[1]伍义文,曹健斌,黄维佳,等.m6A甲基转移酶METTL3介导miR-127调控非小细胞肺癌细胞系自噬的机制研究[J].现代检验医学杂志,2022,37(02):12-16.[doi:10.3969/j.issn.1671-7414.2022.02.003]
　WU Yi-wen,CAO Jian-bin,HUANG Wei-jia,et al.Mechanism of M6A Methyltransferase METTL3 Mediates miR-127 to Regulate Autophagy in Non-small Cell Lung Cancer Cell Lines[J].Journal of Modern Laboratory Medicine,2022,37(02):12-16.[doi:10.3969/j.issn.1671-7414.2022.02.003]
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m6A甲基转移酶METTL3介导miR-127调控非小细胞肺癌细胞系自噬的机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第37卷
期数:
2022年02期
页码:
12-16
栏目:
论　著
出版日期:
2022-03-15

文章信息/Info

Title:
Mechanism of M6A Methyltransferase METTL3 Mediates miR-127 to Regulate Autophagy in Non-small Cell Lung Cancer Cell Lines
文章编号:
1671-7414（2022）02-012-06
作者:
伍义文; 曹健斌; 黄维佳; 何凌云
（柳州市人民医院心胸外科，广西柳州 545006）
Author(s):
WU Yi-wen;  CAO Jian-bin;  HUANG Wei-jia;  HE Ling-yun
（Department of Cardiothoracic, Liuzhou People’s Hospital, Guangxi Liuzhou 545006, China）
关键词:
非小细胞肺癌细胞; 甲基转移酶3; miR-127; 自噬
分类号:
R734.2；R730.43
DOI:
10.3969/j.issn.1671-7414.2022.02.003
文献标志码:
A
摘要:
目的　分析N6 甲基腺苷（m6A）甲基转移酶 3（methyltransferase-like 3, METTL3）和miR-127 在非小细胞肺癌（non small cell lung cancer cells, NSCLC）细胞系中的表达及其相关性，并探究METTL3 介导miR-127 调控非小细胞肺癌自噬的作用机制。方法　采用qRT-PCR 法检测正常肺上皮细胞BEAS-2B 与非小细胞肺癌细胞HCC827，A549 和H460 中METTL3 和miR-127 的表达水平；通过Linked Domics 数据库筛选出肺癌中与miR-127 共表达的基因，并分析METTL3 和miR-127 之间的相关性；选择H460 细胞传代培养至对数生长期后，将浓度接近的细胞随机分为三组，分别转染METTL3-siR，NC-siR 及Control，验证转染后H460 细胞中METTL3 和miR-127 表达；通过吖啶橙染色，Lyso-Tracker Red 染色观察METTL3 对细胞自噬的影响；利用Western blot 检测PTEN，AKT，mTOR，ULK1，Beclin-1等自噬相关蛋白的表达。结果　非小细胞肺癌细胞HCC827，A549，H460 中METTL3 相对表达分别为1.35±0.17，1.54±0.11 和1.78±0.21，明显高于正常肺上皮细胞BEAS-2B 中表达水平（0.91±0.11），差异有统计学意义(F=34.037，P=0.002)。非小细胞肺癌细胞HCC827，A549，H460 中miR-127 相对表达分别为1.56±0.21，1.85±0.19 和2.11±0.25，较正常肺上皮细胞BEAS-2B 中表达（1.02±0.20）亦显著升高，差异有统计学意义（F=28.152，P=0.005）。肺癌中METTL3 与miR-127 共同表达呈正相关性（r=0.452，P ＜ 0.001）。METTL3-siR 组细胞中METTL3 表达水平（0.61±0.15）较Control 组（1.71±0.28） 和NC-siR 组（1.65±0.19） 显著降低， 差异有统计学意义（F=78.357，P ＜ 0.001）。METTL3-siR 组细胞中miR-127 表达水平（0.48±0.15）较Control 组（2.02±0.33）和NC-siR 组（1.97±0.25）亦显著下降，差异有统计学意义（F=105.216，P ＜ 0.001）；吖啶橙和Lyso-Tracker Red 染色分别观察到METTL3-siR 组细胞酸性自噬小泡增多，自噬溶酶体数量也明显增加。与Control 组和NC-siR 组相比，METTL3-siR 组细胞中PTEN，ULK1，Beclin1 蛋白表达水平显著升高，差异均有统计学意义（F=62.420~175.615，均P<0.001）；p-AKT 和p-mTOR 表达水平显著下降，差异均有统计学意义（F=148.781，87.147，均P<0.001）。结论　METTL3 和miR-127 在非小细胞肺癌细胞系中均呈高表达，且它们之间呈正相关性，沉默METTL3 基因可以抑制miR-127 表达，促进非小细胞肺癌H460 细胞发生自噬，其调控机制可能与PTEN/AKT/mTOR 通路有关。
Abstract:
