敲降miR-222-3p靶向PTEN对甲状腺癌131I放疗抵抗的影响及其机制

doi:10.13481/j.1671-587X.20210318

摘要/Abstract

摘要： 目的

探讨miR-222-3p通过人第10号染色体缺失的磷酸酶和张力蛋白合同源基因（PTEN）对甲状腺癌细胞增殖和凋亡的影响，阐明miR-222-3p在甲状腺癌¹³¹I放疗抵抗中的作用及机制。

方法

采用实时荧光定量PCR（RT-qPCR）法检测人甲状腺癌细胞和人正常甲状腺滤泡上皮Nth-ori 3-1细胞中miR-222-3p表达水平。以人甲状腺癌K1细胞和放射抗性K1（K1R）细胞为研究对象，实验分为空白对照组、¹³¹I处理组、¹³¹I+miR-222-3p敲降组、¹³¹I+PTEN过表达组和¹³¹I+PTEN过表达+miR-222-3p过表达组。空白对照组K1和K1R细胞不经任何处理，¹³¹I处理组K1和K1R细胞经1和3 Gy ¹³¹I处理，¹³¹I+miR-222-3p敲降组K1和K1R细胞转染miR-222-3p inhibitor后再经1和3 Gy ¹³¹I处理，¹³¹I+PTEN过表达组K1和K1R细胞转染PTEN过表达载体后再经1 和3 Gy ¹³¹I处理，¹³¹I+PTEN过表达+miR-222-3p过表达组K1和K1R细胞同时转染PTEN过表达载体和miR-222-3p mimic后再经1和3 Gy ¹³¹I处理。克隆形成实验检测K1和K1R细胞克隆形成数，CCK-8实验检测K1和K1R细胞增殖活性，Annexin Ⅴ-FITC/PI双染法流式细胞术检测K1和K1R细胞凋亡率，Western blotting检测K1和K1R细胞中PTEN蛋白表达水平，双荧光素酶报告基因实验验证miR-222-3p与PTEN的靶向关系。

结果

与Nth-ori 3-1细胞比较，甲状腺癌细胞中miR-222-3p表达水平升高（P<0.01）；且甲状腺癌K1R细胞中miR-222-3p表达水平高于甲状腺癌K1细胞（P<0.01）。与¹³¹I处理组比较，¹³¹I+miR-222-3p敲降组K1和K1R细胞克隆形成数减少（P<0.01），细胞增殖活性降低（P<0.01），细胞凋亡率升高（P<0.01）。与miR-NC-PTEN-WT组比较，miR-222-3p-PTEN-WT组HEK-293细胞中荧光素酶活性降低（P<0.01）。与敲降前比较，敲降miR-222-3p后K1和K1R细胞中PTEN蛋白表达水平升高（P<0.01）。与¹³¹I +PTEN过表达组比较，¹³¹I+PTEN＋miR-222-3p过表达组K1和K1R细胞克隆形成数增加（P<0.01），细胞增殖活性升高（P<0.01），细胞凋亡率降低（P<0.01）。

结论

敲降miR-222-3p靶向PTEN并上调其表达水平可以缓解甲状腺癌¹³¹I的放疗抵抗。

关键词: miR-222-3p, 人第10号染色体缺失的磷酸酶和张力蛋白合同源基因, 甲状腺肿瘤, ¹³¹I放疗抵抗

Abstract: Objective

To investigate the effect of miR-222-3p on the proliferation and apoptosis of thyroid cancer cells via regulating gene of phosphatase and tension homolog deleted on chromosome ten（PTEN），and to elucidate the role of miR-222-3p in ¹³¹I radiotherapy resistance of thyroid cancer cells and its mechanism.

