Zheng Xiaochun, Zhang Tingting, Wu Jingfang, Zhang Wenjing, Zhang Jing, Wang Baozhi
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2015 Jul;29(13):1194-8.
To explore the effect on proliferation and invasion of human papillary thyroid carcinoma K1 cells by application of small hairpin RNA (shRNA) silencing TFF3 gene expression.
Using liposome transfection method, TFF3-shRNA targeting of TFF3 gene will be transient transfected to papillary thyroid carcinama K1 cells, inducing the corresponding gene silencing. The experiment set up blank control group (Con group), negative control group (ConNC group) and interference group (TFF3-shRNA group). The TFF3 protein and mRNA expression were evaluated by RT-PCR, Real time-PCR, immunocytochemistry and Western blot in K1 cells after TFF3-shRNA transfected. CCK-8 method and Scratch test were used to detect the change of proliferation ability and invasion ability respectively.
(1) The recombinant plasmid Ca # HSH018037-4-HIVmU6 carrying TFF3-shRNA transfected K1 cells successfully. (2) RT-PCR and Real time-PCR detected the expression of TFF3 mRNA, which was 0.38 ± 0.11 times as many as the blank control group (P < 0.01) after TFF3 gene silenced. But the negative control group was 1.082 times of blank control group (P > 0.05). (3) Western blot show that after TFF3 gene silence induced TFF3 protein expression levels have decreased 59.5% (P < 0.01), The difference was statistically significant compared with the blank control group. (4) Cell scratch detects K1 cell invasion ability. The invasion ability of K1 cells in interference group (TFF3-shRNA group) reduced. The scratch width significantly decreased 57.1% than blank control group (P < 0.01). (5) CCK-8 kit detect cell proliferation ability. K1 cells grow significantly slower in the interference group (TFF3-shRNA group) than the blank control group through the analysis of the growth curve (P < 0.01). In the interference group (TFF3-shRNA group) proliferation inhibition rate of K1 cells at 6 h, 12 h, 24 h and 36 h, 48 h are 16.6%, 26.6%, 33.6%, 33.8%, 35.0% respectively. Compared with negative control group, proliferation ability of K1 cell decreased significantly.
Silenced TFF3 gene can cause the degradation of mRNA, reduce the protein translation , and inhibit the invasion and proliferation ability of K1 cell.
探讨应用小发夹RNA（shRNA）沉默三叶因子3（TFF3）基因表达对人甲状腺乳头状癌K1细胞增殖和侵袭的影响。
采用脂质体转染法，将靶向TFF3基因的TFF3-shRNA瞬时转染至甲状腺乳头状癌K1细胞，诱导相应基因沉默。实验设空白对照组（Con组）、阴性对照组（ConNC组）和干扰组（TFF3-shRNA组）。转染TFF3-shRNA后，采用RT-PCR、实时荧光定量PCR、免疫细胞化学和蛋白质印迹法检测K1细胞中TFF3蛋白和mRNA表达。分别采用CCK-8法和划痕试验检测细胞增殖能力和侵袭能力的变化。
（1）携带TFF3-shRNA的重组质粒Ca#HSH018037-4-HIVmU6成功转染K1细胞。（2）RT-PCR和实时荧光定量PCR检测TFF3基因沉默后TFF3 mRNA表达，其表达量为空白对照组的0.38±0.11倍（P<0.01）。而阴性对照组为空白对照组的1.082倍（P>0.05）。（3）蛋白质印迹法显示，TFF3基因沉默后诱导TFF3蛋白表达水平下降59.5%（P<0.01），与空白对照组比较差异有统计学意义。（4）细胞划痕检测K1细胞侵袭能力，干扰组（TFF3-shRNA组）K1细胞侵袭能力降低，划痕宽度比空白对照组显著降低57.1%（P<0.01）。（5）CCK-8试剂盒检测细胞增殖能力，通过生长曲线分析，干扰组（TFF3-shRNA组）K1细胞生长明显慢于空白对照组（P<0.01）。干扰组（TFF3-shRNA组）K1细胞在6 h、12 h、24 h、36 h、48 h的增殖抑制率分别为16.6%、26.6%、33.6%、33.8%、35.0%。与阴性对照组比较，K1细胞增殖能力明显下降。
沉默TFF3基因可导致mRNA降解，减少蛋白质翻译，抑制K1细胞的侵袭和增殖能力。
