| Gastric cancer is a kind of common malignant tumor.Statistically,it ranks the third among global malignant tumors on incidence rate,and it has a very high mortality rate.In order to investigate whether Chinese herbal monomer gambogic acid was involved in the occurrence and development of gastric cancer by means of adjusting miRNA,we carried out qRT-PCR analysis against gastric cancer cell line treated with gambogic acid,the result of which showed that the expression level of miR-590-5p was increased significantly in treatment group.Then we further made miRNA microarray inspection against the acquired gastric cancer tissues and the tissues adjacent to cancer.We were surprised to find that the expression level of-miR-590-5p was significantly lower than that of control group in tumor tissues,and speculated that miR-590-5p might be potential cancer suppressor gene.miR-590-5p made a targeting regulation for the epithelium-mesenchyme transition of gastric cancer involved in by TGF-β2 by means ofPI3K-AKT-mTOR signal pathway,which was never reported at home and abroad.We further adopted both prediction software,microRNA.org and Targetscan.org,to predict possible target genes for miR-590-5p.In the two prediction software,we simultaneously found that the gene TGF-β2 was possible downstream target point.At present,there is no report on miR-590-5p involved in EMT process for gastric cancer.Our research team speculated that,gambogic acid might cause the changes of miR-590-5p expression level,and then it regulated TGF-β2 and Akt/mTOR signal paths,thus it inhibited the transition process of gastric cancer cell epithelium to mesenchyme.Maybe taken as an important molecular biomarker,miR-590-5p,whose signal transduction mechanism should be determined clearly,combining with a variety of inhibitors or seeking new drugs that act on multiple signal paths and multiple target points,is very important for targeted cancer therapy.This study also provides some theoretical references for the use of Chinese herbal monomer in cancer therapy.Objective:To analyze and study the effect and mechanism,that is,gambogic acid may cause the changes of miR-590-5p expression level,and then it regulates TGF-β2 and Akt/mTOR signal paths,thus it inhibits the transition process of gastric cancer cell epithelium to mesenchyme,in order to clarify the anticancer efficacy of gambogic acid in gastric cancer treatment.The signal transduction mechanism should be determined clearly,and a variety of inhibitors should be combined with or new drugs that act on multiple signal paths and multiple target points should be sought,which will be very important for the targeted cancer therapy.This study will also provide some theoretical references for the use of Chinese herbal monomer in cancer therapy.Methods:1.The effect of gambogic acid on invasion and migration abilities of gastric cancer cell as well as miR-590-5p expression level1.1 Transwell invasion and migration experiments detect the effect of gambogic acid on biological characteristics of gastric cancer cells.1.2 The effect of gambogic acid on expression level of miR-590-5p in gastric cancer cells.2.The screening for gastric cancer miRNA and the detection for gastric cancer miRNA expression in tissues and cells2.1 Construct the expression profile for differential gastric cancer miRNA2.2 The conditions of miR-590-5p expression were verified based on clinical level.2.3 The conditions of miR-590-5p expression were verified based on cellular level.3.The effect of miR-590-5p on gastric cancer cell proliferation,migration and invasion as well as related EMT genes3.1 The conditions of cell proliferation were detected by means of Methyl Thiazolyl Tetrazolium(MTT).3.2 Cell migration capacity was detected by means of Transwell migration experiment after transfecting miR-590-5p mimics and inhibitor.3.3 Cell invasion capacity was detected by means of Transwell invasion experiment after transfecting miR-590-5p mimics and inhibitor.3.4 EMT marker gene,E-cadherin,N-cadherin,Vimentin mRNA and protein expression changes were detected by means of real-time PCR and western blotting.4.Prediction and verification for miR-590-5p target gene4.1 The databases