靶向调控皮肤恶性黑素瘤潜在致病基因CCL18的miRNA预测及表达相关性分析

中华皮肤科杂志 ›› 2017, Vol. 50 ›› Issue (9): 631-635.

靶向调控皮肤恶性黑素瘤潜在致病基因CCL18的miRNA预测及表达相关性分析

宋昊¹,布文博²,倪娜娜³,温斯健⁴,⁵,熊竞舒⁶,戚金亮⁷,徐秀莲⁸,孙建方⁸

1. 中国医学科学院南京皮肤病研究所
2. 中国医学科学院皮肤病研究所（医院）
3. 中国医学科学院皮肤病研究所
4. 广西医科大学第一附属医院皮肤性病科
5. 广西医科大学第一附属医院皮肤性病科（现在本所读研）
6. 中国医学科学院北京协和医学院皮肤病研究所
7. 南京大学生命科学院
8. 南京中国医学科学院北京协和医学院皮肤病研究所

收稿日期:2016-10-24 修回日期:2017-05-29 出版日期:2017-09-15 发布日期:2017-08-31
通讯作者: 徐秀莲 E-mail:xxlqjl@sina.com
基金资助:
国家自然科学基金;国家自然科学基金;江苏省自然科学基金;江苏省自然科学基金

Prediction of miRNA regulating the potential cancer-promoting gene CCL18 in cutaneous malig-nant melanoma and correlation analysis between CCL18 mRNA and miRNA

Received:2016-10-24 Revised:2017-05-29 Online:2017-09-15 Published:2017-08-31
Contact: Xiu-Lian Xu E-mail:xxlqjl@sina.com

摘要/Abstract

摘要： 目的探讨靶向调控皮肤恶性黑素瘤潜在致病基因CCL18的miRNA。方法用miRanda和TargetScan在线软件进行生物信息学分析，预测靶向调控CCL18基因的miRNA。根据人基因序列分析构建CCL18基因3′UTR双荧光素酶报告载体及miRNA载体，共转染293T细胞48 h后裂解细胞并检测荧光素酶活性。其中，CCL18基因3′UTR双荧光素酶报告载体分3′UTR突变型组、3′UTR野生型组和空白对照组，miRNA载体携带筛选的miRNA。应用实时荧光定量PCR检测14例黑素瘤新鲜组织及瘤旁正常皮肤组织（对照）中CCL18和筛选miRNA的表达，并对其相关性进行分析。结果在线软件分析显示，CCL18?3′UTR具有miR?183、miR?128、miR?33a等miRNA的作用靶点。成功构建荧光素酶报告载体及miRNA载体，荧光素酶活性检测显示，miR?183、miR?128的3′UTR突变型组荧光素酶表达量（11.63 ± 0.42；8.80 ± 0.49）明显高于3′UTR野生型组（4.86 ± 0.39；5.01 ± 0.54）及空白对照组（2.41 ± 0.13；2.39 ± 0.05），差异有统计学意义（均P < 0.01），而miR?33a的3′UTR突变型组荧光素酶表达量与3′UTR野生型组差异无统计学意义（6.41 ± 0.47比6.16 ± 0.22，P > 0.05）。实时荧光定量PCR显示，14例黑素瘤肿瘤组织中CCL18基因的相对表达量（3.52 ± 1.68）高于对照组织，而miR?183（0.49 ± 0.32）、miR?128（0.30 ± 0.20）、miR?33a（0.46 ± 0.40）的相对表达量均低于对照组织。CCL18表达水平与miR?128表达水平呈负相关（rs = ?0.548，P < 0.05），而与miR?183、miR?33a表达水平无相关性（P > 0.05）。结论 miR?128可能参与调控皮肤恶性黑素瘤潜在致病基因CCL18。

Abstract: Song Hao, Bu Wenbo, Ni Nana, Wen Sijian, Xiong Jingshu, Qi Jinliang, Xu Xiulian, Sun Jianfang Department of Pathology, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （Song H, Ni NN, Wen SJ, Xiong JS, Xu XL, Sun JF）; Department of Dermatologic Surgery, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China （Bu WB）; State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210023, China （Qi JL） Corresponding authors: Xu Xiulian, Email: xxlqjl@sina.com; Sun Jianfang, Email: fangmin5758@aliyun.com 【Abstract】 Objective To explore the miRNA regulating the potential cancer-promoting gene CCL18 in cutaneous malignant melanoma. Methods Bioinformatics analysis was conducted by using online software miRanda and TargetScan, so as to predict the miRNA targeting CCL18 gene. Three kinds of CCL18 3′UTR dual-luciferase reporter vectors, including mutant 3′UTR vector （mutant 3′UTR group）, wild-type 3′UTR vector （wild-type 3′UTR group） and empty vector （blank control group）, as well as miRNA vectors carring selected miRNAs were constructed according to human gene sequence analysis, and then were used to co-transfect 293T cells. After 48-hour treatment, the cells were lysed for detection of luciferase activity. Real-time fluorescence-based quantitative PCR was performed to measure the of CCL18 and selected miRNA in 14 fresh malignant melanoma tissue specimens and 14 paracancerous normal skin tissue specimens （control tissues）, and their correlations were analyzed. Results Online software analysis showed that some miRNAs were identified to target the 3′UTR of CCL18 gene, including miR-183, miR-128 and miR-33a. Luciferase reporter vectors and miRNA vectors were constructed successfully. As luciferase activity assay showed, when miR-183 and miR-128 were bound to the CCL18 3′UTR, the luciferase activities were significantly higher in their mutant 3′UTR groups （11.63 ± 0.42; 8.80 ± 0.49） than in their wild-type 3′UTR groups （4.86 ± 0.39; 5.01 ± 0.54; both P < 0.05） and blank control groups （2.41 ± 0.13; 2.39 ± 0.05; both P < 0.01）, while there were no significant differences between miR-33a-binding mutant 3′UTR group （6.41 ± 0.47） and miR-33a-binding wild-type 3′UTR group （6.16 ± 0.22, P > 0.05）. Real-time fluorescence-based quantitative PCR revealed higher mRNA of the CCL18 gene （3.52 ± 1.68）, but lower of miR-183 （0.49 ± 0.32）, miR-128 （0.30 ± 0.20） and miR-33a （0.46 ± 0.40） in the malignant melanoma tissues compared with the control tissues. The mRNA of the CCL18 gene was negatively correlated with the of miR-128 （rs = -0.548, P < 0.05）, but showed no significant correlations with the of miR-183 and miR-33a （both P > 0.05）. Conclusion miR-128 may play a role in regulating the potential malignant melanoma-promoting gene CCL18.

宋昊布文博倪娜娜温斯健熊竞舒戚金亮徐秀莲孙建方. 靶向调控皮肤恶性黑素瘤潜在致病基因CCL18的miRNA预测及表达相关性分析[J]. 中华皮肤科杂志, 2017, 50(9): 631-635.

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