Beclin1和HLAⅠ、Ⅱ在人卵巢癌SKOV3细胞中的表达

Expression of Beclin1 and HLA Ⅰ,Ⅱ in human SKOV3 ovarian cancer cells and their correlation

目的探讨转染Beclin1质粒的自噬基因Beclin1和免疫应答效应分子HLAⅠ、Ⅱ在人卵巢癌SKOV3细胞中的表达,分析Beclin1在卵巢癌免疫应答中的作用。方法应用基因转染技术、RT-PCR、Western blot法检测Beclin1质粒转染SKOV3细胞前后Beclin1和HLAⅠ、Ⅱ的mRNA和蛋白表达水平的变化。应用免疫荧光显微镜观察Beclin1质粒转染SKOV3细胞后,其自噬小体的变化。用MTT法检测Beclin1质粒转染SKOV3前后细胞的增殖活性。结果 SKOV3细胞转染Beclin1质粒后,Beclin1的转录和翻译水平分别是空载体组的5、2倍。荧光显微镜观察,转染Beclin1质粒组卵巢癌细胞MDC标记的自噬小体数量显著增多。RT-PCR和Western blot结果显示,转染Beclin1可诱导HLAⅠ、Ⅱ等位基因的转录和翻译。Beclin1质粒组的HLAⅠ-A、B、C和HLAⅡ-DP、DQ、DR的mRNA分别是空载体组的2、1.6、3倍和2、6、3倍;其HLAⅠ、Ⅱ的蛋白表达分别是空载体组的2、1.6倍。MTT结果显示,Beclin1质粒组与空载体组、空白对照组相比,在转染24、48、72、96 h后,其细胞生长抑制率分别为42.6%、37.8%、24.35%、14.81%。结论在人卵巢癌SKOV3细胞中,转染外源性Beclin1可诱导细胞自噬并使免疫应答效应分子HLAⅠ、Ⅱ的表达增加。HLAⅠ、Ⅱ表达的增加可能使卵巢癌细胞免疫应答增强。转染外源性Beclin1可抑制卵巢癌SKOV3的细胞增殖。

Purpose The aim of this study was to investigate the relationship between autophagy gene Beclinl and immune response effector classical HLA Ⅰ,Ⅱ in SKOV3 cells. To explore the role of Beclinl in immunity in ovarian cancer cells which were transfected with the vector of Beclinl. Methods RT-PCR and Western blot were used to detect the expression of Beclinl and HLA Ⅰ,Ⅱ in SKOV3 cells. Fluorescence microscope was carried out to observe the unique autophagosome in SKOV3 cells. MTF was used to analyze the proliferation of the Beclinl over-expressed SKOV3 cells. Results Transfection SKOV3 cells with Beclinl vector could induce Beclinl transcription and translation approximately 5 and 2 times compared with empty vector group respectively. The autophagosome stained by MDC was observed by fluorescence microscope. And much more green fluorescence signal was observed in Beclinl vector group. RT-PCR and Western blot indicated that HLA Ⅰ,Ⅱ induced by transfection with extrinsic Beclinl. The allelic transcriptions of HLA Ⅰ -A, B, C and HLA Ⅱ-DP, DQ, DR in extrinsic Beclinl group were approximately 2, 1.6, 3 and 2, 6, 3 times compared with empty vetcor group or untreated group, respectively. The results of Western blot showed that HLA Ⅰ,Ⅱ in Beclinl vector group induced as much as 2 and 1.6 times compared with empty vetcor group or untreated group,, respectively. The results of MTT showed that the proliferation of SKOV3 cells treated with Beclinl vector was significantly suppressed. The percentage of suppression in Beclinl vector group at 24 h, 48 h, 72 h and 96 h is 42. 6%, 37. 8%, 24. 35%, 14. 81% compared with untreated group or empty vector group respectively. Conclusion The enhancement of autophagy by over-expression of Beclinl could induce HLA Ⅰ,Ⅱ transcription and translation in SKOV5 cells. The expression of HLA Ⅰ,Ⅱ may be responsible for triggering the immune response in ovarian cancer. Over-expression of Beclinl could inhibit the proliferation of SKOV3 cells which were transfected with extrinsic Beclinl.