应用RNA干扰技术阻断PC-3细胞中SRC-1基因表达的意义

Inhibition of Survivin Gene Expression in PC-3 Cells by RNAi

目的:探讨RNA干扰技术沉默SRC-1基因后对PC-3细胞生长及侵袭性的影响。方法:将设计好的siRNA,用脂质体法转染PC-3细胞,通过Q-PCR、蛋白免疫印迹检测SRC-1的表达变化,并用CCK-8法检测细胞生长情况,用transwell小室检测PC-3细胞侵袭力。结果:转染SRC-1siRNA24小时后的前列腺癌PC-3细胞系中SRC-1mRNA的相对表达量下降约42%,48小时后下降约79%,差异具有统计学意义(P<0.05);转染24小时、48小时后PC-3细胞SRC-1蛋白的表达量(24h 0.5536±0.071、48h 0.3419±0.025)与阴性对照组(24h 0.8562±0.092、48h 0.8791±0.076)、转染试剂组(24h 0.7992±0.072、48h 0.9731±0.051)和空白对照组(24h 0.8375±0.054、48h 0.8826±0.043)相比明显减少(P<0.05)。在转染24小时、48小时、72小时、96小时后PC-3细胞生长情况(吸光度A值:24h 0.324±0.0252、48h 0.689±0.0141、72h 1.0032±0.0166、96h 1.4650±0.0327)与阴性对照组(24h 0.3216±0.0152、48h 0.7014±0.017、72h 0.9902±0.0272、96h 1.4596±0.0141)、转染试剂组(24h 0.3222±0.0343、48h 0.6904±0.0301、72h 0.993±0.0383、96h 1.4574±0.0464)和空白对照组(24h 0.3316±0.0192、48h0.7092±0.0265、72h 1.0136±0.0130、96h 1.4596±0.0128)比较没有明显变化(P>0.05),但是PC-3细胞的侵袭能力明显下降,干扰实验组的细胞(38±9.01)与阴性对照组(83.33±10.78)、转染试剂组(83.67±10.016)及空白对照组(84.67±11.24)相比较,穿过小室膜的细胞数量明显减少(P<0.05)。结论:siRNA有效地阻断了PC-3细胞中SRC-1基因的表达,SRC-1基因在mRNA和蛋白水平上的表达明显下调(P<0.01),并使转染后的PC-3细胞的侵袭能力下降,但转染后PC-3细胞生长无明显变化。

Objective： Design the siRNA based on the base sequences and transfect the PC-3 cell line for inhibiting the human SRC-1 gene by RNA interference, and to detect the effect of the silenced SRC-1 gene on PC-3 cells. Meth- ods： The PC-3 cells were transfected with the siRNA and the interference effect was detected by Q--PCR, Western blot. The proliferation of the PC-3 cells was detected by CCK-8 method and their invasion ability was detected by using transwell. Results： The expression of SRC-1 in transfected PC-3 cells was dropped by up to 42% after 24 hours and 79% after 48 hours at mRNA levels as compared with un-treated groups. After transfecting 24 to 48 hours, the expression of SRC-1 protein（24h 0. 5536 ± 0. 071 48h 0. 3419 ± 0. 025 ）was markedly depressed in PC-3 cells as compared with negative control groups（24h 0. 8562 ± 0. 092 48h 0. 8791 ± 0. 076） ,transfection reagent groups （24h 0. 7992 ± 0. 072 48h 0. 9731 ± 0. 051 ）and an-treated groups（24h 0.8375 ±0. 054 48h 0. 8826 ±0. 043）. After transfecting 24 to 96 hours,the growth of the PC-3 cells （ absorbance value A ： 24h 0. 324 ± 0. 0252, 48h 0.689 ± 0. 0141 72h 1. 0032 ± 0. 0166,96h 1. 4650 ± 0. 0327）was not significantly different when compared with negative control groups（24h 0. 3216 ±0. 0152, 48h 0. 7014 ±0. 017,72h 0. 9902 ± 0. 0272,96h 1. 4596 ± 0. 0141 ）,transfection reagent groups （24h 0. 3222 ± 0. 0343,48h 0.6904 ± 0.0301,72h 0. 993 ± 0. 0383,96h 1. 4574 ± 0. 0464） and un-treated groups（24h 0. 3316 ±0. 0192,48h 0. 7092 ±0. 0265,72h 1. 0136 ±0. 0130,96h 1. 4596 ±0. 0128）. Furthermore, the PC-3 cells were observed along with a marked inhibition of invasion as reflected by fewer penetrating cells by hoyden ehamber assay（38 ± 9.01 ）, compared with negative control groups（83.33 ± 10.78） ,transfeefion reagent groups （ 83.67 ± 10. 016 ） and un-treated groups （ 84.67 ± 11.24 ） with significant difference （ P 〈 0.05 ）. Conclusion： After transfeetion, the expressions of SRC-1 gene were inhibited effectively and the invasion ability was reduced obviously by the siR- NA in the prostate cancer cell line PC-3.