摘要
目的探讨LINC01355通过miR-545-5p/叉头盒D1(FOXD1)信号通路对口腔鳞状细胞癌(OSCC)细胞增殖、凋亡和侵袭的影响。方法体外培养人OSCC细胞系Cal-27,随机分为对照组、转染LINC01355 siRNA(si-LINC01355)组、转染LINC01355过表达质粒(pc-LINC01355)组、转染LINC01355 siRNA阴性对照+miR-545-5p阴性对照+空载质粒(si-NC+miR-545-5p-NC+pc-NC)组、转染LINC01355 siRNA+miR-545-5p抑制剂(si-LINC01355+miR-545-5p inhibitor)组。转染后实时定量PCR检测各组细胞LINC01355、miR-545-5p及FOXD1表达;转染后的Cal-27细胞通过皮下接种构建各组移植瘤裸鼠模型,测量移植瘤生长情况;CCK-8法、流式细胞术、Transwell实验分别检测各组细胞增殖、凋亡和侵袭情况;免疫组织化学染色检测细胞上皮-间质转化(EMT)相关蛋白Vimentin、E-cadherin表达;Western blotting检测细胞增殖相关蛋白[增殖细胞核抗原(PCNA)、C-myc]、凋亡相关蛋白[切割型胱天蛋白酶-3(cleaved caspase-3)、BCL-2相关X蛋白(Bax)]与FOXD1表达。双萤光素酶报告实验鉴定LINC01355与miR-545-5p/FOXD1信号通路的靶向关系。结果与对照组比较,si-LINC01355组LINC01355、FOXD1 mRNA表达,增殖活性,侵袭数量,Vimentin蛋白阳性表达,PCNA、C-myc与FOXD1蛋白表达,移植瘤体积均降低(均P<0.05);而miR-545-5p表达,细胞凋亡率,cleaved caspase-3、Bax蛋白表达,E-cadherin蛋白阳性表达均升高(均P<0.05)。pc-LINC01355组各指标变化趋势与si-LINC01355组相反。与si-LINC01355组比较,si-LINC01355+miR-545-5p inhibitor组LINC01355、FOXD1 mRNA表达,增殖活性,侵袭数量,Vimentin蛋白阳性表达,PCNA、C-myc与FOXD1蛋白表达,移植瘤体积均增加(均P<0.05),而miR-545-5p表达、细胞凋亡率、cleaved caspase-3与Bax蛋白表达、E-cadherin蛋白阳性表达均降低(均P<0.05)。Cal-27细胞中LINC01355可靶向下调miR-545-5p表达,且miR-545-5p可靶向下调FOXD1表达。结论LINC01355可通过调控miR-545-5p/FOXD1信号通路促进OSCC细胞增殖和侵袭,抑制细胞凋亡。
Objective To investigate the effects of LINC01355 on the proliferation,apoptosis,and invasion of oral squamous cell carcinoma(OSCC)cells via the miR-545-5p/forkhead box D1(FOXD1)signaling pathway.Methods Cal-27 cells were cultured in vitro and randomly separated into the control group,si-LINC01355(transfected with LINC01355 siRNA)group,pc-LINC01355(transfected with LINC01355 overexpression plasmid)group,si-NC+miR-545-5p-NC+pc-NC(transfected with LINC01355 siRNA negative control,miR-545-5p negative control and empty plasmid)group,and si-LINC01355+miR-545-5p inhibitor(transfected with LINC01355 siRNA and miR-545-5p inhibitor)group.After transfection,quantitative real-time PCR was applied to detect the expression of LINC01355,miR-545-5p,and FOXD1 in cells.The Cal-27 cells transfected into groups were then subcutaneously inoculated to construct nude mouse models of transplanted tumors in each group,and the growth of the transplanted tumors was measured.The CCK-8 method,flow cytometry,and Transwell assay were applied in order to detect cell proliferation,apoptosis,and invasion,respectively.Immunohistochemical staining was applied to detect the expression of epithelial-mesenchymal transition(EMT)related proteins Vimentin and E-cadherin in cells.Western blotting was applied to detect the expression of cell proliferation related proteins(proliferating cell nuclear antigen[PCNA],C-myc),apoptosis related proteins(cleaved cysteinyl aspartate-specific proteases-3[cleaved caspase-3],BCL-2-associated X protein[Bax]),and FOXD1 protein.A dual-luciferase reporter assay was used to identify the relationship between LINC01355 and the miR-545-5p/FOXD1 signaling pathway.Results Compared with the control group,the expression of LINC01355 and FOXD1 mRNA,proliferation activity,number of invasions,positive expression of Vimentin protein,expression of PCNA,C-myc and FOXD1 protein,and transplanted tumor volume reduced(All P<0.05);the expression of miR-545-5p,apoptosis rate,cleaved caspase-3 and Bax protein expression,and positive expression of E-cadherin protein increased(All P<0.05)in the si-LINC01355 group.The trend of changes in the various indicators of the pc-LINC01355 group was opposite to that of the si-LINC01355 group.Compared with the si-LINC01355 group,the expression of LINC01355 and FOXD1 mRNA,proliferation activity,number of invasions,positive expression of Vimentin protein,expression of PCNA,C-myc and FOXD1 proteins,and the transplanted tumor volume in the si-LINC01355+miR-545-5p inhibitor group increased(All P<0.05),whereas the expression of miR-545-5p,apoptosis rate,cleaved caspase-3 and Bax protein expression,and positive expression of E-cadherin protein decreased(All P<0.05).LINC01355 was able to downregulate miR-545-5p expression in Cal-27 cells and miR-545-5p was able to downregulate FOXD1 expression.Conclusion LINC01355 promotes OSCC cell proliferation and invasion,and inhibits apoptosis by targeting the miR-545-5p/FOXD1 signaling pathway.
作者
高静
魏校通
李星晨
闫威
王浩
GAO Jing;WEI Xiaotong;LI Xingchen;YAN Wei;WANG Hao(Medical Examination Center,Cangzhou Central Hospital,Cangzhou 061000,China;Department of Oral and Maxillofacial Surgery,Cangzhou Central Hospital,Cangzhou 061000,China;Department of Dermatology and Venereology,Cangzhou Central Hospital,Cangzhou 061000,China)
出处
《中国医科大学学报》
北大核心
2025年第3期238-245,共8页
Journal of China Medical University
基金
河北省医学科学研究课题(20240509)。
作者简介
高静(1990-),女,主治医师,本科;通信作者:魏校通,E-mail:weixiaotong0820@163.com。
