Abstract
Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21WAF1-mediated signaling to limit proliferation in TEP cells.
Highlights
Abbreviations telomere length maintenance mechanism (TMM) Telomere length maintenance mechanism alternative lengthening of telomeres (ALT) Alternative lengthening of telomeres telomerase positive (TEP) Telomerase-positive APBs ALT-associated promyelocytic leukemia protein nuclear bodies miRNAs MicroRNAs telomeric repeat amplification protocol (TRAP) Telomeric repeat amplification protocol enzyme-linked immunosorbent assay (ELISA) Enzyme-linked immunosorbent assay human umbilical vessel endothelial cells (HUVECs) Human umbilical vessel endothelial cells polyvinylidene fluoride (PVDF) Polyvinylidene fluoride enhanced chemiluminescence (ECL) Enhanced chemiluminescence OE Overexpressing epithelial–mesenchymal transition (EMT) Epithelial-mesenchymal transition structural maintenance of chromosomes 5 (SMC5) Structural maintenance of chromosomes 5 structural maintenance of chromosomes 6 (SMC6) Structural maintenance of chromosomes 6
Assessment of telomere content in both TEP and ALT cells with miR-708 overexpression showed that there was no difference despite the altered miR-708 status. (Fig. 2E) These findings collectively demonstrate that miR-708 is consistently upregulated in ALT-positive cell lines and cancers compared to TEP/C-circle-negative cell lines and tissues, but its level does not influence telomerase activity, and it does not have correlation to C-circle levels in ALT cells
ALT occurs in approximately 10–15% of tumors, many of which are biologically aggressive and currently difficult to treat, so to design new therapeutic modalities it is important to understand any intrinsic differences between cancers that use ALT and those that utilize TEP
Summary
Abbreviations TMM Telomere length maintenance mechanism ALT Alternative lengthening of telomeres TEP Telomerase-positive APBs ALT-associated promyelocytic leukemia protein nuclear bodies miRNAs MicroRNAs TRAP Telomeric repeat amplification protocol ELISA Enzyme-linked immunosorbent assay HUVECs Human umbilical vessel endothelial cells PVDF Polyvinylidene fluoride ECL Enhanced chemiluminescence OE Overexpressing EMT Epithelial-mesenchymal transition SMC5 Structural maintenance of chromosomes 5 SMC6 Structural maintenance of chromosomes 6. Recent studies have indicated that microRNAs (miRNAs), 20–24 base pair non-coding RNAs that silence target genes at the post-transcriptional level by binding to complementary sequences in the 3′UTR to promote transcript degradation or translation inhibition, play major roles in the regulation of cancer[15,16]. They have been reported to be involved in regulation of cell cycle progression, DNA damage response, apoptosis, epithelialto-mesenchymal cell transition, cell motility/invasion, stemness, and in telomere maintenance[17,18,19]. The selected miRNA species were investigated in a large panel of TEP and ALT cells following which the biological significance of the selected miRNA (miR708) in pathways related to the regulation of telomere length, DNA damage, cell proliferation and migration was determined in representative TEP (MG63) and ALT (U2OS) by cellular, biochemical and expression analyses
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