The long non-coding RNA TP73-AS1 modulates HCC cell proliferation through miR-200a-dependent HMGB1/RAGE regulation.
The long non-coding RNA TP73-AS1 modulates HCC cell proliferation through miR-200a-dependent HMGB1/RAGE regulation.
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DOI：
10.1186/s13046-017-0519-z
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发表时间：
2017-04-12
期刊：
影响因子：
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通讯作者：
Fan XG
中科院分区：
文献类型：
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作者：
Li S;Huang Y;Huang Y;Fu Y;Tang D;Kang R;Zhou R;Fan XG
P73反义RNA 1 T（non-protein coding），又称TP 73-AS 1，是一种长链非编码RNA（lncRNA），参与细胞增殖和肿瘤的发生发展。然而，TP 73-AS 1在肝细胞癌（HCC）进展中的确切作用和分子机制仍不清楚。本研究旨在探讨TP 73-AS 1在肝癌细胞增殖调控中的具体功能及其作用机制。采用实时荧光定量PCR方法检测肝癌组织和细胞系中TP 73-AS 1的表达，分析TP 73-AS 1表达与肝癌临床病理特征的相关性。MTT法和BrdU法检测TP 73-AS 1对肝癌细胞增殖的调节作用。采用miRcode、miRWalk、米兰达和Target scan筛选HMGB 1上游候选miRNAs，并采用实时荧光定量PCR方法进行验证。使用荧光素酶报告基因测定证实TP 73-AS 1与miR-200 a之间的相互作用。使用Western blot分析和ELISA测定响应于共加工TP 73-AS 1敲低和miR-200 a抑制的HMGB 1信号相关因子的蛋白质水平。进一步检测HCC组织中miR-200 a、HMGB 1 mRNA和miR-200 a mRNA的表达及其相关性。TP 73-AS 1在HCC组织和细胞系中上调。TP 73-AS 1高表达与临床病理特征、预后和生存期相关。下调TP 73-AS 1基因表达可抑制肝癌细胞增殖，抑制HMGB 1、NF-κ B和NF-κB表达。利用在线工具筛选出HMGB 1上游的几种候选miRNAs，其中miR-200 a过表达对HMGB 1 mRNA表达的抑制作用最显著。通过荧光素酶检测，我们证实了miR-200 a可以直接与TP 73-AS 1和HMGB 1的3 'UTR结合; TP 73-AS 1与HMGB 1竞争miR-200 a的结合。MiR-200 a抑制可上调HMGB 1、RAGE、NF-κB表达以及NF-κB调节的细胞因子水平，si-TP 73-AS 1可部分恢复这一作用。在肝癌组织中，miR-200 a表达下调，HMGB 1和miR-200 a表达上调; TP 73-AS 1与miR-200 a呈负相关，与HMGB 1和miR-200 a呈正相关。提示TP 73-AS 1可能是一种促肝癌细胞增殖的致癌lncRNA，可作为肝癌治疗的靶点。本文的在线版本（doi：10.1186/s13046-017-0519-z）包含补充材料，可供授权用户使用。
P73 antisense RNA 1 T (non-protein coding), also known as TP73-AS1, is a long non-coding RNA (lncRNA) which is involved in cell proliferation and the development of tumors. However, the exact effects and molecular mechanisms of TP73-AS1 in hepatocellular carcinoma (HCC) progression are still unknown. The present study is aimed to investigate the detailed functions and the mechanism of TP73-AS1 in regulation of HCC cell proliferation. TP73-AS1 expression in HCC tissues and cell lines was determined using real-time PCR assays; the correlation of TP73-AS1 expression with clinicopathological features of HCC was analyzed. The functions of TP73-AS1 in regulation of HCC cell proliferation was evaluated using MTT and BrdU assays. The candidate upstream miRNAs of HMGB1 were screened using miRcode, miRWalk, miRanda and Target scan, verified using real-time PCR assays. The interaction between TP73-AS1 and miR-200a was confirmed using Luciferase report gene assays. The proten levels of HMGB1 signaling-related factors in response to co-processing TP73-AS1 knockdown and miR-200a inhibition were determined using Western blot assays and ELISA. Further, miR-200a, HMGB1 mRNA and RAGE mRNA and their correlations in HCC tissues were determined. TP73-AS1 was upregulated in HCC tissues and cell lines. High TP73-AS1 expression was correlated with worse clinicopathological features, poorer prognosis and shorter survival. Knockdown of TP73-AS1 inhibited the HCC proliferation and the expression levels of HMGB1, RAGE and NF-κB in HCC cells. By using online tools, we screened out several candidate upstream miRNAs of HMGB1, among which miR-200a overexpression inhibited HMGB1 mRNA expression the most significantly. By using luciferase assays, we confirmed that miR-200a could directly bind to TP73-AS1 and the 3’UTR of HMGB1; TP73-AS1 competed with HMGB1 for miR-200a binding. MiR-200a inhibition could up-regulate HMGB1, RAGE, NF-κB expression as well as NF-κB regulated cytokines levels, which could be partially restored by si-TP73-AS1. In HCC tissues, miR-200a was down-regulated while HMGB1 and RAGE were up-regulated; TP73-AS1 was inversely correlated with miR-200a, while positively correlated with HMGB1 and RAGE, respectively. Our data indicated that TP73-AS1 might be an oncogenic lncRNA that promoted proliferation of HCC and could be regarded as a therapeutic target in human HCC. The online version of this article (doi:10.1186/s13046-017-0519-z) contains supplementary material, which is available to authorized users.