miR-101-3p靶向E2F2调控黑色素瘤细胞增殖的分子机制探讨

doi:10.11958/20212787

天津医药 ›› 2022, Vol. 50 ›› Issue (11): 1121-1127.doi: 10.11958/20212787

• 细胞与分子生物学 • 下一篇

miR-101-3p靶向E2F2调控黑色素瘤细胞增殖的分子机制探讨

李吻吻(), 朱智鑫, 韩利民, 焦艳林, 翁宗琴, 赵海龙^△()

遵义医科大学基础医学院病理生理学教研室（邮编563000）

收稿日期:2022-01-04 修回日期:2022-04-19 出版日期:2022-11-15 发布日期:2022-11-11
通讯作者: 赵海龙 E-mail:lw20200701@163.com;Hailongzhao@zmu.edu.cn
作者简介:李吻吻（1993），女，硕士在读，主要从事肿瘤靶向治疗方面研究。E-mail：lw20200701@163.com
基金资助:
国家自然科学基金资助项目(82060503);贵州省科技计划项目(黔科合基础[2019]1334号);贵州省卫健委科学技术基金资助项目(gzwjkj2019-1-033)

Molecular mechanism study of melanoma cell proliferation regulated by miR-101-3p-E2F2 targeting pathway

LI Wenwen(), ZHU Zhixin, HAN Limin, JIAO Yanlin, WENG Zongqin, ZHAO Hailong^△()

Department of Pathophysiology, School of Preclinical Medicine, Zunyi Medical University, Zunyi 563000, China

Received:2022-01-04 Revised:2022-04-19 Published:2022-11-15 Online:2022-11-11
Contact: ZHAO Hailong E-mail:lw20200701@163.com;Hailongzhao@zmu.edu.cn

李吻吻, 朱智鑫, 韩利民, 焦艳林, 翁宗琴, 赵海龙. miR-101-3p靶向E2F2调控黑色素瘤细胞增殖的分子机制探讨[J]. 天津医药, 2022, 50(11): 1121-1127.

LI Wenwen, ZHU Zhixin, HAN Limin, JIAO Yanlin, WENG Zongqin, ZHAO Hailong. Molecular mechanism study of melanoma cell proliferation regulated by miR-101-3p-E2F2 targeting pathway[J]. Tianjin Medical Journal, 2022, 50(11): 1121-1127.

摘要/Abstract

摘要：

目的探讨miR-101-3p靶向E2F转录因子2（E2F2）调控黑色素瘤细胞增殖的作用机制。方法使用GEPIA、OSskcm等数据库联合分析E2F2在黑色素瘤中的表达，并评估转录因子E2F2在黑色素瘤预后中的价值；利用短发夹RNA（shRNA）抑制黑色素瘤细胞MV3中E2F2表达后，分别采用PI染色和流式细胞术检测细胞周期的改变；Western blot实验检测细胞周期蛋白E（CyclinE）和细胞周期蛋白依赖性激酶2（CDK2）的表达水平；免疫荧光实验检测细胞中自噬相关蛋白LC3B的表达水平；裸鼠荷瘤实验观察黑色素瘤在体内生长的变化。经miR-101-3p mimics处理后，采用实时荧光定量PCR（qPCR）检测MV3细胞中miR-101-3p的表达水平，同时利用qPCR和Western blot实验检测E2F2的表达水平；通过MTT、BrdU法检测MV3细胞增殖水平。结果 E2F2表达升高的患者预后更差（P<0.05）；与shGFP组相比，shE2F2组中处于G0/G1期的细胞比例显著增多（P<0.05），CyclinE和CDK2的表达水平显著降低，自噬相关蛋白LC3B荧光信号明显减少（P<0.05），体内黑色素瘤形成质量显著降低（P<0.05）；采用miR-101-3p mimics处理后，MV3细胞中miR-101-3p的表达水平明显升高（P<0.05），E2F2的mRNA和蛋白表达均显著低于NC mimics处理组（P<0.05），同时MTT结果表明miR-101-3p mimics组MV3细胞的活力显著降低（P<0.05），BrdU标记试验结果显示miR-101-3p mimics组细胞中BrdU阳性信号明显减少（P<0.05）。结论 miR-101-3p可靶向E2F2并抑制其表达，进而降低黑色素瘤细胞增殖水平。

