摘要目的 探讨沉默轴突导向蛋白4D(Sema4D)基因对卵巢癌SKOV3细胞增殖、侵袭、转移的影响,并验证Sema4D干扰RNA对人卵巢癌裸鼠移植瘤生长的抑制作用.方法 针对Sema4D基因构建短发夹RNA(shRNA)重组载体pshRNA Sema4D-A、pshRNASema4D-B和pshRNASema4D-C质粒,并行酶切鉴定和测序分析.采用逆转录聚合酶链反应(RT-PCR)法筛选RNA干扰效果最佳的重组载体,通过脂质体介导转染卵巢癌SKOV3细胞(重组载体组),并以空载体组(转染空载体pcDNA3.1质粒)和未转染组(未作任何处理)为对照.采用RT-PCR法和Westem blot法分别检测转染前后SKOV3细胞中Sema4D mRNA和蛋白表达,并榆测转染前后SKO3细胞的增殖、侵袭和转移能力.建立人卵巢癌裸鼠移植瘤模型,将满足成瘤标准的15只裸鼠随机分为pshRNASema4D组(重组载体组)、空载体组和未转染组,每组5只,分别向瘤块内注射pshRNASema4D-B(50μg/次)、空载体(50μg/次)和磷酸盐缓冲液(PBS,100μl/次),每3 d注射1次,共3周,观察移植瘤生长情况.结果 酶切鉴定和测序分析证实,靶向Sema4D的3个ShRNA重组载体pshRNASema4D-A、pshRNASema4D-B和pshRNASema4D-C质粒构建成功.RT-PCR法筛选显示,重组载体pshRNASema4D-B质粒干扰效果最佳.重组载体组转染24、48和72 h时,SKOV3细胞中Sema4DmRNA的相对表达水平分别为0.401.4±0.051、0.120±0.035和0.014±0.015,重组载体组转染72 h时的Sema4D mRNA相对表达水平明显低于空载体组(0.521±0.019)和未转染组(0.536±0.040,P<0.05).Western blot法检测显示,重组载体组转染24、48和72 h时,细胞中Sema4D蛋白的相对表达水平分别为0.196±0.023、0.074±0.015和0.040±0.014,重组载体组转染72 h时的Sema4D蛋门相对表达水平明显低于空载体组(0.275±0.009)和未转染组(0.282±0.015,P<0.05).与空载体组和未转染组比较,重组载体组细胞增殖、侵袭和迁移能力受到抑制(P<0.05).注射pehRNASema4D-B后,裸鼠移植瘤生长缓慢,瘤块重量较空载体组和未转染组明显降低(P<0.05).结论 RNA干扰技术能成功沉默SKOV3细胞中Sema4D基因的表达,通过下调Sema4D基因的表达呵抑制卵巢癌细胞增殖、侵袭及转移,Sema4D干扰RNA能抑制人卵巢癌裸鼠移植瘤的生长,Sema4D基因可能成为人卵巢癌基因靶向沉默治疗的重要候选基因之一.

abstractsObjective To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude nice. Methods Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blotwere used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SK0V3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Results Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0. 401 ±0.051, 0. 120 ±0.035 and 0.014 ±0. 015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ±0.019, 0.536 ±0.040,respectively, P<0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0. 196 ± 0. 023, 0. 074 ± 0. 015 and 0. 040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0. 275 ± 0. 009, 0. 282 ± 0. 015, respectively, P < 0. 05 ). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR( inhibitory rate ) of pshRNASema4D-B group was ( 61.0 ± 3.3 ) % ( P < 0.05). Conclusions Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.