Role And Mechanism Of E3 Ubiquitin Ligase RNF183 In The Cell Proliferation And Apoptosis Of Lung Adenocarcinoma

BackgroundLung cancer is the malignant tumour with the highest incidence and mortality rate in men in China.The pathological types of lung cancer can be divided into two major categories: non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),of which NSCLC accounts for about 80%,while NSCLC can be divided into lung adenocarcinoma(LUAD)and lung squamous carcinoma(LUSC).LUAD usually originates from small peripheral bronchial lesions,and is characterised by early clinical symptoms,insensitive screening indicators,unsatisfactory radiotherapy effects and easy recurrence with surgical treatment,so the screening and prognostic diagnosis of LUAD has become a hot topic of current research.RNF183,a member of the RING finger family,contains the remarkable RING finger structural domain and plays an important role in ubiquitin-mediated protein degradation.RNF183 is aberrantly high-expressed in colorectal cancer,uterine corpus endometrial carcinoma and Ewing sarcoma.Besides,the expression of RNF183 is associted with the poor prognosis of tumor patients.High expression of RNF183 has been shown to promote the proliferation of tumor cells and inhibit the apoptosis of tumor cells,thereby considering as a potential oncogene.However,the role and molecular mechanism of RNF183 in LUAD remain unclear,and its in-depth study will help provide new ideas and theoretical basis for the diagnosis and treatment of LUAD.PurposeBy explore the expression pattern of RNF183 in LUAD at the molecular,cellular,tissue and animal levels,we will clarify the role of RNF183 in proliferation and apoptosis of LUAD cells and investigate the molecular mechanism by which RNF183 affects proliferation and apoptosis of LUAD cells through regulating STAT3 signaling pathway,providing new targets for LUAD treatment and prognosis analysis.Methods(1)The Cancer Genome Atlas(TCGA),the Tumor Immune Estimation Resource(TIMER)and the UALCAN database were used to validate of RNF183 expression differences in LUAD tissues and their paraneoplastic tissues.(2)RT-PCR and immunohistochemistry(IHC)were used to detect the expression of RNF183 in LUAD tissues and their paracancerous tissues.The correlation between RNF183 protein expression and clinicopathological characteristics in LUAD patients was analyzed based on IHC scores,and the overall survival curves of RNF183 expression and LUAD patients were plotted using Kaplan-Meier by analyzing the follow-up data of LUAD patients.(3)Western blot and RT-PCR were used to detect the expression of RNF183 in LUAD cell lines to screen the higher or lower RNF183 expression level of cell line.Afterwards,the cell line H358 with low RNF183 expression,and the cell lines H1975 and PC9 with high RNF183 expression were selected to construct RNF183 RNAi and overexpression cell lines by lentiviral vector transfection technique.(4)The effect of RNF183 expression on the proliferation of LUAD cells was examined using CCK-8 assay.Gene Set Enrichment Analysis(GSEA)and flow cytometry assays were used to detect the effects of RNF183 expression on cell cycle,apoptosis and sensitivity to cisplatin(DDP)chemotherapy.A cell-derived xenograft model(CDX)was used to observe the effect of RNF183 knockdown on tumorigenic ability in nude mice in vivo.Western blot assay was used to detect the effect of RNF183 expression on the expression of cell cycle,such as c-myc,Cyclin D1 and apoptotic proteins Survivin and Cleaved caspase3.(5)GSEA and Western blot assays were used to verify the correlation between RNF183 expression and STAT3 signaling pathway.CCK-8 and flow cytometry assays were used to detect the effects of RNF183 overexpression,alone or in combination with JAK/STAT3 signaling pathway inhibitor(AG490)treatment on proliferation,cell cycle and apoptosis of LUAD H358 cells.Western blot assay was used to detect the effects of RNF183 overexpression,alone or in combination with AG490 treatment on the expression of STAT3 signaling pathway downstream target genes including c-myc,Cyclin D1,Survivin and Cleaved caspase3.(6)GSEA,Western blot and RT-PCR experiments were used to verify the correlation between RNF183 expression and SHP2.Immunoprecipitation(Co-IP)and Western blot assays were used to detect the interaction between RNF183 and SHP2.Western blot assay was used to detect the effect of RNF183 overexpression,alone or in combination with proteasome inhibitor(MG132)treatment on SHP2 protein expression.Ubiquitination and Western blot assays were used to detect the effect of interference with RNF183 expression on ubiquitinated degradation of SHP2 protein.(7)The RNF183 overexpressing H358 cell line was selected construct RNF183 and SHP2 double genes overexpressing using the lentiviral vector transfection technique.The effects of RNF183 overexpression,SHP2 overexpression,and RNF183 and SHP2 double gene overexpression on proliferation,cell cycle and apoptosis of LUAD cells were detected by CCK-8and flow cytometry assays,respectively.Western blot assay was used to detect the effects of SHP2 and STAT3 and their downstream target genes such as c-myc,Cyclin D1,Survivin and Cleaved caspase3.Results(1)High expression of RNF183 in LUAD tissues is correlated with poor prognosis of LUADThe expression of RNF183 in LUAD tissues was significantly higher than that in paraneoplastic tissues(P<0.001),and correlated with tumor size and lymph node metastasis in LUAD patients(P<0.05).The overall survival time of patients in the RNF183 high expression group was significantly shorter relative to the RNF183 low expression group(P<0.05).(2)RNF183 promotes proliferation,inhibits apoptosis and reduces sensitivity to cisplatin chemotherapy of LUAD cellsRNF183 was highly expressed in LUAD H1975 and PC9 cells,and lowly expressed in H358 cells.Knockdown of RNF183 inhibited LUAD cell proliferation,induced G0/G1 phase cell cycle arrest,promoted apoptosis,and increased cisplatin chemosensitivity as well as inhibited tumorigenic ability in nude mice.(3)Clarification of the regulatory relationship between RNF183 and STAT3 signaling pathwayThe expression of RNF183 was positively correlated with STAT3 signaling pathway(P<0.001).Knockdown of RNF183 inhibited STAT3 phosphorylation in LUAD cells,while RNF183 overexpression promoted STAT3 phosphorylation.AG490 was able to inhibit RNF183 overexpression mediated STAT3 protein phosphorylation.RNF183 regulates downstream target genes c-myc,Cyclin D1,Survivin and Cleaved caspase3 is involved in cell proliferation,cell cycle and apoptosis of LUAD.(4)Revealed the interaction between RNF183 and SHP2RNF183 expression was negatively correlated with SHP2＿PATHWAY(P<0.001).MG132 inhibited RNF183 overexpression mediated SHP2 ubiquitination.Rescue experiments confirmed that SHP2 overexpression reversed RNF183 overexpression mediated STAT3 signaling pathway regulated LUAD cell phenotype.ConclusionRNF183 is highly expressed in LUAD tissues and is a potential biomarker for diagnosis and poor prognosis of LUAD.The biological function of RNF183 in LUAD cells was clarified and the molecular mechanism of RNF183-SHP2/STAT3 signaling axis mediating proliferation,cell cycle and apoptosis in LUAD cells was revealed.RNF183 may become a new target for the therapy and prognostic analysis of LUAD.