食管鳞癌癌变进程中长链非编码RNAuc010etn调控细胞增殖的功能及机制研究

The long noncoding RNA plays an important role in the development and progression of cancer. Mounting evidence have indentified that the enhancement cell proliferation could accelerate carcinogenesis. Tracking the literature suggests that lncRNA study on the mechanism of regulation of proliferation in the process of esophageal carcinoma has not been reported. We detected the tissues of NEE-LIN-HIN-EEC-I/TEC by microarray and qRT-PCR，and found that lnc-uc010etn raised above 7 times in the process of esophageal carcinoma. Recent studies confirmed that the LncRNA could competitively binding with miRNA,then accelerated the expression of miRNA-targeted-gene and executed related functions.The result of MTT assay showed cell proliferation was significantly decreased after disturbance the expression of lnc-uc010etn in KYSE360. Bioinformatics analysis showed that there are two binding sites of miR-375 of lnc-uc010etn. It was identified that miR-375 was downregulated in esophageal squamous cell carcinoma,and its targete gene AEG-1/MTDH was involved in the function of proliferation regulation. Accordingly, we speculated that the The lnc-uc010etn could combine with miR-375 competitively, relieve inhibiting of the targeted gene, then upregulating the expression of proliferation protein and accelerating the process of esophageal carcinoma.

长链非编码RNA（LncRNA）在肿瘤的发生发展中具有重要功能。已有诸多研究证实细胞的增殖功能的增强可明显加速癌变进程。追踪文献表明在食管癌变进程中lncRNA对细胞增殖的功能及调控机制的研究尚未见报道。课题组经芯片及qRT-PCR检测发现正常食管粘膜-低级别上皮内瘤变-高级别上皮内瘤变-早期食管癌-中晚期食管癌这一癌变进程中lnc-uc010etn上调均在7倍以上。siRNA干扰uc010etn表达可使细胞增殖能力降低。新近研究证实LncRNA可与miRNA竞争性结合，上调miRNA靶基因表达并执行相关功能。生物信息学分析发现uc010etn存在miR-375结合位点。而已有研究证实食管鳞癌中miR-375表达下调，其靶基因AEG-1/MTDH可明显促进细胞增殖。据此课题组推测uc010etn可与miR-375竞争性结合，解除其对靶基因的抑制，使促增殖蛋白表达上调，加速食管癌变进程。

食管癌是严重危害全人类健康的恶性疾病，在全球范围内其发病率和死亡率分别居第八位和第六位。关于 EC 的发病机制的研究已经在诸多领域取得了进展，但其确切的发病机制仍在积极的研究探索之中。LncRNA是近年来被人们所广泛关注的新型调节型ncRNA。课题组研究了PlncRNA-1、SPRY4-IT1在ESCC的表达及功能。首先采用qRT-PCR检测了73 对ESCC癌与癌旁组织中PlncRNA-1的表达水平，其中69.8 %癌组织中PlncRNA-1的表达显著高于癌旁组织，有淋巴结转移的患者较无淋巴结转移患者的PlncRNA-1表达水平明显增（p<0.05），临床III & IV 期较I &II期患者PlncRNA-1的水平也明显增加（p<0.01）；同时qRT-PCR检测PlncRNA-1在ESCC细胞株中的表达水平经，发现PlncRNA-1在KYSE180中表达水平最高；采用siRNA敲降其表达,qRT-PCR检测PlncRNA-1的敲降效果达76.1%；敲降PlncRNA-1后克隆形成实验证实，与对照组相比：敲降PlncRNA-1后细胞的克隆形成能力43.6 % 低于对照组 68.6 % (p<0.01)；MTT检测发现敲降PlncRNA-1后，细胞增殖能力抑制达29.85 %(p<0.05)；流式细胞技术检测细胞周期的变化，G1期细胞数量从65.91%减少到60.22 %；S期数量从17.44%增加到 20.83 %，表明PlncRNA-1可影响细胞G1/S转化；而细胞凋亡率增加6.3 %(p<0.05)。此外研究证实LncRNA SPRY4-IT1在ESCC中的表达及功能。首先采用qRT-PCR检测发现SPRY4-IT1在86.9 % ESCC患者癌组织 (80 of 92) 中表达上调，且其表达显著高于癌旁组织(p<0.01)，有淋巴结转移的患者SPRY4-IT1表达较无淋巴结转移患者升高(p<0.01)，临床分期III & IV 较I &II患者SPRY4-IT1表达明显增加（p<0.01），且高表达SPRY4-IT1患者预后差；并在细胞及活体动物层面证实敲降SPRY4-IT1显著抑制肿瘤细胞增殖、侵袭和转移。研究表明PlncRNA-1、SPRY4-IT1在ESCC发生发展中具有重要作用，可作为ESCC潜在的预后标志物和新的治疗靶点。