关键词: 黑色素瘤, E2F2转录因子, 细胞增殖, miR-101-3p

Abstract:

Objective To explore effect and mechanism of miR-101-3p targeting E2F transcription factor 2 (E2F2) in regulating the melanoma cell proliferation. Methods GEPIA, OSskcm databases were used to analyze the expression of E2F2 in melanoma and evaluate the value of transcription factor E2F2 in the prognosis of melanoma. The expression of E2F2 in melanoma cells MV3 was inhibited by shRNA, and the cell cycle changes were detected by PI staining flow cytometry. The expression levels of CyclinE and Cyclin-dependent kinase 2 (CDK2) were detected by Western blot assay. The expression level of autophagy-associated protein LC3B was detected by immunofluorescence, and the growth changes of melanoma in vivo were observed by tumor-bearing experiments in nude mice. The expression level of miR-101-3p in MV3 cells was detected by real-time fluorescence quantitative PCR, the expression level of E2F2 was detected by qPCR and Western blot assay, and the proliferation level of MV3 cells was detected by MTT and BrdU methods. Results Melanoma patients with increased E2F2 expression had worse prognosis (P<0.05). Compared with the shGFP group, the proportion of cells in G0/G1 phase increased significantly in the shE2F2 group (P<0.05), the expression levels of CyclinE and CDK2 decreased significantly, the fluorescence signal of autophagy-associated protein LC3B decreased significantly (P<0.05), and the weight of melanoma formation in vivo decreased significantly (P<0.05). The expression level of miR-101-3p in MV3 cells increased significantly after treated with miR-101-3p mimics (P<0.05), and the mRNA and protein expression of E2F2 were significantly lower than those in the NC mimics treatment group (P<0.05). Meanwhile, MTT results showed that the vitality of MV3 cells decreased significantly in the miR-101-3p mimics group (P<0.05). BrdU labeling test results showed that the BrdU positive signal decreased significantly in the miR-101-3p mimics group (P<0.05). Conclusion miR-101-3p targets to E2F2 and inhibits its expression, and then reduces the proliferation level of melanoma cells.

Key words: melanoma, E2F2 transcription factor, cell proliferation, miR-101-3p

中图分类号:

R739.5

图/表 13

表1 qPCR引物序列

Tab.1 Primer sequence for qPCR

基因名称	引物序列（5′→3′）	产物大小（bp）
miR-101-3p	上游：TACAGTACTGTGATAACTGAA 下游：GGAGTAG ATGATGGTTAGC	58
U6	上游：CTCGCTTCGGCAGCACA 下游：AACGCTTCACGAATTTGCGT	94
E2F2	上游：CTCTCTGAGCTTCAAGCACCTG 下游：CTTGACGGCAATCACT GTCTGC	118
GAPDH	上游：ACCACAGTCCATGCCATCAC 下游：TCCACCACCCTGTTGCTGTA	452

图1 E2F2不同表达水平的黑色素瘤患者生存曲线 A：GEPIA数据库生存曲线；B：OSskcm数据库生存曲线。

Fig.1 Survival curves of melanoma patients with different expression levels of E2F2

图2 CancerSEA数据库分析黑色素瘤中E2F2的功能图

Fig.2 CancerSEA database analyzes the functional diagram of E2F2 in melanoma

图3 shGFP或shE2F2处理后PI染色流式细胞术检测细胞周期的变化 A：shGFP组细胞周期；B：shE2F2组细胞周期；C：shGFP组和shE2F2组细胞周期的量化图。

Fig.3 The changes of cell cycle detected by PI staining flow cytometry after shGFP or shE2F2 treatment

