LncRNA NEAT1 knockdown attenuates autophagy to elevate 5-FU sensitivity in colorectal cancer via targeting miR-34a
LncRNA NEAT1 knockdown attenuates autophagy to elevate 5-FU sensitivity in colorectal cancer via targeting miR-34a
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DOI：
10.1002/cam4.2746
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发表时间：
2019-12-05
期刊：
影响因子：
4
通讯作者：
Liu, Shao-Jun
中科院分区：
文献类型：
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作者：
Liu, Fen;Ai, Fei-Yan;Liu, Shao-Jun
背景：结直肠癌是一种常见的恶性肿瘤。越来越多的证据表明，结直肠癌对5-氟尿嘧啶(5-FU)耐药，预后不良。在这项研究中，我们旨在研究长非编码RNA核副斑点组装转录本1(LncRNA NEAT1)对结直肠癌细胞存活率、5-FU敏感性和自噬的影响。方法用四甲基偶氮唑盐((3-(4，5-dimethyl-2-thiazolyl)-2，5-diphenyl-2-Htetrazolium)法检测细胞活力，免疫荧光染色检测自噬斑点，荧光素酶报告系统检测miR-34a与NEAT1或可能靶点的结合能力。另外，用qRT-PCR和Western blotting分别检测mRNAs和蛋白质的表达。结果NEAT1在结直肠癌组织和细胞中的表达增加，与miR-34a的表达呈负相关。此外，NEAT1基因敲除显著抑制了结直肠癌细胞的增殖，并增强了5-FU的敏感性。结果表明，NEAT1基因敲除抑制了Lc3点的表达，抑制了Beclin-1、ULK1和LC3II/I的表达，miR-34a的过表达趋势与NEAT1基因敲除相似。MIR-34a被证实可以靶向HMGB1、ATG9A和ATG4B的3‘-UTR端可能的结合位点，这些结合位点参与自噬的激活。抑制miR-34a或过表达HMGB1可有效逆转NEAT1基因敲除后5-FU敏感性的升高。此外，3-MA还逆转了NEAT1过表达诱导的HT29细胞耐药。结论ncRNA NEAT1可靶向miR-34a，促进自噬，促进结直肠癌5-FU耐药。
Backgrounds Colorectal carcinoma (CRC) is a common malignant tumor. Increasing evidences indicated that CRC showed a resistance to 5-fluorouracil (5-FU) and further resulted in a poor prognosis. In this study, we aim to investigate the effect of long noncoding RNA nuclear paraspeckle assembly transcript 1 (LncRNA NEAT1) on cell viability, sensitivity to 5-FU, and autophagy of CRC cell lines. Methods MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide) was used to detect cell viability, immunofluorescent staining was used to detect autophagy puncta, and luciferase reporter system was used to determine binding ability between miR-34a and NEAT1 or putative targets. Additionally, indicated mRNAs and protein expressions were determined by qRT-PCR or western blotting, respectively. Results We found that NEAT1 expression was increased in CRC tissues and cells, which showed a negative correlation with miR-34a expression. In addition, NEAT1 knockdown noticeably inhibited the proliferation of CRC cells and enhanced 5-FU sensitivity. It revealed that NEAT1 knockdown suppressed the LC3 puncta and the expressions of Beclin-1, ULK1, and ratio of LC3II/I. Overexpression of miR-34a showed similar trends with NEAT1 knockdown. miR-34a was validated to target the putative binding sites in 3 '-UTR of HMGB1, ATG9A, and ATG4B, which are involved in the activation of autophagy. Inhibition of miR-34a or overexpression of HMGB1 could effectively reverse elevated 5-FU sensitivity upon NEAT1 knockdown. In addition, 3-MA reversed NEAT1 overexpression-induced resistance in HT29 cells. Conclusion These findings indicate that LncRNA NEAT1 could target miR-34a and promote autophagy to facilitate 5-FU chemoresistance in CRC.