微小RNA-145通过靶向锌指蛋白转录因子5抑制甲状腺乳头状癌K1细胞的增殖及侵袭

MicroRNA-145 inhibits the proliferation and invasion of thyroid papillary carcinoma K1 cells by targeting zinc finger protein transcription factor 5

目的观察微小RNA(miRNA,miR)-145通过调节锌指蛋白转录因子5(KLF5)对甲状腺乳头状癌K1细胞增殖及侵袭能力的影响。方法利用TargetScan和PicTar对人甲状腺乳头状癌K1细胞株的miR-145靶基因进行预测,采用双荧光素酶报告基因验证miR-145对靶基因的直接调控。应用细胞计数试剂盒(CCK-8)检测转染miR-145模拟物及miR-145模拟物同时过表达KLF5的甲状腺乳头状癌K1细胞在4 d的吸光度(A450)值以判断其增殖能力。应用平板克隆形成实验检测转染miR-145模拟物及miR-145模拟物同时过表达KLF5的甲状腺乳头状癌K1细胞孵育2周后克隆细胞的数量以判断其克隆能力。应用Transwell侵袭实验检测转染miR-145模拟物及miR-145模拟物同时过表达KLF5的甲状腺乳头状癌K1细胞孵育48 h后穿膜细胞数以判断其侵袭能力。结果TargetScan和PicTar软件预测显示KLF5基因是miR-145的潜在靶基因。双荧光报告基因的相对荧光值显示,与共转染miR-145模拟物和KLF5-mt组比较,共转染miR-145模拟物和KLF5-wt组荧光素酶活性显著下降(t=14.907,P<0.01)。CCK-8实验结果显示,miR-145模拟物转染组K1细胞在4 d检测到的A450 nm值(0.433±0.003)明显低于阴性对照组及共转染miR-145模拟物和过表达KLF5组(0.534±0.004、0.522±0.005),差异有统计学意义(t=29.954、22.816,P<0.01)。而阴性对照组与共转染miR-145模拟物和过表达KLF5组比较,差异无统计学意义(t=3.035,P>0.05)。平板克隆实验结果显示,miR-145模拟物转染组K1细胞克隆的形成数[(67.00±3.52)个]明显低于阴性对照组及共转染miR-145模拟物和过表达KLF5组[(113.00±4.16)、(109.00±2.52)个],差异有统计学意义(t=10.281、12.124,P<0.01)。而阴性对照组与共转染miR-145模拟物和过表达KLF5组比较,差异无统计学意义(t=2.774,P>0.05)。Transwell侵袭实验结果显示,miR-145模拟物转染组K1细胞穿过基底膜细胞数[(54.00±1.15)个]明显低于阴性对照组及共转染miR-145模拟物和过表达KLF5组[(127.00±4.58)、(119.00±4.51)个],差异有统计学意义(t=35.839、23.579,P<0.01)。而阴性对照组与共转染miR-145模拟物和过表达KLF5组比较,差异无统计学意义(t=1.906,P>0.05)。结论miR-145通过靶向调节KLF5抑制甲状腺乳头状癌K1细胞的增殖及侵袭。

Objective To observe the effect of miRNA (miR)-145 on the proliferation and invasion abilities of thyroid papillary carcinoma K1 cells by regulating zinc finger protein transcription factor 5 (KLF5). Methods The target gene of miR-145 in human papillary thyroid carcinoma K1 cell line was predicted by TargetScan and PicTar. The double luciferase reporter gene was used to verify the direct regulation of miR-145 on the target gene. Cell counting kit (CCK-8) was used to detect the A 450 value of papillary thyroid carcinoma K1 cell with miR-145 mimics and miR-145 mimics combining KLF5 over-expressed at 4d to determine the proliferation ability. Clone formation assay was used to detect the number of cloned cells of papillary thyroid carcinoma K1 cell with miR-145 mimics and miR-145 mimics combining KLF5 over-expressed after incubation for 2 weeks to determine the clone formation ability. Transwell assay was used to detect the number of cells passing through the basement membrane of papillary thyroid carcinoma K1 cell with miR-145 mimics and miR-145 mimics combining KLF5 over-expressed after incubation for 48 h to determine the invasion ability. Results TargetScan and PicTar software predict that KLF5 gene is the potential target gene of miR-145. The relative fluorescence values of the double fluorescent reporter gene showed that the activity of luciferase in the KLF5 wild type group (KLF5-wt) transfected with miR-145 mimics was significantly lower than that of the KLF5mutant type group (KLF5-mt) transfected with miR-145 mimics (t=14.907, P<0.01). CCK-8 assay results showed that the A450 nm value detected on the fourth day of K1 cells in the miR-145 mimics transfected group (0.433±0.003) was significantly lower than that in the negative control group and miR-145 mimics combining KLF5 over-expressed group (0.534±0.004, 0.522±0.005), the difference was statistically significant (t=29.954, 22.816, P<0.01). However, there was no significant difference between the negative control group and miR-145 mimics combining KLF5 over-expressed group (t=3.035, P>0.05). The clone formation assay results showed that the numbers of K1 cells clone in the miR-145 mimics transfected group (67.00±3.52) was significantly lower than that in the negative control group and miR-145 mimics combining KLF5 over-expressed group (113.00±4.16, 109.00±2.52), the difference was statistically significant (t=10.281, 12.124, P<0.01). However, there was no significant difference between the negative control group and miR-145 mimics combining KLF5 over-expressed group (t=2.774, P>0.05). The Transwell assay demonstrated that the numbers of K1 cells passing through the basement membrane in the miR-145 mimics transfected group [(54.00±1.15) cells] was significantly lower than that in the negative control group and miR-145 mimics combining KLF5 over-expressed group [(127.00±4.58),(119.00±4.51) cells], the difference was statistically significant (t=35.839, 23.579, P<0.01). However, there was no significant difference between the negative control group and miR-145 mimics combining KLF5 over-expressed group (t=1.906, P>0.05). Conclusion MiR-145 inhibits the proliferation and invasion of thyroid papillary carcinoma K1 cells by targeting the regulation of KLF5.