图4 MTT法检测shGFP或shE2F2处理后对MV3细胞活力的影响 n=3，培养第1、2、3、4和5天t值分别为2.910、1.663、3.273＊、0.702和3.415＊，＊P<0.05。

Fig.4 The effect shGFP or shE2F2 on cell viability of MV3 cells detected by MTT

图5 shGFP或shE2F2处理后MV3细胞周期相关蛋白的表达

Fig.5 Expression of cycle-related proteins in MV3 cells treated with shGFP or shE2F2

图6 shGFP或shE2F2处理后MV3细胞中p-AKT、AKT和p21表达变化

Fig.6 Expression changes of p-AKT, AKT and p21 in MV3 cells treated with shGFP or shE2F2

图7 shGFP或shE2F2处理后MV3细胞中LC3B荧光信号变化 A：shGFP组LC3B荧光信号图；B：shE2F2组LC3B荧光信号图

Fig.7 The changes of LC3B fluorescence signal of MV3 cells after shGFP or shE2F2 treatment

图8 shE2F2对黑色素瘤体内生长的影响

Fig.8 The effect of shE2F2 on the growth of melanoma in vivo

图9 StarBase预测miR-101与E2F2的3' -UTR序列之间的结合序列图

Fig.9 The binding sequence diagram between miR-101 and the 3'-UTR sequence of E2F2 predicted by StarBase

图10 NC mimics或miR-101-3p mimics处理后MV3细胞中E2F2的蛋白表达水平

Fig.10 Protein expression levels of E2F2 in MV3 cells treated with NC or miR-101-3p mimics

表2 NC mimics和miR-101-3p mimics处理后2组MV3细胞活力比较 (n=3, $\bar{x}±s$)

Tab.2 Comparison of MV3 cell viability after treatment with NC mimics and miR-101-3p mimics between the two groups

组别	1 d	2 d	3 d	4 d	5 d
NC mimics组	0.18±0.02	0.38±0.03	0.74±0.05	1.11±0.16	1.90±0.05
miR-101-3p mimics组	0.19±0.02	0.36±0.03	0.68±0.04	0.87±0.08	1.26±0.12
t	0.692	0.711	1.779	2.336	8.784^＊＊

图11 NC mimics或miR-101-3p mimics处理后MV3细胞中BrdU阳性染色图

Fig.11 BrdU positive staining in MV3 cells treated with NC mimics or miR-101-3p mimics

