低氧对胰腺癌PANC-1细胞株中TWIST表达的影响

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摘要目的: 探讨低氧条件对胰腺癌PANC-1细胞中TWIST表达的影响及可能的机制。方法：低氧浓度下培养PANC-1细胞，蛋白质印迹检测不同时间点TWIST蛋白的表达水平，免疫荧光检测TWIST、E-钙黏蛋白、 α-平滑肌肌动蛋白（αSMA）和波形蛋白的表达；转染TWIST siRNA后，在常氧及低氧条件下培养，蛋白质印迹检测E-钙黏蛋白, α-SMA和波形蛋白的表达。qRT-PCR及蛋白质印迹检测低氧浓度下PANC-1细胞甲基转移酶样分子3（METTL3）的表达，比色法检测细胞总的N6甲基腺嘌呤（m6A）水平；转染METTL3 siRNA后，在常氧及低氧条件下培养，比色法检测细胞总的m6A水平。在低氧条件下，转染METTL3 siRNA后，qRT-PCR及蛋白质印迹检测TWIST的mRNA和蛋白表达，MeRIP-qPCR检测TWIST mRNA m6A水平；转染METTL3野生型和突变型质粒后，蛋白质印迹检测TWIST蛋白的表达。结果：低氧处理胰腺癌细胞，TWIST表达增加，E-钙黏蛋白的表达下降, α-SMA和波形蛋白的表达升高；而同等低氧条件下敲低TWIST表达后E钙黏蛋白的表达增加，α-SMA和波形蛋白的表达下降。低氧处理后，胰腺癌细胞中METTL3的mRNA和蛋白表达均显著升高，同时细胞总的m6A水平也显著升高。常氧条件下敲低METTL3表达后胰腺癌细胞总m6A水平轻度下降，而低氧条件下敲低METTL3则显著抑制细胞总m6A水平。低氧条件下敲低胰腺癌细胞METTL3表达对TWIST mRNA表达无显著影响，但其显著降低TWIST mRNA的m6A水平，抑制TWIST蛋白表达；过表达METTL3显著上调TWIST蛋白的表达，而转入METTL3 突变质粒未见相关改变。结论：低氧诱导METTL3表达，后者通过m6A依赖的方式调控TWIST蛋白表达。

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	毛正发1
	罗钧艺2
	李熙2
	马小艳3

关键词 ：胰腺癌, 低氧, TWIST, 甲基转移酶样分子3

Abstract：Objective: To investigate the effect of hypoxia on the expression of TWIST in PANC-1 cells and its underlying mechanism. Methods: PANC-1 cells were cultured under low oxygen concentration, the expression of TWIST was detected by Western blotting at different time points, and the expression of TWIST, Ecadherin, α-SMA and Vimentin was detected by immunofluorescence; The expression of E-cadherin, α-SMA and Vimentin was detected by Western blotting under hypoxic conditions. After transfection of TWIST siRNA, the expression of E-cadherin, αSMA and Vimentin was detected by Western blotting under normoxic and hypoxic conditions.The expression of METTL3 was detected by qRT-PCR and Western blotting, the total m6A level of cells was detected by colorimetric method under low oxygen concentration; after transfection of METTL3 siRNA, the total m6A level of cells was examined by colorimetric method under normoxia and hypoxia.The expression level of TWIST was detected by qRT-PCR and Western blotting, and the level of TWIST mRNA m6A was detected by MeRIP-qPCR after transfection of METTL3 siRNA under hypoxic conditions; the expression of TWIST protein was detected by Western blotting after transfection of METTL3 wild-type and mutant plasmids. Results: Hypoxia promoted the expression of TWIST, α-SMA and Vimentin while the expression of E-cadherin was decreased in pancreatic cancer cells; at the same time the expression of E-cadherin increased and the expression of α-SMA and Vimentin decreased when knockingdown the expression of TWIST. The mRNA and protein expression of RNA methyltransferase METTL3 were significantly increased under hypoxia treatment, and the total m6A level of the cells was also significantly increased in pancreatic cancer cells. Under normoxia, knockdown of METTL3 expression slightly reduced the m6A level of pancreatic cancer cells, while under hypoxic conditions m6A level was significantly inhibited. Knockdown of METTL3 expression had no significant effect on the expression level of TWIST mRNA in pancreatic cancer cells under hypoxic conditions, but it significantly reduced the m6A level of TWIST mRNA and inhibited the expression of TWIST protein; overexpression of METTL3 significantly increaseed the expression of TWIST protein while not METTL3 mutant plasmid （METTL3-MUT）. Conclusion: Hypoxia could induce the expression of METTL3, which regulates the expression of TWIST protein through an m6A-dependent manner.

收稿日期: 2020-12-28

基金资助:国家自然科学基金资助项目（81502372）；江苏省青年医学人才资助项目（QNRC2016838）

作者简介: 毛正发（1984—），男，博士

引用本文:

毛正发1，罗钧艺2，李熙2，马小艳3. 低氧对胰腺癌PANC-1细胞株中TWIST表达的影响[J]. 江苏大学学报:医学版, 2021, 31(04): 326-332. MAO Zhengfa1, LUO Junyi2, LI Xi2, MA Xiaoyan3. Effect of hypoxia on TWIST expression in PANC-1 cells. Journal of Jiangsu University(Medicine Edition), 2021, 31(04): 326-332.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2021/V31/I04/326

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