MicroRNA-21 Targets the Tumor Suppressor Gene Tropomyosin 1 (TPM1)

Abstract

MicroRNAs are small noncoding RNA molecules that control expression of target genes. Our previous studies show that mir-21 is overexpressed in tumor tissues compared with the matched normal tissues. Moreover, suppression of mir-21 by antisense oligonucleotides inhibits tumor cell growth both in vitro and in vivo. However, it remains largely unclear as to how mir-21 affects tumor growth, because our understanding of mir-21 targets is limited. In this study, we performed two-dimensional differentiation in-gel electrophoresis of tumors treated with anti-mir-21 and identified the tumor suppressor tropomyosin 1 (TPM1) as a potential mir-21 target. In agreement with this, there is a putative mir-21 binding site at the 3-untranslated region (3-UTR) of TPM1 variants V1 and V5. Thus, we cloned the 3-UTR of TPM1 into a luciferase reporter and found that although mir-21 down-regulated the luciferase activity, anti-mir-21 up-regulated it. Moreover, deletion of the mir-21 binding site abolished the effect of mir-21 on the luciferase activity, suggesting that this mir-21 binding site is critical. Western blot with the cloned TPM1-V1 plus the 3-UTR indicated that TPM1 protein level was also regulated by mir-21, whereas real-time quantitative reverse transcription-PCR revealed no difference at the mRNA level, suggesting translational regulation. Finally, overexpression of TPM1 in breast cancer MCF-7 cells suppressed anchorage-independent growth. Thus, down-regulation of TPM1 by mir-21 may explain, at least in part, why suppression of mir-21 can inhibit tumor growth, further supporting the notion that mir-21 functions as an oncogene. MicroRNAs (miRNAs)2 are a class of naturally occurring small noncoding RNAs that regulate gene expression by targeting mRNAs for translational repression or cleavage (1, 2). Like protein-coding mRNAs, miRNAs are transcribed as long primary transcripts in the nucleus. However, unlike protein-coding mRNAs, miRNAs are subsequently cleaved to produce stem-loop-structured precursor molecules of ϳ70 nucleotides in length (pre-miRNAs) by the nuclear RNase III enzyme Drosha (3). The pre-miRNAs are then exported to the cytoplasm, where the RNase III enzyme Dicer further processes them into mature miRNAs (ϳ22 nucleotides). Thus, miRNAs are related to, but distinct from, short inferring RNAs (siRNAs) (4, 5). A key difference between siRNAs and miRNAs is that siRNAs require almost identical sequences to targets to exert their silencing function, whereas miRNAs bind through partial sequence homology to the 3Ј-untranslated region (3Ј-UTR) of target genes. Because of this unique feature, a single miRNA has multiple targets. Thus, miRNAs could regulate a large fraction of protein-coding genes, and as high as 30% of all genes could be miRNA targets (6).As a new layer of gene regulation mechanism, miRNAs have diverse functions, including the regulation of cellular differentiation, proliferation, and apoptosis (7,8). Hence, deregulation of miRNA expression may lead to a variety of disorders. Aberrant expression of miR...

Overexpression of miR-370 and downregulation of its novel target TGFβ-RII contribute to the progression of gastric carcinoma

“…For instance, miR-21 has been shown to be overexpressed in a variety of cancers and targets phosphatase and tensin homolog (PTEN), reversion-inducingcysteine-rich protein with kazal motifs (RECK), programmed cell death and four other genes as well as tropomyosin-1 (Meng et al, 2006;Si et al, 2007;Zhu et al, 2007;Lu et al, 2008;Zhang et al, 2008). In contrast, let-7 is a tumor-suppressive miRNA family and is underexpressed in various tumors; it targets Ras, HMGA2 and others genes (Johnson et al, 2005;Yanaihara et al, 2006;Motoyama et al, 2008).…”

hsa‐miR‐210 is a marker of tumor hypoxia and a prognostic factor in head and neck cancer

“…21 hsa-miR-21 is overexpressed in many cancers, including HNSCC, 22,23 and is associated with a poor prognosis in nonsmall cell lung cancer. 24 Its targets include the tumor suppressors phosphatase and tensin homolog, 25 tropomysin 1, 26 and programmed cell death 4. 27 hsa-miR-21 was shown to be hypoxia-induced in breast cancer cell lines.…”

Interactions between the estrogen receptor, its cofactors and microRNAs in breast cancer

“…A decrease in the expression of let-7 prior to radiation affects cell survival, increasing radioresistance, possibly through an interaction with survival genes and DNA damage response pathways. It has also been demonstrated that the miRNA expression profiles of cholangiocarcinoma cells change in response to the chemotherapy drug gemcitabine, and that the inhibition of miR-21, previously identified as an antiapoptotic miRNA in many cancers [57,[77][78][79][80], increases cellular sensitivity to this drug [81]. Also, miR-214 has been shown to confer cisplatin resistance in ovarian cancer by targeting the PTEN/Akt pathway [82].…”