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

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 by UVA irradiation alone.RNA-seq screened out 141 differential genes upon OPN3 silencing or over-expression in MV3 cells.KEGG analysis further revealed 9 enrichment signaling pathways of differential genes with Hippo signaling pathway as the highest-Log10(FDR)value.With the silencing of OPN3,large tumor suppressor 1(LATS1)expression levels associated with Hippo signaling pathway increased while Phospho-Yes-associated protein(p-YAP),Yes-associated protein(YAP),ras homolog family member A(RhoA)protein expression levels decreased.Conclusion UVA can promote the expression of opsins,specifically OPN3 in human melanoma cell lines(A375 cells and MV3 cells),thereby regulating cell proliferation through the Hippo signaling pathway.