摘要
目的探讨人结节型皮肤基底细胞癌(BCC)病变组织及细胞株中视蛋白3(OPN3)的表达及功能。方法选取31例结节型BCC患者病灶组织及相邻癌旁正常组织(PANST)分别作为病灶组及PANST组,采用免疫荧光染色法检测OPN3的表达,Western blot及实时荧光定量PCR(RT-qPCR)分别检测2组标本组织中OPN3蛋白及mRNA的表达;取对数生长期的人BCC细胞株TE354T,分为对照(Control)组、空白(RNAi-NC)组及实验(RNAi-OPN3)组,并设计针对人OPN3的小干扰RNA转染TE354T细胞,采用Cell Counting Kit-8(CCK-8)检测OPN3下调后0、24、48及72 h RNAi-NC组及RNAi-OPN3组的细胞增殖活性,采用Transwell迁移和细胞划痕实验检测OPN3下调48 h后2组细胞的迁移能力,采用流式细胞术和Western blot分别检测OPN3下调48 h后2组细胞凋亡数量及钙通道相关蛋白环磷腺苷效应元件结合蛋白(CREB)、磷酸化CREB(p-CREB)、钙调蛋白依赖激酶Ⅱ(CAMKⅡ)、磷酸化CAMKⅡ(p-CAMKⅡ)的表达。结果与PANST组比较,病灶组BCC组织中OPN3表达增高(P<0.05);下调OPN3后72 h,RNAi-OPN3组TE354T细胞增殖活性较RNAi-NC组下降(P<0.05);下调OPN3后48 h,与RNAi-NC组相比,RNAi-OPN3组TE354T细胞迁移能力降低(P<0.05),TE354T细胞凋亡细胞数量增加(P<0.05),TE354T细胞钙通道相关蛋白p-CREB和p-CAMKⅡ表达下降(P<0.05)。结论OPN3在人结节型BCC组织中高表达,可调控人BCC系TE354T细胞的增殖、迁移及凋亡,其机制可能与钙依赖G蛋白偶联受体(GPCR)信号通路有关。
Objective To investigate the expression and function of opsin 3(OPN3)in human nodular basal cell carcinoma(BCC)lesions and cell lines.Methods The lesion tissue and paired adjacent normal skin tissue(PANST)of 31 patients with nodular BCC were collected as the lesion group and the PANST group,respectively.The expression of OPN3 was detected by immunofluorescence staining.Western blot and real-time fluorescence quantitative PCR were used to detect the expression of OPN3 protein and mRNA in tissues,respectively.The human BCC cell line TE354T cells in logarithmic growth phase were taken and divided into control group(Control),blank group(RNAi-NC)and experimental group(RNAi-OPN3);small interfering RNA targeting human OPN3 was designed and transfected into human basal cell carcinoma TE354T cells.After successful transfection,the changes of cell proliferation activity of RNAi-NC group and RNAi-OPN3 group at 0,24,48,and 72 h after down-regulated OPN3 were detected by Cell Counting Kit.Changes in cell migration ability of two groups at 48 h before and after OPN3 downregulation were detected by Transwell migration assay and cell scratch assay;flow cytometry and Western blot were used to detect the apoptosis of two groups and the expressions of calcium channel-related protein CREB,p-CREB,CAMKⅡ,p-CAMKⅡat 48 h after OPN3 downregulation.Results Compared with PANST group,OPN3 expression in BCC tissue of lesion group increased(P<0.05).Seventy-two hours after down-regulation of OPN3,the proliferative activity of TE354T cells in the RNAi-OPN3 group was lower than that of RNAi-NC group(P<0.05);48 h after down-regulation of opsin3,TE354T cell migration ability was reduced(P<0.05),number of apoptotic cells increased(P<0.05),and the expression of calcium channel-related proteins p-CREB and p-CAMKⅡdecreased compared with RNAi-NC group(P<0.05).Conclusion OPN3 is highly expressed in human nodular BCC tissues,and is capable to regulate the proliferation,migration,and apoptosis of human BCC line TE354T cells.Such mechanism can be associated with calcium-dependent G protein-coupled receptor signaling pathway.
作者
许佳
韩宁
章俊
兰应华
辜阳广
罗欢欢
杨玉麟
汪宇
陆洪光
XU Jia;HAN Ning;ZHANG Jun;LAN Yinghua;GU Yangguang;LUO Huanhuan;YANG Yulin;WANG Yu;LU Hongguang(Department of Dermatology and Venereology,School of Clinical Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Dermatology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2023年第6期652-660,共9页
Journal of Guizhou Medical University
基金
国家自然科学基金(81972920,82060568,816730609)。
作者简介
许佳,贵州医科大学2019级硕士研究生;通信作者:陆洪光,E-mail:hongguanglu@hotmail.com。
