circMTO1调控Wnt/β-catenin抑制乳腺癌细胞增殖和转移能力的机制研究

doi:10.11958/20220183

摘要/Abstract

摘要：

目的研究环状RNA MTO1（circMTO1）对乳腺癌细胞增殖和转移的影响及其可能机制。方法收集102例浸润性乳腺癌患者的癌组织及癌旁组织，选用乳腺癌细胞系MDA-MB-231、HCC-70、MCF-7和人乳腺正常细胞系MCF-10A，采用荧光定量PCR（qPCR）检测circMTO1在乳腺癌组织和细胞系中的表达水平。分别构建circMTO1过表达和circMTO1敲减质粒转染乳腺癌细胞系，分为NC组、oe-circMTO1组和sh-circMTO1组，qPCR检测转染效果。MTS实验检测各组细胞增殖能力，Transwell实验检测各组细胞转移能力。TOP/FOP荧光素酶实验检测各组细胞Wnt/β-catenin信号通路活性，Western blot检测各组细胞Wnt/β-catenin信号通路相关蛋白Wnt3a、β-catenin、cMyc、Cyclin D1和基质金属蛋白酶7（MMP7）的表达。结果与癌旁组织和人正常乳腺细胞系相比，circMTO1在乳腺癌组织和乳腺癌细胞系中的相对表达量降低（P<0.05）。有淋巴结转移和TNM分期为Ⅲ期的乳腺癌患者组织中circMTO1的相对表达水平较低（P<0.05）。与NC组相比，oe-circMTO1组circMTO1相对表达量增加，细胞的增殖、转移能力以及Wnt/β-catenin信号通路活性减弱，Wnt3a、β-catenin、cMyc、Cyclin D1和MMP7蛋白表达水平降低（P<0.05）；sh-circMTO1组circMTO1相对表达量降低，细胞的增殖、转移能力以及Wnt/β-catenin信号通路活性增强，Wnt3a、β-catenin、cMyc、Cyclin D1和MMP7蛋白表达水平增加（P<0.05）。结论 circMTO1在乳腺癌细胞中呈低表达，circMTO1通过抑制Wnt/β-catenin信号通路活性，从而抑制乳腺癌细胞的增殖和转移能力。

关键词: 乳腺肿瘤, 基因表达调控，肿瘤, 细胞增殖, 肿瘤转移, circMTO1, Wnt/β-catenin

Abstract:

Objective To study the effect of a circRNA derived from MTO1 gene (circMTO1) on the proliferation and metastasis of breast cancer and its possible mechanism. Methods A total of 102 patients with invasive breast cancer were included in this study. Breast cancer cell lines MDA-MB-231, HCC-70, MCF-7 and human normal breast cell line MCF-10A were selected, and fluorescence quantitative PCR (qPCR) was used to detect the expression level of circMTO1 in breast cancer tissues and cell lines. The circMTO1 overexpression and circMTO1 knockdown plasmids were respectively constructed to transfect breast cancer cell lines, and which were divided into the NC group, the oe-circMTO1 group and the sh-circMTO1 group. The transfection effect was tested by qPCR. The MTS experiment was used to detect the cell proliferation ability of each group, and the Transwell experiment was used to detect the cell metastasis ability of each group. TOP/FOP luciferase assay was used to detect the activity of Wnt/β-catenin signaling pathway in each group of cells, and Western blotting was used to detect the expression of key proteins Wnt3a,β-catenin, cMyc, Cyclin D1 and matrix metalloproteinase 7 (MMP7) in Wnt/β-catenin signaling pathway. Results Compared with adjacent tissues and human normal breast cell lines, the relative expression of circMTO1 was significantly decreased in breast cancer tissues and breast cancer cell lines (P<0.05). The relative expression level of circMTO1 was lower in breast cancer patients with lymph node metastasis and TNM stage Ⅲ (P<0.05). Compared with the NC group, the relative expression of circMTO1 was significantly increased in the oe-circMTO1 group, the cell proliferation and metastasis ability and the activity of the Wnt/β-catenin signaling pathway were weakened, and the protein expressions of Wnt3a, β-catenin, cMyc, Cyclin D1 and MMP7 were also decreased (P<0.05). In the sh-circMTO1 group, the relative expression of circMTO1 was decreased, the proliferation and metastasis ability of cells and the activity of Wnt/β-catenin signaling pathway were enhanced, and the protein expression levels of Wnt3a, β-catenin, cMyc, Cyclin D1 and MMP7 were increased (P<0.05). Conclusion circMTO1 is down-expressed in breast cancer cells. circMTO1 inhibits the proliferation and metastasis of breast cancer cells by inhibiting Wnt/β-catenin.