参考文献 20

[1]	SCHADENDORF D, VAN AKKOOI A C J, BERKING C, et al. Melanoma[J]. Lancet, 2018, 392(10151):971-984. doi:10.1016/S0140-6736(18)31559-9.
[2]	AUDRITO V, MANAGÒ A, GAUDINO F, et al. Targeting metabolic reprogramming in metastatic melanoma: The key role of nicotinamide phosphoribosyltransferase (NAMPT)[J]. Semin Cell Dev Biol, 2020, 98:192-201. doi:10.1016/j.semcdb.2019.05.001.
[3]	ZHAO G, GREEN C F, HUI Y H, et al. Discovery of a highly selective NAMPT inhibitor that demonstrates robust efficacy and improved retinal toxicity with nicotinic acid coadministration[J]. Mol Cancer Ther, 2017, 16(12):2677-2688. doi:10.1158/1535-7163.MCT-16-0674.
[4]	LIANG B, ZHAO J, WANG X. Clinical performance of E2Fs1-3 in kidney clear cell renal cancer,evidence from bioinformatics analysis[J]. Genes Cancer, 2017, 8(5/6):600-607. doi:10.18632/genes andcancer.143.
[5]	AN X, MA H, LIU Y, et al. Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1[J]. J Anim Sci Biotechnol, 2020, 11:102. doi:10.1186/s40104-020-00506-6.
[6]	VAN ZEIJL M C, WAN DEN EERTWEGH A J, HAANEN J B, et al. (Neo)adjuvant systemic therapy for melanoma[J]. Eur J Surg Oncol, 2017, 43(3):534-543. doi:10.1016/j.ejso.2016.07.001.
[7]	GOODY D, GUPTA S K, ENGELMANN D, et al. Drug repositioning inferred from E2F1-coregulator interactions studies for the prevention and treatment of metastatic cancers[J]. Theranostics, 2019, 9(5):1490-1509. doi:10.7150/thno.29546.
[8]	ZHAO H, TANG W, CHEN X, et al. The NAMPT/E2F2/SIRT1 axis promotes proliferation and inhibits p53-dependent apoptosis in human melanoma cells[J]. Biochem Biophys Res Commun, 2017, 493(1):77-84. doi:10.1016/j.bbrc.2017.09.071.
[9]	ZENG Z, CAO Z, TANG Y. Increased E2F2 predicts poor prognosis in patients with HCC based on TCGA data[J]. BMC Cancer, 2020, 20(1):1037. doi:10.1186/s12885-020-07529-2.
[10]	HUANG Y L, NING G, CHEN L B, et al. Promising diagnostic and prognostic value of E2Fs in human hepatocellular carcinoma[J]. Cancer Manag Res, 2019, 11:1725-1740. doi:10.2147/CMAR.S182001.
[11]	SUN C C, LI S J, HU W, et al. Comprehensive analysis of the expression and prognosis for E2Fs in human breast cancer[J]. Mol Ther, 2019, 27(6):1153-1165. doi:10.1016/j.ymthe.2019.03.019.
[12]	YANG C, ZHANG Z C, LIU T B, et al. E2F1/2/7/8 as independent indicators of survival in patients with cervical squamous cell carcinoma[J]. Cancer Cell Int, 2020, 20:500. doi:10.1186/s12935-020- 01594-0.
[13]	LIU X, HU C. Novel potential therapeutic target for E2F1 and prognostic factors of E2F1/2/3 /5/7/8 in human gastric cancer[J]. Mol Ther Methods Clin Dev, 2020, 18:824-838. doi:10.1016/j.omtm.2020.07.017.
[14]	GAO J, CHEN X, SHAN C, et al. Autophagy in cardiovascular diseases: role of noncoding RNAs[J]. Mol Ther Nucleic Acids, 2020, 23:101-118. doi:10.1016/j.omtn.2020.10.039.
[15]	YAN X, ZHOU R, MA Z. Autophagy-cell survival and death[J]. Adv Exp Med Biol, 2019, 1206:667-696. doi:10.1007/978-981-15-0602-4_29.
[16]	CUI G, WANG H, LIU W, et al. Glycogen phosphorylase B is regulated by miR101-3p and promotes hepatocellular carcinoma tumorigenesis[J]. Front Cell Dev Biol, 2020, 8:566494. doi:10.3389/fcell.2020.566494.
[17]	HUANG Z, WU X, LI J. miR-101 suppresses colon cancer cell migration through regulation of EZH2[J]. Rev Esp Enferm Dig, 2021, 113(4):255-260. doi:10.17235/reed.2020.6800/2019.
[18]	WU R S, QIU E H, ZHU J J, et al. MiR-101 promotes nasopharyngeal carcinoma cell apoptosis through inhibiting Ras/Raf/MEK/ERK signaling pathway[J]. Eur Rev Med Pharmacol Sci, 2020, 24(16):8240. doi:10.26355/eurrev_202008_22580.
[19]	HUANG Y, ZOU Y, LIN L, et al. miR-101 regulates cell proliferation and apoptosis by targeting KDM1A in diffuse large B cell lymphoma[J]. Cancer Manag Res, 2019, 11:2739-2746. doi:10.2147/CMAR.S197744.
[20]	WANDLER A, RIBER-HANSEN R, HAGER H, et al. Quantification of microRNA-21 and microRNA- 125b in melanoma tissue[J]. Melanoma Res, 2017, 27(5):417-428. doi:10.1097/CMR.0000000000000374.

miR-101-3p靶向E2F2调控黑色素瘤细胞增殖的分子机制探讨

Molecular mechanism study of melanoma cell proliferation regulated by miR-101-3p-E2F2 targeting pathway

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