MALAT1靶向miR-142-3p在卵巢癌化疗耐药中的机制研究

doi:10.3760/cma.j.issn.1673-422X.2020.02.004

摘要/Abstract

摘要：

目的研究肺腺癌转移相关转录本1(MALAT1)靶向调控微小RNA-142-3p(miR-142-3p)导致卵巢癌化疗耐药的机制。方法收集2016年2月至2019年2月陕西省人民医院80对卵巢癌组织及对应非癌正常组织标本,通过实时荧光定量PCR法检测卵巢癌组织和非癌正常组织中MALAT1及miR-142-3p的相对表达量,并分析二者表达的相关性;通过CCK-8实验验证异常表达的MALAT1及miR-142-3p对卵巢癌Hey细胞增殖和化疗药物5-氟尿嘧啶及顺铂敏感性的影响;通过双荧光素酶报告基因实验(分4组:MALAT1 wt组、MALAT1 wt+miR-142-3p mimic组、MALAT1 mut组和MALAT1 mut+miR-142-3p mimic组)检测miR-142-3p与MALAT1的靶向关系;通过RNA免疫沉淀反应实验验证MALAT1与miR-142-3p的靶点。结果在卵巢癌组织和对应非癌正常组织中,MALAT1的相对表达量分别为0.000 52(0.002 56)、0.000 47(0.000 89),差异有统计学意义(Z=2.365,P=0.018);miR-142-3p的相对表达量分别为0.001 19(0.002 69)、0.001 61(0.008 48),差异有统计学意义(Z=2.935,P=0.003);二者在卵巢癌组织中的表达呈负相关(r=-0.474,P<0.001)。miR-142-3p在miR-142-3p mock组的相对表达量明显低于MALAT1+miR-142-3p mimic组(0.004 18±0.001 24 vs. 0.006 51±0.000 28;t=3.174,P=0.017)。MALAT1 wt组与MALAT1 wt+miR-142-3p mimic组的相对荧光富集度分别为2.27±0.86、31.10±6.05,差异具有统计学意义(t=8.172,P<0.001)。Hey细胞转染MALAT1过表达质粒48、72、96 h后,MALAT1过表达组和对照组的吸光度(A)值(0.522±0.021 vs. 0.433±0.021;0.644±0.012 vs. 0.544±0.051;0.887±0.055 vs. 0.698±0.042)差异均有统计学意义(均P<0.05)。Hey细胞过表达MALAT1后,5-氟尿嘧啶0.10 ng/μl浓度时,MALAT1过表达组的细胞增殖速度明显较对照组快(0.615±0.036 vs. 0.506±0.042;t=4.432,P=0.002),5-氟尿嘧啶1.00、10.00、100.00 ng/μl浓度时显示出同样的趋势(均P<0.05);顺铂0.01 ng/μl浓度时,MALAT1过表达组的细胞增殖速度明显较对照组快(0.777±0.015 vs. 0.733±0.039;t=2.355,P=0.023),顺铂0.10、1.00、10.00、100.00 ng/μl浓度时显示出同样的趋势(均P<0.05)。Hey细胞过表达miR-142-3p后,5-氟尿嘧啶0.10 ng/μl浓度时,miR-142-3p过表达组的细胞增殖速度明显较对照组细胞慢(0.512±0.051 vs. 0.744±0.119;t=4.028,P=0.004),5-氟尿嘧啶1.00、10.00、100.00 ng/μl浓度时显示出同样的趋势(均P<0.05);顺铂0.10 ng/μl浓度时,miR-142-3p过表达组的细胞增殖速度明显较对照组慢(0.520±0.043 vs. 0.674±0.096;t=3.441,P=0.009),顺铂1.00、10.00、100.00 ng/μl浓度时显示出同样的趋势(均P<0.05)。采用0.10、1.00、10.00、100.00 ng/μl浓度的5-氟尿嘧啶和顺铂分别处理卵巢癌Hey细胞后,MALAT1过表达组、MALAT1+miR-142-3p组与对照组间的细胞增殖差异均具有统计学意义(均P<0.05),进一步两两比较发现,MALAT1+miR-142-3p组均显著慢于MALAT1过表达组(均P<0.05)。结论 MALAT1通过靶向调控miR-142-3p使卵巢癌细胞对5-氟尿嘧啶及顺铂的敏感性降低,导致卵巢癌化疗耐药。

