Effect Of MiR-138 On Proliferation?invasion And Apoptosis Of Brain Glioma Cells U-87 MG Through Targeting FAK

Background and Purpose The tumor originated from the neural epithelium is called the brain glioma(glioma), which accounts for 40-50% of the brain tumor, and is the most common malignant tumor in the brain. The main features include cell heterogeneity, rapid proliferation, angiogenesis, extensive invasion, hypoxia and necrosis. The surgery, radiotherapy and chemotherapy has been applicated as the primary treatment of brain glioma and has certain effects, but the clinical curative effects and the prognosis are poor, so it is necessary to find a new therapeutic target and treatment method. the abnormal expression of small molecule RNA(micro RNAs, mi RNAs) in brain glioma makes it possible to become a new diagnostic marker of brain glioma. Recently, a large number of related researches have been published. The mi RNA can be divided into carcinogenic mi RNA and tumor suppressor mi RNA in the regulation of tumor development, studies showed that the upregulation or downregulation of mi RNA make effects on glioma cell proliferation, invasion and apoptosis. FAK is a tyrosine kinase, participate in a variety of biological responses, including cell survival, proliferation and migration, and increasing the expression of FAK is closely related to cells invasion of this kind of tumor. Studys found that FAK was highly expressed in glioma. The results of our pre-experiments also showed that abnormally high expression of FAK existed in glioma tissues. Bioinformatics software were used to indicate that there were specific binding sites between mi R-138 and FAK 3'UTR, FAK may be one of the targets of mi R-138. Next we carried out a series of experiments aimed to further analyze the expression level of mi R-138 and FAK in brain glioma tissues and normal tissues, to explore the effects of abnormal upregulation of mi R-138 expression on the proliferation, invasion and apoptosis of glioma cells, and to verify the target and regulation mechanism of mi R-138.Methods 1. 20 cases of clinical samples which had been identified as glioma were collected from the First Affiliated Hospital of Zhengzhou University and Luoyang Central Hospital Affiliated to Zhengzhou University. The samples were obtained from surgical treatment from August 2014 to July 2015. 6 cases of normal brain tissues were collected from patients with brain trauma surgery in the First Affiliated Hospital of Zhengzhou University. 2. Western blot assay was used to detect the expression of FAK protein in tissue samples. The expression levels of mi R-138 and FAK m RNA were detected by q RT-PCR 3. Brain glioma U-87 MG cells were divided into three groups according to different kinds of transfection. Group a was mi R-138 group, cells were transfected with liposome complex and mi R-138 agomir; group b was negative control, cells were transfected with liposome complex and mi R-138 agomir negative control; group c was Blank control, cells were transfected with only liposome complex. After transfection, the cells were cultured at 37?,the CO2 concentration was 5%. After 24 h, the expression of FAK protein were examed by Western blot assay, the expression of mi R-138 and FAK m RNA were detected by q RT-PCR, as a result, we can verify the effect of transfection. 4. CCK-8 experiment was used to detect the proliferation of cells in groups a?b?c. 5. Transwell experiment was used to detect the invasion ability of cells in groups a?b?c. 6. Flow cytometry was used to detect the apoptosis of cells in groups a?b?c. 7. After establishing the nude mice transplanted tumor model, the proliferation of mices in three groups was detected by small animal imaging technology in vivo. 8. Bioinformatics analysis was used to explore whether there is a complementary Seedregion region between mi R-138 and FAK. Vectors of reportor gene of wild and mutant FAK 3 'UTR region was constructed. Then were co-transfected into U-87 MG cells with mi R-138 agonist(agomir) or mi R-138 agomir negative control, respectively. Sual-luciferase assay was conducted to verify if FAK is one of the targets of mi R-138. 9. Compared the effect of FAK downregulation and mi R-138 upregulation acting on U-87 MG cells. FAK-si RNAs and mi R-138 agomir were transfected into U-87 MG cells, then compare the effects on proliferation, invasion and apoptosis of U-87 MG cells. Western blot assay was used to detect the expression of relevant g Western blot assay was used to detect the expression of FAK protein in tissue samples. The expression levels of mi R-138 and FAK m RNA were detected by q RT-PCRResults 1. The expression level of mi R-138 in glioma tissues was significantly lower than that in normal brain tissue(P<0.05), the FAK m RNA and FAK protein expression in glioma tissues was significantly higher than that in normal brain tissues(P<0.05). 2. After U-87 MG cells transfection, compared with the Blank group and NC group, the expression of mi R-138 was significantly higher(P<0.05), while the expression of FAK m RNA was significantly lower(P<0.05); Western Blotting results showed compared with Blank group and NC group, the expression of FAK protein in mi R-138 group was significantly lower(P<0.05), demonstrating that the expression of mi R-138 upregulated and the expression level of FAK downregulated in U-87 MG cells transfected with mi R-138 agomir. 3. CCK-8 assay showed that compared to the Blank group and NC group, the OD value at 450 nm decreased after 24 h, 48 h and 72 h transfection, demonstrating that the proliferationrate of the U-87 MG cells have significant decreased(P<0.05). 4. Small animal imaging was used to detect xenograft proliferation ability in vivo, the results showed that, fluorescence signal of mi R-138 group were significantly decreased compared with Blank group and NC group from the second week(P<0.05), and with the extension of time, difference of the fluorescence signal in mi R-138 group and Blank group or NC group were more pronounced(P<0.05). 5. Transwell assay was used to detect the invasion ability of U-87 MG cells in the three groups. The results showed that the number of cells invading the matrigel notably decreased significantly in mi R-138 group cells compared with Blank group and NC group(P<0.05). 6. The results of flow cytometry showed that the apoptotic rate in cells transfected with mi R-138 agomir and liposome complex was significantly higher than that of Blank group and NC group(P<0.05), demonstrating that the upregulation of mi R-138 could induce cell apoptosis in U-87 MG cells. 7. Bioinformatics information detection showed that the FAK 3'UTR may be a direct downstream target of mi R-138. Wild and mutant type of FAK 3 'UTR dual luciferase reporter vectors were successfully constructed. Double luciferase report experimental results showed that: the luciferase activity was significantly lower in cells co-transfected with mi R-138 and wild type of FAK 3 'UTR dual luciferase reporter vector than the other 3 control group, indicating that FAK is one of the targets of mi R-138. 8. Compared the effect of FAK downregulation and mi R-138 upregulation acting on glioma cells and detected the expression of mi R-138 and FAK. The results showed similar effects of FAK downregulation and mi R-138 upregulation, indicating the inhibitive effect of mi R-138 on glioma was at least partly due to acting on FAK.Conclusions 1. The expression level of mi R-138 downgraduates, while the expression level of FAK upgraduates in glioma tissues. 2. Upregulation of mi R-138 level can inhibit the proliferation and invasion of U-87 MG cells, and can promote its apoptosis. 3. mi R-138 conversely regulates expression level of FAK through acting on 3'UTR region thereby exerting its biological function.