Functional elucidation of MiR-34 in osteosarcoma cells and primary tumor samples

https://doi.org/10.1016/j.bbrc.2009.07.101Get rights and content

Abstract

MiR-34s have been characterized as direct p53 targets, which induce apoptosis, cell cycle arrest, and senescence. MiR-34s were found to associate with tumorigenesis. Thus far, there is no study on the role of MiR-34s in osteosarcoma. In the current study, we intensively investigated the function of MiR-34s in two osteosarcoma cell lines: U2OS (p53+/+) and SAOS-2 (p53−/−). We found that MiR-34s affect the expression of its target genes partially in a p53-dependent manner. And p53 also partially contributes to the MiR-34s induced cell cycle arrest and apoptosis. Finally, we examined the expression, genetic and epigenetic alterations of MiR-34 gene in 117 primary osteosarcoma samples. Expression of MiR-34s was decreased in tumor samples, and MiR-34 genes underwent minimal deletions and epigenetic inactivation in osteosarcomas.

Introduction

Micro-RNAs (MiRNAs) are small non-coding RNAs that regulate gene expression mainly through affecting productive translation and mRNA stability [1], [2], [3]. Nascent MiRNA transcripts (pri-MiRNAs) are transcribed by RNA polymerases II or III to yield primary transcripts (pri-MiRNAs), which are processed by the nuclear RNase III enzyme Drosha to form hairpin products (pre-MiRNAs). These pre-MiRNAs are transported to the cytoplasm where the RNase III enzyme Dicer cleaves off the double-stranded (ds) portion of the hairpin and generates a short-lived dsRNA of about 20–25 nucleotides in size [1], [2], [3]. After maturation, these small RNAs are incorporated into the RNA-induced silencing complex (RISC). The MiRNA complex binds to the partially complementary binding sites located in the 3′ untranslated region (UTR) of target mRNAs by imperfect base pairing that depends mainly on the “seed” sequences comprising bases 2–7 of the mature MiRNA [2], [3]. MiRNAs mediate post-transcriptional gene silencing of specific mRNA targets by inhibiting translation or destabilizing target mRNAs [4], [5]. However, recent findings indicate that MiRNA-mediated repression can be reversed, prevented or even act as translational activators [6].

MiRNAs play important roles in several cellular processes, such as proliferation, differentiation, apoptosis and development, by simultaneously controlling the expression levels of hundreds of genes [2], [7]. Similar to mRNA-encoding genes, several MiRNA-encoding genes have been meanwhile classified as oncogenic or tumor-suppressive genes according to their function in cellular transformation and expression in tumors [8], [9], [10], [11]. In human cancer, recent studies have shown that MiRNA expression profiles differ between normal tissues and derived tumors and between tumor types [12], [13]. MiRNAs can act as oncogenes or tumor suppressors, exerting a key function in tumorigenesis [10], [14]. Furthermore, tumor cells seem to undergo a general loss of MiRNA expression, and forced reduction of global MiRNA expression promotes transformation [15]. Interestingly, MiRNAs cluster within fragile sites and other genomic regions frequently altered in cancers [16], [17]. Besides their causal involvement in tumor formation, MiRNAs may be very useful for the classification, diagnosis, prognosis, and therapy of malignancies [8], [9], [12].

Recently, reports from several laboratories surfaced that the MiR-34 family are direct p53 targets, which induce apoptosis, cell cycle arrest, and senescence [18], [19], [20], [21], [22], [23], [24]. In vertebrates, MiR-34 diverged into a family of three homologous MiRNAs: MiR-34a, MiR-34b and MiR-34c. The mature MiR-34a sequence is located within the second exon of its non-coding host gene which contains a predicted p53 binding site [18], [19], [22], [23], [25]. Both MiR-34b and MiR-34c are located within a single non-coding precursor (MiR-34b/c), whose transcriptional start site is adjacent to a predicted p53 binding site [18], [25]. Ectopic expression of MiR-34a and MiR-34b/c caused a cell cycle arrest in the G1 phase [18], [23], [25], and inhibited proliferation and colony formation in soft agar [20]. Introduction of MiR-34a and MiR-34b/c into primary human diploid fibroblasts induced cellular senescence [25]. Interestingly, tumor cells also showed signs of senescence after introduction of ectopic MiR-34a [24]. Furthermore, expression of MiR-34a induced apoptosis [19], [22], [23], [26].