关键词: 卵巢肿瘤, 氟尿嘧啶, 顺铂, 转移相关肺腺癌转录物1, 微小RNA-142-3p

Abstract:

Objective To study the mechanism of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) targeting microRNA-142-3p (miR-142-3p) in ovarian cancer chemotherapy resistance. Methods A total of 80 ovarian cancer tissues and paired normal tissues were collected in Shaanxi Provincial People's Hospital from February 2016 to February 2019. The relative expression levels of MALAT1 and miR-142-3p in ovarian cancer tissues and paired normal tissues were detected by real-time fluorescence quantitative polymerase chain reaction (PCR), and the correlation between MALAT1 and miR-142-3p was analyzed. The effects of abnormal expressions of MALAT1 and miR-142-3p on proliferation and chemotherapy sensitivity of 5-fluorouracil (5-FU) and cisplatin of ovarian cancer Hey cells were verified by CCK-8 assay. Dual luciferase reporter gene experiment was used to detect the targeted relationship between miR-142-3p and MALAT1 (Hey cells were divided into four groups: MALAT1 wt, MALAT1 wt+miR-142-3p mimic, MALAT1 mut, MALAT1 mut+miR-142-3p mimic). RNA immunoprecipitation assay was use to confirm the binding site of MALAT1 and miR-142-3p. Results In the ovarian cancer tissues and paired normal tissues, the relative expression levels of MALAT1 were 0.000 52 (0.002 56) and 0.000 47 (0.000 89), with a statistically significant difference (Z=2.365, P=0.018); the relative expression levels of miR-142-3p were 0.001 19 (0.002 69) and 0.001 61 (0.008 48), with a statistically significant difference (Z=2.935, P=0.003). The relative expression level of MALAT1 was negatively correlated with miR-142-3p in the ovarian cancer tissues (r=-0.474, P<0.001). The relative expression level of miR-142-3p in the miR-142-3p mock group was statistically lower than that of MALAT1+miR-142-3p mimic group (0.004 18±0.001 24 vs. 0.006 51±0.000 28; t=3.174, P=0.017). The relative fluorescence concentrations of MALAT1 wt group and MALAT1 wt+miR-142-3p mimic group were 2.27±0.86 and 31.10±6.05 respectively, with a statistically significant difference (t=8.172, P<0.001). After 48, 72 and 96 hours of ovarian cancer Hey cells being transfected with MALAT1 overexpression plasmid, the absorbance (A) values of cells in the MALAT1 overexpression group were significantly greater than those in the control group (0.522±0.021 vs. 0.433±0.021; 0.644±0.012 vs. 0.544±0.051; 0.887±0.055 vs. 0.698±0.042), with statistically significant differences (all P<0.05). After MALAT1 being overexpressed in Hey cells, at 0.10 ng/μl concentration of 5-FU, the proliferation rate of cells in the overexpression group was significantly faster than that in the control group (0.615±0.036 vs. 0.506±0.042; t=4.432, P=0.002), and the cells at 1.00, 10.00, 100.00 ng/μl concentrations of 5-FU showed the same trends (all P<0.05). At 0.01 ng/μl concentration of cisplatin, the proliferation rate of cells in the overexpression group was significantly faster than that in the control group (0.777±0.015 vs. 0.733±0.039; t=2.355, P=0.023), and the cells at 0.10, 1.00, 10.00, 100.00 ng/μl concentrations of cisplatin showed the same trends (all P<0.05). After miR-142-3p being overexpressed in Hey cells, at 0.10 ng/μl concentration of 5-FU, the proliferation rate of cells in the overexpression group was significantly slower than that in the control group (0.512±0.051 vs. 0.744±0.119; t=4.028, P=0.004), and the cells at 1.00, 10.00, 100.00 ng/μl concentrations of 5-FU showed the same trends (all P<0.05). At 0.10 ng/μl concentration of cisplatin, the proliferation rate of cells in the overexpression group was significantly slower than that in the control group (0.520±0.043 vs. 0.674±0.096; t=3.441, P=0.009), and the cells at 1.00, 10.00, 100.00 ng/μl concentrations of cisplatin showed the same trends (all P<0.05). After ovarian cancer Hey cells being treated with 0.10, 1.00, 10.00, 100.00 ng/μl concentrations of 5-FU and cisplatin, the proliferation rates of cells in the MALAT1 overexpression group, MALAT1+miR-142-3p group and control group showed statistically significant differences (all P<0.05). Further pairwise comparisons revealed that the proliferation rates of cells in the MALAT1+miR-142-3p group were significantly slower than those in the MALAT1 overexpression group (all P<0.05). Conclusion MALAT1 can reduce the sensitivity of ovarian cancer cells to 5-FU and cisplatin by targeted miR-142-3p, leading to chemotherapy resistance of ovarian cancer.

