Identification And Characterization Of Human ARIP2 And It's Relation With Breast Cancer

Activins are member of the transforming growth factorβ(TGF-β) superfamily of growthand development of differentiation factors and have numerous biological functions and widelybiological actions. Activin regulates the development of cells and tissues and keep thehomeostasis of organisms. Especially activin A, always plays important roles for organismsfrom new born to adult. The biological actions of activin on target cells are tissue-specific,which included regulating the cell proliferation and inhibiting the development of tumor cells.Activin signaling has been associated with mammary cell growth inhibition as well asregulation of mammary glandular development.Studies showed that activin and its receptorsare expressed during postnatal mouse mammary development and glandular formation duringthe lactating phase. Activin receptors and signaling molecules are also present in humanbreast cancer cell lines. These results suggest that the expression of activin A has closerelationship with breast tumorigenesis and progression.Then signal transduction pathway is as followed: activin binds directly to its typeⅡreceptor, leading to the recruitment, phosphorylation and subsequent activation of the typeⅠreceptor. On activation, the type I receptor binds and then phosphorylates a subset ofcytoplasmic Smad protein-Smad 2/3. The phosphorylated Smad 2/3 can bind to Smad 4.These Smads translocate to the nucleus, where they control the transcription of target genes.In the course of signal transduction, the expression level of typeⅡreceptor in membrane willaffect the intensity of activin signal. The recent investigation indicated that mouse ARIPs canregulate activin signal through control the expression level of typeⅡreceptor on membrane.But to date, there is no report in this aspect.In this study, we obtained a novel member of ARIPs from 293 cells by RT-PCR. The novelcDNA clone encodes the full length is 546bp. The novel gene encodes a 182 amimo acidprotein. Because of high homology with mouse ARIP2, we named the novel protein as humanactivin receptor interacting protein 2. Like murine ARIP2, hARIP2 has a PDZ domain in itsNH2-terminal region and can interact specifically with ActRIIs.Our study further tested the effect of hARIP2 on activin-induced signal transductionpathway, the CAGA-lux construct which provides luciferase reporter gene and specificallyresponds to activin was selected. CAGA-lux, pcDNA3-FLAG-hARIP2 or pSilencer-hARIP2were co-transfected into human breast adenocarcinoma MCF-7 cells and MDA-MB-231 cellsin vitro. The results showed that the overexpression of hARIP2 decreased activin-inducedtranscriptional activity. However, inhibiting the expression of hARIP2 augmentedactivin-induced transcriptional activity.To determine a role of hARIP2 for the proliferation of tumor cells, the control,recombinant plasmid Flag-pcDNA3-hARIP2, control siRNA and pSilencer-hARIP2 weretransfected into human breast adenocarcinoma MCF-7 cells and MDA-MB-231 cellsrespectively. Through MTT assay and plate clonality formation assay, we found that thelevels of cell proliferation increased when hARIP2 was over-expressed compared with control. However, when the cells were transfected with pSilencer-hARIP2, the levels of cellproliferation decreased compared with control siRNA. Meanwhile, similar results wereobtained in other tumor cells. These results indicated that overexpressing hARIP2 promotedbreast tumor cell proliferation.Moreover, we prepared anti-hARIP2 polyclonal antibody by ourselves. Byimmunohistochemistry method, we determined the distribution of hARIP2 in the tissuesincluding breast benign hyperplasia, fibroadenoma, mucous adenocarcinoma, trachealcarcinoma and simple carcinoma using the antibody, and further investigated the associationof the expression of hARIP2 with malignant progression, tumor size, tumor grade andmetastases by Immunohistochemistry method. The result showed significant correlations inbreast cancer specimens between an increase of hARIP2 abundance and high tumor malignantprogression, high tumor grade, and high tumor metastases. Moreover, hARIP2 was expressedmore frequently and much more intensely in malignant breast tissues such as simplecarcinoma, invasive ductal carcinoma and mucinous adenocarcinoma than in benignhyperplasia or fibroadenoma cases. These results suggesting that the expression level ofhARIP2 has close relationship with breast tumorigenisis, tumor malignant progression, tumorgrade, and tumor metastases.This study strengthened the bases of activin-mediated breast cancer diseases development,progression and therapies with theoretical and experimental evidence. Further more, this studyhad a significant value for the diagnosis and treatment of breast cancer.