沉默lncRNA HOTTIP通过上调miR-663a表达增加非小细胞肺癌细胞系放射敏感性

目的探讨lncRNA HOTTIP通过调控miR-663a表达对4个非小细胞肺癌细胞系放射敏感性影响。方法用0、2、4、6、8 Gy分别照射H838、H157、A549、H1299细胞系,采用克隆形成实验检测细胞存活情况。qRT-PCR检测细胞中HOTTIP和miR-663a表达水平。以A549、H1299细胞为研究对象,沉默HOTTIP表达、过表达miR-663a后用克隆形成实验检测细胞存活情况。流式细胞术检测细胞凋亡情况,Western blot检测Cleaved caspase-3、Cleaved PARP和γ-H2AX表达。双荧光素酶报告基因实验和qRT-PCR检测验证HOTTIP和miR-663a的靶向关系。结果HOTTIP在放射耐受的H157、A549、H1299细胞中表达上调,miR-663a表达下调。沉默HOTTIP或过表达miR-663a均可抑制A549、H1299细胞存活(放射增敏比分别为1.562、1.507),促进Cleaved caspase-3、Cleaved PARP和γ-H2AX表达,促进放射照射诱导细胞凋亡。miR-663a是HOTTIP的靶基因,HOTTIP可负性调控miR-663a的表达。抑制miR-663a表达可逆转沉默HOTTIP对肺癌细胞系放射敏感性的影响。结论沉默HOTTIP通过上调miR-663a表达,抑制肺癌细胞系存活,促进其凋亡,从而提高肺癌细胞系的放射敏感性。

Silencing lncRNA HOTTIP affects radiosensitivity of non-small cell lung cancer cell lines by up-regulating miR-663a

Objective To investigate the effect of lncRNA HOTTIP on the radiosensitivity of four non-small cell lung cancer cell lines cultured in vitro by regulating the expression of miR-663a. Methods Four non-small cell lung cancer cell lines (H838, H157, A549, and H1299) were irradiated with different radiation intensities (0, 2, 4, 6, and 8 Gy). Cell survival was detected by colony formation assay. The expression levels of HOTTIP and miR-663a were detected by qRT-PCR. A549 and H1299 cells were selected for the subsequent experiment. After silencing HOTTIP and/or over-expressing miR-663a, cell survival was detected by colony formation assay. Cell apoptosis was detected by flow cytometry. The expression levels of Cleaved caspase-3, Cleaved PARP andγ-H2AX were quantitatively measured by Western blot. The targeting relationship between HOTTIP and miR-663a was vefiried by dual luciferase reporter assay and qRT-PCR. Results The expression of HOTTIP was up-regulated, whereas that of miR-663a was down-regulated in the radiation-resistant H157, A549 and H1299 cells. Silencing HOTTIP or over-expressing miR-663a inhibited the survival of A549 and H1299 cells (radiosensitization ratios were 1.562 and 1.507, respectively), promoted the expression of Cleaved caspase-3, Cleaved PARP and γ-H2AX, and accelerated cell apoptosis induced by radiation exposure. miR-663a was a target gene of HOTTIP, and HOTTIP negatively regulated the expression of miR-663a. The inhibition of miR-663a reversed the effect of silencing HOTTIP on the radiosensitivity of non-small cell lung cancer cells. Conclusion Silencing HOTTIP can suppress the survival of non-small cell lung cancer cells and promotes cell apoptosis by up-regulating the expression of miR-663a, thereby enhancing the radiosensitivity of non-small cell lung cancer cell lines.