flow cytometry.Results: SUZ12 m RNA and protein were highly expressed in A549 cell line,moderately expressed in NCI-H1650 cell line and low expressed in NCI-H23 cell line.Knockdown of SUZ12 significantly inhibited the proliferation,cloning formation,invasion and migration of A549 cells,promoted apoptosis and induced G1/S phase cycle arrest(P<0.05),while overexpression of SUZ12 had the opposite effect in NCI-H23 cells(P<0.05).Conclusion: SUZ12 is expressed in lung adenocarcinoma cell lines in various degrees.SUZ12 promoted cell proliferation,cloning formation,invasion,migration,antagonized apoptosis,induced G1/S phase cycle conversion,and played the biological function of cancer gene.Chapter 4: Study on the downstream molecular mechanism of SUZ12 in lung adenocarcinomaMethods: q RT-PCR and western bolt were used to detect the effect of knockdown and overexpression of SUZ12 on the expression of downstream target molecules in terms of proliferation,apoptosis,invasion,metastasis and immunity.Results: sh-SUZ12 group compared with the sh-NC group,the q RT-PCR results showed that the m RNA expressions of CDK3,p57,Bcl-2,MMP14 and E-cadherin were significantly decreased,Bax and MMP1/2/3/9 were significantly increased(P<0.05);the western bolt results showed that the protein expressions of CDK2/3,cyclin D1,p18,p19,p-p53,Bcl-2,MMP1/9/14,TIMP3,E-cadherin,ITGB1/5 and PD-L1 were significantly decreased;cyclin E1,p57,Rb,p Rb,Bax,TIMP1/2 and Vimentin were significantly increased(P<0.05).Oe-SUZ12 group compared with the oe-NC group,the q RT-PCR results showed that the m RNA expressions of CDK3,CDK6,Bcl-2,MMP14 and E-cadherin were significantly increased,Bax and TIMP2 were significantly decreased(P<0.05);the western bolt(only some of the above indexes were selected)results showed that the protein expressions of CDK3,Bcl-2,MMP14 and PD-L1 were significantly increased,while Bax and TIIMP1/2 were significantly decreased(P<0.05).Conclusion: SUZ12 plays an important role in the regulation of tumor related molecules such as proliferation,apoptosis,invasion,metastasis and immunity.Chapter 5: The study of SUZ12 tumor formation model in nude miceMethods: The stable A549 cell line with SUZ12 knockdown was inoculated subcutaneously into nude mice to observe the growth of transplanted tumor.The expressions of SUZ12,Bcl-2 and Bax in tumor tissues were detected by western bolt method.Results: The growth rate of tumor size in sh-SUZ12 group was significantly slower than that in sh-NC group,and the tumor volume was significantly lower than that in sh-NC group at day 7(P=0.041),day 10(P=0.031)and day 25(P=0.037).At day 25,the tumor weight in the sh-SUZ12 group was significantly lower than that in sh-NC(P=0.047).Western blot results showed that sh-SUZ12 group significantly reduced the protein expression of SUZ12(P=0.001)and Bcl-2(P<0.001),while increased Bax(P=0.005).Conclusion: Knockdown of SUZ12 inhibited the graft tumor growth in nude mice,and downregulated Bcl-2 and up-regulated the protein expression of Bax.Chapter 6: The binding of SUZ12 to the target gene Bax promoter and the correlation between the two expressionsMethods: The potential binding sites of SUZ12,EZH2 and Bax promoters were predicted using h TFtarget human transcription factor binding sites database.Since H3K27me3 was not included in the transcription factor database,the primers that bind to Bax promoters were found in the literature.The predicted sites were verified by chromatin immunoprecipitation assay.The expression of Bax in human lung adenocarcinoma was detected by immunohistochemistry and its correlation with SUZ12 expression was analyzed.Results: The chromatin immunoprecipitation results showed that SUZ12 is able to bind to the predicted sites 1 and 2 in the Bax promoter region.In addition,EZH2,H3K27me3 can also bind to the Bax promoter region.Sh-SUZ12 significantly reduced the enrichment of SUZ12 at predicted site 1(P <0.001)and predicted site 2(P<0.001),and also significantly reduced the enrichment of EZH2(P=0.002)and H3K27me3(P<0.001)in the Bax promoter region.Immunohistochemical results showed that SUZ12 and Bax protein expression was negatively correlated in human lung adenocarcinoma tissues(R=-0.352,P=0.013).Conclusion: SUZ12 specifically binds to the Bax promoter region of the target gene in the form of the PRC2 complex,silencing Bax expression at the epigenetic level by forming H3K27me3.There was a negative correlation between SUZ12 and Bax protein expression in human lung adenocarcinoma tissues,which further confirmed that SUZ12 negatively regulated Bax expression.