长波紫外线对人黑素瘤细胞中视蛋白3的表达及细胞增殖的影响  

Effects of long-wave ultraviolet on the expression of opsin 3 in human melanoma cells and cell proliferation

作　　者：张玉蕾 曾雯 张伟 董仙 冯江龙 靳姝琦 罗欢欢 刘婷 陆洪光 ZHANG Yulei;ZENG Wen;ZHANG Wei;DONG Xian;FENG Jianglong;JIN Shuqi;LUO Huanhuan;LIU Ting;LU Hongguang(Department of Dermatology and Venereology,School of Clinical Medicine,Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Dermatology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Pathology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学临床医学院皮肤病与性病学教研室,贵州贵阳550004 [2]贵州医科大学附属医院皮肤科,贵州贵阳550004 [3]贵州医科大学附属医院病理科,贵州贵阳550004

出　　处：《贵州医科大学学报》2024年第1期1-13,共13页Journal of Guizhou Medical University

基　　金：国家自然科学基金(82260615);贵州省自然科学基金项目(黔科合基础-ZK[2022]一般449,黔科合基础-ZK[2022]一般413);2023-2024年贵州省卫生健康委省级医学重点学科建设项目(黔卫健科[2023]2)。

摘　　要：目的研究长波紫外线对人黑素瘤细胞中视蛋白3(OPN3)的表达及对细胞增殖的影响。方法利用不同剂量的UVA分别照射人黑素瘤细胞系(A375细胞和MV3细胞)后,通过5-乙炔基-2′脱氧尿嘧啶核苷(EdU)实验及细胞增殖与活性检测试剂(CCK-8)检测照射前后细胞的增殖情况,实时荧光定量(RT-qPCR)分别检测细胞中视蛋白(OPN)的mRNA表达水平;利用慢病毒技术分别沉默和过表达A375细胞和MV3细胞中的OPN3,采用EdU实验及CCK-8方法检测细胞的增殖水平;沉默A375细胞和MV3细胞中OPN3后进行UVA照射,通过EdU及CCK-8方法检测照射前后细胞增殖的变化;采用转录组测序技术检测过表达及沉默OPN3的MV3细胞中差异基因的表达,并进行KEGG富集相关信号通路分析,免疫印迹法(Western blot)验证Hippo信号通路相关蛋白的表达情况。结果与Control组相比,UVA照射组中的细胞增殖能力明显增强(P<0.05);RT-qPCR结果显示,OPN1、OPN2、OPN3、OPN4以及OPN5在A375和MV3细胞中均有表达,其中OPN3的转录表达水平水平均明显高于其他OPN(P<0.05);UVA照射组中A375和MV3细胞OPN3蛋白表达水平明显升高,沉默OPN3的表达后,A375和MV3细胞的增殖能力减弱,过表达OPN3后A375和MV3细胞的增殖能力增强(P<0.05);沉默人黑素瘤细胞(A375细胞和MV3细胞)OPN3后经UVA照射,细胞增殖能力较单纯照射UVA时降低(P<0.05);沉默及过表达MV3细胞中OPN3的表达后,RNA-seq测序结果筛选出141个差异基因,KEGG分析筛选差异基因富集信号通路9条,其中-Log10(FDR)值最高的为Hippo信号通路;沉默OPN3后,检测到LATS1表达水平增加,p-YAP、YAP、RhoA蛋白表达水平降低(P<0.05)。结论UVA可促进人黑素瘤细胞系(A375细胞和MV3细胞)OPN3的表达,并通过Hippo信号通路调控细胞的增殖过程。Objective To investigate the effect of long-wave ultraviolet on the expression of opsin 3 in human melanoma cells lines(A375 cells and MV3 cells)and its impact on cell proliferation,as well as to explore the molecular mechanism of opsin 3 regulation on human melanoma cell proliferation.Methods Human melanoma cell lines(A375 cells and MV3 cells)were exposed to various doses of UVA,and cell proliferation was assessed by the 5-ethynyl-2'-deoxyuridine(EdU)and Cell Counting Kit-8(CCK-8)experiments.The transcription levels of opsin in human melanoma cell lines(A375 cells and MV3 cells)were assessed by real time quantitative PCR(RT-qPCR)and Western blot.Lentivirus infection were utilized to silence or overexpress opsin 3 in A375 cells and MV3 cells,with subsequent assessment of cell proliferation levels by the EdU method and CCK-8 assay.After A375 cells and opsin 3 in MV3 cells were silenced,the changes of cell proliferation before and after ultraviolet A(UVA)irradiation by EdU method and CCK-8 assay.RNA-seq was used to detect differential gene expression in OPN3-overexpressing or silenced MV3 cells,with subsequent analysis conducted on KEGG enrichment-related signaling pathways.Western blot was used to verify purposes regarding the expression of Hippo signaling pathway-related proteins.Results Compared with the control group,the cell proliferation ability of UVA group was significantly enhanced,and this difference was statistically significant(P<0.05).RT-qPCR revealed that opsins 1,2,3,4,and 5 were expressed in A375 and MV3 cells.The transcriptional expression and protein expression of opsin 3 were significantly higher than those of other opsins(P<0.05).Long wave ultraviolet irradiation significantly increased the expression of opsin 3 protein in A375 and MV3 cells.With the silencing of opsin 3,the proliferation ability was weakened while overexpression enhanced it(P<0.05).The proliferation ability of A375 cells and MV3 cells after the silence of OPN3 by UVA irradiation was significantly attenuated compared with that b

关 键 词：视蛋白3 黑素瘤 增殖 长波紫外线 转录组测序技术 Hippo信号通路 

分 类 号：R739.5[医药卫生—肿瘤]