| ObjectiveGlioma is highly malignant and aggressive and is the most common primary malignant brain tumor. Although it has been investigated for decades, but the prognosis remains poor because of rapid proliferation, its aggressive potential, and its resistance to chemotherapy or radiotherapy. The average survival of glioblastoma(GBM) is 15 to 18 months. A growing body of researches suggests that in malignant tumors, micro RNAs(mi RNAs) abnormal expression is closely related to tumor progression, recurrence and prognosis. Mi RNAs are small non-coding RNAs that regulate gene transcription and translation via up-regulating or down-regulating the levels of mi RNAs. It is reported that mi R-199a-3p has a significant difference in many malignant tumors and affect tumor proliferation, apoptosis, invasion and other biological function by targeting the mammalian target of rapamycin(m TOR). However, the study of mi R-199a-3p in glioma is limited. Thus, this study was conducted to explore the expression in glioma by q PCR. Subsequently, molecular functions of mi R-199a-3p in glioma cell lines were studied.MethodsWe detected the expression of mi R-199a-3p in 61 glioma samples which were collected from the First Affiliated Hospital of Soochow University by quantitative PCR(q PCR). Then, we transfected the U87 and U251 cell lines with mi R-199a-3p. Cellular proliferation, invasion, and apoptosis were assessed to explain the function of mi R-199a-3p. The expression of protein related to m TOR signaling pathway was analyzed by Western blot.Results1. Expression of mi R-199a-3p, m TOR, and its downstream effector molecules in glioma samplesTo gain further insight into the expression of mi R-199a-3p in glioma samples and non-cancerous brain tissues, q PCR was conducted in 6 non-cancerous brain tissues and 61 gliomas samples. Results showed that the expression of mi R-199a-3p was lower in glioma samples than non-cancerous brain tissues(P < 0.01), and there was no significant difference between low-grade glioma(gradeⅡ) and high-grade(grade Ⅲ, grade Ⅳ) glioma(P﹥0.05). The expression of m TOR in glioma samples was higher combined with non-cancerous brain tissues and increased with the increasing degree of malignancy in glioma(low-grade glioma vs. high-grade glioma, P < 0.01). In addition, P70S6 k expressions in glioma samples were higher than non-cancerous brain tissues(P < 0.01) and had no significant association in low-grade and high-grade glioma samples(P﹥0.05).2. Up-regulation of mi R-199a-3p suppress proliferation in glioma cellsWe conducted cellular proliferation assays in U87 and U251 cells to assess whether the up-regulation of mi R-199a-3p has a suppressive effect on the growth of glioma cells. As a result, we found that cell proliferation was distinctly decreased in glioma cells after transfection with mi R-199a-3p(P < 0.01). To gain further insight into cell cycle modulation, flow cytometry analysis was performed in U87 and U251 cells. Results revealed that the G1 phase(P < 0.01) was increased, and the S phase was decreased(P < 0.05) in cells with the over-expression of mi R-199a-3p.3. Mi R-199a-3p had no effect on invasion and apoptosis capabilityTo determine whether the suppression of cellular proliferation was due to decreased invasion capability or increased apoptosis by the over-expression of mi R-199a-3p, we carried out a cell invasion assay and apoptosis assay in vitro. At the same time, we detected the expression of matrix metallopeptidase 2(MMP2)and caspase-3, which were related to invasion and apoptosis capability by Western blot analysis. Results indicated that no significant difference was found between glioma cells’ overexpression of mi R-199a-3p and those with negative control oligonucleotide(P﹥0.05).4. Up-regulation of mi R-199a-3p suppresses m TOR and its downstream geneThe expression of m TOR in U87 and U51 cell lines obviously decreased after transfection with mi R-199a-3p(P < 0.01). In addition, to estimate the effect of mi R-199a-3p on m TOR, we also found m TOR protein levels and the phosphorylation of AKT, P70S6 K, and 4E-BP1 decreased in mi R-199a-3p groups by Western blot analysis and immunofluorescence staining(P < 0.05). We further assessed the biological significance of mi R-199a-3p in the regulation of m TOR expression in glioma cells. Cells treated with NVP-BEZ235 to inhibit m TOR activity and further transfected with mi R-199a-3p did not have a significant difference in inhibiting cell proliferation(P﹥0.05).ConclusionResults indicated that the expression of mi R-199a-3p in glioma samples and glioma cell lines(U87,U251)was lower comparing to non-cancerous brain tissues. Cellular proliferation was inhibited via regulating the m TOR signaling pathway by elevating levels of mi R-199a-3p in glioma cells. Mi R-199a-3p in glioma cell lines has effects similar to the tumor suppressor gene on cellular proliferation via the m TOR signaling pathway. |
PDF Full Text Request