miR-141-5p影响黏膜黑色素瘤细胞生物学行为的机制研究

Study on the mechanism of miR-141-5p affecting the biological behavior of mucosal melanoma cells

目的探讨miR-141-5p在黏膜黑色素瘤(MM)组织及细胞中的表达及其意义,分析其影响MM细胞生物学行为的机制。方法回顾性收集2015年1月至2020年12月温州医科大学附属第一医院手术切除的MM和色素痣组织标本。qRT-PCR法检测并比较MM和色素痣组织、MM细胞株A375和人角质形成细胞株HaCaT中miR-141-5p、细胞外信号调节激酶(ERK)1/2 mRNA的相对表达量。采用Pearson相关分析miR-141-5p与ERK1/2 mRNA相对表达量的相关性。A375分别用miR-141-5p模拟物(模拟物组)、miR-141-5p模拟物对照(模拟物对照组)、miR-141-5p抑制物(抑制物组)和miR-141-5p抑制物对照(抑制物对照组)处理;采用qRT-PCR检测并比较4组细胞中miR-141-5p相对表达量;采用细胞计数(CCK)-8法、克隆形成实验、流式细胞术、划痕实验和Transwell侵袭实验观察并比较4组细胞存活率、增殖能力、细胞凋亡比例和细胞迁移、侵袭能力;采用Western blot法观察并比较4组细胞ERK1/2、磷酸化的ERK1/2(p-ERK1/2)蛋白相对表达量。采用HE染色、免疫组化染色观察MM和色素痣组织形态并检测p-ERK1/2、ERK1/2表达水平,比较不同p-ERK1/2、ERK1/2表达情况MM患者临床病理特征差异。结果miR-141-5p和ERK1/2 mRNA在MM组织和A375中表达下调(均P<0.01);Pearson相关分析显示,miR-141-5p与ERK1/2 mRNA相对表达量呈正相关(P<0.05)。相比对照组,模拟物组细胞增殖和迁移、侵袭能力显著降低,细胞凋亡比例升高,抑制物组细胞增殖和迁移、侵袭能力升高,细胞凋亡比例降低(均P<0.05)。相比对照组,模拟物组细胞p-ERK1/2蛋白相对表达量显著升高,抑制物组显著降低(均P<0.05)。MM组织中ERK1/2与p-ERK1/2蛋白表达明显减少(均P<0.05)。p-ERK1/2阴性组和阳性组、ERK1/2阴性组和阳性组不同临床病理特征比较,差异均无统计学意义(均P>0.05)。结论miR-141-5p可能通过ERK1/2通路影响MM细胞生物学行为。

Objective To explore the expression of miR-141-5p and its significance in mucosal melanoma(MM)tissues and A375 cells,and to analyze the mechanism by which it affects the biological behavior of MM cells.Methods MM and pigmented nevus specimens surgically excised from the First Affiliated Hospital of Wenzhou Medical University were collected from January 2015 to December 2020.qRT-PCR was performed to detect the expression levels of miR-141-5p,and extracellular signal-regulated kinase(ERK)1/2 mRNA in MM tissues and pigmented nevus tissue,MM cell line A375 and human keratinocyte cell line HaCaT,and Pearson correlation analysis was used to anaiyze the correlation between miR-141-5p and the relative expression of ERK1/2 mRNA.A375 was treated with miR-141-5p mimic(mimic group),miR-141-5p mimic blank sequence(mimic control group),miR-141-5p inhibitor(inhibitor group),and miR-141-5p inhibitor blank sequence(inhibitor control group),respectively;and qRT-PCR was performed to detect the expression levels of miR-141-5p in the cells of the four groups;Cell counting kit(CCK)-8 method,clone formation assay,flow cytometry,scratch assay and Transwell invasion assay were used to observe and compare the survival rate,proliferation ability,apoptosis rate and cell migration and invasion ability of the four groups of cells;Western blot was used to observe and compare the relative expressions of ERK1/2 and phosphorylated ERK1/2(p-ERK1/2)proteins in A375 cells of the four groups.HE staining and immunohistochemical staining were used to observe the morphology and detect the expressions of p-ERK1/2 and ERK1/2.The differences in p-ERK1/2 and ERK1/2 expression between MM patients with different clinicopathological characteristics were compared,respectively.Results miR-141-5p and ERK1/2 mRNA expression were down-regulated in MM tissues and A375 cells(all P<0.01);Pearson correlation analysis showed that miR-141-5p was positively correlated with the expression level of ERK1/2 mRNA(P<0.05).Compared with the control group,the cell proliferation and migration and invasion abilities were significantly reduced and apoptosis rate was elevated in the mimic group,while the cell proliferation and migration and invasion abilities were elevated and apoptosis rate was reduced in the inhibitor group(all P<0.05).Compared with the control group,the relative expression levels of p-ERK1/2 proteins in the mimic group were significantly increased,while significantly lowered in the inhibitor group(all P<0.05).The expressions of ERK1/2 and p-ERK1/2 proteins in MM tissues were significantly reduced(both P<0.05).There were no significant differences in clinicopathological features between p-ERK1/2 negative group and positive group or ERK1/2 negative group and positive group(all P>0.05).Conclusion miR-141-5p may inhibit the biological behavior of MM cells through the ERK1/2 pathway.