Research On The Molecular Mechanisms Of Hsa-miR-139Modulating Invasion, Metastasis And Multidrug Resistance In Human Colorectal Cancer

Colorectal cancer (CRC), a kind of malignant tumor, is the second leading cause of cancer-related death. CRC is the third most commonly diagnosed cancer in males and the second in females worldwide. The high mortality rate of CRC is mainly due to the frequent metastasis and multidrug-resistance (MDR). In the context of cancer research, it has become more and more important to find effective treatments and anti-tumor drugs for CRC. MicroRNA (miRNA), a kind of recently discovered non-coding RNA, modulate target gene expression through sequence-specific binding to their mRNA targets, resulting in translational inhibition or mRNA degradation. So far, a great number of evidences showed that miRNA take part in almost all the cellular biological process. Dysregulation of miRNA is closely related to diseases. A better understanding of roles that miRNA play in cancer may lead to a better understanding of cancer initiation and progression.To identify critical miRNA in CRC, the gene signature of873miRNAs in6CRC tissues and their corresponding non-tumor tissuses were screened using Agilent human miRNA array (V.12.0). Among them,13miRNAs were found dysregulated (p<0.05, Fold Change>2) in CRC tissues and the most down-regulated miRNA, miR-139-5p, was selected for further validation. Quantitative PCR was then conducted to confirm the miRNA array by detecting miR-139-5p expression levels in34CRC tissues and their corresponding non-tumor tissues. The low expression levels of miR-139-5p indicated that miR-139-5p dysregulation is frequent in34human CRC tissues. On this basis, the in vitro and in vivo analysis were performed, indicating that miR-139-5p have inhibiting effect on CRC cell invasion and metastasis (p<0.001in vitro, p=0.0079in vivo). However, its anti-proliferation effect is minimal (p>0.05in vitro, p>0.05in vivo). The bio-informatic prediction and dual-luciferase analysis showed that Type I Insulin like Growth Factor (IGF1R) is a direct target gene of miR-139-5p. MiR-139-5p inhibits IGF1R translation through binding to its3’-untranslation region (3’UTR), consequently inactivating the down-stream MEK/ERK pathway, inhibiting the transcription of MMP-2, eventually regulating the invasive and metastatic ability of CRC cells. MiR-139-5p expression level was also found closely related to aggressive pathologic features including tumor staging (p=0.02), lymph-node involvement (p=0.02), vascular invasion (p<0.01) and metastasis (p=0.03). This study also unveiled the mechanism underlying miR-139-5p under-expression in CRC. MiR-139located on chromosome11and resides within intron-2of its host gene Phosphodiesterase2(PDE2A). However, linear regression analysis indicated that neither miR-139precursor (pre-miR-139) nor mature miR-139s (miR-139-3p and miR-139-5p) expression levels were directly correlated with PDE2A mRNA level in CRC tissues. It was also found that cellular miR-139expression was not PDE2A activator inducible. The chromatin immunoprecipitation analysis revealed that miR-139owns an independent translational starting site. Furthermore, miR-139expression is also histone acetyltransferase inhibitor inducible. The expression levels of pre-miR-139, IGF1R, ROCK2and RAP1B in34CRC tissues indicated that the over-expression of miR-139-5p target genes may also be the culprits of miR-139-5p dysregulation in CRC. Over-expression of these3’UTRs inhibited other targeting miRNA expression, indicating that post-transcriptional regulation also plays important role in miR-139-5p dysregulation. MiR-139-5p target genes are involved in a miR-139-5p mediated reciprocal regulatory network, in which they can regulate each others’ protein levels through miR-139-5p competition. To our knowledge, it is the first time for discovering the biofunction of3’UTR of IGF1R, ROCK2and RAP1B in CRC.MiR-139-5p and IGF1R were found down-regulated and up-regulated in CRC drug-resistant sublines respectively (p<0.001,p<0.01). Silencing of IGF1R, which has the similar effect to miR-139-5p over-expression, led to the inactivation of downstream PI3K/AKT/Nrf2/MRP-2pathway, consequently inhibiting drug-resistant cell in vitro proliferation and reversing the drug-resistant phenotype. The high expression level of cancer stem cell (CSC) associated biomarkers CD44and CD133in drug-resistant cell showed that these cells are CSC like cell. The bio-informatic prediction and dual-luciferase analysis showed a de novo miR-139-5p target gene Notch-1. MiR-139-5p regulates CRC cell drug resistance through IGF1R and Notch-1modulation simultaneously.This study unveiled the molecular mechanisms of miR-139-5p modulating CRC cell invasion, metastasis and MDR. The tumor suppressive effect of miR-139-5p was validated in cellular, tissue and animal levels. These findings indicate the potential of using miR-139-5p as biomarker, drug target and novel anti-cancer drug. Our results provide a theoretical basis and new strategy for CRC diagnosis, targeted therapy and prognosis.