Studies On The Significance Of Hes1 And Hes5 Expressions Related To The Proliferation Of Human Glioma Cells

Objective:1,To investigate the significance of the expressions of Notch1,Hes1,Hes5 and their relationships in human gliomas.2,To construct RNAi lentivirus expressive vectors specific to Hes1,Hes5 gene and establish Hes1,Hes5 knockdown glioma cell lines.3,To observe the effect of Hes1,Hes5 gene silencing on proliferation of gliomas cells and find out the possible mechanisms of Notch-Hes signal pathway involved in proliferation of gliomas.Methods:1,Expressions of the Notch1, Hes1 and Hes5 protein in human astrocytoma tissue specimens and normal human brain samples were detected by immunohistochemical method.2,Several pairs of double-strand oligos encoding shRNA of Hes1 and Hes5 were designed and synthesized, then cloned into pENTR/U6 entry vector use Gateway technology. Transfect the entry clones into U251 cells to perform transient RNAi analysis by western blot and screening the effective target sequences. Perform LR recombination reactions using the pLenti6/BLOCK-iT?-DEST vector and the pENTR entry clones containing effective target sequences to generate expression clones. Expression clones co-transfect with the pLenti6/BLOCK-iT?-DEST expression construct and the ViraPower? Packaging Mix into the 293FT cell line to produce lentiviral stock. U251 cells were infected by lentiviral stock and U251 cell lines with stable silencing were screened and established.3,MTT assay, plate clone formation assay and flow cytometry were performed to assay the proliferation ability and cell cycle changes of U251 cells before and after Hes1,Hes5 gene knockdown. Results:1,Effective shRNA target sequences of Hes1,Hes5 gene were screened.2,Lentiviral vectors expressing shRNA of Hes1,Hes5 gene were constructed and lentivirus were produced in 293FT cells. The titer of Hes1,Hes5 lentiviral stock is 8.4×104 TU/ml and 7.2×104 TU/ml respectively.3,U251 cell lines with stable Hes1,Hes5 gene silencing were established.4,Proliferation and colony formation of U251 cells can be effectively inhibit by Hes1 or Hes5 gene silencing.Conclusion:1,Notch1 is involved in the tumorigenesis of human astrocytomas, the expression level of Notch1 is not correlated with pathological grade of astrocytoma. The role of Notch1 in astrocytoma may be via its effector Hes1 but not Hes5.2,The constructed RNAi lentivirus expressive vectors specific to Hes1,Hes5 gene can inhibit the expression of Hes1,Hes5 efficiently in U251 cell line. They are important tools in studying the functions of Notch-Hes signal pathway and their relationship with glioma.3,Hes1 and Hes5 closely correlate with the proliferation of glioma cells. RNAi targeted to Hes1 or Hes5 could inhibit the proliferation of glioma cell U251 in vitro.