ceRNA调控miR-330-3p、Ereg在肿瘤淋巴道转移中的作用机制及临床相关性研究

Lymphatic metastasis is a leading problem for malignant tumor. Competing endogenous RNA (ceRNA) regulation is one of the cutting edge research fields in tumor, however, its association with tumor lymphatic metastasis has been rarely reported. Previous work indicated that the levels of lnc045874, Ereg and miR-330-3p were differentially expressed between murine hepatocarcinoma cells Hca-F and Hca-P with high and low lymphatic metastasis potentials with statistical significances. Through the binding with the 3’-UTRs of lnc045874 and Ereg, miR-330-3p inhibited the migration and invasion of Hca-F cells. Lnc045874, Ereg and miR-330-3p were found deregulated by matching the established ceRNA regulation pathway in the cancerous tissues of the patients suffering with hepatocarcinoma and renal carcinoma with clinical significance. This proposal aims to clarify the expression level changes and the mutual responding mechanism of lnc045874, Ereg and miR-330-3p on the in vitro proliferation, migration and invasion capacities, and in vivo tumorigenic and lymph node metastatic abilities of Hca-F and Hca-P cells. The role of lnc045874 in regulating Ereg expression through sponging miR-330-3p and the corresponding regulation mechanism on lymphatic metastasis will be demonstrated. The downstream molecules linked to the proposed ceRNA pathway will be screened and identified. Clinical analysis of the deregulations of lnc045874, Ereg and miR-330-3p patients’ tissues will provide their correlations with the development and metastases as well as their potential use as early warning markers for liver and renal carcinomas. This proposal aims to provide a novel ceRNA regulation pathway and new clues into the academic research, the diagnosis and treatment of malignant tumors.

淋巴道转移是恶性肿瘤难题，竞争性内源RNA(ceRNA)调控为肿瘤研究前沿，但二者相关性研究匮乏。本组前期发现lnc045874、miR-330-3p和Ereg在肝癌高低淋巴道转移力细胞株Hca-F和Hca-P中差异表达显著，符合ceRNA调控关系；miR-330-3p通过与lnc045874和Ereg 3′-UTR结合抑制Hca-F迁移和侵袭；相关分子在肝/肾癌患者组织中表达水平异常，临床意义明显。本课题拟明确三者表达水平变化对Hca-F和Hca-P体外增殖、侵袭和迁移、体内致鼠成瘤和淋巴结转移影响及相互应答；阐明lnc045874通过对miR-330-3p“海绵”性作用调节Ereg表达，进而调控肿瘤淋巴道转移的机制；获得该ceRNA通路调控下游分子；阐明三者在患者组织中表达水平差异性、作为预警肝/肾癌进展分子的临床意义，建立调控肿瘤进展的新ceRNA途径，为恶性肿瘤研究诊治提供新线索。

竞争性内源RNA(ceRNA)调控肿瘤转移研究匮乏。项目建立一条新的由鼠lnc045874(人lnc021545)、miR-330-3p和Ereg构成的调控肝/肾/乳腺癌进展中的ceRNA途径。研究内容：1)lnc021545、miR-330-3p和Ereg在ccRCC、HCC和BC患者癌组织中表达降低、升高和降低，相关性明显；2)三者水平变化一致与HCC分期，ccRCC分期和Fuhrman分级，BC分期、肿瘤大小和浸润性相关；3) miR-330-3p含有与lnc045874/021545和Ereg的3-UTR直接结合位点,负调控前两者表达并影响肿瘤细胞的恶性表型；4) 三者表达水平变化对肿瘤细胞体外增殖力影响不大，主要通过影响体外其侵袭和转移能力而影响肿瘤细胞转移力；5)人肝癌细胞裸鼠体内成瘤表明：miR-330-3p过表达，裸鼠原位瘤、腹股沟、肾内和腋下淋巴结质量明显增加，肿瘤细胞向淋巴转移升高；抑制miR-330-3p表达，原位瘤瘤重变化不明显，但腹股沟、肾内和腋下淋巴结质量明显降低，肿瘤细胞向淋巴转移降低；6)鼠Hca-P细胞株致小鼠表明：miR-330-3p下调促进Hca-P细胞成瘤速度和瘤重，淋巴结转移增强，原位瘤中miR-330-3p水平明显减低，Ereg和lnc045874表达增加；lnc045874促进Hca-P细胞成瘤速度和瘤重，淋巴结转移增强，原位瘤中lnc045874表达升高，miR-330-3p表达降低，Ereg表达增加；7) miR-330-3p表达水平变化在肝癌/乳腺癌细胞系中负向调控Ereg和lnc045874/lnc021545的表达；lnc045874/lnc021545表达水平变化在肝癌细胞系中分别负向和正向调控miR-330-3p和Ereg的表达；Ereg表达变化在肝癌细胞系中正向调控lnc021545表达，对miR-330-3p表达无影响；8) lnc045874/021545、miR-330-3p和Ereg形成的ceRNA通过EMT的关键分子snail、slug、E-cadherin、N-cadherin和vimentin等调控肿瘤的转移。结果表明：lnc021545、miR-330-3p和Ereg表达异常，致三者间ceRNA调控失衡，促进多种肿瘤(肝/肾/乳腺癌)转移，在肿瘤基础研究、肿瘤转移预警和诊治中有重要意义。