Effects And Mechanism Of RNA Interference-mediated Notch1 Or Notch2 Knockdown On Proliferation Of Human Glioma U251 Cells

Gliomas are the most common intracranial tumors. The prognosis for patients with high-grade gliomas is generally poor. It is very important to analyze the molecular mechanisms of gliomas and to develop new treatments. Recently, the relationship of Notch signaling and gliomas was concerned and the Notch signaling was seemed to be a new target of glioma therapies. Therefore, we had interest of the role of Notch signaling in gliomas. We knocked down the Notch1 by lentivirus-mediated RNA interference technology and screened the stable transduced cell clones. After that we measured the cell proliferation of Notch1 knockdown clones in vitro and in vivo. We analyzed the different gene expression between the Notch1 knockdown clone and the control clone by cDNA microarray technology. We also knocked down the Notch2 expression by lentivirus-mediated RNA interference and measured the cell proliferation of Notch2 knockdown clones in vitro, in order to compare the Notch1 and Notch2 function in gliomas.In the first part of our study we screened effective Notch2 siRNA sequence by real-time PCR technology and we constructed Notch2-shRNA lentiviral vector for virus packaging. We succeeded in packing the Notch2-knockdown and negative control lentivirus. The titer of the viruses was 3.7×10~5 TU/ml and 2.5×10~5 TU/ml respectively.In the second part of our study we screened the cell clones of Notch1-knockdown and Notch2-knockdown. The human glioma U251 cells were infected with lentivirus expressing Notch1-shRNA or Notch2-shRNA. The knockdown effects of Notch1 or Notch2 of transduced U251 cell clones was detected by real-time PCR and Western blot.In the third part of our study we investigated the effects of Notch1 knockdown by RNA interference on proliferation of human glioma U251 cells. The cell growth viability was evaluated by CCK-8 assay. Colony formation assay and soft-agar colony formation were used to measure the colony formation ability of stably transduced cells. The influence of Notch1-knockdown on the implanted tumor growth was observed. The ability of growth in Notch1-knockdown cells was decreased significantly. The plate colony formation rate of Notch1-knockdown cells was (18.6±2.5)%, whereas the rate of control cells was (37.0±3.3)%. The soft-agar colony formation rate of Notch1-knockdown cells was (51±5)%, whereas the rate of control cells was (80±4)%. Implanted glioma mouse model was successively established. The tumor weight in Notch1-knockdown group was markedly lower than that in control group(0.31±0.09g vs 2.35±0.71g, P<0.01). But the Notch2 knockdown had no effect on cell proliferation.In the fourth part of our study we analyzed the different gene expression between the Notch1 knockdown clone and the control clone by cDNA microarray technology. The results showed that there are total of 775 differentially expressed genes related to cell proliferation and differentiation, cell cycle, cell communication and adhesion, lipid metabolism, MAPK signal transduction, TGF-βsignal transduction, etc. The inhibition of cell proliferation of Notch1 knockdown cell clone might be mediated by cell cycle regulation and TGF-βsignaling.