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 hor-mone 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 siR-NA 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 长链非编码RNA TRHDE-AS1可以通过促进交互蛋白SPRED1的转录和翻译水平,降低MAPK/ERK通路活性,抑制黑色素瘤的增殖、侵袭转移.
Study on mechanism of SPRED1-mediated MAPK/ERK signaling pathway mediated by long non-coding RNA TRHDE-AS1 affecting metastasis of melanoma
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 fluores-cence 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 in-creased(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 ex-pression 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 significantly higher than those in control group(P＜0.05).The expression level of SPRED1 in TRHDE-AS1 siRNA group was significantly decreased(P＜0.05).Conclusion Long non-coding RNA TRHDE-AS1 can reduce the activity of MAPK/ERK pathway and in-hibit the proliferation,invasion and metastasis of melanoma by promoting the transcription and translation level of interacting protein SPRED1.
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