反义HSC70与mtP53和肿瘤耐药关系的探讨

ASSOCIATION BETWEEN ANTISENSE HSC70、INSTABILITY OF mtP53 PROTEIN AND CHEMOSENSITIVITY IN HUMAN BREAST CANCER CELL LINE

目的 观察反义HSC 70RNA对MCF7/Adr人乳腺癌细胞耐药性影响 ,探讨其与突变型p5 3稳定性和肿瘤耐药间的关系。方法 利用DNA重组技术将人HSC70cDNA片段反向克隆至真核表达载体pLXSN中 ,构建反义人HSC70真核表达质粒pX AHSC70。用脂质体方法将重组质粒转染至MCF7/Adr人乳腺癌细胞内 ,以PCR确定转染细胞中外源DNA存在后 ,Westernblot检测HSC70蛋白的抑制程度。MTT法检测细胞耐药性的影响。p5 3稳定性实验分析反义HSC70RNA对突变型p5 3半衰期的改变。结果 经G418筛选获得稳定表达反义HSC70RNA的细胞株MAc70 ,与空载体MAX细胞相比 ,HSC70蛋白表达下降 42 % ,细胞耐药性显著下降。p5 3稳定性实验表明 ,其胞内突变型p5 3含量下降 ,半衰期缩短 ,稳定性明显下降。结论 反义HSC70RNA能够明显降低HSC70蛋白表达 ,导致乳腺癌细胞的耐药性下降 ,提示HSC70可能通过增强突变型p5 3稳定性的途径而参与MCF7/Adr乳腺癌细胞的耐药。

Objective This study was to find out the relationship between HSC70?instability of mtP53 protein and chemosensitivity in human breast cancer cell, and to explore the anti drug mechanism.Methods The antisense HSC70 retroviral recombinant plasmid was constructed by using DNA recombination technique, and then transfected into MCF7/Adr cells with lipofectin method. After the existence of foreign DNA was identified by PCR, the protein level of HSC70 was detected by western blot. The chemosensitivity of MAc70 cell to adriamycin was tested by MTT assay. The half life of mutant p53 was detected by using p53 stability assay. Results The transfectant cells were obtained through G418v selection. The inhibition rate of expression of HSC70 protein was 42% in MAc70 cell. Drug resistance to Adr in MAc70 cell declined obviously. Furthermore, The stability of mutant p53 protein decreased significantly. Conclusion Antisense HSC70 RNA significantly declined the drug resistance of MCF7/Adr cell through inhibition of HSC70 protein expression and increasing the instability of mutant p53.$$$$