RNF19A Promotes The Proliferation And Inhibits Apoptosis Of Non-small Cell Lung Cancer Cells By Ubiquitination Degradation Of P53

Objective: Lung cancer is one of the most common malignant tumors in humans and the leading cause of death from malignant tumors worldwide.China is a big country with lung cancer,with the highest morbidity and mortality rates in the world and a heavy burden of disease.Lung cancer can be divided into small cell lung carcinoma and non-small cell lung carcinoma(NSCLC)according to histological types,of which NSCLC is a major histological type,accounting for about 80-85%.Despite the development of surgery,chemoradiotherapy,and targeted therapy,the 5-year survival rate for patients with advanced NSCLC is still less than 5%.Identification of new early diagnosis and prognostic markers and a deeper understanding of its pathogenesis are of great importance for early diagnosis and clinical treatment of NSCLC patients.p53 gene is the tumor suppressor gene that has the highest correlation with human tumors.Under normal conditions,p53 acts as a "genomic guardian",inspecting DNA damage sites and monitoring the integrity of the genome during the G1 phase of the cell cycle.If the p53 gene is inactivated,the normal proliferation of cells loses control,leading to the occurrence of cancer.The mechanism of p53 gene inactivation mainly includes two aspects: one is mutation of p53 gene;the other is inactivation through interaction with other proteins.Although mutations in p53 are common in cancers,in most cancers that retain wild type p53,the protein can be inactivated through interactions with other proteins,resulting in loss or reduction of its tumor suppressor function.For example,E3 ubiquitin ligases such as MDM2 and MDMX can bind p53 and degrade p53 through the ubiquitination pathway to make it inactivated.It has been one of the long-term research directions in the academic circles to study the mechanisms involved in the regulation of p53 ubiquitination and degradation and how to restore the tumor suppressor function of p53.RBR E3 ligases are named because they contain the RBR(RING-IBR-RING)domain,which mediates the protein-protein interaction.RBR E3 ligases are widely found in eukaryotes and are involved in many cellular events such as transcription and RNA metabolism,translation,post-translation modification,regulation of protein stability,cell and stress signaling,and cell cycle control.RBR E3 s also plays an important role in the occurrence and development of tumors.For example,Parkin and RNF144 A are involved in the occurrence and progression of lung cancer,breast cancer and other tumors through ubiquitin-mediated degradation of tumor promoters or inhibitors.RNF19 A is a member of the RBR E3 ligase family and has a typical RBR three-step domain,which can play the role of E3 ubiquitin ligase independently.It has been reported that mRNA accumulation expression of RNF19 A was increased in serum of prostate cancer patients.However,the function of this gene is unknown in NSCLC at present,so we intend to explore the expression of RNF19 A in NSCLC and the mechanism of its involvement in the regulation of the occurrence and development of NSCLC,in order to provide new ideas and directions for the diagnosis and treatment of NSCLC.Methods: 1.Collect the 1st affiliated hospital of China medical university archive of nonsmall cell lung cancer with paraffin specimens of 136 cases and 30 cases of tissue adjacent to carcinoma.Immunohistochemical S-P method was used to detect the expression difference of RNF19 A protein in cancer tissues and paracancerous tissues,and the correlation between the expression of RNF19 A protein in cancer tissues and the clinicopathological features of lung cancer was analyzed.In addition,8 pairs of fresh NSCLC and adjacent normal tissue samples were collected to detect the expression of RNF19 A protein in NSCLC tissues by Western blot.2.The expression of RNF19 A mRNA in human NSCLC tissues was analyzed using the transcription level of Oncomine database,and the relationship between RNF19 A gene and the prognosis of NSCLC patients was analyzed online using GEPIA database.3.After knockdown or overexpression of RNF19 A gene in p53 wild-type human NSCLC cell lines A549 and H460,respectively,the viability of lung cancer cells was detected by MTT assay,cell proliferation ability was detected by plate colonization assay,Annexin VFITC staining,and the number of apoptosis was detected by flow cytometry.To clarify the biological function of RNF19 A in NSCLC cells.4.The expression changes of p53 protein and its downstream proteins p21,BAX,Cyclin D1,CDK4,CDK6 and BCL2 after RNF19 A gene knockdown or overexpression were detected by Western blot.Real-time PCR was used to detect the changes of p53 mRNA levels after bidirectional manipulation of RNF19 A gene.The