Silence Of ASPP2Induced By MiR-205Promotes Metastasis And EMT In Esophageal Carcinomas

Background and Aims：ASPP2, a member of the ankyrin-repeat-, SH3-domain-and proline-rich-region-containing protein (ASPP) family is frequently decreased in various cancers.Clinical data reveals that reduced expression of ASPP2is related with poor clinicaloutcomes in diffuse large B-cell lymphoma and tumor metastasis and poorrecurrence-free survival in breast cancer patients. The effects and underlying mechanismof ASPP2on tumor metastasis are remains unknown.Previous studies have suggested that the expression of ASPP1and ASPP2could beactivated by E2F or inactivated by DNA methylation. Hypermethylation of ASPP1andASPP2promoters is found in several tumor cell lines expressing wildtype p53. wepreviously showed that ASPP1and ASPP2were frequently methylated in HCCs. What’sthe regulation mechanism of ASPP2in Esophageal Squamous Cell Carcinoma (ESCC)remains unknown.In order to metastasis to distant oranges, tumor cells must acquire motility andinvasiveness properties to disseminate from primary tumors. Recent evidences havesuggested that a subset of epithelial tumor cells acquires such abilities by undergoingepithelial-mesenchymal transition (EMT), characterized by the loss of cell polarity, andgain of mesenchymal differentiation properties. A key step in EMT is down-regulation ofE-cadherin. Recently, ASPP2is found to be involved in the regulation of cell polaritythrough binding with the PAR complex protein Par-3at cell junctions in neuralprogenitor cells or epithelial cells. The importance of cell polarity and adherens junctionin EMT indicates a possible role of ASPP2in the regulation of EMT and tumormetastasis.Methods and Results：In current study; we analyzed ASPP2expression in132Esophageal Squamous CellCarcinoma (ESCC) samples and revealed that ASPP2expression was down-regulated inprimary tumor and lymph metastasis compared with the normal esophageal tissue. Further,decreased ASPP2expression correlated with tumor metastasis poor prognosis in ESSCpatients.MicroRNAs (miRNAs) inhibit translation or induce mRNA degradation in general by binding to the3’ untranslational region (3’UTR) of target mRNAs. Since ASPP2wasregulated at protein level in ESSC, we analyzed potential miRNA target ASPP2throughtargetscan，Microlnspector and targetscan, the results showed that miR-205, miR-221andmiR-222may target ASPP2. Luciferase report systerm demonstrats that miR-205can bindwith ASPP23’UTR. We detect expression of miR-205in ESCC cell lince, the data showsthat expression of miR-205is sharply increased in ESSC cell lines compared with that inthe normal esophageal cell line. Further, The expression of miR-205in ESCC cells weredeceased by miR-205inhibitor or increased by miR-205mimics, then mRNA and proteinlevels of ASPP2are detected, the results show miR-205decreased ASPP2expression byinhibits it’s translation.The expression of ASPP2and miR-205were deceased by RNA interference orincreased by transected with over expression plasmid, then tumorigenicity and metastasispotentials of ESSC cells were studied in vitro and in nude mice. Wound-healing,migrationg and invasion assays show silence of ASPP2incerase migration and invasionpotentials of ESSC cells; overexpression miR-205may increase migration and invasionpotentials of ESSC cells as well, which can be inhibit by ASPP2. Futher, silence of ASPP2or overexpression of miR-205promot tumor metastasis in vivo. RT-PCR,Western blotand Immunofluorscence were used to analysis EMT. The results show silence of ASPP2induce morphological and molecular changes of EMT, overexpression of miR-205aslomay induce morphological and molecular changes of EMT and that can be inhibit byASPP2.Colusion：Our findings present functional and mechanistic insight into the critical role of ASPP2in the progression and metastasis of cancer.