MiRNNA-200b Controls Anoikis And Is Regulated By PEA3and ELK-1

MicroRNAs (miRNAs) are a class of19-to23-nucleotide (nt) length, small non-coding RNA molecules which can regulate gene expression post-transcriptionally by interacting with the3’untranslated regions (3’UTR) of target mRNAs. Presumably, about40%of the genes are subject to miRNAs regulation, so it is not hard to understand that miRNAs are widely involved in a variety of physiological and pathological processes. A large number of studies have shown that abnormal expression of miRNAs are closely related to tumor development.miRNA-200s family includes five members and can be grouped into two subfamilies according to their function:miRNA-200a, miRNA-141and miRNA-200b, miRNA-200c, miRNA-429. MiRNA-200s regulate the expression of E-cadherin through interacting with the3’UTR of ZEB1/ZEB2which are the transcriptional repressor of E-cadherin. Increased expression of miRNA-200s resulted in downregulation of E-cadherin which caused the occurrence of EMT (Epithelial-mesenchymal transition). As an epithelial adhesion molecule, E-cadherin mediates cell-cell and cell-matrix interactions, reduced expression of E-cadherin makes cancer cells easily detached from the primary tumor which provides a prerequisite for metastasis.Under normal circumstances, detaching from the extracellular matrix, cells generally will undergo anoikis process. This is an important mechanism to maintain physiological homeostasis in the body. Anoikis is believed in acting as a barrier to metastasis, thus cancer cells must acquire the ability to resist anoikis in the process of metastasis with homelessness status. However, whether and how miRNA-200s play a role in the regulation of anoikis has not been reported.In this study, we find that overexpression of miRNA-200b in MDA-MB-231cells can induce the occurrence of anoikis. Several computational algorithms, including TargetScan and miRbase, show that Pinl (Peptidyl-prolyl cis/trans isomerase, PPIase) is one of the target genes of miRNA-200b. We demonstrated that miRNA-200b can regulate the expression of Pinl in translational level using Western blot, luciferase report and etc. The experiment of coexpression miRNA-200b and Pinl demonstrated that the function of miRNA-200b regulates anoikis, at least in partly, depends on its role of regulation of Pinl. Meanwhile, we also demonstrated that miRNA-200b controls anoikis through Pinl-pERK/pAKT pathway.Promoter analysis shows that the expression of miRNA-200b maybe regulated by PEA3and ELK-1—two members of ETS family. We then used siRNA to knockdown the expression of PEA3and ELK-1, and found that knockdown of PEA3decreased whereas knockdown of ELK-1increased the expression of miRNA-200b. It was reported that pERK can promote the phosphorylation of ELK-1and the sumoylation of PEA3, we speculated that overexpression of miRNA-200b may influence the phosphorylated ELK-1and sumoylated PEA3. To test this hypothesis, miRNA-200b mimics were transfected into MDA-MB-231cells and supplemented with EGF treatment. The results of Western blot and IP established a positive feedback loop between miRNA-200b and pELK-1and a negative feedback loop between miRNA-200b and sumoylated PEA3through Pinl-pERK pathway. Thus, miRNA-200b can regulate its own expression through the two feedback loops.Cancer cell is in a homelessness status during the process which it detached from primary tumor and moved to metastases. It has been reported that circulating tumor cells (CTCs) are in a balance between apoptosis and proliferation. We wonder whether the expression of miRNA-200b is changed when cancer cells are in homelessness status. MCF-7cells were suspension cultured in poly-HEMA coated plates. The results showed that the expression of miRNA-200b was decreased whereas the expression of Pinl was increased. It was reported that miRNA-200b can regulate proliferation and we also detected the downregulation of Cyclin D1expression which was also regulated by miRNA-200b. Based on these, we speculated that miRNA-200b may play a role in the balance between apoptosis and proliferation of CTCs.We detected the expression level of miRNA-200b and Pinl protein in human breast cancer tissues using Real-time quantitative PCR and Western blot. Statistical analysis showed that the expression of miRNA-200b was lower in cases with lymph node metastasis (p<0.01), while Pinl was higher (p<0.01) in these cases compared with those without lymph node metastasis. Moreover, the expression of miRNA-200b and Pinl protein in human breast cancer samples has a significant negative correlation (r=-0.431, p=0.014), suggesting that the correlation between reduced expression of miRNA-200b and breast cancer metastasis is closely related to the regulation of miRNA-200b to Pinl in these breast cancer specimens.