Oncol2012: 靶向黑色素瘤细胞RhoC基因慢病毒表达载体的构建及初步鉴定

背景 恶性黑色素瘤是体表肿瘤中死亡率最高的恶性肿瘤，高度侵袭转移性是其重要的生物学特征之一，亦是其预后不良的主要因素。肿瘤转移相关因子RhoC可通过多种转移相关机制参与肿瘤的侵袭与转移，是控制肿瘤转移的分子开关，RhoC有望成为抑制肿瘤转移的重要靶位。目前应用RNAi技术靶向RhoC基因对黑色素瘤侵袭转移能力的影响尚未见报道。本研究拟利用RNAi技术，以慢病毒作为基因载体，以人黑色素瘤A375细胞作为研究对象，以转移相关基因RhoC作为靶基因，研究靶向RhoC基因慢病毒干扰载体的构建并检测对RhoC的沉默效应。方法 根据RhoC编码区基因信息设计构建三条pGPU6/GFP/Neo-shRNA表达载体，通过检测其对A375细胞RhoC 的沉默效应筛选出最有效的靶序列。将RNAi效应最佳序列进行慢病毒包装构建形成靶向RhoC 的pLenti6.3-EGFP-453慢病毒表达载体，并感染A375细胞，进一步应用荧光定量PCR和Western blot检测对RhoC mRNA及蛋白表达的沉默效应,应用Transwell小室细胞侵袭实验检测 A375细胞侵袭能力。结果 构建的三对shRNA表达载体分别为pGPU6/GFP/Neo-shRNA336、pGPU6/GFP/Neo-shRNA453及pGPU6/GFP/Neo-shRNA680，应用荧光定量PCR和Western blot检测转染人黑色素瘤A375细胞后靶基因RhoC mRNA和蛋白表达水平分别为1.47±0.26、1.13±0.16、1.39±0.11和70.98±9.21、50.67±6.06、65.77±4.06，RhoC453为最佳干扰序列。成功构建了靶向RhoC的pLenti6.3-EGFP-453慢病毒表达载体，经测序证实序列正确；将构建的pLenti6.3-EGFP-453慢病毒表达载体感染A375细胞，应用荧光定量PCR和Western blot检测RhoC mRNA和蛋白的表达水平分别为1.05?0.05、62.04?15.86，而阴性对照慢病毒组、正常对照组RhoC mRNA和蛋白分别为4.21±0.24、220.86±24.07和4.63±0.32、257.39±12.30，Transwell小室细胞侵袭实验结果显示pLenti6.3-EGFP-453慢病毒组穿膜细胞数为40.41±8.88，阴性对照慢病毒组、正常对照组分别为64.82±13.48和77.46±6.47，慢病毒表达载体组较对照组相比较差异具有统计学意义（P＜0.05）。结论 成功构建的靶向RhoC基因的pLenti6.3-EGFP-453慢病毒表达载体可高效感染体外培养的人黑色素瘤A375细胞，并可显著抑制靶基因RhoC的表达和细胞的侵袭能力。

Construction and identification of RNA interference lentivirus vector targeting RhoC gene of melanoma cells

Background Melanoma has the highest mortality among all superficial malignant tumors. The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets. As a molecular switch that controls tumor metastasis, Ras homology C (RhoC) has been correlated with tumor progression, especially tumor invasion and metastasis. However, little research has been done about the effects of RNA interference (RNAi) targeting RhoC on the invasion and metastasis of melanoma. In this study, we constructed an RNAi lentivirus vector targeting RhoC gene of melanoma cells and identified its silencing effects on RhoC gene.Methods Based on RhoC gene encoding information, 3 pGPU6/GFP/Neo-short hairpin (shRNA) plasmids were constructed. After detecting their silencing effects on RhoC gene of A375 cells, the most effective pGPU6/GFP/Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453, which was used to infect A375 cells, and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting, meanwhile, the invasive capacity of cells were detected by Transwell assay.Results The plasmids pGPU6/GFP/Neo-shRNA 336, pGPU6/GFP/Neo-shRNA 453, and pGPU6/GFP/Neo-shRNA 680 were constructed. After they were transfected into A375 cells, the expressions of RhoC mRNA and protein were 1.47±0.26, 1.13±0.16, 1.39±0.11 and 70.98±9.21, 50.67±6.06, 65.77±4.06, respectively. pGPU6/GFP/Neo-shRNA 453 was the most effective sequence, and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC. pLenti6.3-EGFP-453 was used to infect A375 cells. The expression of RhoC mRNA and protein were 1.05±0.05 and 62.04±15.86 in lentivirus group, 4.21±0.24 and 220.86±24.07 in negative lentivirus control group, and 4.63±0.32 and 257.39±12.30 in the normal control group, respectively, in addition, the migrated cells were 40.41±8.88, 64.82±13.48 and 77.46±6.47 in the pLenti6.3-EGFP-453 group, negative lentivirus and normal control groups, with the difference between the lentivirus group and control groups statistically significant (P<0.05).Conclusions The successfully constructed pLenti6.3-EGFP-453 targeting RhoC can effectively infect human melanoma A375 cells in vitro, and significantly inhibit RhoC expression and invasion of A375 cells.