Methods

The expression levels of miR-222-3p in the human thyroid cancer cells and human normal thyroid follicular epithelial Nth-ori 3-1 cells were determined by Real-time fluorescence quantitative PCR（RT-qPCR） method.The thyroid cancer K1 cells and ¹³¹I resistance of thyroid cancer K1R cells were used as the subjects and were divided into blank control group， ¹³¹I treatment group， ¹³¹I+miR-222-3p knockdown group， ¹³¹I+PTEN over-expression group，and ¹³¹I+PTEN over-expression+miR-222-3p over-expression group. The K1 cells and KIR cells in blank control group didn’t receive any treatment； both the K1 and K1R cells in ¹³¹I treatment group were treated with ¹³¹I at doses of 1 and 3 Gy； the cells in ¹³¹I+miR-222-3p knockdown group were transfected with miR-222-3p inhibitor prior to ¹³¹I treatment at dose of 1 and 3 Gy； the cells in ¹³¹I+PTEN over-expression group were transfected with pcDNA3.1-PTEN prior to ¹³¹I treatment with doses of 1 and 3 Gy； the cells in ¹³¹I+PTEN over-expression+miR-222-3p over-expression group were simultaneously transfected with pcDNA3.1-PTEN and miR-222-3p mimic prior to ¹³¹I treatment with doses of 1 and 3 Gy. Clony formation assay was used to detect the clony formation number of K1 and K1R cells；CCK-8 assay was used to detect the proliferation activities of K1 and K1R cells；Annexin Ⅴ-FITC / PI double staining method was used to detect the apoptotic rates of K1 and K1R cells；Western blotting method was used to detect the expression levels of PTEN protein in K1 and K1R cells； Double luciferase reporter gene assay was used to verify the targeting relationship between miR-222-3p and PTEN.

Results

Compared with the Nth-ori 3-1 cells，the expression levels of miR-222-3p in thyroid carcinoma cells were increased （P<0.01）； the expression level of miR-222-3p in the thyroid cancer K1R cells was higher than that in thyroid cancer K1 cells （P<0.01）. Compared with ¹³¹I treatment group， the clony formation number of the K1 and K1R cells in ¹³¹I + miR-222-3p knockdown group was decreased（P<0.01）， the proliferation activities of K1 and K1R cells were decreased （P<0.01），and the apopotic rates were increased（P<0.01）. Compared with miR-NC-PTEN-WT group， the luciferase activity of HEK-293 cells in miR-222-3P-PTEN-WT group was decreased （P<0.01）. Compared with before knockdown， the expression level of PTEN protein in K1 and K1R cells were significantly increased after knockdown（P<0.01）. Compared with ¹³¹I + PTEN over-expression group， the colony formation number of K1 and K1R cells in ¹³¹I +PTEN over-expression+miR-222-3p over-expression group was increased （P<0.01）， the proliferation activities were increased （P<0.01）， and the apoptotic rate was decreased （P<0.01）.

Conclusion

Knockdown of miR-222-3p targeting PTEN and up-regulating its expression level can alleviate the ¹³¹I radiotherapy resistance of thyroid cancer cells.

Key words: miR-222-3p, gene of phosphatase and tension homolog deleted on chromosome ten, thyroid neoplasms, ¹³¹I radiotherapy resistance

中图分类号:

R736.1

冯曜宇, 张承磊, 侯丽娟, 王益夫, 吴秀玲, 马云海. 敲降miR-222-3p靶向PTEN对甲状腺癌¹³¹I放疗抵抗的影响及其机制[J]. 吉林大学学报(医学版), 2021, 47(3): 677-686.

Yaoyu FENG, Chenglei ZHANG, Lijuan HOU, Yifu WANG, Xiuling WU, Yunhai MA. Effect of miR-222-3p knockdown targeting PTEN on ¹³¹I radiotherapy resistance of thyroid cancer and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2021, 47(3): 677-686.

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Primer	Sequence
miR-222-3p	F: 5'-GGGGAGCTACATCTGGCT-3'
miR-222-3p	R: 5'-TGCGTGTCGTGGAGTC-3'
U6	F: 5'-GCTTCGGCAGCACATATACTAAAAT-3'
U6	R: 5'-CGCTTCACGAATTTGCGTGTCAT-3'

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敲降miR-222-3p靶向PTEN对甲状腺癌¹³¹I放疗抵抗的影响及其机制

Effect of miR-222-3p knockdown targeting PTEN on ¹³¹I radiotherapy resistance of thyroid cancer and its mechanism

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