C/EBP-? Induced Co-activation Of Mir-31 And Mir-223 To Promote Colorectal Cancer Tumorigenesis Through Targeting RASA1

Colorectal cancer(CRC)is a common malignant tumor with seriously threat to human health.Meanwhile the RAS protein and RAS-related signaling pathway are considered to play an important role in the pathogenesis of CRC because about 30%?50%of reported CRC cases show mutations in K-RAS.Recent studies provide evidence that microRNAs(miRNAs)are known to play a vital role in a wide variety of tumors,but the biological function of miRNAs in RAS protein related signaling pathways remains unclear and the use of these aberrantly expressed miRNA precursors or inhibitors to treat CRC in vivo has not yet been rqported.In this paper,we studied that miR-31 and miR-223 could regulate RAS p21 GTPase activating protein 1(RASA1)which played an important part in the RAS signaling pathways.In the first part of the experimental section,we found a widespread disruption in miRNA expression in CRC and paired adjacent,non-tumor tissue(NAT)using microarray and quantitative real-time PCR(qRT-PCR)analysis;of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples,miR-31 was the most significantly dysregulated.We identified candidate targets of miR-31 using bioinrormatics approaches and validated RASA1 as a direct target.First,we found an inverse correlation between miR-31 and RASA1 protein levels in vivo.Second,in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV,whereas anti-miR-31-LV treatment increased RASAI protein levels.Third,a Luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3-untranslated region of RASA1 transcripts.Furthermore,the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo.Taken together,our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation,thereby improving colorectal cancer cell growth and stimulating tumorigenesis.In the second part of the experimental section,RASA1-targeted miRNA expression profiles were examined in 24 CRC tissues using qRT-PCR.MiRNA expression analysis revealed that miR-223 was significantly up-regulated in 24 CRC tissues relative to NAT.In addition,expression of RASA1,a target of miR-223,was significantly decreased in CRC tissues,as shown using western blotting and in situ immunofluorescence analysis.Furthermore,the direct inhibition of RASA1 translation by miR-223 binding to the“seed region”of the RASA1 3'UTR were evaluated in Caco-2 and HT-29 cells which induced colorectal cancer cell proliferation through the activated the RAS-MAPK pathway by the use of MEKI/2 inhibitor.An in vivo xenograft model suggested that the activation of miR-223 could promote tumor growth and that the inhibition of miR-223 might prevent solid tumor growth.On the whole,our results studied the role of miR-223 in CRC development process for the first time,which enriched our understanding of function duality of miR-223 in cancers.MiR-223-targeted inhibitors significantly inhibited the growth of colorectal tumors in nude mice,Providing a promising approach that may be valuable for CRC treatment.Finally,we studied the relationship between miR-31 and miR-223 in the third part of the experimental section and we discovered that miR-31 and miR-223 were co-activated by C/EBP-? in CRC.Firstly,C/EBP-? and miR-31,miR-223 were significantly up-regulated in 24 CRC tissues relative to NAT using qRT-PCR,which showed that there was a positive correlation between C/EBP-? and miR-31,miR-223.Secondly,when the expression of C/EBP-? was up-regulated,phosphorylation of the C/EBP-? enhanced and the levels of miR-31 and miR-223 also increased in Caco-2 cells while the same situation occurred when C/EBP-? was down-regulated.Thirdly,characterization and experimental analysis of the promoters demonstrated that C/EBP-? could bind to the promoters of miR-31 and miR-223 respectively in Caco-2 cells,affecting the expressions of miR-31 and miR-223,which suggested C/EBP-?might be a positive regulator of miR-31 and miR-223.Taken together,in the development of CRC,C/EBP-? in the cytoplasm will be phosphorylated and enter the nucleus,then bind to the promoters of miR-31 and miR-223 respectively,so as to maintain miR-31 and miR-223 high expression,resulting in miR-31 and miR-223 mediated promoting cell proliferation of colon cancer cells and tumor growth.