Abstract:

Abstract:Objective To construct and screen shRNA plasmids of HSPA8 with high interfering ability and  provide the material for further  researching on the effect of HSPA8 gene on breast cancer.Methods In accordance with the sequence of HSPA8 gene in GenBank,four interfering gene fragments and four shRNA interfering plasmids（pGPU6/GFP/Neo-349,pGPU6/GFP/Neo-818,pGPU6/GFP/Neo-931,pGPU6/GFP/Neo-1180）were constructed.The plasmids were transfected  into  human breast cancer cell MCF-7 respectively,and negative control group and blank control group were set up.The HSPA8 positive expressing cells were screened by G418.The interfering efficiencies of the plasmids on HSPA8 gene  were detected  at the mRNA and  protein levels by RT-PCR and Western blotting.Results The RT-PCR results   showed  that at the mRNA  level  in four transfected groups the expression levels were decreased by 25.0%,37.5%,43.7%,and 68.5%,respectively,compared with untransfected group.The Western blotting results showed that the differences of the protein expression levels between transfected groups and blank control group were significant(P<0.05).The interfering efficiency of  pGPU6/GFP/Neo-1180 plasmid was the highest.Conclusion The high ability shRNA interfering plasmids of HSPA8 gene are constructed successfully.The pGPU6/GFP/Neo-1180 interfering plasmid may provide a basis  for  further study on the occurrence and  development of breast cancer.

Key words: HSPA8 gene； breast neoplasms； RNA interference;MCF-7