The Sensitizing Effects And Mechanisms Of RNA Interference Targeting CircPVT1 On 5-fluorouracil Chemosensitivity In Gastric Cancer

Gastric cancer(GC)is a kind of malignant tumor that develops from the lining of the stomach.GC is one of the common digestive tract tumors threatening human health.Since GC clinically has no characteristic manifestations in its early stages,most GC patients were diagnosed at advanced stage,which is one of the main reasons for its high mortality and poor prognosis.At present,palliative chemotherapy is widely used for the treatment of GC.5-FU is the most commonly used in GC chemotherapy.However,most GC cells are insensitive to 5-FU,which leads to the failure of chemotherapy.Thus,seeking a therapeutic target for overcoming the chemoresistance is of great significance to improve the efficancy of chemotherapy for the patients with GC.Circular RNAs(circRNAs)are considered as a novel type of ncRNAs that are hallmarked by covalently closed continuous loops.Unlike linear RNA,circRNAs are generated by back-splicing without 3'-end and 5'-end.Owing to its unique closed loop structure,circRNAs are resistant to exonuclease-mediated degradation.CircRNAs have been shown to serve as a key regulator in the occurrence and development of many kinds of tumors,including GC.Notably,emerging evidence suggests that circRNAs play an important role in the development of chemoresistance.circPVT1 is a kind of circRNAs,multiple studies have shown that the abnormal expression of circPVT1 is closely related to the occurrence and development of cancer.However,the functional role of circPVT1 in 5-FU resistance of GC has not been adequately studied.Objective: We silence circularPVT1(circPVT1)in 5-fluorouracil(5-FU)-resistant gastric cancer cells to explore its functional role in 5-FU resistance of gastric cancer cells.Its molecular mechanism was also investigated in5-FU-resistant gastric cancer cells,which provide an experimental basis for circPVT1 as a novel therapeutic target of gastric cancer chemoresistance.Methods: The gastric cancer tissues were collected from patients showing resistance or sensitivity to 5-FU.Real-time polymerase chain reaction(RT-PCR)was performed to determine the expression of circPVT1.Meanwhile,the expression of circPVT1 was evaluated in gastric cancer cells(BGC823)and 5-FU-resistant gastric cancer cells(BGC823/5-FU).We silence circPVT1 in BGC823/5-FU cells and measured cell viability using cell count kit-8(CCK-8)assay.BGC823/5-FU cells were treated with different 5-FUconcentration(0,1,2,4,8,16 and 32?M)and then the cell activity was analyzed by CCK-8 to select the dose for the subsequent test.BGC823/5-FU cells were randomly assigned into four groups: control group,5-FU group(16?M),5-FU(16 ?M)+ si-circPVT1 group and 5-FU(16 ?M)+ si-negative control(NC)group.CCK-8 assay,colony formation assay,TUNEL apoptosis assay kit and flow cytometry were applied to assess the effects of silencing circPVT1 on cell viability,proliferation and apoptosis in BGC823/5-FU cells treated with 5-FU.RT-PCR and western blot was performed to determine the protein levels of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax)and caspase-3.Results: Compared with gastric cancer patients showing sensitivity to 5-FU,the expression of circPVT1 was much higher in gastric cancer tissues of patients showing resistance to 5-FU.The expression of circPVT1 was higher in BGC823/5-FU cells as compared with that in BGC823 cells.3.The cell activity of BGC823/5-FU decreased with the increase of 5-FU concentration,and the cell activity decreased significantly when the dose was 16?M,which was used as the dose concentration for the subsequent experiment.Both si-circPVT1-1 and si-circPVT1-2 can reduce the expression level of circPVT1 significantly,and si-circpvt1-1 has a higher silencing efficiency,which can be used as a sequence for the silencing of circPVT1.The cell activity of BGC823/5-FU decreased with the increase of 5-FU concentration,and the cell activity decreased significantly when the 5-FU concentration was 16?M.Silencing circPVT1 enhanced 5-FU-induced cell viability inhibition in BGC823/5-FU cells.Furthermore,silencing circPVT1 promoted the apoptosis of BGC823/5-FU cells induced by 5-FU.Besides,silencing of circPVT1 enhanced the cytotoxic effect of 5-FU BGC823/5-FU cells through downregulation of Bcl-2 and upregulation of Bax and caspase-3.Conclusion: Silencing of circPVT1 enhanced the cytotoxic effect of 5-FU in BGC823/5-FU cells through downregulation of Bcl-2 and upregulation of Bax and caspase-3.