An Experimental Study Of SiRNA Targeting P75^NTRto Induce Apoptosis Of U251Glioma Cells

[Objective] Glioblastoma is the most common primary brain tumor. Despite advances in imaging techniques and multimodal treatment options, the overall prognosis of patients with GBM remains grim. The median duration of patient survival is estimated to be between12and18weeks with maximal treatment, but those without any intervention die soon after diagnosis. Glioblastoma has an unfavorable prognosis mainly due to its high propensity for tumor recurrence. It has been suggested that GBM recurrence is inevitable after amedian survival time of32to36weeks. P75neurotrophin receptor (P75NTR) is a newly found gene which related to accelerating the proliferation of the glioblastoma and restraining the apoptosis. This research adopts RNA interfere (RNAi) technology to silence the expression of P75NTR in the glioblastoma strain U251, and observes the effect of accelerating the apoptosis of the glioblastoma, so as to provide new alternative method for the gene theraphy of the glioblastoma.[Methods]①U251glioma cell is cultured in DMEM culture medium with10%fetal calf serum, and it is generated for one time every3days. Adopt U251glioma cell with sound growth status, and change the culture medium for standby application24h before experiment. The experiment is divided into5groups. Control group:commonly cultured U251glioma cell; no-load group:transfect U251glioma cell of no-load plasmid; siRNA1-3group:transfect U251glioma cell of Sequence (1)(3) respectively.②Detect mRNA expression of P75NTR by means of RT-PCR to determine the gene silencing effect: adopt Primer Premier5.0Software to design relevant primers. The primer sequence of P75is:upstream "5’-CGTATTCCGACGAGGCCAACC-3’", downstream "5’-CCACAAGGCCGACAACCACAGG-3’", and product size "286bp"; the primer sequence of GAPDH is:upstream "5’-CGGAGTCAACGGATTTGGTAT-3’", downstream "5’-AGCCTTCTCCATGGTGGTGAAGA C-3’", and product size "598bp".③Draw cell growth curve: take U251cell transfected by the stable gene which gains the best gene silencing effect as RNAi group and put it in the cell incubator for culture. Count the cells at the24th,48th,72nd,96th,120th, and144th during the culture, and draw cell growth curve.④In Situ cell apoptosis experiment is to detect the apoptosis status of the cells.⑤Western blot is to detect the protein expression of P75NTR, B apoptosis gene Caspase-3and apoptosis inhibitor gene Bcl-2on the third day since from the stable transfection.[Results]①RT-PCR detection results: U251glioma cell of both no-load group and control group presents high expression of P75NTR; mRNA expression level of U251cell P75NTR after transfecting P75NTR siRNA plasmid decreases remarkably.②Cell growth curve: the cell proliferation of the control group and no-load group is vigorous, and the cell proliferation speed of RNAi is obviously lower than that of the control group and no-load group in several detection time points, namely the cell reproductive capacity decreases.③The detection results of TUNEL apoptosis:it turns out that the control group and detection group almost do not have apoptotic cells (the red cell nucleus is positive)48h after transfection, but the quantity of cell apoptosis in RNAi group obviously increases.④Western blot results:compared with the control group and no-load group, the protein expression level of P75NTR and Bcl-2decreases remarkably in the RNAi group, while that of Caspase-3increases remarkably.[Conclusion]①It turns out that effective RNAi transfection can obviously reduce the cell proliferation speed through drawing the cell growth curve, and U251cell apoptosis quantity transfected by stable gene increases obviously through the cell apoptosis detection.②After the gene expression of P75NTR in target silencing U251glioblastoma, it may induce the over-expression of B apoptosis gene Caspase-3and meanwhile restrain the protein expression of apoptosis inhibitor gene Bcl-2through direct or indirect mechanism, and control and induce the apoptosis progress of the cancer cell at the access level of cell apoptosis.③This research discovers and verifies that3RNAi fragments of target silencing P75NTR can effectively mRNA and protein expression of apoptosis inhibitor gene P75NTR, so as to provide new gene target selection for the molecular therapy of the future glioblastoma.