MiR-34 was found to associate with tumorigenesis. For example, loss of MiR-34a expression was observed in neuroblastoma, which may be due to the relatively common deletion of a region on chromosome 1p36, which encompasses MiR-34a [26]. 1p36 deletions frequently encompass very large numbers of genes; however, primary neuroblastomas and cell lines often showed low MiR-34a expression [26]. Artificial elevation of MiR-34a in these cells inhibited proliferation and activated cell death pathways [26]. Furthermore, expression of MiR-34a was low or undetectable in 11 of 15 pancreatic cancer cell lines [19]. The expression level of MiR-34b was decreased by more than 90% in 6 out of 14 non-small-cell lung cancers (NSCLCs) [18]. Minimal deletions containing MiR-34b and MiR-34c have also been found in breast and lung cancer [17]. Epigenetic inactivation of MiR-34b/c was also found to associate with cancer metastasis [27]. A combination of bioinformatic predictions and experimental analysis led to the notion that MiR-34s control broad programs of targets involved in cell cycle control, apoptosis, and DNA repair, among which CDK4/6, Cyclin E2, E2F3/5, cMET, and Bcl-2 were shown as possible candidates [18], [24], [25].

Thus far, there is no study on the role of MiR-34 in osteosarcoma. In the current study, we intensively investigated the function of MiR-34 in two osteosarcoma cell lines: U2OS (p53+/+) and SAOS-2 (p53−/−). We also examined the genetic and epigenetic alterations of MiR-34 gene in primary osteosarcoma samples, providing more evidence on understanding MiR-34 in osteosarcoma.

Section snippets

Materials and methods

Cell culture, reagents and tumor samples. Human osteosarcoma cell lines U2OS and SAOS-2 were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen). Antibodies against CDK6, E2F3, Cyclin E2, Bcl-2 and Actin were from Santa Cruz. MiR-34 mimics, antagonists and control MiRNA mimic were obtained from Dharmacon with the following sequences: hsa-MiR-34a-5′-uggcagugucuuagcugguugu-3′; hsa-MiR-34b-5′-caaucacuaacuccacugccau-3′; hsa-MiR-34c-5′-aggcaguguaguuagcugauugc-3′.

Expression and induction of MiR-34s in osteosarcoma cells

Since Mir-34s are direct p53 targets, we took advantage of two osteosarcoma cell lines, U2OS (p53+/+) and SAOS-2 (p53−/−), to investigate the function of MiR-34s in osteosarcoma cells. We examined the expression of MiR-34s in these cells and found that both cell lines express MiR-34s at similar levels (Fig. 1A), suggesting that basal Mir-34s levels in these cells are p53-independent.

The transcriptional activity of p53 can be readily induced in cells by exposure to genotoxic stress, such as

Conclusion

Our study showed that: (a) both U2OS (p53+/+) and SAOS-2 (p53−/−) cells express MiR-34s at similar levels; (b) irradiation and adriamycin induce MiR-34s expression in U2OS cells but not in SAOS-2 cells; (c) MiR-34s affect the expression of CDK6, E2F3, Cyclin E2 and Bcl-2 partially in a p53-dependent manner; (d) MiR-34s induce G1 arrest and apoptosis partially in a p53-dependent manner; (e) expression of MiR-34s in osteosarcoma samples was decreased; and (f) MiR-34s undergo genetic and

References (31)

  • W. Filipowicz et al.

    Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?

    Nat. Rev. Genet.

    (2008)
  • L. He et al.

    MicroRNAs: small RNAs with a big role in gene regulation

    Nat. Rev. Genet.

    (2004)
  • G.A. Calin et al.

    MicroRNA signatures in human cancers

    Nat. Rev. Cancer

    (2006)
  • J.M. Cummins et al.

    Implications of micro-RNA profiling for cancer diagnosis

    Oncogene

    (2006)
  • A. Esquela-Kerscher et al.

    Oncomirs – microRNAs with a role in cancer

    Nat. Rev. Cancer

    (2006)
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