Circular RNA Circ₀026359 Enhances Cisplatin Resistance In Gastric Cancer Via Targeting MiR-1200/POLD4 Pathway

OBJECTIVE: To investigate the relationship between circ₀026359 and cisplatin resistance in gastric cancer tissues and cells.The roles and regulatory relationships of circ₀026359-related miRNA-1200 and POLD4 were studied.To research the relationship between the role of circ₀026359 in miR-1200/POLD signaling axis and cisplatin resistance in gastric cancer.Methods: In the first part,In this study,55 fresh gastric cancer tissues and 55 normal gastric tissues were collected.RT-q PCR was carried out to examine expression levels of circ RNA.Besides,the expression levels of circ₀026359 in gastric cancer cell lines and normal gastric cell lines were examined.For further study,CDDP-resistant gastric cells were developed.The expression level of circ₀026359 in CDDP gastric cells and normal control group was detected by qRT-PCR.Part 2: Silent circ₀026359 was constructed by transfection of lentiviral si RNA into SGC-7901/CDDP and MKN-45/CDDP cells.The expression level of circ₀026359 in cisplatin-resistant gastric cancer cells after transfection of lentiviral si RNA was detected by qRT-PCR.MTT assay was used to determine the survival rate and IC50 value of cisplatin resistant gastric cancer cells.In this study,MTT assay and IC50 analysis was carried out to examine cell sensitivity to CDDP.After 1000 cells per well were seeded into 6-well plates and treated with 1 μg/ml CDDP for 48 hours,Cell colony formation was examined 10 days later.Caspase-3/7 activity assay and flow cytometric analysis was carried out to examine cell apoptosis.Part 3: The localization and expression of circ₀026359 in cells were analyzed by karyoplasma isolation experiment and qRT-PCR.The related miRNA and target gene m RNAs were found in bioinformatics database;Circ₀026359 and miR-1200 were understood by double luciferase reporter gene assay,RNA pull down and qRT-PCR assay.Expression and biological function of miR-1200 and target gene POLD4.To certify our prediction through qRT-PCR and Western blot assay,the gene expression profile of POLD4 after transfecting miR-200 were detected.The si-NC + miRNA-NC group,si-circ₀02635 +NC-inhibitor group and si-circ₀02635 + miRNA-1200-inhibitor group were set up by transfecting circ RNA（si-circ₀026359）,miRNA inhibitor（miRNA-1200-inhibitor）and blank control group（si-NC,NC-inhibitor）,respectively.The transcription and protein expression levels of m RNA POLD4 in cisplatin-resistant gastric cancer cells SNC-7901/CDDP were measured by qRT-PCR and WB.The viability of gastric cancer cells transfected into SNC-7901/CDDP was measured by MTT assay.Cisplatin resistance in SNC-7901/CDDP was measured by IC50.The proliferation ability of SNC-7901/CDDP was measured by clonal formation assay.The apoptotic effect of SNC-7901/CDDP cells was investigated by Capase3/7 assay other methods.Results: Part I: The circ₀026359 levels in gastric cancer tissue were significantly higher than normal gastric tissues.Meanwhile,compared with normal gastric cell GES-1,the expression levels of circ₀026359 were extremely higher in gastric cancer cells.The expression levels of circ₀026359 were much higher in their CDDP cells compared with their parental SGC-7901 and MKN-45 cells,respectively.gastric cancer patients with high circ₀026359 expression exhibited both lower OS rates（P = 0.0057）and RFS rates（P = 0:0335）.Compared with patients with low circ₀026359 expression.Therefore,high levels of circ₀026359 were associated with poor OS rates and RFS rates in gastric cancer patients.Part II: The gene profiles of circ₀026359 in SGC-7901/CDDP and MKN-45/CDDP transfected with si RNA was decreased by qRT-PCR assay.Silencing circ₀026359 could inhibit CDDP cell proliferation by MTT assay and clone formation assay.IC50 values showed that silencing circ₀026359 could reduce the resistance of CDDP gastric cancer cells.Silencing circ₀026359 could promote CDDP gastric cancer cell apoptosis by multiple cell apoptotic experiments.Part III: Nucleoplasmic separation assay showed that circ₀026359 was located in the cytoplasm.Circ₀026359 could bind and interact with miR-1200 and miR-1200 and POLD4 in a targeted manner by Luciferase Reporter assay and RNA Pull down assay.Silencing circ₀026359 could increase the gene profiles of miR-1200 in CDDDP gastric cancer cells.Mi R-1200 inhibited the transcription and translation levels of POLD4 in CDDP gastric cancer cells.After co-transfecting si-circ₀026359 and NC-inhibitor into SGC-7901/CDDP cells,The expression level of miR-1200 and the target gene POLD4 were radically increased by qRT-PCR and WB assay.The increased gene profiles of miR-1200 and the decreased gene profiles of POLD4 were reversed the results after co-transfecting si-circ₀026359 and miR-1200-inhibitor into SGC-7901/CDDP cells.MTT assay and IC50 values showed that miR-1200-inhibitor could reverse the decreased cell viability and IC50 values induced by si-circ₀026359transfection.Cloning and formation experiments showed that miR-1200-inhibitor could reverse the decreased cell value induced by circ₀026359 silencing by transfection.Through the above experiments,Silencing circ₀026359 could increase the apoptosis rate of CDDP gastric cancer cells,and miR-1200-inhibitor could reverse the apoptosis of CDDP gastric cancer cells induced by si-circ₀026359.CONCLUSIONS: Circ₀026359 Was Overexpressed in Gastric Cancer and Associated with CDDP Resistance and Poor Survival Rates in Gastric Cancer Patients.Silencing circ₀026359 inhibited CDDP resistance in gastric cancer cells.Circ₀026359binds and interacts with miR-1200 and POLD4 targeting.Mi R-1200-inhibitor was able to reverse the decrease of cisplatin resistance induced by circ₀026359 silencing in CDDP gastric cancer cells.