miRNA expression profile in human osteosarcoma: Role of miR-1 and miR-133b in proliferation and cell cycle control

Abstract

Abstract. miRNA profile deregulation affecting downstream signaling pathways activates endpoints that represent potential biomarkers for prognosis and treatment of tumor patients. In the past 20 years conventional therapy for osteosarcoma (OS) reached a survival plateau, highlighting the need for new therapeutic approaches. In this study, microarray unsupervised and supervised analysis identified, respectively, 100 and 40 differentially expressed miRNAs in OS samples with different grades of malignancy compared to normal bone. When analyzing low-grade and high-grade OS by unsupervised analysis, 12 miRNAs were found to be differentially expressed. Real-time PCR performed on a larger series of OS confirmed a significant lower expression of miR-1, miR-133b and miR-378 * in tumors with respect to control, also showing lower mRNA levels in 31 high-grade OS than in 25 low-grade and in metastatic versus non-metastatic patients. We demonstrated that miR-1 and miR133b were downregulated in OS cell lines compared to normal osteoblasts. Secondly, by transfection with miRNA precursor molecules, we demonstrated that the ectopic expression of miR-1 and miR-133b in U2-OS cell lines significantly reduced cell proliferation and MET protein expression and negatively regulated cell invasiveness and motility in a short-term assay. Cell cycle distribution revealed block in G 1 and delay of cell cycle progression associated with increased apoptosis in miR-1-and miR-133b-transfected cells, respectively. Our data assessed specific miRNA profiling deregulation in OS clinical samples and suggest that the expression of miR-1 and miR-133b may control cell proliferation and cell cycle through MET protein expression modulation. IntroductionOsteosarcoma (OS) is a rare malignant bone neoplasm for which biologic and pathologic information are still largely incomplete. OS has multiple genetic risk factors (1) and is characterized by complex chromosomal abnormalities and genetic cell heterogeneity that in over 70% provide a complex karyotype and drug resistance (2). MicroRNAs (miRNAs) are endogenous non-coding RNAs of 19-24 nucleotides that interacting with the 3' untranslated region (UTR) of mRNA target induce mRNA cleavage when pairing is complete and protein synthesis repression when pairing is incomplete. The relationship between miRNAs and their targets shows combinatorial complication, in terms of both target multiplicity and signal integration (3). MiRNAs are differentially expressed in relation to the developmental state, cell type and tissue (4-6). In literature, there are approximately 1,000 miRNA molecules per cell, with some cells exceeding 50,000 molecules (7). miRNA expression follows a dynamic range and this underscores the regulatory functional importance of miRNAs (3). Recent studies indicate that miRNAs called 'oncomirs' can function either as tumor suppressors or as oncogenes (8).Interconnection between miRNA and cancer is even more evident when analyzing the genomic location of known miRNA genes, more than 50% are in cancer-as...

MiR-133b Acts as a Tumor Suppressor and Negatively Regulates TBPL1 in Colorectal Cancer Cells

“…We also found that cells transfected with miR-133b mimics inhibited the cell proliferation, which strongly suggests that miR-133b acts as tumor suppressor in CRC. At the same time, similar results had also been observed in osteosarcoma (Novello et al, 2013), gastrointestinal stromal tumor (Yamamoto et al, 2013), gastric cancer (Wen et al, 2013) as well as bladder cancer (Yamasaki et al, 2012). However, the targets of miR-133b that regulates in CRC have not been established previously.…”

“…However, current studies indicate a broader expression pattern of miR-133b in diverse tissues. Moreover, most of the studies showed that miR-133b was down-regulated in some human malignancies, such as osteosarcoma (Novello et al, 2013), gastrointestinal stromal tumor (Yamamoto et al, 2013), gastric cancer (Wen et al, 2013) as well as bladder cancer (Yamasaki et al, 2012). Therefore, up-regulating miR-133b or exogenously providing its analogous pharmaceutical compounds might provide effective cancer therapies for tumors.…”

Decreased miR-134 expression and its tumor-suppressive function in human osteosarcoma

“…In terms of osteosarcoma, abnormal expression of several miRs such as miR-206 (Bao et al, 2013), miR-34a (Wu et al, 2013), and miR-145 have been reported. Novello et al (2013) found that miR-1 and miR-133b may regulate cell proliferation of osteosarcoma cells (Novello, et al, 2013). Cai et al (2013) indicated that overexpression of miR-210 is strongly associated with large tumor size, chemotherapy resistance, positive metastasis, and decreased overall survival in pediatric osteosarcoma .…”

miR-574-3p acts as a tumor promoter in osteosarcoma by targeting SMAD4 signaling pathway

“…In the present study, it was suggested that SMAD4 is a direct target of miR-574-3p and the associated genes CDKN1A, CDKN2B as well as ID3 were strongly suppressed by miR-574-3p transfection. SMAD4 is often mutated in numerous cancers and it acts as a tumor suppressor that is involved in the regulation of the TGF-β signal transduction pathway, which negatively regulates growth of epithelial cells and the extracellular matrix (28)(29)(30)(31). Therefore, the inhibition of SMAD4 and its downstream proteins may be an important part of the mechanism of miR-574-3p in osteosarcoma.…”