抑制口腔癌和乳癌細胞轉移的微型核醣核酸

轉移是癌症患者治療失敗的主要臨床因數。微RNA分子在癌症進展和轉移扮演一個重要的角色。此項研究是深入探討微RNA分子與高侵入性的口腔癌及乳癌細胞之間的關係。我們分別使用了體外和體內的篩選方式建立了高度侵入性的口腔鱗狀細胞癌（OSCC）及乳癌細胞，並且藉由微小RNA微陣列技術分析這些高度轉移細胞株與其低侵入性母代細胞株的轉錄圖譜。我們以基因表現差異兩倍為門檻，並且使用gene ontology軟體分析找出與侵入相關的微RNA分子以及其下游標的基因。研究顯示所篩選出的全部高侵入口腔癌細胞株及乳癌細胞株其miR-491-5p或miR-149分別表現顯著減少。同時研究也顯示當miR-491-5p或miR-149分別大量表達在高侵入性口腔癌或乳癌細胞時會抑制其爬動和侵入能力，在老鼠模式中也發現大量表達這些微RNA分子會抑制癌細胞轉移的能力。藉由3'UTR報告基因分析證實G protein-coupled receptor kinase-interacting protiein 1（GIT1）同時是miR-491-5p及miR-149的目標基因。另一方面在口腔癌細胞中大量表達GIT1可以回復miR-491-5p及miR-149所抑制的爬動、侵入及轉移的能力。若抑制GIT1的表現則會抑制口腔癌細胞的爬動、侵入和肺轉移的能力。這個研究也證實miR-491-5p藉由抑制GIT1的表現進而減少口腔癌細胞的focal adhesion，同時導致paxillin蛋白的降解及減少paxillin、FAK及EGF/EGFR調控ERK1/2的活性，也抑制了MMP2/9的表現量與活性。在乳癌方面，miR-149同樣藉由抑制GIT1的表現進而促使paxillin及α5β1 integrin蛋白分別走向proteasome及lysosome的分解路徑。此外，我們發現miR-491-5p和FOCAD同時位於染色體9p21.3，我們的實驗證明miR-491-5p是一個intronic微RNA分子，其座落在FOCAD的intron 4。實驗證明當FOCAD的表現被抑制時也會導致口腔癌細胞癌轉移及侵入的能力減少。進一步我們也證實FOCAD與miR-491-5p會一同被表現出來，並且藉由抑制GIT1調控路徑來減少口腔癌細胞的爬動、侵入和轉移的能力。因此，我們的結論是miR-491-5p及miR-149可以同時藉由GIT1的調控路徑有效的抑制口腔癌及乳癌細胞的轉移能力，這些發現暗示著本研究探討的調控機制可能成為臨床診斷和治療的標的。

關鍵字

口腔癌；乳癌；轉移；微型核醣核酸；局部黏著斑激酶；口腔鱗狀上皮細胞癌

並列摘要

Metastasis is an important clinical parameter for patient prognosis and the major cause of treatment failure for cancer. MicroRNAs are molecules that could play an abstrusive role in cancer progression and metastasis. The study is to identify relevant microRNAs associated with invasive phenotype of oral and breast cancers. We have established isogenic highly invasive oral squamous cell carcinoma (OSCC) lines and breast cancer lines from their respective low invasive parental lines via in vitro and in vivo selection protocols. MicroRNA array analysis was used for transcriptome profiling between each pair of the parental and the highly invasive subline. Using threshold of 2-fold change of gene expression, we analyzed the microarray data by gene ontology enrichment of Partek and identify genes as well as microRNAs revealed to be significantly associated with invasive phenotype. We found that miR-491-5p and miR-149 level were significantly decreased in the selected OSCC invasive lines and highly invasive breast cancer lines respectively. Overexpression of miR-491-5p or miR-149 in those highly invasive cells suppressed their migration/invasion in vitro and metastatic ability in a xenograft mouse model. The G protein-coupled receptor kinase-interacting protiein 1 (GIT1) is a direct target of miR-491-5p and miR-149 as revealed by 3'UTR reporter assays. The miR-491-5p- and miR-149-mediated inhibition of migration/invasion and lung metastasis could be rescued by overexpression of GIT1. Depletion of GIT1 inhibited migration/invasion and lung metastasis of OSCC and breast cancer cells. MiR-491-5p-mediated GIT1 repression reduced focal adhesion and concurrently decreased steady state levels of paxillin, phospho-paxillin,phospho-FAK, EGF/EGFR-mediated ERK1/2 activation as well as decreased MMP2/9 levels and activities in OSCC cells. Overexpression of miR-149 or depletion of GIT1 led to enhanced protein degradation of paxillin and α5β1 integrin via proteasome and lysosome pathways respectively. In addition, we found that miR-491-5p and focadhesin (KIAA1797/FOCAD) gene are located on chromosome 9p21.3 together. Our data suggest that miR-491-5p is an intronic miRNA processed form FOCAD intron 4 rather than being transcribed as a separate RNA. Depletion of FOCAD promoted cell migration/invasion abilities in human oral cancer cells. Furthermore, FOCAD/miR-491-5p were co-expressed in OSCC cells and they suppresses OSCC cell migration/invasion and metastasis, suggesting potential application of the miR-491-5p/GIT1 pathways in OSCC prognosis and therapy. Therefore, we conclude that miR-491-5p and miR-149 suppresses migration/invasion and metastasis of OSCC and breast cancer cells, respectively, by targeting GIT1, suggesting potential application of the miR-491-5p/GIT1 and miR-149/GIT1 pathways in clinical diagnosis and therapeutics.

並列關鍵字

oral cancer ； breast cancer ； metastasis ； microRNA ； focal adhesion kinase ； OSCC

參考文獻

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抑制口腔癌和乳癌細胞轉移的微型核醣核酸

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