downstream signaling pathway of p53 regulated by RNF19 A was determined.5.In p53 null NSCLC cell line H1299 and p53 mutation cell line SK-MES-1,RNF19 A gene was knocked down or overexpressed.Cell proliferation was detected by MTT and plate cloning-formation assay,and cell apoptosis was detected by Annexin V-FITC staining and flow cytometry.In order to further clarify this point of view,a rescue experiment was designed,that is,on the basis of transfection of RNF19 A siRNA knockdown p53 gene,to observe the response of knockdown RNF19 A to the inhibition of cell proliferation.At the same time,we observed whether p53 knockdown could reverse the effect of RNF19 A knockdown on p53 downstream protein expression.6.RNF19 A knockdown A549 and H460 cells were treated with proteasome inhibitor MG132,and p53 expression was detected by Western blot to determine whether RNF19 A promoted the degradation of p53 through the proteasome pathway.7.72 h after transfection of RNF19 A siRNA,CHX was added to collect the cells at 6 time points of 0,0.5,1,1.5,2 and 2.5h,respectively.The endogenous p53 protein half-life in A549 and H460 cells was detected by Western blot.8.The interaction between RNF19 A and p53 protein was determined by endogenous and exogenous immunoprecipitation experiments.9.RNF19 A siRNA and overexpressed plasmid were transfected into A549 cells,respectively.24 h after transfection,proteasome inhibitor MG132 was added to inhibit protein degradation.The effect of RNF19 A on the ubiquitination degradation of p53 protein was detected.Results: 1.The high expression of RNF19 A in NSCLC was associated with poor prognosis.RNF19 A protein expression in 8 fresh paired NSCLC tissues was significantly higher than that in the paired adjacent normal tissues.Immunohistochemical detection of NSCLC paraffin specimens and adjacent normal tissues showed that RNF19 A was highly expressed in NSCLC tissues,and its high expression was positively correlated with NSCLC tumor size and TNM staging.Searching the data sets from the Oncomine database,we found that RNF19 A mRNA expression was significantly higher in NSCLC tissues than in paracancerous tissues.GEPIA database analysis showed that high expression of RNF19 A mRNA in NSCLC was positively correlated with poor prognosis.2.RNF19 A promotes the proliferation of NSCLC cells.In p53 wild-type cell lines A549 and H460,MTT and plate colony formation experiments showed that RNF19 A could promote the proliferation of NSCLC cells.3.RNF19 A inhibited apoptosis of NSCLC cells.Annexin V-FITC staining and flow cytometry showed that RNF19 A could inhibit NSCLC cell apoptosis.4.RNF19 A inhibits the expression of p53 protein and regulates the downstream signaling pathway of p53.Western blot results showed that RNF19 A inhibited the expression of p53 and its target protein p21 and BAX,and up-regulated the expression of p53 downstream proteins Cyclin D1,CDK4,CDK6 and BCL2.However,the p53 mRNA level did not change after knockdown or overexpression of RNF19 A gene.5.The role of RNF19 A in promoting cancer depends on p53.The knockdown or overexpression of RNF19 A had no significant effect on the proliferation and apoptosis of p53 null cell line H1299 and p53 mutation cell line SK-MES-1,which indicated that the role of RNF19 A in promoting cancer may be dependent on p53.In order to further confirm this view,a recovery experiment was designed.We found that p53 knockdown not only reversed the inhibitory effect of RNF19 A knockdown on cell proliferation,but also reversed the effect of p53 downstream protein expression.6.RNF19 A interacts with p53 to promote p53 ubiquitination.The proteasome inhibitor MG132 rescinded the increased p53 expression caused by knockdown of RNF19 A gene,suggesting that RNF19 A may promote p53 degradation through the proteasome pathway.The results of the CHX assay showed that the endogenous p53 half-life in RNF19 A knockdown cells was significantly longer than that in the control group.Endogenous and exogenous immunoprecipitation experiments confirmed that RNF19 A protein can interact with p53 protein.Overexpression of RNF19 A significantly induced p53 ubiquitination,while knockdown of RNF19 A significantly decreased p53 ubiquitination.Conclusion: 1.RNF19 A,as a novel cancer-promoting gene,is highly expressed in NSCLC,and its high expression is correlated with tumor size,TNM stage and poor prognosis.2.RNF19 A promoted proliferation and inhibited apoptosis of NSCLC cells.3.In NSCLC,RNF19 A plays a pro-cancer role by promoting p53 ubiquitination and degradation,thereby inhibiting the downstream p53 signaling pathway.Therefore,this study contributes to a deeper understanding of the mechanism of p53 inactivation during the occurrence and development of NSCLC,and provides a new idea for the diagnosis and targeted therapy of patients with wild-type p53.