E3 Ligase RNF38 Promotes The Invasion And Metastasis Of Non-small Cell Lung Cancer By Ubiquitin Degrading DSG1

The incidence and mortality rate of lung cancer were at the top of the first among different malignant tumors.In all cases of malignant tumor death,lung cancer ranked first worldwide.According to the latest cancer report from Chinese National Cancer Center in 2018,there were 781000 new cases and 626000 death cases in China.Based on the biological characteristics,the lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Among them,the NSCLC accounts for 80 % ~ 85 % of all cases.Although the basic research and treatment of lung cancer has made some breakthrough,the 5-year survival rate of lung cancer patients remains at around 15 % in recent 30 years.The ability of invasion and metastasis of lung cancer is the main culprit leading to a bad effective treatment of lung cancer.Clinically,nearly 80 % of NSCLC have presented with the regional lymph node or distant organ metastasis at original clinical diagnosis.Thus,it has important theoretical and clinical application to further reveal the molecular mechanisms of invasion and metastasis of lung cancer.By the detection of these molecules,we can not only predict the metastasis and prognosis of lung cancer,but also provide valuable molecular targets for blocking the invasion and metastasis of lung cancer.Ubiquitin protein degradation pathway is a degradation process of ubiquitin protein by the synergy of ubiquitin and a series of related enzymes.These series of related enzymes in this process is mainly composed of E1 Ubiquitin-activating enzyme,E2 Ubiquitin-conjugating enzyme,and E3 Ubiquitin ligase.E3 ligase has the function of identifing the target protein specifically,so it has the extremely important role in the ubiquitin-proteasome pathway.As we all know,the E3 ubiquitin ligase mainly including two categories: HECT domain family and RING domain family.A large number of studies have shown that E3 ligase was closely related to the origination,development and metastasis of tumor.By the analysis of GEO Data Set,we found that the expression of E3 ligase RNF38(Ring Finger Protein 38)in lung cancer increased obviously;RNF38 gene locates on chromosome 9 short arm 13 zone2(9 p13.2),the gene is found to have a number of different transcription variant coding subtypes.As one of E3 ligase,the function of RNF38 is still unclear.Research has shown that the ubiquitin substrate of RNF38 included the tumor suppressor gene p53,RNF38 could adjust the subcellular localization of p53 by ubiquitin pathway,so it is possible that RNF38 is related to tumorigenesis,intracellular signal transduction and apoptosis,etc.In our study,q RT-PCR,Western blot and Immunohistochemical staining were used in NSCLC and the corresponding adjacent normal lung tissues,then the RNF38 expression differences between the two groups in m RNA and protein level were analyzed.By building 208 cases of NSCLC tissue microarrays,immunohistochemical detection of RNF38 expression combined with clinical pathological characteristics of NSCLC patients,we analyzed the relationship between RNF38 expression and clinical pathological features of NSCLC patients.Then,we detected the RNF38 expression in different NSCLC cell lines.With the RNA interference,we investigated the change of NSCLC cell invasion and proliferation after downregulating RNF38 expression.Moreover,we also studied the influence of RNF38 expression on epithelial and mesenchymal markings expression of NSCLC cells.Co-immuneprecipitation combined with mass spectrometry analysis were used to determine the interacting proteins of RNF38.After further validating the relationship between RNF38 and its interacting proteins,we modified the expression of these proteins and detected the functional effect of the latter on the former.The relationship of RNF38 and its interacting proteins in NSCLC and tool cells was detected,and was further validated in the tissue level.Finally,we detected the expression of the interacting proteins of RNF38 in the same tissue microarrays using immunohistochemical staining,analyzed its relationship with clinical pathological characteristics of NSCLC patients,and made clear out the prognostic significance of the two in NSCLC patients combining with RNF38 expression analysis.Chapter 1 The relationship between the expression of E3 ligase RNF38 in NSCLC and the clinicopathological features of NSCLCObjective: A variety of experimental methods were used to study the expression of RNF38 protein in NSCLC and its corresponding adjacent tissues and to clarify its relationship with the clinicopathological parameters of NSCLC patients.Methods: Real-time fluorescence quantitative PCR and protein immunoblotting(Western blot)were used to detect the expression of RNF38 in 30 NSCLC and the adjacent tissues,and the expression of RNF38 in NSCLC was preliminarily determined.And the large sample in tissue immunohistochemical staining was further to confirm the above results.Combined with the clinical pathological features of patients,SPSS software was used to analyze the relationship between RNF38 expression and clinical pathological parameters of the 208 NSCLC patients.Results: The expression of RNF38 in NSCLC tissues was higher than that in the corresponding adjacent tissues(RNF38 m RNA,3.43 ± 0.33 vs.1.83 ± 0.21,RNF38 protein,2.98 ± 0.36 vs.1.63 ± 0.21).The positive expression of RNF38 in 208 cases of non-small cell lung cancer was 48.8%(102/208),whereas the corresponding expression in the adjacent tissues was only 10.1%(p < 0.05).High level of RNF38 protein expression was related to tumor size(p = 2.09 e-04),TNM stage(p = 0.011),lymph node metastasis(p = 4.43 e-05),tumor classification(p = 0.030)and vascular invasion(p = 0.001),but without related to ages(p = 0.155),gender(p = 1.000)and tumor differentiations(p = 0.403).Conclusion: RNF38 is over-expressed in NSCLC,and its expression is positively correlated with NSCLC malignant phenotypes.Chapter 2 RNF38 induces epithelial-mesench-ymal transition and promotes the invasion and metastasis of NSCLCObjective: To study the effect and mechanism of RNF38 expression on the biological function of NSCLC cells.Methods: The expression differences of RNF38 in multiple NSCLC cell lines were detected by fluorescence quantitative PCR and Western blot.The RNF38 expression was regulated by RNA interference technique in NSCLC cells with RNF38 over-expression,and stable cell lines were established.The capacity of cell proliferation were detected by CCK-8;Transwell and cell wound scratch assay were used to study the changes of cell invasion and mobility.Finally,the molecular mechanism of RNF38 inducing invasion and metastasis of NSCLC was investigated.Results: RNF38 was highly expressed in 95-D and A549 cells,while was lowly expressed in 95-C,H460 and 16 HBE cells.After building stable RNF38 interference cells,we found that high expression of RNF38 promoted the invasion and migration of NSCLC cells as well as cell proliferation.Western blot and immunofluorescence detection showed that the epithelial marker E-cadherin was up-regulated and the mesenchymal marker Vimentin was down-regulated after RNF38 expression interference.Conclusion: RNF38 induces NSCLC epithelial-mesenchymal transition and promotes the invasion and migration of NSCLC.Chapter 3 RNF38 ubiquitin degradation of DSG1 protein involved in NSCLC invasion and metastasisObjective: To explore the molecular mechanism of ubiquitin ligase RNF38 inducing the progression of NSCLC cells.Methods: Co-immuneprecipitation combined with liquid mass spectrometry and bioinformatics were used to screen the proteins interacted with RNF38,and further determine the substrate.The mechanism of RNF38 inducing NSCLC progression was determined by interference,western blot and metastasis test.Results: Thirteen proteins related to RNF38 were selected by the co-immuneprecipitation combined with liquid-phase mass spectrometry and bioinformatics analysis.Finally,DSG1 was determined as the substrate of RNF38 by the bioinformatic analysis of cell biology function after RNF38 interference and western blot analysis as well as Co-immuneprecipitation.Meanwhile,we found that RNF38 influenced NSCLC cell invasion by adjusting the stability of DSG1.Conclusion: E3 ligase RNF38 promotes NSCLC progression by ubiquitin degradation of DSG1.Chapter 4 RNF38 and DSG1 expression were related to prognosis of NSCLC patientsObjective: To detect the expression of DSG1 in NSCLC and its corresponding adjacent normal tissues,and analyze the relationship between DSG1 and the clinicopathological features of NSCLC.And analyze the relationship between RNF38 and DSG1 expression as well as the prognosis of NSCLC patients.Methods: Immunohistochemical detection of DSG1 in tissue microarrays was performed,and the relationships between DSG1 expression and RNF38 as well as the clinicopathological features of NSCLC were all analyzed.Finally,the relationship between RNF38 and DSG1 and NSCLC prognosis was analyzed with the data from follow-up study.Results: The level of DSG1 expression in NSCLC tissues was lower than that in the corresponding adjacent tissues,and DSG1 expression in non-small cell lung cancer tissues was negatively correlated to RNF38.The DSG1 expression in 208 cases of NSCLC was 43.3%(90/208),while the corresponding expression in the adjacent tissues was 60.1%(p <0.05).DSG1 expression level was related to TNM stage(p = 0.011),lymph node metastasis(p = 3.7 E-05)and tumor size(p = 0.001),but without related to ages(p = 0.094),gender(p = 0.877)and tumor differentiations(p = 0.261).Kaplan-meier survival analysis showed that the prognosis of patients with high RNF38 was lower than that of lower RNF38 patients(p=0.012),while the prognosis of low DSG1 expression patients was lower than that of high DSG1 expression patients(p = 0.032).The Cox analysis showed that the influence factors of prognosis of lung cancer survival including: tumor size(p = 9.76 E-06),lymph node metastasis(p = 3.47 E-09),tumor stage(p = 4.65 E-08),RNF38 expression(p =0.013)and lack of DSG1(p = 0.033).After Cox multivariate analysis,we found that lymph node metastasis(p =0.001),tumor size(p = 0.002),tumor stage(p = 0.046)and RNF38/DSG1 expression(p = 0.043)were independent prognostic factorsaffecting the overall survival rate.Conclusion: The overall survival rate of high RNF38 and low DSG1 expression patients was low,and high RNF38 and low DSG1 expression were the prognostic factors of NSCLC patients.Therefore,RNF38 and DSG1 protein can be served as new targets for predicting the prognosis and biological treatment of NSCLC patients.