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

发布时间：2018-04-05 22:35

本文选题：慢病毒载体　切入点：短发夹RNA　出处：《重庆医科大学学报》2014年11期

【摘要】：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。
[Abstract]:Aim: to investigate the effect of specific silencing on the expression of 1(phosphatase of regenerating liver cell-1 (PRL-1) gene on the migration and invasion of tongue cancer cells.Methods: five interference sequences of short hairpin RNA(short hairpin RNAs targeting PRL-1 gene were designed and synthesized, and were constructed into lentivirus vector plasmids by polymerase chain reaction electrophoresis and gene sequencing.Western blot was transfected into 293T cells to screen the best interference sequence, and the lentivirus was produced by packaging.After transfection of TCA8113 cells, a stable transfection cell line real-time PCR was established to detect the silencing efficiency of PRL-1 gene. Scratch assay and Transwell assay were used to compare the effect of PRL-1 gene silencing on lentivirus-transfected cells (KD group, empty virus transfected cell group (NC group) and TCA8113 cell group (Con group)).Change in invasiveness.Results 5 sh RNA sequences were inserted into the plasmid of lentivirus vector. Western blot showed that PRL-1-sh RNA-1 gene silencing efficiency was the highest, and the lentivirus titer was 7 脳 108TU / ml.Conclusion: silencing the expression of PRL-1 gene can effectively inhibit the migration and invasion of tongue cancer cells.
【作者单位】：重庆医科大学附属口腔医院口腔疾病与生物医学重庆市重点实验室;
【基金】：重庆市卫生局重点资助项目(编号:2013-1-030)
【分类号】：R739.86

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