类固醇生成因子-1基因沉默对人肾上腺皮质腺癌H295R细胞的作用及其临床意义

Effect and significance of steroidogenic factor-1 gene silencing on proliferation of human adrenocortical H295R cells

目的研究类固醇生成因子-1（SF-1）基因下调对人肾上腺皮质腺癌H295R细胞的影响，并探讨SF-1在肾上腺肿瘤发病中的作用。方法用含SF-1特异性短发夹（shRNA）序列的质粒载体pGenesill—SF1shRNA及含非特异性序列的阴性对照质粒pGenesill—negative-shRNA分别转染H295R细胞。48h后用蛋白质印迹法和荧光定量PCR检测SF-1表达水平。WST-1法及细胞计数法检测转染后细胞增殖情况，免疫组化SABC法检测转染后各细胞组中Ki67表达，TUNEL法检测细胞凋亡情况。结果SF-1-shRNA有效地抑制了SF—1的表达，在蛋白水平和mRNA水平的抑制率分别为69．07％和71．02％（P〈0．01）。WST-1及细胞计数法均显示SF-1基因沉默组细胞增殖缓慢，增殖明显受到抑制（P〈0．01）；而TUNEL结果恰好相反，荧光镜下SF-1基因沉默组凋亡细胞的数目及形态均比较明显，凋亡细胞数约为阴性对照组的3．7倍（P〈0．01）；SF-1基因干扰后，其Ki-67阳性细胞率要低于阴性对照组细胞，分别为（16．90±2．17）％和（33．48±3．16）％（P〈0．01）。结论SF-1基因沉默可抑制肾上腺皮质腺癌细胞增殖，有望成为研究肾上腺肿瘤发病机制及其治疗的重要分子。

Objective To study the influence of inhibited steroidogenic factor-1 on human adrenocortical H295R cells, and explore its role in the pathogenesis of adrenal tumors. Methods The plasmids pGenesill-SF-l-shRNA which containing U6 promoter and SF-l-specific short hairpin RNA （shRNA） and pGenesill-negative-shRNA containing unspecific shRNA were transfeeted into H295R cell. The expression of SF-1 was measured by Western blot and real-time polymerase chain reaction （RT-PCR）. Cell proliferation was analyzed by WST-1 assay and cell count. Ki 67 expression was detected by immunohistochemistry and cell apoptosis was examined by TUNEL assay. Results Compared with those in control cells, the protein and mRNA level of SF-1- transfected cells were reduced by 69.7% and 71.2%（P〈0.01）. WST-1 and cell count method showed that SF-1 gene silencing obviously inhibited cell proliferation（P〈0.01）. By contrast, there was a 3. 7-fold increase in the percentage of apoptotic H295R ceils in SF 1-inhibited group than that of control group （P〈0.01）. Im munohistochemistry showed that Ki-67 positive cells in SF 1 inhibited cells were lower than the negative control cells （16.90±2.17） % and （33. 48±3.16）%, （P〈0.01）. Conclusion SF-1 gene silencing can inhibit the proliferation of adrenocortical cells, and it is expected to become a key protein in understanding pathogenesis of adrenal tumors or treating them.