长链非编码RNA TRHDE-AS1调控SPRED1介导MAPK/ERK信号通路影响黑色素瘤转移的机制  

Study on mechanism of SPRED1-mediated MAPK/ERK signaling pathway mediated by long non-coding RNA TRHDE-AS1 affecting metastasis of melanoma

作　　者：买买提艾力·哈斯木[1] 籍素芝 杨文鹏 江仁兵[1] Maimaitiaili Hasimu;JI Suzhi;YANG Wenpeng;JIANG Renbing(Department of Bone and Soft Tissue Tumor and Melanoma,the Affiliated Tumor Hospital,Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学附属肿瘤医院骨与软组织肿瘤及黑色素瘤科,乌鲁木齐830011

出　　处：《新疆医科大学学报》2024年第9期1193-1199,共7页Journal of Xinjiang Medical University

基　　金：新疆维吾尔自治区自然科学基金项目(2022D01C538)。

摘　　要：目的探讨TRHDE-AS1对A375细胞侵袭转移的调控作用及内在机制。方法采用CCK-8、流式细胞术检测细胞活性,Transwell检测细胞侵袭能力,划痕实验检测细胞的愈合能力,实时荧光定量PCR检测促甲状腺激素释放激素降解酶反义RNA1(Thyrotropin releasing hormone degrading enzyme antisense RNA1,TRHDE-AS1)和含侧支发芽因子相关EVH域蛋白1(Sprouty related EVH1 domain containing 1,SPRED1)的mRNA表达,Western blot检测SPRED1、磷酸化的有丝分裂活化蛋白激酶(phosphoric mitogen-activated protein kinase 1,p-ERK)、RAS原癌基因(RAS proto-oncogene,RAS)、磷酸化的Raf蛋白激酶(phosphoric Raf protein kinase,p-RAF)、Raf蛋白激酶(Raf protein kinase,RAF)的蛋白表达,RNA pulldown检测TRHDE-AS1和SPRED1的结合。结果细胞增殖活性结果显示,与Control组(100.00±0.00)%比较,TRHDE-AS1组细胞增殖活性(71.75±10.60)%显著降低(P<0.05),而TRHDE-AS1 siRNA组的细胞增殖活性(125.46±11.20)%显著升高(P<0.05)。流式细胞凋亡结果显示,与Control组(6.08±0.47)%比较,TRHDE-AS1组细胞凋亡率(22.39±0.47)%显著升高(P<0.05);而TRHDE-AS1 siRNA组的细胞凋亡率(3.47±1.07)%显著降低(P<0.05)。细胞划痕实验结果显示,与Control组(41.53±3.00)%比较,TRHDE-AS1组的细胞愈合能力(22.74±0.96)%显著降低(P<0.05);而TRHDE-AS1 siRNA组的细胞愈合能力(68.76±2.41)%显著升高(P<0.05)。Transwell实验结果显示,与Control组(135.20±9.01)比较,TRHDE-AS1组的细胞侵袭数量(66.00±4.85)显著降低(P<0.05);而TRHDE-AS1 siRNA组的细胞侵袭数量(204.80±9.83)显著升高(P<0.05)。Western blot结果显示,TRHDE-AS1组的p-ERK、Ras、p-Raf的蛋白表达水平较Control组显著降低(P<0.05);而TRHDE-AS1 siRNA组p-ERK、Ras、p-Raf的蛋白表达水平较Control组显著升高(P<0.05)。此外,TRHDE-AS1可以交互结合SPRED1,TRHDE-AS1组SPRED1的mRNA和蛋白表达水平较Control组显著升高(P<0.05);TRHDE-AS1 siRNA组SPRED1的表达水平显著降低(P<0.05)。结论长链非Objective To investigate the regulation and mechanism of TRHDE-AS1 on A375 cell invasion and metastasis.Methods CCK-8 and flow cytometry were used to detect cell activity,Transwell was used to detect cell invasion ability,scratch assay was used to detect cell healing ability,and real-time fluorescence quantitative PCR was used to detect mRNA expression of TRHDE-AS1 and SPRED1.The protein expressions of SPRED1,p-ERK,RAS,p-RAF and RAF were detected by Western blot,and the binding of TRHDE-AS1 and SPRED1 was detected by RNA pulldown.Results Compared with control group(100.00±0.00)%,the cell proliferation activity in TRHDE-AS1 group was significantly decreased(71.75±10.60)%(P<0.05).The cell proliferation activity in TRHDE-AS1 siRNA group was significantly increased(125.46±11.20)%(P<0.05).Flow cytometry showed that compared with control group(6.08±0.47)%,the apoptosis rate of TRHDE-AS1 group was significantly increased(22.39±0.47)%(P<0.05).The apoptosis rate in TRHDE-AS1 siRNA group was significantly decreased(3.47±1.07)%(P<0.05).The results of cell scratch test showed that the healing ability of cells in TRHDE-AS1 group was significantly decreased(22.74±0.96)%compared with(41.53±3.00)%in control group(P<0.05).The cell healing ability of TRHDE-AS1 siRNA group was significantly increased(68.76±2.41)%(P<0.05).The results of Transwell experiment showed that compared with control group(135.20±9.01),the number of cell invasion in TRHDE-AS1 group(66.00±4.85)was significantly decreased(P<0.05).The number of cell invasion in TRHDE-AS1 siRNA group(204.80±9.83)was significantly increased(P<0.05).Western blot results showed that the protein expression levels of p-ERK,Ras and p-Raf in TRHDE-AS1 group were significantly decreased compared with control group(P<0.05).The protein expression levels of p-ERK,Ras and p-Raf in TRHDE-AS1 siRNA group were significantly increased(P<0.05).In addition,TRHDE-AS1 could interactively bind SPRED1,and the mRNA and protein expression levels of SPRED1 in TRHDE-AS1 group were significantl

关 键 词：黑色素瘤 长链非编码RNA 侵袭 机制 

分 类 号：R739.5[医药卫生—肿瘤]