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目的:探讨小干扰RNA(siRNA)介导的核糖核苷酸小亚基M2(ribonucleotide reductase M2,RRM2)沉默对顺铂(DDP)诱导的卵巢癌耐药细胞株SKOV3/DDP敏感性的影响。方法:采用间歇浓度梯度递增法诱导SKOV3/DDP;将RRM2基因的特异性siRNA转染SKOV3/DDP细胞,另设空白组和SKOV3/DDP-RRM2-neg(阴性组)做为对照;荧光PCR技术和蛋白质印迹法分别检测转染后各组细胞中RRM2基因mRNA和蛋白表达;MTT法检测RNAi对SKOV3/DDP细胞药物敏感性的影响。结果:成功诱导出卵巢癌DDP耐药细胞株SKOV3/DDP细胞,其耐药系数达到3.6,属低度耐药,但对吉西他滨(Gem)仍敏感。DDP对干扰组、阴性组和空白组细胞48h的IC50分别为(3.09±0.11)、(9.88±0.37)和(10.35±0.03)μg/mL,3者间差异有统计学意义,P<0.001。干扰组细胞对DDP的敏感度提高了约3倍,其RF为3.3。SKOV3/DDP-RRM2-RNAi 667(Ⅰ)组RRM2 mRNA水平下调为74.1%,P=0.010;SKOV3/DDP-RRM2-RNAi480(Ⅱ)组RRM2 mRNA水平下调为55.9%,P=0.016;SKOV3/DDP-RRM2-RNAi1179(Ⅲ)组RRM2mRNA水平下调为43.1%,P=0.001。其中,以转染Ⅰ组基因沉默率(82.33%)最高,P<0.001;阴性组(0.008 6%)与空白组(0.008 2%)比较,差异无统计学意义,P=0.133。转染RRM2的不同靶序列72h后RRM2蛋白的表达量最低,Ⅰ组为0.062±0.006,Ⅱ组为0.314±0.002,Ⅲ组为0.123±0.002,与阴性组(0.715±0.034)和空白组(0.516±0.040)比较均明显降低,但以转染Ⅰ组细胞的蛋白相对表达量下降最明显,F=658.6,P=0.003 3。Gem和DDP联合作用72h时,转染组细胞凋亡率为(96±3.0)%,与其各组比较,差异均有统计学意义,P值均<0.001。结论:通过RNAi技术可有效抑制RRM2基因在卵巢癌中的转录和翻译水平,增加DDP耐药细胞的药物敏感性,并促进DDP诱导的卵巢癌耐药细胞凋亡,在一定程度上逆转DDP耐药性;RNAi技术联合Gem及DDP作用可有效提高卵巢癌DDP耐药细胞的凋亡率,有望成为铂类耐药的卵巢癌晚期患者一线治疗方案。
OBJECTIVE: To investigate the effect of silencing siRNA-mediated ribonucleotide reductase M2 (RRM2) on the sensitivity of cisplatin (DDP) -induced ovarian cancer cell line SKOV3 / DDP. Methods: SKOV3 / DDP was induced by intermittent concentration gradient method. Specific siRNA of RRM2 gene was transfected into SKOV3 / DDP cells, and blank control group and SKOV3 / DDP-RRM2-neg (negative control group) And Western blotting were used to detect the expression of RRM2 mRNA and protein in each group after transfection. The effect of RNAi on the drug sensitivity of SKOV3 / DDP cells was detected by MTT assay. Results: The ovarian cancer cell line DDP SKOV3 / DDP was successfully induced with resistance coefficient up to 3.6, which was low resistant but still sensitive to gemcitabine (Gem). The IC50 values of DDP for interfering group, negative group and blank group for 48h were (3.09 ± 0.11), (9.88 ± 0.37) and (10.35 ± 0.03) μg / mL, respectively, with significant difference between the three groups (P <0.001). The sensitivity of the interfering cells to DDP was increased about 3-fold with an RF of 3.3. The level of RRM2 mRNA in SKOV3 / DDP-RRM2-RNAi 667 (Ⅰ) group was down-regulated to 74.1%, P = 0.010. The level of RRM2 mRNA in SKOV3 / DDP-RRM2-RNAi480 group was 55.9% The level of RRM2 mRNA in RRM2-RNAi1179 (Ⅲ) group was down-regulated to 43.1%, P = 0.001. Among them, the gene silencing rate (82.33%) in transfected group I was the highest (P <0.001). There was no significant difference between the negative group (0.008 6%) and the blank group (0.008 2%), P = 0.133. The expression of RRM2 protein was the lowest in 72h after transfected with different target sequences of RRM2, which was 0.062 ± 0.006 in group Ⅰ, 0.314 ± 0.002 in group Ⅱ, 0.123 ± 0.002 in group Ⅲ, and 0.715 ± 0.034 in group Ⅲ and 0.516 ± 0.040). However, the relative expression of protein in group Ⅰ transfected cells decreased most obviously, F = 658.6, P = 0.003 3. The apoptosis rate of the transfection group was (96 ± 3.0)% at 72h after Gem combined with DDP, the difference was statistically significant (P <0.001). Conclusion: RNAi can effectively inhibit the transcription and translation of RRM2 gene in ovarian cancer, increase the drug sensitivity of DDP-resistant cells and promote the apoptosis of drug-resistant ovarian cancer cells induced by DDP, to a certain extent, reverse DDP resistance The combination of RNAi and Gem and DDP can effectively increase the apoptosis rate of DDP resistant cells in ovarian cancer and is expected to be the first-line treatment for patients with advanced stage ovarian cancer.