The Study Of Regulatory Mechanism And Clinical Value Of MiR-222 In Colorectal Cancer And Hepatocellular Carcinoma Cell-derived Exosome Promoting Transition Of Adipose-derived Mesenchymal Stem Cells To Cancer-associated Myofibroblasts

In the first part,we investigated the effects of miR-222 on migration and invasion of CRC cell lines and its downstream target,then the expression pattern of miR-222 and its target gene in CRC and their clinical significance.Colorectal cancer(CRC)is the third most common cancer and cancer-related death worldwide.Resection can cure early stage of colorectal cancer,but the 5-year survival of metastatic colorectal cancer is less than 12%.The incidence and mortality of CRC in China have continued to increase annually in recent years.Therefore,it is still essential to study mechanisms of colorectal cancer initiation and progression,especially those of tumor recurrence and metastasis.MicroRNAs(miRNAs)are 19-25 nucleotide single-stranded RNA which can bind to target mRNA with partial or perfect complementarity and consequently mediate translational repression and mRNA degradation.Recently,many miRNAs were found differentially expressed in CRC,suggesting they might act as either oncogenes or tumor suppressors.Our previous study had revealed that the expression of miR-222 was significantly higher in CRC tumor tissues compared to corresponding normal tissues.However,there was no evidence showing the role of miR-222 in CRC.Methods Human CRC cell lines(HCT8,HCT116,LOVO and SW480)were transfected with synthesized miR-222 mimics,miR-222 inhibitor and corresponding control respectively using lipotransfectine reagent.The transfection efficiency was confirmed by real-time PCR(RT-PCR).Cell proliferation was assessed using MTS assay.The effect of miR-222 on cell migration and invasion was determined by in vitro transwell assays and in vivo mice tumor model.MiR-222 target genes were predicted by TargetScan.Dual-luciferase reporter assay system was performed to verify whether or not miR-222 bind 3’UTR of these candidate target mRNA.In CRC cells,western blot and RT-PCR were used to reveal whether or not miR-222 regulate the expression of target genes at post-transcriptional level.si-RNA technique was performed for functional analysis of candidate target genes in CRC cells.We then evaluated the effect of MST3 over-expression.Furthermore,Clinico-pathological factors and follow-up data of 38 CRC patients with Stage Ⅲ cancer were collected retrospectively.All patients accepted radical surgeries and their paraffin-embedded samples of tumor tissues and corresponding paracancerous normal tissues were also colleced.MiR-222 expression in 21 CRC tumor tissues was assessed using real-time PCR.Local miR-222 target gene protein expression was assessed using immunohistochemical(IHC)staining.The relation of miR-222 with its target gene expression,target gene expression pattern between tumor tissues and normal tissues,and the relation of miR-222 target gene protein expression with clinico-pathological factors and prognosis were planned to be discussed.Results We found that over-expression of miR-222 significantly promoted migration and invasion ability of CRC cells both and in vivo compared to its control group(p<0.05).On the contrast,miR-222 down-regulation could significantly prohibit cell migration and invasion compared to its control group in vitro(p<0.05).Bioinformatics and dual luciferase reporter assay revealed that miR-222 specifically targeted the 3’-UTR of MST3.The expression level of MST3 protein was down-regulated in miR-222 mimic group and up-regulated in miR-222 inhibitor group compared to corresponding control groups using western blot.But there was no significant difference in MST3 mRNA level between these groups(p>0.05),suggesting miR-222 mediated MST3 expression at post-transcriptional level.Block MST3 using si-RNA was shown to significantly to reduce cell migration and invasion ability in HCT8 and HCT 116 cells(p<0.05).MST3 over-expression significantly reduced migration and invasion of SW480 and HCT116(Figure 5).To further confirm miR-222 effect was mediated by MST3,we performed an RNA interference rescue assay.Over-expression of MST3 could rescue the promoting effect of miR-222 over-expression on CRC migration and invasion In clinical CRC tissues,miR-222 expression level was inversely correlated with MST3,whose expression was lower in CRC tissue than in pericarcinous tissue.Higher expression of MST3 in CRC patients predicted a longer disease-free survival(DFS).Conclusion High expression level of miR-222 correlated to CRC migration and invasion.Our study showed for the first time that miR-222,by down-regulation MST3,enhances migration and invasion in CRC cells.And the different expression of MST3 between tumor and matched pericarcinous tissue,may predict prognosis of CRC patients.Taken together,these findings demonstrate that miR-222 modulates MST3 and thus plays a critical role in regulating CRC cell migration and invasion.Therefore,miR-222 may be a novel therapeutic target for CRC.In the second part,we study the influence of human hepatocellular carcinoma HepG2 cell-derived Exosome on the process of mesenchymal stem cells(MSC)differentiate into Cancer-Associated Myofibroblasts(CAF)and the impacts of CAF on liver cancer cell proliferation,migration and invasion.Methods The protein expression of HepG2 cell-derived Exosome was detected by western blot.MSC were separated from human adipose tissue and cultured with HepG2 cell-derived Exosome(100ng/nl)to initiate differentiation.Then,the expression of mesenchymal markers and several interleukin were detected by Western blot.HepG2 cells were co-cultured with the conditioned media(CM)which HepG2 Exosome induced MSC differentiation CAF.The expressions of epithelial and mesenchymal markers were detected by real time PCR and Western blot.Cell proliferation was assessed using MTS assay.Transwell chambers were used in the in vitro migration and invasion assay.Results HepG2 cell-derived particles expressed CD63,HSP70 and HSP90 which have the typical characteristics of Exosome.With the treatment of HepG2 cell-derived Exosome,the expression of mesenchymal marker a-SMA,FAPA,IL-6,IL-8,IL-1β was up-regulated,while VEGF had no significant change.These are the characterization of CAFs.The conditioned media which HepG2 Exosome induced MSC differentiation CAF(CAF-CM)could significantly promote HepG2 cells proliferation,compared to the MSC-CM.Moreover the conditioned medium which HepG2 Exosome induced MSC differentiation into CAF could also promote expression of mesenchymal related genesβ-catenin,Vimentin,FN and Sip1,in contrast,inhibit the expression of epithelial related genes ZO-1.HepG2-Exosome-MSC conditioned media could significantly enhance cell migration and invasion compared to its control group(p<0.05).Conclusion Exosome extracted from Hepatoma cells can induced Human adipose-derived MSC to differentiate into cancer-associated myofibroblasts.CAF-like cell can promotes migration of the liver cancer cell line HepG2.