稳定沉默Rictor表达的前列腺癌CWR22RV1的构建

Construct a prostate cancer cell line CWR 22RV1 with Rictor-shRNA

目的应用RNA沉默技术下调前列腺癌CWR22RV1细胞株的Rictor基因表达,并筛选具有稳定转染Rictor-shRNA的细胞株,为进一步研究Rictor在前列腺癌中的作用打下基础。方法针对Rictor基因设计合成siRNA序列;鉴定、测序并转染293T细胞观察基因表达情况;构建Rictor-shRNA慢病毒载体;进行Rictor-shRNA慢病毒包装及滴度测定;筛选稳定表达Rictor-shRNA的前列腺癌CWR22RV1细胞株;Western blot检测稳定转染前列腺癌CWR22RV1细胞株Rictor的表达水平。结果 Rictor-shRNA慢病毒成功包装后转染前列腺癌CWR22RV1细胞,进行嘌呤霉素筛选,获得沉默Rictor基因的前列腺癌CWR22RV1细胞株,Western blot检测显示该细胞株Rictor水平明显降低(P<0.01)。结论成功构建了稳定沉默Rictor表达的前列腺癌CWR22RV1细胞株,为后续的实验了打下坚实的基础。

Objective To establish a prostate cancer cell line CWR22RV1 with Rictor stably silenced by using RNAi, and provide a basis to further study the role of Rictor in prostate cancer. Methods We have designed and synthesized the subsequence of siRNA aimed to Rictor,and identify, sequence the subsequence of siRNA , and transfect 293T cells to observe the expression of Rictor. Construct a lentiviral vector carrying the Rictor-shRNA. We have packaged a lentiviral vector carry- ing the Rictor-sbRNA and viral titer is determined. Screening a prostate cancer cell line CWR22RV1 with Rictor stably silenced by using RNAi. The expression of Rictor in prostate cancer cell line CWR22RV1 with Rictor shRNA was examined by Western blot. Results We had successfully constructed a lentiviral vector carrying the Rictor-shRNA, and transfected into prostate cancer cell line CWR22RV1, screening prostate cancer cell line CWR22RV1 with Rictor-shRNA with puromycin, and obtained prostate cancer cell line CWR22RV1 with Rietor was stably silenced, the expression of Rictor in prostate cancer cell line CWR22RV1 with Rictor-shRNA was significant deceased （P〈0.01）. Conclusions We had successfully established a prostate caneer cell line CWR22RV1 with Rictor was stably silenced, which provided a basis to further study the prostate cancer.