CXCL10和miR-34c-5p在乳腺浸润性导管癌中的靶向作用

摘要/Abstract

摘要：

目的探讨乳腺浸润性导管癌中CXC趋化因子配体10(CXCL10)与miR-34c-5p的靶向调控关系。方法收集2016年3月至2017年3月在中山大学附属第三医院行手术切除且经病理确诊的乳腺浸润性导管癌组织(n=56)和乳腺良性组织(n=14),采用HTA2.0和miRNA4.0芯片检测CXCL10和miR-34c-5p在两组中的表达并用反转录实时荧光定量PCR(RT-qPCR)进行验证;采用Pearson相关分析两者表达的相关性并用IPA软件预测靶向关系;采用双荧光素酶报告基因系统验证二者的作用靶点,最后在乳腺癌细胞株MCF-7、ZR-75-30中进一步验证。结果基因芯片检测结果显示,CXCL10 mRNA在乳腺浸润性导管癌组织和乳腺良性组织中的表达量分别为7.135±1.350和4.348±0.722,miR-34c-5p mRNA在两组中的表达量分别为1.309(1.001,2.055)和3.948(3.278,4.371),差异均有统计学意义(t=7.628,P<0.001;Z=-4.405,P<0.001);Pearson相关分析显示,CXCL10 mRNA和miR-34c-5p mRNA的表达呈负相关(r=-0.327,P=0.004)。CXCL10 mRNA的表达与乳腺浸润性导管癌患者人表皮生长因子受体-2(t=2.415,P=0.019)和Ki-67(t=2.483,P=0.016)的表达有关;miR-34c-5p mRNA的表达与乳腺浸润性导管癌患者组织学分级(χ²=8.626,P=0.013)和雌激素受体/孕激素受体(Z=-2.195,P=0.028)的表达有关。RT-qPCR结果显示,CXCL10 mRNA在乳腺浸润性导管癌组织和乳腺良性组织中的相对表达水平分别为6.059±1.714和1.000±0.232,miR-34c-5p的相对表达水平分别为0.093±0.004和1.000±0.244,差异均有统计学意义(t=3.484,P=0.002;t=5.112,P<0.001),该结果与芯片检测结果一致。双荧光素酶报告基因结果表明,过表达miR-34c-5p后,在MCF-7细胞中WT-CXCL10和MUT-CXCL10报告载体的荧光素酶活性分别为1.117±0.040和1.647±0.031;在HeLa细胞中,两者的荧光素酶活性分别为0.885±0.051和1.430±0.020,差异均有统计学意义(t=18.120,P<0.001;t=24.040,P<0.001)。分别转染miR-34c-5p和miR-NC后,ZR-75-30细胞中CXCL10的相对表达水平分别为0.008±0.000和0.012±0.000;MCF-7细胞中CXCL10的相对表达水平分别为0.315±0.000和0.386±0.004,差异均有统计学意义(t=20.384,P<0.001;t=3.473,P=0.026)。结论miR-34c-5p在乳腺浸润性导管癌中表达下调,而CXCL10表达上调;CXCL10是miR-34c-5p的靶基因。

关键词:乳腺肿瘤,CXC趋化因子配体10,miR-34c-5p

Abstract:

