URI Promotes Gastric Cancer Cell Resistance To Adriamycin And Motility Ability

Background:Gastric cancer(GC) is one of the most common cancer in the world,and the second leading cause of cancer-related death. Over 70% of new cases and deaths occur in developing countries, especially in China.Surgical resection was often taken to cure early stage of gastric cancer,but many GC patients were diagnosed at locally advanced or metastatic stage, then missed the best time to surgical treatment. GC patients with advanced stage have a poor prognosis, with a five-year of survival rate less than 15%. In order to improve the quality of life and prolong survival,combined chemotherapy remains the main method for treating locally advanced gastric cancer(AGC) and metastatic GC. Due to drug resistance, recurrence and metastasis, the prognosis of gastric cancer remains poor. Therefore, effective and feasible therapy was needed to solve metastasis and chemotherapy drug resistance. Unconventional prefoldin RPB5 interactor(URI), a RNA polymerase II Subunit5-Interacting protein, is known to participate in the regulation of nutrient-sensitive m TOR-dependent transcription programs. Some studies have recently demonstrated that URI functions as an oncoprotein.However, whether and how URI plays a role in gastric oncogenesis has not been elucidated. PI3K/Akt/m TOR pathway is overexpressed in gastric cancer, with potential prognostic significance. Here, we mainly investigated the effects of URI on gastric cancer cell lines MGC-803 and HGC-27, with a focus on drug resistance to adriamycin, migratory ability and the related mechanism.Objective:This study aims to explore the effects of URI on biological characteristics of gastric cancer cell lines MGC-803 and HGC-27,with a focus on its influence on the resistance to adriamycin and the migratory ability of gastric cancer cells.Materials and Methods:(1)Cell culture Human gastric carcinoma MGC-803 cells were grown in Dulbecco’s Modified Eagle Medium(DMEM, Corning, USA) medium and HGC-27 cells were grown in RPMI-1640(Corning, USA) medium. The culture media used were all supplemented with 10% fetal bovine serum(Gibco,New Zealand) and 1% penicillin-streptomycin mixture(Invitrogen, CA,USA). Exponentially growing cell cultures were maintained in a humidified atmosphere of 5% CO2 at 37℃.(2)Overexpression or knockdown of URI Western blotting was performed to detect URI expression in gastric cancer SGC-7901, BGC-823, HGC-27 and MGC-803 cells. MGC-803 and HGC-27 cells were chosen for the next experiment. To overexpress URI, URI expression plasmid p CMV6-URI were transiently transfected into gastric cancer cells using the Lipofectamine 2000 transfection reagent and opti-MEM. Meanwhile, to knockdown URI, three candidate URI si RNA sequences(si RNA-A,-B, and-C) were used for transfection.The real-time PCR and western blotting were taken to measure URI expression in the level of m RNA and protein.(3)Cell proliferation assay A CCK-8 assay was performed to detect cell proliferation after URI overexpression and knockdown in cells with or without adriamycin treatment.(4)Apoptosis related proteins expression and apoptosis analysis Western blotting was used to measure apoptosis related protein after URI overexpression or knockdown, and apoptotic cells were detected via flow cytometer after knockdown URI under adriamycin treatment.(5)Cell migration assay Transwell assay was used to monitor the migratory ability of cells after transfection, EMT related protein Vimentin and Snail expression were detected by western blotting.Results:(1)URI expression of gastric cancer cells Western blot results showed that URI expressed in all four gastric cancer cell lines. We then chose the poorly differentiated MGC-803 and the undifferentiated HGC-27 cells for further experiments. The expression of URI was significantly increased in cells transfected with p CMV6-URI. URI expression was markedly decreased in cells transfected with URI si RNA, the si RNA-A sequence showed the strongest interfering effect for URI expression.(2)URI promotes gastric cancer cell proliferation Overexpression of URI by transient transfection of p CMV6-URI remarkably promoted cell proliferation in two cell lines. Accordingly,knockdown of URI significantly decreased the cell proliferation.(3)URI antagonizes the cell proliferation inhibitory of doxorubicin and inhibits adriamycin-induced apoptosis Cell proliferation was evaluated by CCK-8 cell viability assay.The mean IC50 of adriamycin was significantly higher in two p CMV6-URI transfected cell lines and lower in two URI knockdown cell lines. After adriamycin treatment, URI overexpression resulted in reduction of the relative ratio of Bax/Bcl-2, Cleaved PARP-1, Cleaved Caspase-3expression. On the contrary, URI knockdown increased the level of relative ratio of Bax/Bcl-2, cleaved PARP-1, cleaved Caspase-3 level in two cell lines. After adriamycin treatment, the ratio of early apoptotic cell(Annexin V+/PI-) was significantly increased in two cell lines of URI knockdown compared with the control groups.(4)URI enhanced cell migratory ability and increased Vimentin and Snail expression Cell migratory ability was enhanced in p CMV6-URI transfected cells and weakened in cells transfected with URI si RNA in two gastric cancer cell lines. The expression of migration-related EMT markers Vimentin and Snail significantly increased in p CMV6-URI transfected cells, while URI knockdown caused significantly decreased expression of Vimentin and Snail in two gastric cancer cell lines.Conclusions:(1)URI can promote cell proliferation in gastric cancer cells(2)URI enhances drug resistance by promoting cell proliferation and inhibiting apoptosis in a caspase dependent manner.( 3) URI promotes cell migratory ability perhaps through up-regulation EMT related protein Vimentin and Snail expression.