Mechanism Of Proliferation And Epithelial-to-mesenchymal Transition By CISD2 In Pancreatic Cells

Pancreatic cancer is highly lethal and aggressive which has an very poor prognosis.Currently,the treatment options for pancreatic cancer are limited that mainly include surgical removal,adjuvent chemotherapy and radiation.However,most patients with advanced pancreatic cancer are not eligible for operation.In the meanwhile,chemotherapy is not very effective in advanced pancreatic cancer due to drug resistance which causes low overall 5 years survival rate < 5%.Thus,it is imperative to develop new strategy and identify new molecular target to treat pancreatic cancer.The CDGSH iron sulfur domain2(CISD2)is primarily located in the mitochondrial outer membrane and participates in regulation of mitochondrial integrity.However,recent studies have revealed the involvement of CISD2 in tumorigenesis that CISD2 promotes tumor growth in human breast cancer.In addition,high level of CISD2 has been found to be related with metastasis and poor prognosis in early stage cervical cancer.Moreover,CISD2 was identified to promote proliferation through AKT pathway in gastric cancer cells which could be an independent prognostic factor in human gastric cancer.In pancreatic cancer,epithelial-to-mesenchymal transition(EMT)contributes to the metastasis and chemotherapy resistance.Epithelial-to-mesenchymal transition(EMT)is characterized by losing epithelial phenotypes and gaining mesenchymal characteristics in epithelial cells,as evidenced by the decrease of E-cadherin,γ-cadherinand increase of vimentin,N-cadherin.Patients with high EMT have worse prognosis and more remote metastasis.Wnt/β-catenin pathway has been found to be one of the pathways that are involved in regulation of EMT.Wnt/β-catenin pathway regulates E-cadherin via promoting the expression of repressors of this adhesion molecule including transcriptional factors SNAI1 and SNAI2,and ZEB1,which are demonstrated to play important roles in regulating EMT.Recently,some studieshave revealed that the Wnt/β-catenin/EMT pathway is involved in the carcinogenesis.Moreover,CISD2 level is highly related with the clinical characteristics as well as prognosis of pancreatic cancer.In our study,40 fresh pairs of pancreatic cancer tissues and their adjacent non-pancreatic cancer tissues were collected after surgery between January 1st,2015 and January 1st,2016 at China-Japan Union Hospital of Jilin University.120 paraffin-embedded pancreatic cancer samples were collected in China-Japan Union Hospital of Jilin University from March 1st,2008 to January 1st,2014.All these 120 patients have been followed up for 25 months after surgery.In order to detect the m RNA and protein level of CISD2 insamples,Western Blot,Real-time Quantitative PCR,Immunohistochemistry staining were used.The human pancreatic cancer cell lines PANC-1,SW1990,Capan-1,Capan-2 and Hs766 T were cultured and tested as well as normal pancreatic cell line h TERT-HPNE was used as control.Lentivirus carrying sh CISD2 or Lv-oe CISD2 to silence or overexpress CISD2 in PANC-1 and SW1990 cells.The viability of cells was determined by MTT assay and TUNEL assay was used to detect apoptosis.Flow cytometrywas used to analyze the ratio of apoptotic cells.In our animal model,PANC-1 cells were transfectedwith Lv-scramble or Lv-sh CISD2 or Lv-oe CISD2 and then injected subcutaneously into the right groin of nude mice.On thefortieth-day post-inoculation,the tumors were collected and subjected to analysis.The weight of tumors were scaled and the volumes were calculated(length x width2 x π/6).Analysis between categorical variables were performed with χ2 test.Multivariate analysis was based on the Cox proportional hazards regression model.Survival analysis was conducted with Kaplan-Meier plot and log-rank tests.Data among or between groups were analyzed by one-way ANOVA or Student’s t-test.P<0.05 was considered to be statistically significant.All the analyse were done using SPSS 18.0.All experiments were repeated in triplicate and the results were expressed as mean±SD.Our results showed that both the m RNA and protein level of CISD2 were increased in pancreatic cancer samples and cell lines.High CISD2 expression was significantly positive correlationwith advanced clinical stage(p<0.05),advanced T-stage(p<0.05),positive vascular invasion(p<0.05),positive distant metastasis(p<0.05)and larger tumor size(p<0.05).High CISD2 expression was negative correlation with the overall survival time of patients with pancreatic cancers and was an independent prognostic factor(p=0.02).Also,our results demonstrated distant metastasis(p<0.001),clinical stage(p=0.019)and vascular invasion(p=0.021),were independent prognostic factors for pancreatic cancer.In the other aspect,we tested the molecular mechanism of the regulation of proliferation by CISD2,Wnt/β-catenin pathway.Our results demonstrated that High CISD2 expression was significantly positive correlationwith advanced clinical stage and was negative correlation with the overall survival time of patients with pancreatic cancers.In conclusion,our study demonstrated that CISD2 regulated the EMT through Wnt/β-catenin pathway.CISD2 overexpression groups promoting epithelial-to-mesenchymalto promote proliferation and migration.CISD2 could be an independent prognostic factor for pancreatic cancer,hinting a new promising molecular target for pancreatic cancer therapy.