慢病毒介导shRNA特异性沉默促肝细胞再生磷酸酶1基因表达抑制舌癌细胞迁移侵袭能力

Suppressing migration and invasion potencies of tongue carcinoma cells by lentiviral victor mediated sh RNA specially silencing of phosphatase of regenerating liver cell-1 gene expression

目的:探讨特异性沉默促肝细胞再生磷酸酶1(phosphatase of regenerating liver cell-1,PRL-1)基因表达对舌癌细胞迁移、侵袭能力的影响。方法:设计、合成5条针对PRL-1基因的短发夹RNA(short hairpin RNA,sh RNA)干扰序列,构建于慢病毒载体质粒中,PCR电泳及基因测序验证。转染293T细胞,Western blot筛选最佳干扰序列,包装产生慢病毒。转染TCA8113细胞,筛选、建立稳定转染细胞株;real-time PCR检测PRL-1基因沉默效率;划痕实验和Transwell实验分别比较慢病毒转染细胞(KD组)、空病毒转染细胞(NC组)及TCA8113细胞(CON组)迁移、侵袭能力变化。结果:PCR电泳及基因测序证实5条sh RNA序列定向插入慢病毒载体质粒中。Western blot筛选出PRL-1-sh RNA-1基因沉默效率最高,包装获得慢病毒滴度为7×108 TU/ml。通过转染、筛选,建立了TCA8113细胞稳定转染细胞株;real-time PCR测得PRL-1基因m RNA表达明显下降(F=809.120,P=0.000);PRL-1基因沉默后细胞迁移能力降低,穿过人工基底膜的细胞数KD组(25.5±0.4)明显少于NC组(81.5±2.0)和CON组(88.5±2.3)(F=1 092.970,P=0.000)。结论:沉默PRL-1基因表达能有效抑制舌癌细胞迁移、侵袭能力。

Objective：To study the effect of specially silencing of phosphatase of regenerating liver cell-1 （PRL-1 ） gene expression on migration and invasion potencies of tongue carcinoma cells. Methods：Five interfering sequences of shRNA targeting PRL-1 gene were designed,synthesized and constructed in corresponding plasmids and were confirmed by PCR electrophoresis and DNA sequencing. The most effective sequence of shRNA was screened by Western blot and the lentivirus was produced and packaged. TCA8113 cells were infected with recombinant lentivirus and the stably transfected cells were filtered and obtained. The silencing efficiency of PRL- 1 gene was assessed by real-time PCR. The migration and invasion potencies of PRL-1 gene silenced cells were analyzed by wound healing and Transwell invasion assay. Results：PCR electrophoresis and DNA sequencing revealed that 5 shRNA sequences were directionally constructed in plasmids. PRL-1-shRNA-1 was the most effective interference sequence selected by Western blot. The stably infected TCA8113 cells were established after interfering and filtering. The expression of PRL-1 gene mRNA was decreased significantly （F=809.120,P=0.000）. After silencing the PRL-1 gene expression, the migration and invasion potencies of TCA8113 ceils were significantly suppressed（F=1 092.970,P=0.000）. Conclusion：Silencing of PRL-1 gene expression can inhibit the migration and invasion potencies of TCA8113 cells significantly.