ObjectiveTo explore the targeted relationship between the CXC chemokine ligand 10 (CXCL10) and miR-34c-5p in invasive ductal carcinoma of breast.MethodsA total of 56 cases of invasive ductal carcinoma of breast and 14 cases of benign breast tissues surgically resected and pathologically confirmed in the Third Affiliated Hospital of Sun Yat-sen University were collected from March 2016 to March 2017. The expressions of CXCL10 and miR-34c-5p in the two groups were detected using HTA2.0 and miRNA4.0 chips and verified by reverse transcription quantitative real-time PCR (RT-qPCR). The correlation between the two expressions was analyzed using Pearson correlation analysis and the targeted relationship was predicted by IPA software. The dual luciferase reporter gene system was used to verify the targets of the two and finally verified in breast cancer cell lines MCF-7 and ZR-75-30.ResultsThe results of gene microarray showed that the mRNA expression values of CXCL10 in invasive ductal carcinoma of breast and benign breast tissues were 7.135±1.350 and 4.348±0.722, and the mRNA expression values of miR-34c-5p in the two groups were 1.309 (1.001, 2.055) and 3.948 (3.278, 4.371), with a statistically significant difference (t=7.628,P<0.001;Z=-4.405,P<0.001). Pearson correlation analysis showed that the mRNA expressions of CXCL10 and miR-34c-5p were negatively correlated (r=-0.327,P=0.004). The expression of CXCL10 mRNA was related to the expressions of human epidermal growth factor receptor-2 (t=2.415,P=0.019) and Ki-67 (t=2.483,P=0.016), and the expression of miR-34c-5p mRNA was related to the histological grading (χ²=8.626,P=0.013) and estrogen receptor/progesterone receptor (Z=-2.195,P=0.028) in patients with invasive ductal carcinoma of breast. RT-qPCR showed that the relative expressions of CXCL10 mRNA in invasive ductal carcinoma of breast and benign breast tissues were 6.059±1.714 and 1.000±0.232, the relative expression levels of miR-34c-5p in the two groups were 0.093±0.004 and 1.000±0.244, with statistically significant differences (t=3.484,P=0.002;t=5.112,P<0.001), which was consistent with the results of microarray. The results of double luciferase reporter gene showed that, after overexpression of miR-34c-5p, the luciferase activities of WT-CXCL10 and MUT-CXCL10 were 1.117±0.040 and 1.647±0.031 in MCF-7 cells, and the luciferase activities of them were 0.885±0.051 and 1.430±0.020 in HeLa cells, with statistically significant differences (t=18.120,P<0.001;t=24.040,P<0.001). After transfection with miR-34c-5p and miR-NC respectively, the relative expression levels of CXCL10 were 0.008±0.000 and 0.012±0.000 in ZR-75-30 cells, and the relative expression levels of CXCL10 were 0.315±0.000 and 0.386±0.004 in MCF-7 cells, with statistically significant differences (t=20.384,P<0.001;t=3.473,P=0.026).ConclusionThe expression of miR-34c-5p in invasive ductal carcinoma of breast is down-regulated, while the expression of CXCL10 is up-regulated. CXCL10 is the target gene of miR-34c-5p.

Key words:Breast neoplasms,CXC chemokine ligand 10,miR-34c-5p

高建, 金亦, 林洁, 林圣, 段山. CXCL10和miR-34c-5p在乳腺浸润性导管癌中的靶向作用[J]. 国际肿瘤学杂志, 2020, 47(7): 391-396.

Gao Jian, Jin Yi, Lin Jie, Lin Sheng, Duan Shan. Targeting role of CXCL10 and miR-34c-5p in invasive ductal carcinoma of breast[J]. Journal of International Oncology, 2020, 47(7): 391-396.

图/表2

表1

56例浸润性导管癌患者CXCL10和miR-34c-5p的表达与临床病理特征的相关性分析"

临床病理特征^a	例数	CXCL10 mRNA( $x ˉ$ ±s)	t/F值	P值	miR-34c-5p mRNA[M(P₂₅,P₇₅)]	Z/χ²值	P值
年龄(岁)
≤50	26	6.861±1.431	-1.472	0.147	1.448(1.077,2.614)	-0.378	0.706
<50	30	7.373±1.175			1.286(0.946,2.047)
组织学分级
Ⅰ	7	7.814±1.700			0.808(0.600,1.122)
Ⅱ	34	6.867±1.310	2.369	0.104	1.466(1.151,2.205)	8.626	0.013
Ⅲ	13	7.559±1.003			1.291(1.005,2.117)
ER/PR
单/双阳性	40	6.954±1.238	-1.656	0.103	1.466(1.142,2.055)	-2.195	0.028
双阴性	16	7.588±1.427			1.027(0.746,1.959)
Her-2
阳性	22	7.656±1.325	2.415	0.019	1.244(0.914,1.704)	-1.320	0.187
阴性	32	6.803±1.242			1.497(1.124,2.504)
Ki-67
≥14%	45	7.358±1.315	2.483	0.016	1.291(0.942,2.055)	-0.458	0.647
<14%	10	6.270±0.889			1.365(1.151,2.261)

表1

图1

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