mir-375/206协同抑制喉鳞癌发生发展的分子机制研究

Laryngeal squamous cell carcinoma (LSCC) is one of the malignant tumors in the head and neck region. Despite improvements in diagnoses and treatments, most deaths from cancer result from the metastases that are resistant to conventional therapies. However, mechanisms underlying the invasion and metastasis of LSCC remain unclear..Endogenous microRNAs (miRNAs) are small, non-coding RNAs that negatively regulate mRNA translation or stability. miRNAs are dysregulated in a broad spectrum of malignancies including LSCC. By microarray analysis, we have found that miR-375 and miR-206 were among the most dysregulated genes. These results have been previously observed by other groups; however, we extend these findings with the unique observation that restoring the expression of miRNA-375 or miR-206 inhibited tumor proliferation and induced the apoptosis of LSCC Hep-2 cells. Furthermore, a significant tumor reduction was observed in nude mice subcutaneously injected with the miR-375 or miR-206 transfectants. These findings prompted us to raise some intriguing questions, such as whether miR-375 and miR-206 are involved in the regulatory networks of the pathological process of LSCC and whether concomitantly expressing miR-375 and miR-206 offers an improved approach in inhibiting the aggressiveness of LSCC. .To answer these questions, we will develop a new method to simultaneously express both miR-375 and miR-206 and further identify shared target genes of the two mRNAsas potential mediators of the pathological process of LSCC..We will firstly investigate the correlation between the expressions of miR-375/206 and the clinical parameters of LSCC. The shared miRNA target genes will be further functionally characterized for their potential involvement in LSCC..Then, as a main goal of the study, we will engineer a single unit (oligonucleotide, ODN) that is capable of simultaneously expressing MTg-mODN （MTg:Multi-target, mODN: miRNA oligonucleotide) by lentivirus, such as MTg-miR-375/206-Lenti and verified the ability of MTg-mODN to antagonize their target mRNAs using luciferase reporter activity assays and to specifically knock down the levels of their target mRNAs using real-time RT-PCR methods. .To better understand the tumorigenesis of LSCC, we will next investigate in vitro the possible signal pathways by examining the expression of the target genes SP1，JUND，KLF4,PDGFA and YWHAZ after the transfection of MTg-miR-375/206-Lenti..Finally, multiple methods will be employed to investigate the efficiency of the MTg-mODN technology in the inhibition of invasion and metastasis of LSCC in vivo and to demonstrate the interactive miRNA target gene network. .The main objectives of this research are to demonstrate the mechanisms of tumorigenesis of LSCC, scrutinize the efficiency of MTg-mODN in suppressing the cancer invasive properies, which will be essential for developing improved treatment plans for LSCC.

内源性小分子RNAs(miRNAs)在喉癌发生发展过程中的机制尚不清楚。我们前期工作发现miR-375和miR-206的喉癌组织中表达明显下调，而体外转染miR-375和miR-206可抑制喉癌细的胞生长，促进肿瘤凋亡,干扰裸鼠成瘤。我们推测miR-375和miR-206可能共同参与喉癌发生发展的信号传导通路。本课题拟应用分子克隆、报告基因、定量PCR等多项技术从体内外及转基因水平系统研究miR-375和miR-206抑制喉癌发生发展的分子机制，通过对miR-375和miR-206以及共同靶基因SP1,JUND,KLF4,PDGFA以及YWHAZ的功能分析，阐明miR-375/206及下游信号分子之间的调控关系，最终揭示以miR-375/206为核心的信号调控网络在抑制喉癌细胞增殖，迁移，凋亡以及新血管形成过程中的作用。该课题的完成将为此研究领域提供全新信息，为喉癌生物靶向治疗提供新思路。

喉癌是头颈部常见的恶性肿瘤之一，95%为鳞状细胞癌。尽管化疗和放疗在一定程度上能够控制局部复发、阻止远处转移进而提高生存率，但其毒性和耐药作用影响着肿瘤治疗的临床应用和疗效。因此，如何有效地抑制喉癌侵袭和转移是研究人员一直努力解决的关键问题。内源性小分子RNA（miRNAs）在肿瘤增殖、分化、凋亡以及细胞周期调控发挥着重要作用，在喉癌发生发展过程中的机制尚有待发现。通过前期工作发现在喉癌组织中表达明显下调的miR-375和miR-206分别有抑癌的作用，我们构建了具有miR-375和miR-206的“多序列慢病毒载体”，并转染入Hep-2细胞，通过流式细胞仪、PCR及MTT等检测方法，发现miR-375和miR-206联合能明显抑制喉癌细胞的生长及诱导喉癌细胞的凋亡。并通过体内实验验证了miR-375和miR-206共同的联合作用，与体外实验结果相符合。此外，我们还发现miR-375和miR-206联合作用的可能的靶基因SP1和KLF4，发现miR-375/206负向调控SP1、KLF4，可能存在靶向关系。根据本研究，我们初步得出结论：miR-375/ 206可以联合抑制喉癌细胞增殖和诱导喉癌细胞凋亡，较单一的miR-375或miR-206作用强，此作用过程可能通过调控SP1和KLF4完成。该课题的结果可以为此研究领域提供全新信息，为喉癌生物靶向治疗提供另一条途径。