The Inhibitory Effects Of Survivin Expression Vector Of HL-60 Cells By RNAi

Objective: Leukemia is malignant hematopoietic tumor, which is associated with the dysregulation of cell proliferation and apoptosis. Survivin gene is a new member of inhibitors of the apoptosis protein(IAP)family. Its distribution characteristics in tussues are differrent from other apoptosis inhibitory factors, it did not express in most adult tissues but highly expressed in most human cancers. The expression of survivin gene directly relates to the histological classification of carcinoma and prognosis. RNA interference(RNAi) is a genetic interference phenomenon mediated by the double-stranded RNA(dsRNA) . It could degrade homologous mRNA specifically and efficiently, resulting in post-transcriptional gene silencing(PTGS), which is a natural mechanism in organisms underlying the resistance to virus invasion and inhibition of transposon mobility .Gene silencing by RNAi has become a quick and efficient experimental method to study gene function and gene therapy. In this experiment, to construct the short hairpin RNA(shRNA) expression vector of survivin and investigate its effect on the proliferation and the apoptosis of leukemia cell line HL-60, which set a foundation for further research on gene therapy for tumors such as leukemia.Method:1. Eukaryotic expression vector of survivin gene was constructed ,named as pSIN/shRNA.2. Experiment groups: pSIN/shRNA transfected group;negative control group;blank control group3. The expressions of the survivin gene was assessed by RT-PCR and the protein of Survivin was observed by immuno-histochemistry in HL-60 cells treated with pSIN/shRNA .4. The propagation of the HL-60 cells was evaluated by MTT assay after the transfered of pSIN/shRNA.5. 5. Cell apoptosis population was analysed by Annexin v-FITC/PI flow cytometry.6. Software SPSS 10.0 was applied to statistics the data. The standard of significant level was a=0.05.Result:1. Identification of recombinant plasmids digested with restriction enzymes and sequence analysis: After digested by restriction enzymes BamH I and Cla I,the recombinant vector appeared 5606bp and 63bp bands,the empty vector appeared 6522bp band by agarose gel electrophoresis. The sequence analysis completely coincided with the designs,the recombinant vector constructed successfully.2. The effects of pSIN/shRNA on expression of survivin mRNA and protein: After HL-60 cells were transfered with 4.0μg pSIN/shRNA for 24h,48h,72h,96h, expression of survivin gene mRNA was decreased as assayed by RT- PCR. Compared with two control groups, survivin gene mRNA were decreased significantly in 24h,48h,72hand 96h transfected groups(P<0.01),but there was no difference between two control group(P>0.05). Comparison among the transfected groups at 24h,48h, 72h and 96h , survivin gene mRNA was not decreased significantly in 24h transfected group as compared with 48,72 and 96h transfected groups(P<0.05).There was no difference among 48,72 and 96h transfected groups(P>0.05). When the HL-60 cells were treated with pSIN/shRNA for 48h,the mean score of survivin protein in positive cells was (50.33±7.33), which was significant lower than that in their corresponding blank control group(147.32±13.71 ) and negative control group (142.33±14.43 ) (P<0.01). But there was no significant difference between the two control groups.(P>0.05)3. Inhibitory effect of pSIN/shRNA on the proliferation of HL-60 cells: After transfered with 4.0μg pSIN/shRNA for 24h,48h and 72h, , the inhibitory effect was significant higher in the transfected groups at 24,48 and 72h compared with two control groups (P<0.01).There was no difference between the two control groups (P>0.05). The inhibitory effect of 24h transfected group was not significant compared with 48h and 72h transfected groups(P<0.05). There was no difference between 48 and 72h transfected groups. The results demonstated that pSIN/shRNA was capable ofinhibiting the HL-60 cells proliferation by time and sequence specificity-dependentmanners with the optimal time at 48h.4. Apoptosis of HL-60 cells induced by pSIN/shRNA: With Flow cytometer, anapoptosis population(20.21±0.75)% was observed when the HL-60 cells were treatedwith 4.0ug pSIN/shRNA for 48h. Low apoptosis populations were observed in thenegative control group(2.58±0.48)% and the blank control group(1.26±0.30)%. Theapoptosis rate of the transfected group significantly increased compared with that inthe two control groups(P<0.01),and there was no difference between two controlgroups(P>0.05).Conclusion:1. Eukaryotic expression vector of survivin gene was constructed and named as pSIN/shRNA2. pSIN/shRNA can down-regulate expression of survivin gene in mRNA and protein level in HL-60 cells significantly and continually.3. pSIN/shRNA can inhibit the proliferation of HL-60 cells in a time and sequence specificity-dependent manners with the optimal time at 48h.4. pSIN/shRNA induced the HL-60 cells apoptosis.