Key words: Ovarian neoplasms, Fluorouracil, Cisplatin, Metastasis associated lung adenocarcinoma transcript 1, MicroRNA-142-3p

樊阳阳, 王影, 袁峰, 杜善平, 贺荣荣, 加燕. MALAT1靶向miR-142-3p在卵巢癌化疗耐药中的机制研究[J]. 国际肿瘤学杂志, 2020, 47(2): 82-89.

Fan Yangyang, Wang Ying, Yuan Feng, Du Shanping, He Rongrong, Jia Yan. Study on the mechanism of MALAT1 targeting miR-142-3p in ovarian cancer chemotherapy resistance[J]. Journal of International Oncology, 2020, 47(2): 82-89.

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组别	5-氟尿嘧啶浓度(ng/μl)
组别	0.01	0.10	1.00	10.00	100.00
MALAT1过表达组	0.893±0.018	0.615±0.036	0.451±0.041	0.376±0.089	0.282±0.067
对照组	0.881±0.039	0.506±0.042	0.228±0.017	0.124±0.051	0.076±0.031
t值	0.625	4.432	11.143	5.481	6.242
P值	0.275	0.002	<0.001	<0.001	<0.001

组别	顺铂浓度(ng/μl)
组别	0.01	0.10	1.00	10.00	100.00
MALAT1过表达组	0.777±0.015	0.510±0.030	0.337±0.031	0.280±0.066	0.197±0.013
对照组	0.733±0.039	0.456±0.042	0.228±0.017	0.124±0.051	0.067±0.027
t值	2.355	2.372	6.865	4.418	9.626
P值	0.023	0.045	<0.001	0.003	<0.001

组别	5-氟尿嘧啶浓度(ng/μl)
组别	0.01	0.10	1.00	10.00	100.00
miR-142-3p过表达组	0.683±0.107	0.512±0.051	0.306±0.058	0.162±0.043	0.099±0.052
对照组	0.750±0.075	0.744±0.119	0.552±0.068	0.455±0.046	0.228±0.086
t值	1.147	4.028	6.198	10.348	2.868
P值	0.858	0.004	<0.001	<0.001	0.021

组别	顺铂浓度(ng/μl)
组别	0.01	0.10	1.00	10.00	100.00
miR-142-3p过表达组	0.701±0.071	0.520±0.043	0.219±0.092	0.169±0.0038	0.128±0.029
对照组	0.821±0.133	0.674±0.096	0.556±0.107	0.437±0.065	0.271±0.041
t值	1.780	3.441	5.363	8.007	6.277
P值	0.935	0.009	<0.001	0.003	<0.001

组别	5-氟尿嘧啶浓度(ng/μl)
组别	0.01	0.10	1.00	10.00	100.00
MALAT1过表达组	0.778±0.055	0.723±0.035^ab	0.596±0.063^ab	0.494±0.046^ab	0.418±0.053^ab
MALAT1+miR-142-3p组	0.735±0.098	0.635±0.040^b	0.458±0.062^b	0.351±0.064^b	0.233±0.057^b
对照组	0.620±0.073	0.413±0.064	0.280±0.064	0.188±0.043	0.114±0.035
F值	1.528	55.700	31.952	43.890	48.666
P值	0.322	<0.001	<0.001	<0.001	<0.001