Lü Qiong-ying, Zhang Wei, Cheng Jing, Zhang Wen-ting, Zhong Ya-juan
Department of Gynecology, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Zhonghua Fu Chan Ke Za Zhi. 2013 Jul;48(7):532-7.
Design and synthesize short hairpin RNA (shRNA) expression vector of RNA for specific silencing of heparanase (HPA) gene, screened plasmid which silence effects is the best. Observe the function of cell invasion after inhibiting the expression of HPA in cervical carcinoma cell lines (HeLa).
The genomic sequence of HPA gene was retrieved from GenBank database. Designed four pairs of specific oligonucleotide sequences and a negative control according to the shRNA design principles. They were inserted into the vector pYr-1.1, vectors, and transfected into HeLa cells via lipofectamine. Reverse transcription(RT)-PCR and immunofluorescence were employed to detect the expression of HPA gene in the transfected cells at the mRNA and protein levels, respectively. The plasmid were screened and transfected into HeLa cells, then transwell small room stromal invasion experiment were employed to observe the cervical carcinoma cell invasion.
RT-PCR results of transfected HeLa cells shown that the mRNA amplification multiples were 0.54 ± 0.05 in the HPA-592 group, 0.89 ± 0.18 in HPA-995 group, 0.82 ± 0.22 in the HPA-1351 group, 0.91 ± 0.47 in HPA-1658 group. While, they were 1.31 ± 0.72 and 1.09 ± 0.16 in negative control and blank control group, respectively. Green fluorescence was visible in the cytoplasm, which indicated that the HPA protein was expressed in the cytoplasm, of them the weakest green fluorescence in the HPA-592 group . The relative numbers of invasive cells among the HeLa cells were as follows: 182 ± 6 in the blank control group, 258 ± 17 in the negative control group, and 44 ± 4 in the HPA-592-specific interference group (P < 0.01) .
Successfully screened shRNA vector targeting human HPA, efficiently inhibit expression of HPA gene when transfected into HeLa cells, and significantly reduced the invasion capacity of cervical carcinoma cells.
设计并合成针对乙酰肝素酶（HPA）基因特异性沉默的RNA短发夹RNA（shRNA）表达载体，筛选沉默效果最佳的质粒。观察宫颈癌细胞系（HeLa）中HPA表达被抑制后细胞侵袭功能的变化。
从GenBank数据库检索HPA基因的基因组序列。根据shRNA设计原则设计四对特异性寡核苷酸序列和一个阴性对照。将它们插入载体pYr-1.1中，构建载体，并通过脂质体转染HeLa细胞。分别采用逆转录（RT）-PCR和免疫荧光法在mRNA和蛋白质水平检测转染细胞中HPA基因的表达。筛选质粒并转染HeLa细胞，然后采用Transwell小室基质侵袭实验观察宫颈癌细胞的侵袭情况。
转染HeLa细胞的RT-PCR结果显示，HPA-592组mRNA扩增倍数为0.54±0.05，HPA-995组为0.89±0.18，HPA-1351组为0.82±0.22，HPA-1658组为0.91±0.47。而阴性对照组和空白对照组分别为1.31±0.72和1.09±0.16。细胞质中可见绿色荧光，表明HPA蛋白在细胞质中表达，其中HPA-592组绿色荧光最弱。HeLa细胞中侵袭细胞的相对数量如下：空白对照组为182±6，阴性对照组为258±17，HPA-592特异性干扰组为44±4（P<0.01）。
成功筛选出靶向人HPA的shRNA载体，转染HeLa细胞后能有效抑制HPA基因表达，并显著降低宫颈癌细胞的侵袭能力。