Key words: breast neoplasms, gene expression regulation, neoplastic, cell proliferation, neoplasm metastasis, circMTO1, Wnt/β-catenin

中图分类号:

R737.9

图/表 6

表1 不同临床病理参数患者乳腺癌组织中circMTO1表达量比较 $\bar{x}\pm s$

Tab.1 Comparison of circMTO1 expression in the breast cancer tissue of patients with different clinical pathological parameters

临床参数	n	circMTO1相对表达量	t
年龄（岁）
≤60	72	0.89±0.33	1.051
>60	30	0.82±0.24	1.051
肿瘤大小（cm）
≤2	43	0.93±0.34	1.172
>2	59	0.83±0.24	1.172
组织分级
Ⅰ-Ⅱ	38	0.91±032	1.012
Ⅲ	64	0.85±0.27	1.012
淋巴结转移
有	58	0.78±0.26	3.478^＊＊
无	44	0.99±0.35	3.478^＊＊
TNM分期
Ⅰ-Ⅱ	42	1.00±0.34	3.767^＊＊
Ⅲ	60	0.78±0.25	3.767^＊＊
ER
阳性	32	0.84±0.26	0.650
阴性	70	0.88±0.30	0.650
PR
阳性	35	0.81±0.26	1.468
阴性	67	0.90±0.31	1.468
HER-2
阳性	61	0.84±0.27	1.382
阴性	41	0.92±0.31	1.382

表2 各组细胞中circMTO1相对表达水平、转移情况和Wnt/β-catenin信号通路活性的比较 ($\bar{x}\pm s$)

Tab.2 Comparison of relative expression level, metastasis and Wnt/β-catenin of catenin signaling pathway between the three groups of cells

组别	n	circMTO1 相对表达水平	穿膜细胞数（个/视野）	TOP/FOP荧光素酶活性
NC组	3	1.01±0.02	69.33±6.81	0.97±0.03
oe-circMTO1组	3	3.57±0.55^a	32.67±4.25^a	0.49±0.03^a
sh-circMTO1组	3	0.41±0.11^ab	98.33±5.34^ab	1.69±0.12^ab
F		80.472^＊＊	104.829^＊＊	202.667^＊＊

图1 circMTO1对乳腺癌细胞转移能力的Transwell实验检测结果（苏木素染色，×200）

Fig.1 Effect of circMTO1 on breast cancer cell metastasis detected by Transwell experiment (Hematoxylin staining, ×200)

图2 MTS检测circMTO1对各组乳腺癌细胞增殖能力的影响＊＊P<0.01；24 h、48 h及72 h的F值分别为5.034、127.620＊＊、240.398＊＊；a与NC组比较，b与oe-circMTO1组比较，P<0.05。

Fig.2 MTS detection of the effect of circMTO1 on the proliferation of breast cancer cells in each group

图3 circMTO1对乳腺癌细胞Wnt/β-catenin信号通路相关蛋白的影响

Fig.3 Effects of circMTO1 on proteins related to Wnt/β-catenin signaling pathway in breast cancer cells

表3 各组细胞中Wnt/β-catenin信号通路相关蛋白相对表达水平的比较 (n=3, $\bar{x}\pm s$)

Tab.3 Comparison of Wnt/β-catenin signaling pathway-related protein expressions between the three groups of cells

组别	cMyc		Cyclin D1		MMP7
NC组	0.52±0.02		0.64±0.05		0.45±0.06
oe-circMTO1组	0.17±0.02^a		0.28±0.02^a		0.33±0.03^a
sh-circMTO1组	1.47±0.20^ab		1.89±0.11^ab		1.97±0.18^ab
F	99.816^＊＊		428.420^＊＊		203.837^＊＊
组别		Wnt3a		β-catenin
NC组		0.42±0.04		0.48±0.05
oe-circMTO1组		0.15±0.02^a		0.19±0.03^a
sh-circMTO1组		1.02±0.09^ab		1.56±0.18^ab
F		176.703^＊＊		131.036^＊＊

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