Experimental study on the inhibitory effect of silencing hTERT gene′s short hairpin RNAs on the growth of human glioma subcutaneous xenograft in nude mice

Objective To explore the inhibitory effect of silencing human telomerase reverse transcriptase(hTERT) gene′s short hairpin RNAs(shRNAs) on the growth of human glioma in nude mice by RNA interference technique to afford an experimental basis for concerned gene therapy of glioma.Methods Human glioma U251 cells,cultured in vitro,were used to establish models of human glioma by subcutaneous injection into nude mice.The 36 mouse models formed were divided into 3 groups for varied purposes(n=12 each): the hTERT shRNA therapy group was given in situ injection of hTERT shRNAD plamid(pshRNA) 20 μg + lipid 60 μl + PBS;the PBS group,given that of PBS 150 μl;the blank plasmid group,given that of blank plasmid 20 μg + lipid 60 μl + PBS.H-E stained specimens of the xenograft tumor were prepared for pathohistological study after the plasmid therapy,Western blot method was used to determoine the protein level of hTERT,flow cytometry was for assessing the apoptosis of tumor cells.Results Satisfactory subcutaneous xenograft glioma model of nude mice could be constructed within 15 days,with an average size of 132 mm3.Compared with that in the blank plasmid and PBS groups,the tumor growth in the therapy group was evidently depressed down to 58.2%.Through light microscopy,it was found in the hTERT shRNA group that the tumor growth was inhibited obviously,there were relatively few tumor cells,but there were numbers of necrotic and apoptotic tumor cells everywhere.Western blot showed the expression of hTERT protein was restrained.Flow cytometry showed the apoptosis ratio of the tumor was higher in hTERT shRNA group than in the control groups.The differences in these indexes were very significant between the therapy group and the control groups(P0.01).Conclusion hTERT shRNA can inhibit the transcription and translation level of hTERT in the xenografted glioma in nude mice,inhibiting effectively the tumor growth and accelerating the apoptosis of tumor cells.