Yurt/Mosaic Eyes-like1(YMO1) Suppresses The Invasion And Metastasis Of Hepatocelluar Carcinoma By Inhibiting RhoC Signaling

Hepatocellular carcinoma (HCC) is one of the clinically common malignancies. With the great development of hepatic surgery technics, resectability of HCC has been improved. However, the therapeutic effectiveness of HCC is still very low, because of the high rate of recurrence and metastasis after hepatectomy. Thus, metastasis and recurrence have seriously restricted the longterm survival of HCC. Studies on the mechanisms of invasion and metastasis of HCC and effective methods of prevention or controlling will be of great importance in the HCC therapies.Yurt/Mosaic eyes-like1(YMOl), also known as EPB41L5, is a recently identified protein engaged in gastrulation, widely expressed in the mesoderm and ectoderm of mammalian embryos. It has been reported a series of YMO1functions such as regulation of cytoskeletal structure system, modifacation the cell adhesion molecule, formation of basement membrane and cell polarity, moreover, YMO1expressed location consisted with adult liver, suggesting the important role of YMOl in HCC invasion and metastasis. Therefore, in the present study, YMOl expression in human HCC tissues and cell lines was measured. Prognostic value was analyzed in combination with clinical data. The role of YMO1in invasion and metastasis HCC was investigated in vitro and in vivo by upregulation the YMO1expression with the recombinant plasmid. Additionally, molecular regulatory mechanisms of YMO1in the invasion and metastasis of HCC was demonstated. Our result was shown below:1. YMO1is significantly reduced in HCC tissues:Expression of EPB41, EPB41L1, EPB41L2, EPB41L3, EPB41L4A, EPB41L4B, EPB41L5was measured by quantitative Realtime-PCR in a well-characterized set of30HCC tissues and adjacent non tumor liver tissues. We discovered that YMO1(EPB41L5) expression showed a notable decrease in HCC tissues(p<0.001). YMO1mRNA and protein were measured by RT-PCR, real-time PCR and Western blot in30matched cases of HCC fresh tissues, adjacent noncancer liver tissues (ANLT), and6normal liver tissues (NL). Our results showed that both YMO1mRNA and protein levels in HCC tissues were significantly lower than those in the corresponding ANLT and NL tissues. YMO1mRNA and protein have significantly higher levels in solitary large HCC (SLHCC) and small HCC (SHCC) compared to nodular HCC (NHCC). Furthermore, YMO1mRNA and protein were measured in normal liver cell line L02and four stepwise metastatic HCC cell lines HepG2、 MHCC97-L、MHCC97-H and HCCLM3. The results indicated that the YMO1expression was highest in L02cell than other HCC cell lines. YMO1expression was down regulated according to priority of HepG2、 MHCC97-L、MHCC97-H and HCCLM3, which was in accordance with the increasing metastatic potential.2. The correlation of YMO1expression with the clinicopathologic features for HCC:We further investigated the YMO1protein expression in155cases by immunohistochemical staining. A cytoplasmic and membranic distribution of YMO1protein was observed, and the positive expression rate of YMO1was in HCC (78/155,40.6%), significantly lower than that in ANLT (131/155,84.5%).The YMO1low expression was significantly related to multiple nodules, without capsule formation and vein invasion.We found that the patients in YMO1low expression group had the poorer prognosis of disease free survival and overall survival than those in YMO1high expression group. Multivariate analyses revealed that YMO1was a significant predictor for overall survival, together with multiple nodules and vein invasion.3. YMOl inhibits HCC cell invasion and migration ability rather than cell proliferation in vitro:To further define the correlation of YMO1expression and HCC metastasis, we employed plasmid transfection approach to up-regulate the expression of YMO1in HCCLM3cells. We designed and constructed the recombinant plasmid pcDNA3-YMO1and transfected into HCCLM3cell lines with Lipofactamine LTX. After selection with G418, stably YMO1-expressing HCCLM3cell lines and control vector HCCLM3cell lines were named as HCCLM3YMO1+and HCCLM3vector