of Targetscan and microRNA.org predict that the downstream target gene of miR-590-5p is TGF-β2.4.2 Direct targeting effects of miR-590-5p and TGF-β2 were detected by luciferase reporter genes.4.3 The changes of TGF-β2 expression of two gastric cancer cell lines were detected by real-time PCR and western blotting after transfecting miR-590-5p mimics and inhibitor.5.A study on the mechanism of miR-590-5p regulating and controlling gastric cell EMT5.1 The changes of TGF-β2 protein expression were detected after respectively transfecting or co-transfecting miR-590-5pNC,mimics and PMIR-GLOTM-TGF-β2(3’-UTR mutated)or empty PMIR-GLOTM plasmid into BGC-823 and SGC-7901 cell.5.2Cell migration capability was detected by Transwell migration experiment after respectively transfecting or co-transfecting miR-590-5pNC,mimics,PMIR-GLOTM-TGF-β2(3’-UTR mutated)or empty PMIR-GLOTM plasmid into BGC-823 and SGC-7901 cell.5.3Cell invasion capability was detected by Transwell migration experiment after respectively transfecting or co-transfecting miR-590-5pNC,mimics,PMIR-GLOTM-TGF-β2(3’-UTR mutated)or empty PMIR-GLOTM plasmid into BGC-823 and SGC-7901 cell.5.4The changes of PI3K/AKT/mTOR pathway proteins were detected after respectively transfecting or co-transfecting miR-590-5pNC,mimics and inhibitor into BGC-823 and SGC-7901 cell.5.5 The changes of PI3K/AKT/mTOR pathway proteins were detected after respectively transfecting or co-transfecting miR-590-5pNC,mimics,PMIR-GLOTM-TGF-β2(3’-UTR mutated)or empty PMIR-GLOTM plasmid into BGC-823 and SGC-7901 cell.5.6 The changes of E-cadherin,N-cadheiin and Vimentin expression of EMT marker gene were detected by means of Western blotting after respectively transfecting or co-transfecting miR-590-5pNC,mimics,PMIR-GLOTM-TGF-β2(3’-UTR mutated)or empty PMIR-GLOTM plasmid into BGC-823 and SGC-7901 cell.6.The effect of miR-590-5p on gastric cancer cell tumorigenicity was verified by in vivo experiment.6.1 Gastric cancer cell lines with stable miR-590-5p expression were constructed by means of lentiviral infection method.6.2 Weight and tumor forming conditions for nude mice were observed by implanting gastric cancer cell lines with stable miR-590-5p expression in the subcutaneous part of nude mice.Results:1.The expression level of miR-590-5p can be altered by gambogic acid.2.miR-590-5p was differentially expressed in normal gastric mucosa cell and gastric cancer cell line, that is,lowly expressed in gastric cancer cell and highly expressed in normal gastric mucosa cell.3.miR-590-5p mimics inhibited gastric cancer cell proliferation,and weakened gastric cancer cell migration and invasion.miR-590-5p(inhibitor)promoted gastric cancer cell proliferation,and enhanced gastric cancer cell migration and invasion.4.miR-590-5p was involved in the invasion and metastasis of gastric cancer by targeting TGF-P2.5.TGF-β2 was downstream target gene of miR-590-5p.Therefore,after miR-590-5p mimic was added,the expression quantity of TGF-β2 was lowered,cell migration and invasion were dropped,and PI3K/AKT/mTOR pathway proteins expression was reduced.The expression quantity of TGF-β2 was not affected by miR-590-5p-NC.The expression quantity of TGF-β2 was constant after miR-590-5p-NCH+PMIR-GLOTM.The expression quantity of TGF-(32 was reduced after miR-590-5p +PMIR-GLOTM.The expression quantity of TGF-P2 was recovered after miR-590-5p+PMIR-GLOTM-TGF-β2.6.The tumorigenicity in vivo of the BGC-823 cells and SGC-7901 cells with high expression of miR-590-5p declined obviously,the tumor volume was significantly lower than that of the NC group,Compared with NC group,there was significant difference.Conclusion:1.The expression level of miR-590-5p in gastric cancer can be enhanced by gambogic acid.2.miR-590-5p has low expression in gastric cancer and is potential cancer suppressor gene.3.miR-590-5p regulates and controls TGF-β2 of gastric cancer cells to regulate and control EMT progress.4.Gambogic acid causes the changes of miR-590-5p expression level,and then it regulates TGF-β2 and Akt/mTOR signal pathway,thus it inhibits the epithelium-mesenchyme transition process of gastric cancer cell. |
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