A Study Of RNF139 In Anti-apoptosis Of Pancreatic Cancer Under Hypoxia Micro-Environment

BackgroundRNF139(Ring Finger Protein 139)is a multiple transmembrane protein containing Ring-H2.The protein is localized in the endoplasmic reticulum and possesses ubiquitin ligase activity.Further studies have demonstrated that the abnormality of this gene is associated with hereditary renal disease and non-genetic thyroid cancer.Research on Drosophila shows that the gene interacts with the tumor suppressor protein VHL and the protein COPS5/JAB1,which is responsible for degrading the tumor suppressor CDKN1B/P27 KIP.There are few studies on the gene in tumors.It has been reported that the expression of RNF139 in glioblastoma and oral squamous cell carcinoma is significantly up-regulated.Another study indicates that RNF139 is involved in Barrett's esophageal cancer.Sequential progress.In a recent histology study of paclitaxel resistance in non-small cell lung cancer,RNF139 expression was significantly increased in albumin-resistant paclitaxel-resistant tumor cell lines.Albumin paclitaxel is the most important chemotherapy drug for pancreatic cancer with the highest objective response rate.Chemotherapy resistance has always been a major problem that plagues clinical practice.Therefore,we envisage whether RNF139 will cause tumor cells through certain anti-apoptosis related molecular mechanisms in pancreatic cancer.Resistance to albumin paclitaxel has been reported and there is currently no report of RNF139 in pancreatic cancer.Through preexperimental results and online database analysis,we found that compared with pancreatic tissue,RNF139 mRNA and protein levels were significantly increased in surgical specimens of pancreatic ductal adenocarcinoma;compared with normal pancreatic ductal cell lines,pancreatic cancer The expression of RNF139 in cell lines was also significantly up-regulated;RNF139 was positively correlated with apoptosis-related genes.Under hypoxic conditions,the expression of RNF139 was significantly up-regulated in pancreatic cancer cell lines.Thus,we further designed experiments to explore the relationship between elevated RNF139 expression in patients with pancreatic cancer and the survival,prognosis,and clinical pathological parameters of patients,and the role of RNF139 in anti-apoptosis of pancreatic cancer and its regulatory mechanism,with the aim of setting the clinical level.Different levels of research,such as molecular biology level,cellular level and animal experiment level,have explored the clinical significance and molecular mechanism of RNF139 involvement in the anti-apoptosis of pancreatic cancer and the possible feedback regulation of hypoxia in pancreatic ductal adenocarcinoma under hypoxic conditions.To provide a theoretical basis and application basis for the search for new therapeutic targets for pancreatic cancer.method1.To further collect tissue paraffin specimens from patients with pancreatic ductal adenocarcinoma to further study the sample size,perform serial sections,and perform colocalization immunohistochemical staining on HIF-1? and RNF139,and then express the expression amount from the level of tissue protein expression.The correlation between the expression level of RNF139 and various clinicopathological parameters was analyzed comprehensively in combination with the clinicopathological parameters of the patient.2.Western Blot(WB)and Quantitative Real-time Polymerase Chain Reaction(RTqPCR)were used to detect RNF139 and HIF-1? in pancreatic cancer cell lines and normal pancreatic ductal cell lines.The expression levels of mRNA and protein levels were analyzed and their correlations were analyzed;Immunofluorescence technique was used to detect the cellular localization of RNF139 in pancreatic cancer cells.3.RNF139 and HIF-1? overexpression plasmids and shRNA down-expression plasmids of RNF139 and HIF-1? were constructed by molecular cloning methods to establish stable transfected cell lines;stable RNF139 and HIF-II were constructed by WB,RT-qPCR and detection.1? expression level.4.The TUNEL(TdT-mediated dUTP Nick-End Labeling),flow cytometry and other experimental methods were used to detect the apoptosis rate of the stably transfected cell line constructed above.Transient transfection technique was used to transiently transfect