Li Yan, Zhao Qiaoshi, Yao Jinyin, Lv Chunpeng, Gao Yanhui, Sun Dianjun, Yang Yanmei
Center for Endemic Disease Control, Chinese Center for Disease Control and Prevention, Harbin Medical University, Harbin 150081, China.
Key Lab of Etiology and Epidemiology, Education Bureau of Heilongjiang Province & Ministry of Health (23618504), Harbin Medical University, Harbin 150081, China.
Toxics. 2023 Dec 1;11(12):978. doi: 10.3390/toxics11120978.
Long-term exposure to arsenic has been linked to a variety of cancers, among which skin cancer is the most prevalent form. However, the mechanism underlying arsenic carcinogenesis is unclear, and there is still limited information on the role of miRNAs in arsenic-induced skin cancer. This study aims to explore the role of miR-96-5p in the arsenite-induced proliferation and malignant transformation of human HaCaT keratinocytes. The GEO database (accession numbers GSE97303, GSE97305, and GSE97306) was used to extract mRNA and miRNA expression profiles of HaCaT cells treated with or without 0.1 μmol/L sodium arsenite for 3 and 7 weeks. In this paper, according to the CCK8 assay result, HaCaT cells exposed to 0.1 μmol/L sodium arsenite for 48 h were finalized. CCK8, MTT, EdU incorporation, and colony formation assays were used to determine the viability and proliferation of HaCaT cells and transformed HaCaT (T-HaCaT) cells. The subcellular localization and relative expression levels of DTL, as well as miR-96-5p in HaCaT cells induced by arsenite, were determined via immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was performed to identify miR-96-5p bound directly to DTL. Transfection of miR-96-5p mimics or DTL siRNA was conducted to verify the arsenite-induced viability of HaCaT cells and T-HaCaT cells. T-HaCaT cells and nude mice were used to construct arsenite-induced malignant transformation and an in vivo xenograft model to demonstrate the over-expressed effect of miR-96-5p. The results showed that DTL was the target gene of miR-96-5p. Meanwhile, we also found that 0.1 μmol/L sodium arsenite upregulated DTL by decreasing the miR-96-5p level, leading to the proliferation and malignant transformation of HaCaT cells. MiR-96-5p agomir treatment slowed the growth of transplanted HaCaT cells transformed by arsenite in a manner associated with DTL downregulation in the nude mice xenograft model. Taken together, we confirmed that miR-96-5p, as a potent regulator of DTL, suppressed arsenite-induced HaCaT cell proliferation and malignant transformation, which might provide a novel therapeutic target for the treatment of arsenic-induced skin cancer.
长期接触砷已与多种癌症相关联，其中皮肤癌是最常见的形式。然而，砷致癌的潜在机制尚不清楚，关于miRNA在砷诱导的皮肤癌中的作用的信息仍然有限。本研究旨在探讨miR-96-5p在亚砷酸盐诱导的人HaCaT角质形成细胞增殖和恶性转化中的作用。利用GEO数据库（登录号GSE97303、GSE97305和GSE97306）提取用或不用0.1μmol/L亚砷酸钠处理3周和7周的HaCaT细胞的mRNA和miRNA表达谱。在本文中，根据CCK8检测结果，确定用0.1μmol/L亚砷酸钠处理48小时的HaCaT细胞。采用CCK8、MTT、EdU掺入和集落形成试验来测定HaCaT细胞和转化的HaCaT（T-HaCaT）细胞的活力和增殖情况。通过免疫荧光、RT-qPCR和蛋白质免疫印迹法测定亚砷酸盐诱导的HaCaT细胞中DTL以及miR-96-5p的亚细胞定位和相对表达水平。进行双荧光素酶报告基因检测以鉴定直接与DTL结合的miR-96-5p。转染miR-96-5p模拟物或DTL siRNA以验证亚砷酸盐诱导的HaCaT细胞和T-HaCaT细胞的活力。使用T-HaCaT细胞和裸鼠构建亚砷酸盐诱导的恶性转化和体内异种移植模型，以证明miR-96-5p的过表达效果。结果表明DTL是miR-96-5p的靶基因。同时，我们还发现0.1μmol/L亚砷酸钠通过降低miR-96-5p水平上调DTL，导致HaCaT细胞增殖和恶性转化。在裸鼠异种移植模型中，miR-96-5p激动剂处理以与DTL下调相关的方式减缓了亚砷酸盐转化的移植HaCaT细胞的生长。综上所述，我们证实miR-96-5p作为DTL的有效调节剂，抑制了亚砷酸盐诱导的HaCaT细胞增殖和恶性转化，这可能为砷诱导的皮肤癌治疗提供新的治疗靶点。