respectively. The transfection efficiency expression of pcDNA3-YM01was verified by RT-PCR and Western blot. The wound-healing assay showed that migration of HCCLM3YMO1+cells had decreased than that of HCCLM3vector cells, significantly. Transwell assay showed a decreases invasion ability of HCCLM3YMO1+cells than HCCLM3vector cells. Adhesion assay confirmed the result that homogeneity adhesion ability of HCCLM3YMO1+cells was significantly higher and heterogeneity adhesion ability was significantly lower than HCCLM3vector cells. However, no marked difference was observed between the cell growth curves of HCCLM3YMO1+cells and HCCLM3vector cells. All above indicated a important role of YMO1in invasion and metastasis ability of HCCLM3cell, rather than regulation of proliferation.4. YMO1suppresses tumor formation and intrahepatic and pulmonary metastasis in vivo:We further examined biological function of YMO1in vivo by construct HCC subcutaneous xenograft model and orthotopic xenograft tumor model in nude mice. Our result showed that in HCCLM3YMO1+group had a lower rate of tumor formation and the average size of subcutaneous tumor was dramatically smaller than that of HCCLM3vector group. The orthotopic xenograft of HCCLM3YMO1+group showed a smaller diameter tumor size than HCCLM3vector group. Furthermore, a relative lower intrahepatic and pulmonary metastasis was observed in HCCLM3YMO1+group. Our data support an important role of Ymo in inhibition of HCC tumorgenesis and metastasis in vivo.5. YMOl upregulated RhoC and suppresses invasion and metastasis in HCC:Related references reported the protein binding between the PDZ domain of YMO1and GTPase or adhesion molecule.We further screened the downstream molecule in a series of GTPase by co-immunoprecipita-tions. Direct interaction of YMO1and RhoC proteins in HCCLM3cells was confirmed in our experiment. Next, we transfected HCCLM3YMOli+cell with recombinant plasmid pEGFP-C1-RhoC, served HCCLM3YMOli+and HCCLM3vector as the control. We compare the the RhoC expression by western blot, GTPase activity by pulldown assay, invasion and migration by wound healing assay and trans well assay, cytoskeletons distribution by immunofluroescence. The results showed that RhoC expression and GTPase activity significantly decreased, which caused a decline of invasion and migration ability and standardized cytoskeletons after pcDNA3-YMO1transfection. Whereas no markly difference was found between HCCLM3vector and pEGFP-C1-RhoC transfection groups in HCCLM3YMO1+cell. The results confirmed that YMO1inhibits RhoC expression and activity, which contribute to inhibition of HCC invasion and metastasis. Expression and phosphorylation level of ROCK1, PTEN, AKT, FAK, ERK1/2were detected by Western blot. Decreased of ROCK1, p-PTEN, p-AKT, p-FAK, p-ERK were observed in HCCLM3YMO1+cell. Discripancy of PTEN, AKT, FAK, ERK1/2expression among three groups did not reach stastistic significant.6. YMO1transcription is regulated by PAX5:Analysis result from DECODE database (DECipherment Of DNA Elements from SABiosciences) indicates that PAX5may be the transcription factor regulating YMO1. We further validate the result with Dual-Luciferase reporter assay system, chromatin immunoprecipitation assay (ChIP), and Western blot. All the result support that PAX5directly interact with the promoter sequence and up-regulate the expression of YMO1. Immunohistochemical staining of PAX5, YMO1and RhoC in the155cases above confirmed that low expression of YMO1was significantly and positively associated with PAX5underexpression but negatively associated with RhoC overexpression in human HCCs, respectively.In conclusion, low expression levels of YMO1have been shown in HCC tissues and cell lines. Our study also suggests that low expression of YMO1reflects multiple nodules, without capsule formation and with vein invasion, and thus indicates a poor prognosis for HCC patients. Upregulation of YMO1can inhibit of HCC invasion and metastasis in vitro and in vivo, by regulation of RhoC expression and activity. Therefore, YMO1may be of great value in predicting the prognosis of HCC patients and acting as a therapeutic target for HCC.