RNF139 siRNA into a stably transfected cell line over-expressing HIF-1? and detect changes in its apoptosis rate;transient transfection of RNF139 was transiently transfected in a stably transfected cell line that down-expressed HIF-1?.The plasmid and compare its changes in the proportion of apoptosis.5.The expression levels of VHL and HIF-1? in the RNF139 stable cell line were detected by WB,RT-qPCR,and the like.VHL-overexpressing plasmids were transiently transfected into a stably transfected cell line that was down-expressing RNF139,and VHL-siRNAs were transiently transfected into RNF139-overexpressing stable transfectant cells and the changes in apoptosis ratio were compared.6.A subcutaneous tumor model of pancreatic cancer was constructed using NODSCID mice,and subcutaneous tumor growth curves were recorded.After 3 weeks,the tumor was removed and the effect of RNF139 on the anti-apoptosis of pancreatic cancer was verified in vivo.result1.It was found that RNF139 was higher in pancreatic cancer tissues than matched pancreatic normal tissues.The level of expression,and the high expression of RNF139 was negatively correlated with the overall survival of patients with pancreatic cancer,the difference was statistically significant(P<0.05).The data of TCGA(The Cancer Genome Atlas)database was used as the verification cohort,and the results were consistent.2.We found that overexpression of RNF139 group apoptosis rate was significantly reduced,down-expression of RNF139 group apoptosis rate was significant Increased.Detection of apoptosis-related proteins by WB revealed significant changes in Caspase-12,Caspase-3,and the like.It was found that the tumor volume decreased significantly in the RNF139 group compared with the control group;further studies confirmed that the proportion of apoptosis in the RNF139 group was significantly increased.3.Using WB technology to detect transiently overexpressing and down-regulating RNF139 pancreatic cancer cell lines,it was found that the proteins related to endoplasmic reticulum stress such as eIF2? and CHOP changed significantly,that is,RNF139 could affect the pancreas by affecting the endoplasmic reticulum stress of pancreatic cancer cells.Apoptosis of cancer cells.After pancreatic cancer cells were treated with hypoxia,RNF139 was significantly up-regulated at both mRNA and protein levels.At the tissue and cellular levels,the expression of HIF-1? is consistent with that of RNF139.Database analysis found HIF-1? in the promoter region of RNF139.The specific binding site.Chromatin immunoprecipitation experiments and dual luciferase reporter systems have shown that HIF-1? can bind to the hypoxia response element of the RNF139 promoter region and initiate transcription.The results showed that RNF139 is a direct transcriptional regulation target of HIF-1.5.WB detection revealed that HIF-1? was significantly up-regulated and VHL expression was down-regulated in stable transfected cell lines overexpressing RNF139.In RNF139 down-regulated cell lines,HIF-1? was significantly downregulated and VHL expression was significantly up-regulated.VHL siRNA was transiently transfected in a stably transfected cell line down-expressing RNF139,and no significant change in RNF139 was detected by WB assay,and the expression level of HIF-1? was significantly up-regulated.The above results indicate that RNF139 can affect the degradation of HIF-1? through the VHL pathway.6.RNF139 is one of the important ways that HIF-1 exerts its anti-apoptosis effect in pancreatic cancer.By detecting the over-expression of HIF-1? in a stably transfected cell line and overexpression of HIF-1?,the apoptosis of a cell line that was transiently transfected with RNF139 was found to be significantly lower than that of the former.in conclusion1.RNF139 expression is elevated in pancreatic cancer,and high expression of RNF139 has important clinical significance2.RNF139 participates in anti-apoptosis of pancreatic cancer cells by affecting endoplasmic reticulum stress in pancreatic cancer cells3.HIF-1? can affect the apoptosis of pancreatic cancer by regulating the expression of RNF139;the expression of HIF-1? is related to the therapeutic effect of albumin paclitaxel4.RNF139 can upregulate the expression of HIF-1? by reducing the expression of VHL to form a positive feedback loop.