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

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pGenesil1-SF-1-shRNA及含非特异性序列的阴性对照质粒pGenesil1-negative-shRNA分别转染H295R细胞.48 h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Ki-67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.

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

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 pGenesil1-SF-1-shRNA which containing U6 promoter and SF-1-specific short hairpin RNA (shRNA) and pGenesil1-negative-shRNA containing unspecific shRNA were transfected 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 cells in SF-1-inhibited group than that of control group (P＜0. 01). Immunohistochemistry 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.