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目的探讨 X 染色体相关凋亡抑制蛋白(XIAP)的小分子干扰 RNA 对卵巢上皮性癌(卵巢癌)细胞系 SKOV3细胞生长的影响。方法设计和合成长度为64 nt 的脱氧寡核苷酸链,克隆至 pSUPER 质粒,转化大肠埃希菌 DH5α菌株,构建重组质粒 pSUPER-siXIAP,对其进行双酶切和DNA 序列分析鉴定。间接免疫荧光染色法鉴定 SKOV3细胞 XIAP 蛋白的表达。实验分为4组:分别用重组质粒 pSUPER-siXIAP(pSUPER-siXIAP 组)及阴性无关序列重组对照质粒 pSUPER-nsRNA(pSUPER-nsRNA 组)及空载体质粒 pSUPER(pSUPER 组)转染 SKOV3细胞,以未转染质粒的 SKOV3细胞为空白对照组。RT-PCR 技术检测各组细胞 XIAP mRNA 的表达,蛋白印迹法检测各组细胞XIAP 蛋白的表达,流式细胞仪分析各组细胞细胞周期的变化,四甲基偶氮唑蓝(MTT)比色法观察各组细胞生长的变化。结果本实验成功构建了 XIAP 特异性的 RNA 干扰载体 pSUPER-siXIAP 质粒。间接免疫荧光染色法检测证实 SKOV3细胞中存在 XIAP蛋白的过度表达。转染后72 h,SKOV3细胞XIAP 蛋白表达水平降低,空白对照组、pSUPER 组、pSUPER-nsRNA 组及 pSUPER-siXIAP 组的相对密度值分别为3584±124、2138±65、1973±80和110±12,各组间比较,差异有统计学意义(P=0.0334);转染后72 h,SKOV3细胞 XIAP mRNA 表达水平明显下降,空白对照组、pSUPER 组、pSUPER-nsRNA 组及 pSUPER-siXIAP 组各组的相对密度值分别为6674±274、4532±107、2322±57和1864±78,各组间比较,差异有统计学意义(P=0.0127)。流式细胞仪检测结果显示,pSUPER-siXIAP组 G_1期细胞较空白对照组、pSUPER 组明显增加(P<0.05);pSUPER-siXIAP 组 S 期细胞较空白对照组、pSUPER 组明显减少(P<0.05)。MTT 比色法检测结果显示,pSUPER-siXIAP 组细胞生长增殖被抑制,生长速度较空白对照组细胞明显减慢(P=0.0237);pSUPER-nsRNA 组和 pSUPER 组细胞生长速度较空白对照组细胞也明显减慢(P=0.0441,P=0.0472)。结论本研究成功构建了 XIAP RNA干扰载体 pSUPER-siXIAP 质粒,将其转染 SKOV3细胞后可有效降低其 XIAP mRNA 及蛋白的表达水平,并将 SKOV3细胞阻滞于 G_1期,造成转染细胞生长速度减慢,这有望为卵巢癌基因治疗提供一个新的有希望的分子靶点。
Objective To investigate the effect of XIAP small interfering RNA (RNAi) on the growth of human ovarian epithelial ovarian cancer cell line SKOV3. Methods A 64 nt deoxy-oligonucleotide was designed and synthesized. It was cloned into pSUPER plasmid and transformed into Escherichia coli DH5α strain. The recombinant plasmid pSUPER-siXIAP was constructed and identified by double enzyme digestion and DNA sequence analysis. Indirect immunofluorescent staining was used to identify the expression of XIAP protein in SKOV3 cells. The experiment was divided into 4 groups: SKOV3 cells were transfected with the recombinant plasmid pSUPER-siXIAP (pSUPER-siXIAP group) and the negative control plasmid pSUPER-nsRNA (pSUPER-nsRNA group) and empty vector plasmid pSUPER (pSUPER group) The untransfected SKOV3 cells were blank control group. The expression of XIAP mRNA in each group was detected by RT-PCR, the expression of XIAP protein in each group was detected by Western blotting, the cell cycle was analyzed by flow cytometry, MTT colorimetric Law observed changes in cell growth in each group. Results XIAP specific RNA interference vector pSUPER-siXIAP plasmid was successfully constructed. Indirect immunofluorescence staining confirmed the presence of XIAP protein overexpression in SKOV3 cells. The expression of XIAP in SKOV3 cells decreased 72 h after transfection, and the relative density values in the blank control group, pSUPER group, pSUPER-nsRNA group and pSUPER-siXIAP group were 3584 ± 124, 2138 ± 65, 1973 ± 80 and 110 ± (P = 0.0334). At 72 h after transfection, the expression of XIAP mRNA in SKOV3 cells was significantly decreased. The expression of XIAP mRNA in SKOV3 cells was significantly decreased in the control group, pSUPER group, pSUPER-nsRNA group and pSUPER-siXIAP group The relative density values of the two groups were 6674 ± 274, 4532 ± 107, 2222 ± 57 and 1864 ± 78, respectively. The differences among the groups were statistically significant (P = 0.0127). The results of flow cytometry showed that the number of cells in G_1 phase of pSUPER-siXIAP group was significantly higher than that of pSUPER group and pSUPER group (P <0.05), and the number of S phase cells in pSUPER-siXIAP group was significantly lower than that of blank control group and pSUPER group ). The results of MTT assay showed that the proliferation of pSUPER-siXIAP group was inhibited and the growth rate was significantly slower than that of blank control group (P = 0.0237). The growth rate of pSUPER-nsRNA group and pSUPER group was also higher than that of blank control group Significantly slowed down (P = 0.0441, P = 0.0472). Conclusion The plasmid pSUPER-siXIAP containing XIAP RNA interference vector was successfully constructed and transfected into SKOV3 cells. The expression of XIAP mRNA and protein was effectively reduced, and SKOV3 cells were arrested in G 1 phase, resulting in the growth of transfected cells Slowed down, which is expected to provide a new promising molecular target for ovarian cancer gene therapy.