Li Hong-yu, Zhu Tao, Zhou Jin-hua, Xu Qian, Wang Shi-xuan, Bai Xiang-yang, Lu Yun-ping, Ma Ding
Molecular Cancer Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhonghua Fu Chan Ke Za Zhi. 2006 Jun;41(6):417-21.
To study the effect of short hairpin RNA (shRNA) on the expression of Pin1 mRNA and protein and its influence on the proliferation and apoptosis in HeLa cell lines.
The recombinant plasmid pSIREN-Pin1 expressing Pin1-targeted shRNA was constructed and then transfected into cervical cancer cell lines by lipofectamine (HeLa/p-shRNA), the control vector pSIREN-Con (HeLa/p-Con) and culture media (HeLa) as control, respectively. The Pin1 mRNA and protein expression were detected by RT-PCR and immunoblotting. The proliferation of cells after transfection was detected by methyl thiazolyl tetrazolium (MTT) and colony formation in soft agar. Flow cytometry (FCM) was used to test the apoptosis of cells marked with fluorescein isothiocyanate (FITC)-annexin V.
After transfected with pSIREN-Pin1 for 48 h, the expression of Pin1 mRNA and protein in HeLa cells were 0.19 +/- 0.05 and 0.33 +/- 0.14 respectively. Compared with HeLa/p-Con cells (0.84 +/- 0.16, 0.79 +/- 0.17) and HeLa cells (0.89 +/- 0.11, 0.81 +/- 0.15), the difference was significant respectively (P < 0.05). The proliferation was suppressed significantly in HeLa/p-shRNA cells. The colony ratios are as follows: HeLa/p-shRNA, (12 +/- 3)%; HeLa/p-Con, (20 +/- 5)%; HeLa, (24 +/- 4)% (P < 0.05). The apoptosis ratio was (24.3 +/- 5.7)% in HeLa/p-shRNA cells. It was significantly higher than that in HeLa/p-Con cells (5.0 +/- 1.4)% and HeLa cells (1.8 +/- 0.4)% (P < 0.05).
RNA interference through Pin1-targeted shRNA can effectively inhibit the expression of target gene, and might be potentially useful in gene therapy of Pin1 related cervical cancers. Silencing Pin1 gene can suppress the proliferation and promote the apoptosis in HeLa cells.
研究短发夹RNA（shRNA）对Pin1 mRNA和蛋白表达的影响及其对HeLa细胞系增殖和凋亡的作用。
构建表达靶向Pin1的shRNA的重组质粒pSIREN-Pin1，然后分别用脂质体转染至宫颈癌细胞系（HeLa/p-shRNA），以对照载体pSIREN-Con（HeLa/p-Con）和培养基（HeLa）作为对照。采用逆转录-聚合酶链反应（RT-PCR）和免疫印迹法检测Pin1 mRNA和蛋白表达。转染后通过甲基噻唑基四氮唑（MTT）法和软琼脂集落形成实验检测细胞增殖。采用流式细胞术（FCM）检测异硫氰酸荧光素（FITC）-膜联蛋白V标记的细胞凋亡情况。
用pSIREN-Pin1转染HeLa细胞48小时后，HeLa细胞中Pin1 mRNA和蛋白表达分别为0.19±0.05和0.33±0.14。与HeLa/p-Con细胞（0.84±0.16，0.79±0.17）和HeLa细胞（0.89±0.11，0.81±0.15）相比，差异均有统计学意义（P<0.05）。HeLa/p-shRNA细胞的增殖受到显著抑制。集落比例如下：HeLa/p-shRNA为（12±3）%；HeLa/p-Con为（20±5）%；HeLa为（24±4）%（P<0.05）。HeLa/p-shRNA细胞的凋亡率为（24.3±5.7）%，显著高于HeLa/p-Con细胞（5.0±1.4）%和HeLa细胞（1.8±0.4）%（P<0.05）。
通过靶向Pin1的shRNA进行RNA干扰可有效抑制靶基因表达，可能在Pin1相关宫颈癌的基因治疗中具有潜在应用价值。沉默Pin1基因可抑制HeLa细胞增殖并促进其凋亡。
