Curcumin Inhibits Cell Growth And Induces Cell Apoptosis Through Upregulation Of MiR-33b In Gastric Cancer

Background and objectiveGastric cancer is one of the malignant tumors that seriously endangers the health and lives of human beings in the world.China is a high incidence area of gastric cancer with high morbidity and mortality.The clinical treatment of gastric cancer is mainly through surgery and chemotherapy,but patients after treatment are prone to recurrence and metastasis,which lead to a relatively lower 5-year survival rate.Therefore,looking for a new approach to improve the treatment effect of gastric cancer is necessary at this stage.Curcumin was a kind of polyphenols compounds weight of 368.39,and was extracted from turmeric.Curcumin has been found to inhibit the inflammatory response,anti-oxidation,anti-rheumatoid,anti-infective effect,and plays a tumor suppressor role in gastric cancer,pancreatic cancer,non-small cell lung cancer and melanoma and other tumor diseases.With the deepening of the research,the anti-tumor effect of curcumin has received extensive attention,but its mechanism in tumor disease remains to be further explored.In the study of anti-tumor mechanisms,microRNAs play an important role.MiRNAs can downregulate the expression of target genes by interfering with the translation of mRNAs and being involved in many basic signal pathways in vivo,including the expression of important tumor oncogenes or tumor suppressor genes.Curcumin analog EF24 has been found to inhibit the migration of melanoma cells by upregulating the expression of miR-33b.However,whether the two sides have similar effects in gastric cancer is not yet clear.XIAP,a member of the IAP gene family,has access to inhibit caspase activity and cell apoptosis.XIAP was reported to be aberrantly expressed in many types of tumors.Moreover,studies have shown that downregulation of XIAP expression in gastric cancer could induce cell apoptosis.This study was applied to explore the effect of curcumin on the expression of miR-33b and XIAP in gastric cancer cells,and then to analyze the relationship between miR-33b and XIAP,which finally to enrich and improve the relevant regulatory mechanisms of curcumin exerting anti-tumor effect in gastric cancer.Methods1.GES-1,BGC-823 and SGC-7901 cells were cultured in a 5%CO₂ incubator at37?.2.Curcumin was dissolved in dimethylsulfoxide to prepare curcumin solution with the concentration of 0?mol/L,5?mol/L,10?mol/L,15?mol/L,20?mol/L and40?mol/L according to the experimental requirements.3.To study the effect of different concentrations of curcumin on the proliferation of GC cells,the cells were divided into six groups,which are 0?mol/L,5?mol/L,10?mol/L,15?mol/L,20?mol/L and 40?mol/L groups respectively.The CCK-8assay was used to detect the proliferation of cells in each group.4.In order to study the effect of different concentrations of curcumin on apoptosis ofGC cells,curcumin was diluted to 0?mol/L,5?mol/L,15?mol/L and 20?mol/L.The cells were further divided into the above 4 groups,and the apoptosis of cells in each group was detected by flow cytometry.5.The expression levels of mi R-33b and XIAP mRNA in SGC-7901 and BGC-823cells treated with curcumin at concentration of 0?mol/L,5?mol/L,10?mol/L,15?mol/L,20?mol/L and 40?mol/L were detected by qRT-PCR.6.Western blot assay was utilized to assess the expression of XIAP protein in SGC-7901 and BGC-823 cells treated with curcumin at concentration of 0?mol/L,5?mol/L,10?mol/L,15?mol/L,20?mol/L and 40?mol/L.7.Using Pearson correlation analysis to analyze the correlation between mi R-33b and XIAP mRNA expression levels in GC cells.8.Bioinformatic analysis was used to predict the potential target genes of miR-33b.Overlap PCR was applied to amplify wild and mutant XIAP 3'UTR sequence.The target of miR-33b was verified by western blot and a dual-luciferase assay.9.To investigate the effect of upregulated expression of miR-33b on proliferation and apoptosis of GC cells,the liposome complex,miR-33b mimic and miR-33b scramble were transfected into SGC-7901 and BGC-823 cells respectively through Lipofectamine^TM2000.As a result,the cells were divided into Blank group,miR-33b mimic group and miR-33b scramble group.The proliferation rates of cellsin three groups were detected by CCK-8 assay,and the number of apoptotic cellswas detected by flow cytometry.10.The effects of 20?mol/L curcumin treatment and the upregulation of miR-33b and the downregulation of XIAP of GC cells were compared.Western blot was used to detect the expression of XIAP protein.CCK-8 assay and flow cytometry were used to compare the proliferation and apoptosis of gastric cancer cells in each group.11.Restore assay of XIAP was then performed.Recombinant vector of pcDNA3.1-XIAP not containing 3'UTR was successfully conducted and then transfect GC cells alone or together with 20?mol/L curcumin or miR-33b mimic.Western blot was used to detect the expression of XIAP protein.Flow cytometry was used to detect cell apoptsis.So that we can further analysis the anti-tumor mechanisms of curcumin regulating miR-33b in gastric cancer.12.SPSS 21.0 software was used for statistical analysis of the experimental data,?=0.05 was served as a significant control.Results1.According to the results of our CCK-8 assay,both SGC-7901 and BGC-823 cells showed a significantly decreased survival rate along with the increased concentration of curcumin?P<0.05?.2.The results from the flow cytometry assay showed that curcumin could promote the apoptosis of SGC-7901 and BGC-823 cells,and the apoptosis induction was obviously promoted with the increase of dose?P<0.05?.3.The results from qRT-PCR showed that miR-33b expression was significantly upregulated with the increased concentration of curcumin?P<0.05?.On the contrary,XIAP expression from qRT-PCR and western blotting showed the opposite trend in which increased concentration of curcumin is associated with significant downregulation?P<0.05?.Meanwhile,we analysed the data of miR-33b and XIAP mRNA and found that the expression of mi R-33b and XIAP mRNA were inversely related?R²=0.694,P<0.05?.4.Bioinformatics analysis showed that miR-33b and XIAP had complementary binding regions in the 3'UTR region.Western blotting demonstrated an obvious decrease in XIAP expression with the presence of miR-33b mimics compared to the control group in two cells?P<0.05?.The dual-luciferase reporter assay confirmed that XIAP is one of the target genes of miR-33b.5.The results of CCK-8 assay and flow cytometry suggested that the upregulation of miR-33b can inhibit cell proliferation and induce cell apoptosis in SGC-7901 and BGC-823 cells?P<0.05?.6.Results of different treatment to SGC-7901 and BGC-823 cells?20?mol/L curcumin,miR-33b mimics and si-XIAP?demonstrated similar effects on XIAP protein expression,cell proliferation and apoptosis.7.The results of restore assay indicated that XIAP without 3'UTR possibly rescuedthe effect of 20?mol/L curcumin or mi R-33b mimic on the apoptosis of GC cells.ConclusionCurcumin suppresses cell proliferation and enhances cell apoptosis in gastric cancer,and it does so apparently by upregulating miR-33b,which in turn targets XIAP.