| Gastric cancer is one of the most common gastrointestinal tumors in China[1].Gastric cancer is the fourth most common malignant disease and second leading cause of cancer-related deaths worldwide.Approximately 1,154,000 newly diagnosed gastric cancer cases and 752,500 deaths occur per year.Poor prognosis of gastric cancer patients mainly involves unlimited growth and strong metastatic capacities of gastric cancer cells[2].Mechanism of gastric cancer oncogenesis remains largely unclear in spite of extensive clinical and basic research efforts.Therefore,studies should focus on elucidating molecular mechanisms underlying gastric cancer occurrence and development and exploring novel therapeutic targets for gastric cancer treatments.MicroRNAs(miRNAs)represent a large group of highly conserved and small RNA molecules of spanning 22 nucleotides[3,4].MicroRNAs regulate the expression of target genes by silent compounds in differentiation,proliferation and development of cancer cells[5-7].A large number of miRNAs contribute to gastric cancer tumourigenesis and tumour development by regulating expression of specific target genes[8-11].miR-28,is an important microRVA we studied,It includes two structures,miR-28-5p and miR-28-3p,in which miR-28-3P is closely related to the development of many types of human malignant tumors.PI3K/AKT pathway is related to the development of many kinds of tumors.The pathway can phosphorylate the downstream AKT protein through the activation of PI3K,and then activate many protein molecules of downstream,so as to promote cell proliferation and inhibit apoptosis[20].PTEN is a tumor Suppressor gene that blocks the activation of PI3K and inhibits phosphorylation of AKT to block the uncontrolled proliferation of tumor cells.To clarify the relationship between miR-28-3P and PTEN/PI3K/AKT pathway will provide important direction for the treatment and prevention of gastric cancer.Material and methods1.Tissue samples and cell lines Selection of cell lines:gastric cancer cell lines:MGC-803,MKN-1,SGC-7901,BGC-823,AGS and normal gastric epithelial cells GES-1 cells.These cells were all from the ATCC Cell Bank of the United States(United States).Sources of tissue specimens:Tissue specimens from June 2015 to December 2015 were taken to gastric cancer patients undergoing subtotal gastrectomy at the Zhujiang Hospital of Southern Medical University,a total of 31 pairs of gastric cancer tissues and normal adjacent tissues.None of the patients received radiotherapy and chemotherapy.Specimens were collected with the consent of patients and their families;Q-RT-PCR was used to detect the expression of miR-28 in 31 cases of gastric cancer and its corresponding adjacent tissues.Analysis of miR-28 expression in gastric cancer tissues was conducted by differential analysis of GEO database;2.Both sh-miR-28-3P and the corresponding negative control(ie,NC group)were synthesized by Shanghai Jema Biosciences Co.,Ltd.The cells are then transfected to create a stable cell line;CCK8 cell proliferation experiments,plate cloning experiments and subcutaneous tumorigenesis experiments in nude mice were used to test the effect of miR-28 gene on proliferation,apoptosis,and subcutaneous tumorigenicity of gastric cancer cells.Cell invasion and invasion experiments and scratch experiments were performed to detect the effect of miR-28-3P on migration and invasion of gastric cancer cells.The expression of invasion-associated proteins MMP2 and MMP9 was detected in each group by Western blotting.3.The effects of miR-28-3P gene on cell proliferation,apoptosis and ability of subcutaneous tumor growth were detected in nude mice,and the changes of cell malignant behavior and angiogenesis were verified by immunohistochemistry and HE.4.To investigate the specific target and mechanism of miR-28-3P in gastric cancer cells by fluorescence enzyme report and bioinformatics analysis and functional recovery experiment.Experimental results:1.The differential GO analysis performed by the GEO database yields the following results:In the microarray data analysis of gastric cancer tissues and paracancerous tissues,miR-28-3P was highly expressed in gastric cancer tissues;The results of PCR in patients with gastric cancer tissues and adjacent tissues of gastric cancer showed that miR-28-3P was highly expressed in gastric cancer tissues.The results of western blot and Q-PCR in gastric cancer cell lines and normal cell lines showed that miR-28-3P was highly expressed in gastric cancer cell lines.2.After Sh-miR-28-3P interfered with gastric cancer cells,the results of cell function tests showed that:after inhibiting the expression of miR-28-3P,the proliferation ability of the cells decreased significantly,and the expression of Bcl2,which promotes apoptosis,increased.The expression of Caspase3 increased significantly.After miR-28-3P gene expression was inhibited,the migration and invasion ability of gastric cancer cells and wound healing ability were significantly decreased,and the expression of MMP protein associated with migration and invasion was also significantly decreased.3.The tumorigenesis experiments in nude mice showed that the tumorigenic capacity of gastric cancer cells was significantly decreased after miR-28-3P gene was inhibited,that is,the growth rate was decreased,and the final tumor volume was significantly reduced;HE staining was performed to find out that miR-28-3P was inhibited.In the subcutaneous tumors expressed,the malignant phenotypes of the tissues were significantly decreased,and the expression of the gene CD31+,which was significantly associated with tumor microvessel formation,showed low expression in immunohistochemistry.4.Bioinformatics analysis and luciferase report showed that there is a close relationship between miR-28-3P gene and PTEN gene.Western blotting results show that PTEN is present in cell lines that inhibit miR-28-3P gene expression.Overexpression status;PTEN expression was suppressed by small interfering RNAs in cell lines that inhibited miR-28-3P gene expression.PTEN gene expression levels were restored and sh-miR-28-3P interfered with cell migration induced.The decrease of invasive ability has enhanced the ability of migration and invasion after PTEN gene interference,indicating that PTEN gene may be the downstream target gene of miR-28-3P;We detected the expression of PI3K/AKT pathway in gastric cancer cell lines.We found that PI3K and p-AKT are highly expressed in gastric cancer cells.After inhibiting the expression of miR-28-3P,they are in gastric cancer cell lines.The phenomenon of low expression may be the inhibition of miR-28-3P gene,which can inhibit the expression of PI3K and the phosphorylation of AKT,and thus correlate with the malignant biological behavior of gastric cancer.Conclusion:miR-28-3P is highly expressed in gastric cancer tissues and a variety of gastric cancer cell lines.High expression of miR-28-3P promotes cell migration and invasion and promotes malignant transformation of cells.After inhibiting the high expression of miR-28-3P in gastric cancer cell lines,the migration and invasion ability of the cells was significantly reduced,and the proliferation ability of the cells was also significantly inhibited.This role is through the PTEN/PI3K/AKT pathway. |
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