Objective　To analyze the expression and correlation of m6A methyltransferase METTL3 and miR-127 in NSCLC cell lines, and to explore the mechanism of METTL3 mediated miR-127 regulating autophagy in NSCLC. Methods　qRT-PCR method to detect the expression levels of METTL3 and miR-127 in normal lung epithelial cells BEAS-2B and non-small cell lung cancer cells HCC827, A549 and H460. The genes co expressed with miR-127 in lung cancer were screened by linked domics database, and the correlation between METTL3 and miR-127 was analyzed. After selecting H460 cells to be subcultured to the logarithmic growth phase, the cells with close concentrations were randomly divided into three groups, and they were transfected with METTL3-siR, NC-siR and Control, respectively, to verified the expression of METTL3 and miR-127 in H460 cells after transfection. The effect of METTL3 on autophagy was observed by cridine orange staining and Lyso-Tracker Red staining. Western blot was used to detect the expression of autophagy-related proteins such as PTEN, AKT, mTOR, ULK1 and Beclin-1. Results　The relative expression of METTL3 in HCC827, A549 and H460 cells were 1.35±0.17, 1.54±0.11 and 1.78±0.21, respectively, which was significantly higher than that in beAS-2B cells (0.91±0.11)，the difference was statistically significant(F=34.037, P = 0.002). The relative expression of miR-127 in non-small cell lung cancer cells HCC827, A549 and H460 were 1.56±0.21, 1.85±0.19 and 2.11±0.25, respectively, which was significantly higher than that in normal lung epithelial cells BEAS-2B (1.02±0.20) ，the difference was statistically significant (F=28.152, P= 0.005). The co-expression of METTL3 and miR-127 was positively correlated in lung cancer (r=0.452, P< 0.001). The expression level of METTL3 in METTL3-siR group (0.61±0.15) was significantly lower than that in Control group (1.71±0.28) and NC-siR group (1.65±0.19)，the difference was statistically significant (F=78.357, P < 0.001). The expression level of miR-127 in METTL3- siR group (0.48±0.15) was significantly lower than that in Control group (2.02±0.33) and NC-siR group (1.97±0.25), the difference was scatiscically significant (F=105.216, P< 0.001). Acridine orange and Lyso-Tracker Red staining showed that acidic autophagic vesicles and the number of autophagic lysosomes increased significantly in METTL3-siR group, respectively. Compared with the Control group and the NC-siR group, the protein expression levels of PTEN, ULK1 and Beclin1 in METTL3- siR group were significantly increased, the differences were statistically significant (F=62.420~175.615, all P<0.001). The expression levels of P-Akt and P-MTOR were significantly decreased，the differences were statistically significant (F=148.781, 87.147, all P<0.001). Conclusion　METTL3 and miR-127 were highly expressed in non-small cell lung cancer cell lines, and there was a positive correlation between them. Silencing the METTL3 gene can inhibit the expression of miR-127 and promote autophagy in non-small cell lung cancer H460 cells. Its regulatory mechanism may be related to the PTEN/AKT/mTOR pathway.

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备注/Memo

备注/Memo:
基金项目：广西壮族自治区卫生健康委科研项目（Z20200665）。
作者简介：伍义文（1986-），男，硕士研究生，研究方向：胸部肿瘤的综合治疗，E-mail:ldiguy53@163.com。
更新日期/Last Update: 1900-01-01