Role And Mechanism Of MiR-143 In Regulating Tumor Growth, Angiogenesis And Chemoresistance Of Colorectal Cancer

Colorectal cancer(CRC)is one of the most common digestive malignant tumors.It is the third leading cancer-related deaths in the world.In China,CRC is the third gastrointestinal cancer following gastric cancer and esophageal carcinoma.The incidence of CRC in our country arises because of changes in diet,environmental pollution and other factors.No obvious symptoms in early stage of CRC render it very difficult to diagnose at early stage.When diagnosed,CRCs are often in the advanced stages,even metastatic to liver.The principle therapeutic method is surgery.Even though the surgical techniques have been developed rapidly,the five-year survival rate hasn't been improved yet.Though chemotherapy and radiotherapy improve CRC treatment in some extent;drug-resistance,metastasis and side effects still challange medical doctors and researchers.Therefore,it is urgent and important to investigate molecular mechanisms of CRC,and to look for novel biomarkers for diagnosis,treatment and prognosis.MicroRNAs(miRNAs)are a class of small non-coding RNAs that were discovered more than 20 years ago.miRNAs have been among one of the most active research fields in the recent 10 years.miRNAs posttranscriptionally regulate a wide variety of genes by targeting their mRNAs,leading to mRNA degradation or inhibition of protein translation.miRNAs are multi-functional in cell proliferation,differentiation,apoptosis,development of organs,including tumor formation and development.Growing evidence has shown that a large amount of human miRNAs are located in cancer related genomic regions and function as tumor promoters or tumor suppressors.And miRNA expression patterns were found to be abnormal,even specific in cancers.Expression levels of microRNA-143(miR-143)are found to be downregulated in many types of cancers,including CRC.The expression profiles and biological roles of miR-143 in CRC have been preliminarily reported by some researchers,but the role and mechanisms of miR-143 in regulating tumorigenesis,angiogenesis and drug resistance remain to be elucidated.We detected miR-143 expression levels in 62 pairs of CRC specimens and corresponding adjacent normal tissues by qRT-PCR method,and combined with clinical features for further analysis.In CRC,miR-143 is downregulated in tumor tissues compared with controls.Lower expression levels of miR-143 are positively correlated with late Duke's stages(C and D)and with lymph node metastasis.Furthermore,qRT-PCR analysis was performed to detect miR-143 expression in plasma samples from 41 CRC patients and 10 healthy subjects.In accordance with expression pattern in CRC tissues,miR-143 levels were significantly decreased in plasma of CRC patients.These results indicated possibilities of using miR-143 as a novel biomarker in early diagnosis and prognosis for CRC.miRNAs directly inhibit expression of their target genes,consequently modulate the biological functions.Thus,it is important to find novel targets of miRNAs to investigate their roles in carcinogenesis.Bioinformatics analysis suggested that miR-143 may directly target insulin-like growth factor-1 receptor(IGF-IR)and insulin receptor substrate-1(IRS-1),who are usually overexpressed in CRC for regulating many signaling cascades including phosphatidylinositol-3-kinase(PI3K)/protein kinase B(AKT)and mitogen-activated protein kinases(MAPK)/extracelluar regulated protein kinase(ERK)pathways.In this study,IGF-IR and IRS-1 were validated as novel targets of miR-143 by luciferase reporter assay and western blotting,and were found to be negatively correlated with miR-143 expression levels in CRC samples,indicating that miR-143 and its targets,IGF-IR and IRS-1 may play important roles in CRC.In order to investigate roles of miR-143 in CRC tumor growth and angiogenesis,we established a stable cell line that overexpressed miR-143 which was originally with low levels of miR-143.Upon forced expression of miR-143,cell proliferation and migration were decreased.And also,IGF-IR/IRS-1/PI3K/AKT signaling pathway was inactived,leading to inhibition of hypoxia-inducible factor 1?(HIF-1?)and vascular endothelial growth factor(VEGF),which were proved to be vital inducers in tumorigenesis and angiogenesis.Next,we explored the in vivo functions of miR-143 in CRC.Chorioallantoic membrane(CAM)system was employed to show that overexpression of miR-143 greatly impaired angiogenic ability of CRC cells.Furthermore,xenograft tumor models in nude mice indicated that forced expression of miR-143 decreased tumor growth,and slowed down the progression of CRC.We then investigated whether miR-143 regulated CRC via its targets.We constructed lentivirus and adenovirus carrying IGF-IR and IRS-1 mRNAs without 3'-UTR regions,respectively,to restore the expression levels of IGF-IR or IRS-1 in those stable cells overexpressing miR-143.After gain-of-function of IGF-IR or IRS-1,the miR-143-overxpressing cells were “rescued” characterized by elevated levels of HIF-1? and VEGF,increased cell proliferation in vitro and tumor growth in vivo when compared with the control.Drug resistance is acquired in the process of CRC chemotherapy and will diminish treatment effect of CRC.We firstly found that overexpression of miR-143 sensitized in vivo chemo-effect of oxaliplatin in treating CRC xenografts.After a period of 3-day i.p.administration of oxaliplatin,the xenografts shrank day by day.We further found that miR-143 increased chemo-sensitivity of CRC cells by inducing apoptosis via its target IGF-IR.In this study,we identified two novels targets of miR-143,IGF-IR and IRS-1,and found that,via these two targets,miR-143 modulated tumor growth and angiogenesis through HIF-1? and VEGF,and increased chemosensitivity of oxaliplatin in CRC;besides these,we also showed that miR-143 levels were reduced in tumor tissues and plasma of CRC patients,and that low levels of miR-143 were correlated with CRC staging and metastasis.Collectively,these findings suggest that miR-143 may be a novel biomarker for diagnosis and prognosis of CRCs,and provide new information for developing miR-143-based therapeutic strategies for CRC treatment in the future.