lncRNA TUG1在儿童肝母细胞瘤中的表达及其与miR-204介导的JAK2-STAT3通路关系

Expression of lncRNA TUG1 pediatric in hepatoblastoma and its association with miR-204-mediated JAK2-STAT3 pathway

背景与目的:近年来,儿童肝母细胞瘤(HB)的治疗方面取得一定的进步,但整体临床预后仍然较差,因此探索其发病机制和有效治疗靶点具有重要意义。本研究探讨长链非编码RNA(lncRNA)牛磺酸上调基因1(TUG1)与JAK2-STAT3通路相关分子在HB组织中的表达,初步分析HB中TUG1与miR-204介导的JAK2-STAT3血管生成信号通路之间的关系。方法:选取2017年3月—2018年4月湖南省儿童医院收治的60例HB患儿为研究对象,收集所有患儿HB肿瘤组织及其远端瘤旁正常组织,分别采用免疫组化与Western blot法检测组织中JAK2、STAT3及下游血管生成相关分子蛋白的表达,用qRT-PCR法检测组织中TUG1、miR-204与JAK2、STAT3及下游血管生成相关分子的RNA表达,并分析HB组织中TUG1与miR-204的表达的相关性。此外,在人HB细胞系HepG2中,观察TUG1敲减或miR-204过表达后,JAK2、STAT3及下游血管生成相关分子的RNA表达的变化。结果:免疫组化结果显示,HB组织中JAK2与STAT3蛋白的阳性表达率明显高于瘤旁正常组织(JAK2:40.1%vs.16.9%;STAT3:55.7%vs.19.8%,均P<0.05)。qRT-PCR结果显示,HB组织中TUG1、JAK2、STAT3及血管生成相关分子VEGF、VEGFR2、HIF-1α的RNA表达均较瘤旁组织明显上调(均P<0.05);HB组织中,TUG1与miR-204的表达呈明显负相关(r=-0.962,P=0.014)。Western blot结果显示,HB组织中JAK2、STAT3及下游血管生成相关分子的蛋白表达均较瘤旁组织明显上调(均P<0.05)。HepG2中,TUG1敲减或miR-204过表达后,JAK2、STAT3及下游血管生成相关分子的RNA与蛋白表达均明显下调(均P<0.05)。结论:HB患儿肿瘤组织内TUG1的表达上调,并伴有JAK2-STAT3通路的活性升高,且TUG1与miR-204的表达呈负相关,这提示在HB中,TUG1可能通过抑制miR-204表达,从而激活JAK2-STAT3通路,促进HB的血管生成。

Background and Aims:Although a certain progress has been made in the treatment of hepatoblastoma(HB)in children,the overall clinical prognosis is still poor.So,exploring its pathogenesis and effective therapeutic targets are of great importance.This study was conducted to investigate the expressions of the long non-coding RNA taurine-upregulated gene 1(TUG1)and the molecules associated with JAK2-STAT3 pathway in HB tissue and preliminarily analyze the relationship between TUG1 and miR-204-mediated JAK2-STAT3 angiogenic signaling pathway.Methods:Sixty pediatric patients with HB admitted in Hunan Children’s Hospital from March 2017 to April 2018 were enrolled as study subjects.The HB tumor tissues along with the tumor-adjacent normal tissues of the patients were collected.The protein expressions of JAK2,STAT3 and their downstream angiogenesis-related molecules in the tissue samples were detected by immunohistochemical staining and Western blot,respectively.The RNA expressions of TUG1 and miR-204 as well as JAK2 and STAT3 and their downstream angiogenesisrelated molecules in the tissue samples were determined by qRT-PCR method,and the correlation between TUG1 and miR-204 expressions in HB tissue was analyzed.Moreover,in human HB cell line HepG2,the changes in RNA and protein expression levels of JAK2,STAT3 and their downstream angiogenesis-related molecules were analyzed after TUG1 knockdown or miR-204 overexpression.Results:The results of immunohistochemical staining showed that the positive expression rates of both JAK2 and STAT3 HB tissue were significantly higher than those in tumor-adjacent normal tissue(JAK2:40.1%vs.16.9%;STAT3:55.7%vs.19.8%,both P<0.05).The results of qRT-PCR showed that the RNA expression levels of TUG1,miR-204 as well as JAK2 and STAT3 and their downstream angiogenesis-related molecules that included VEGF,VEGFR2 and HIF-1αwere significantly up-regulated in HB tissue than those in tumor-adjacent normal tissue(all P<0.05),and there was a significant negative correlation between TUG1 and miR-204 expressions in HB tissue(r=–0.962,P=0.014).The results of Western blot showed that the protein expression levels of JAK2 and STAT3 and their downstream angiogenesis-related molecules were significantly upregulated in HB tissue than those in tumor-adjacent normal tissue(all P<0.05).In HepG2 cells after TUG1 knockdown or miR-204 overexpression,the RNA and protein expressions of JAK2 and STAT3 and their downstream angiogenesis-related molecules were significantly down-regulated(all P<0.05).Conclusion:In the tumor tissues from HB children,the expressions of TUG1 is up-regulated,accompanied with the increased activity of JAK2-STAT3 pathway,and there is also a negative correlation between TUG1 and miR-204 expressions.These findings suggest that TUG1 can probably activating the JAK2-STAT3 pathway and thereby promote the angiogenesis through inhibiting miR-204 expression in HB.