低氧对胰腺癌PANC-1细胞株中TWIST表达的影响

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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.

Received: 28 December 2020

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	Articles by authors
	MAO Zhengfa1
	LUO Junyi2
	LI Xi2
	MA Xiaoyan3

Cite this article:

MAO Zhengfa1,LUO Junyi2,LI Xi2等. Effect of hypoxia on TWIST expression in PANC-1 cells[J]. Journal of Jiangsu University(Medicine Edition), 2021, 31(04): 326-332.

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http://zzs.ujs.edu.cn/xbyxb/EN/ OR http://zzs.ujs.edu.cn/xbyxb/EN/Y2021/V31/I04/326

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