中华肿瘤杂志
中華腫瘤雜誌
중화종류잡지
CHINESE JOURNAL OF ONCOLOGY
2011年
5期
324-330
,共7页
陈颖%张磊%吴卉娟%刘文欣%郝权
陳穎%張磊%吳卉娟%劉文訢%郝權
진영%장뢰%오훼연%류문흔%학권
卵巢肿瘤%Semaphorin4D%RNA干扰%细胞增殖%侵袭%转移
卵巢腫瘤%Semaphorin4D%RNA榦擾%細胞增殖%侵襲%轉移
란소종류%Semaphorin4D%RNA간우%세포증식%침습%전이
Ovarian neoplasms%Semaphorin4D%RNA interference%Cell proliferation%Invasion%Metastasis
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.
目的 探討沉默軸突導嚮蛋白4D(Sema4D)基因對卵巢癌SKOV3細胞增殖、侵襲、轉移的影響,併驗證Sema4D榦擾RNA對人卵巢癌裸鼠移植瘤生長的抑製作用.方法 針對Sema4D基因構建短髮夾RNA(shRNA)重組載體pshRNA Sema4D-A、pshRNASema4D-B和pshRNASema4D-C質粒,併行酶切鑒定和測序分析.採用逆轉錄聚閤酶鏈反應(RT-PCR)法篩選RNA榦擾效果最佳的重組載體,通過脂質體介導轉染卵巢癌SKOV3細胞(重組載體組),併以空載體組(轉染空載體pcDNA3.1質粒)和未轉染組(未作任何處理)為對照.採用RT-PCR法和Westem blot法分彆檢測轉染前後SKOV3細胞中Sema4D mRNA和蛋白錶達,併榆測轉染前後SKO3細胞的增殖、侵襲和轉移能力.建立人卵巢癌裸鼠移植瘤模型,將滿足成瘤標準的15隻裸鼠隨機分為pshRNASema4D組(重組載體組)、空載體組和未轉染組,每組5隻,分彆嚮瘤塊內註射pshRNASema4D-B(50μg/次)、空載體(50μg/次)和燐痠鹽緩遲液(PBS,100μl/次),每3 d註射1次,共3週,觀察移植瘤生長情況.結果 酶切鑒定和測序分析證實,靶嚮Sema4D的3箇ShRNA重組載體pshRNASema4D-A、pshRNASema4D-B和pshRNASema4D-C質粒構建成功.RT-PCR法篩選顯示,重組載體pshRNASema4D-B質粒榦擾效果最佳.重組載體組轉染24、48和72 h時,SKOV3細胞中Sema4DmRNA的相對錶達水平分彆為0.401.4±0.051、0.120±0.035和0.014±0.015,重組載體組轉染72 h時的Sema4D mRNA相對錶達水平明顯低于空載體組(0.521±0.019)和未轉染組(0.536±0.040,P<0.05).Western blot法檢測顯示,重組載體組轉染24、48和72 h時,細胞中Sema4D蛋白的相對錶達水平分彆為0.196±0.023、0.074±0.015和0.040±0.014,重組載體組轉染72 h時的Sema4D蛋門相對錶達水平明顯低于空載體組(0.275±0.009)和未轉染組(0.282±0.015,P<0.05).與空載體組和未轉染組比較,重組載體組細胞增殖、侵襲和遷移能力受到抑製(P<0.05).註射pehRNASema4D-B後,裸鼠移植瘤生長緩慢,瘤塊重量較空載體組和未轉染組明顯降低(P<0.05).結論 RNA榦擾技術能成功沉默SKOV3細胞中Sema4D基因的錶達,通過下調Sema4D基因的錶達呵抑製卵巢癌細胞增殖、侵襲及轉移,Sema4D榦擾RNA能抑製人卵巢癌裸鼠移植瘤的生長,Sema4D基因可能成為人卵巢癌基因靶嚮沉默治療的重要候選基因之一.
목적 탐토침묵축돌도향단백4D(Sema4D)기인대란소암SKOV3세포증식、침습、전이적영향,병험증Sema4D간우RNA대인란소암라서이식류생장적억제작용.방법 침대Sema4D기인구건단발협RNA(shRNA)중조재체pshRNA Sema4D-A、pshRNASema4D-B화pshRNASema4D-C질립,병행매절감정화측서분석.채용역전록취합매련반응(RT-PCR)법사선RNA간우효과최가적중조재체,통과지질체개도전염란소암SKOV3세포(중조재체조),병이공재체조(전염공재체pcDNA3.1질립)화미전염조(미작임하처리)위대조.채용RT-PCR법화Westem blot법분별검측전염전후SKOV3세포중Sema4D mRNA화단백표체,병유측전염전후SKO3세포적증식、침습화전이능력.건립인란소암라서이식류모형,장만족성류표준적15지라서수궤분위pshRNASema4D조(중조재체조)、공재체조화미전염조,매조5지,분별향류괴내주사pshRNASema4D-B(50μg/차)、공재체(50μg/차)화린산염완충액(PBS,100μl/차),매3 d주사1차,공3주,관찰이식류생장정황.결과 매절감정화측서분석증실,파향Sema4D적3개ShRNA중조재체pshRNASema4D-A、pshRNASema4D-B화pshRNASema4D-C질립구건성공.RT-PCR법사선현시,중조재체pshRNASema4D-B질립간우효과최가.중조재체조전염24、48화72 h시,SKOV3세포중Sema4DmRNA적상대표체수평분별위0.401.4±0.051、0.120±0.035화0.014±0.015,중조재체조전염72 h시적Sema4D mRNA상대표체수평명현저우공재체조(0.521±0.019)화미전염조(0.536±0.040,P<0.05).Western blot법검측현시,중조재체조전염24、48화72 h시,세포중Sema4D단백적상대표체수평분별위0.196±0.023、0.074±0.015화0.040±0.014,중조재체조전염72 h시적Sema4D단문상대표체수평명현저우공재체조(0.275±0.009)화미전염조(0.282±0.015,P<0.05).여공재체조화미전염조비교,중조재체조세포증식、침습화천이능력수도억제(P<0.05).주사pehRNASema4D-B후,라서이식류생장완만,류괴중량교공재체조화미전염조명현강저(P<0.05).결론 RNA간우기술능성공침묵SKOV3세포중Sema4D기인적표체,통과하조Sema4D기인적표체가억제란소암세포증식、침습급전이,Sema4D간우RNA능억제인란소암라서이식류적생장,Sema4D기인가능성위인란소암기인파향침묵치료적중요후선기인지일.
Objective 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.
