Multiple tumor suppressor microRNAs regulate telomerase and TCF7, an important transcriptional regulator of the Wnt pathway

Abstract

The human TERT (hTERT) gene encodes the telomerase catalytic subunit which plays a role in telomerase regulation. Telomerase is activated in more than 90% of all human malignancies and understanding how telomerase is regulated is necessary for implementation of successful anti-cancer therapies. microRNAs (miRNAs) are important regulators of gene expression in eukaryotic cells but evidence of their role in telomerase regulation has not been documented. To determine whether hTERT activity is regulated by multiple miRNAs, eight miRNAs which have putative binding sites in the hTERT 3'UTR together with miR-138-5p were evaluated in luciferase assays with a reporter containing the hTERT 3'UTR. Six miRNAs (let-7g*, miR-133a, miR-138-5p, miR-342-5p, miR-491-5p, and miR-541-3p) specifically inhibited the expression of the reporter luciferase-driven constructs and let-7g*, miR-133a, miR-138-5p, and miR-491-5p also downregulated endogenous telomerase activity in cells. Moreover, all six miRNAs significantly inhibited cell proliferation. miRNAs (miR-133a, miR-138-5p, 342-5p, 491-5p, 541-3p) also have predicted binding sites within the 3'UTR of three genes involved in Wnt signaling (TCF7, MSI1, and PAX5). These miRNAs inhibited the expression of the luciferase reporter constructs containing 3'UTRs of these genes and downregulated protein expression of the TCF7 transcription factor, which mediates the canonical Wnt pathway. Together, these results suggest the existence of a miRNA regulatory network involving the hTERT and Wnt pathway.

Figure 1. Selection of miRNA target sites in the 3′UTR of human TERT.

(a) miRNA target sites predicted by TargetScanHuman 5.2 (http://www.targetscan.org/). The horizontal bar on the right upper corner represents 100 kb scale. (b) miRNAs selected for experimental evaluation. The table identifies the miRNA gene, hairpin strand encoding the mature targeting strand (Str), the evolutionary conservation (EC) of the specific miRNA family: B - conserved in Bilateria, V - across vertebrates, P - in placental mammals. Number of targeting sites in the hTETR 3′UTR is indicated (#) together with the position in the 3′UTR (Pos). Evolutionary conservation of the sites is shown in the chimpanzee (Ptr), rhesus macaque (Mml), and common marmoset (Cja) indicated by black boxes (absolute conservation) or gray boxes (conserved when wobble G-U pairing is considered). Presence of the sites in the 3′UTR of the three genes of the Wnt pathway is indicated by a number in the gray field.

Figure 2. Several miRNAs target the hTERT 3′UTR.

Downregulation of luciferase activity of the hTERT 3′UTR reporter in HeLa cells transfected with miRNA mimic molecules (Table S1). The results are expressed as percent of reporter activity in cells transfected with a scrambled control (SC). The specificity of miRNA effect was evaluated by comparing the luciferase activity of wild type hTERT 3′UTR reporter (WT, gray bars) with a set of mutant 3′UTR reporters (Mut, black bars) in HeLa cells transfected with miRNA mimics. The reporter activity of the mutated construct in cells transfected with miRNA mimics is expressed as percent of mutated reporter activity in parallel cell cultures transfected with a scrambled control. Means and standard errors were calculated from 3–7 values (numbers are below bars) from 4 independent experiments. Statistically significant differences relative to wild type 3′UTR are indicated. P values for differences were determined by two-tailed Student′s t test (*P<0.05, **P<0.01, ***P<0.001, **** P<0.0001).

Figure 3. Multiple miRNAs decrease telomerase activity.

(A) The cells were transfected by miRNA mimics or transfection reagent alone (TR). The mixtures of miRNAs as well as scrambled control (SC) were always at a concentration of 60 nM; MIX1 (miR-491, miR-541, and miR-342), MIX2 (let-7g*, miR-133a, and, miR-138), MIX3 (MIX1+MIX2). The telomerase activity in these cells determined 4 hours post transfection by a TRAP assay. Telomerase activity was evaluated by the intensity of the telomerase product ladder as measured using the Typhoon Trio scanner and Quantify software. Means and standard errors were calculated from 5 independent experiments. Downregulation of telomerase activity in HeLa cells transfected with miRNA mimic molecules is expressed as percent of telomerase activity in cells transfected with a scrambled control. Statistically significant differences relative to the negative control are expressed as in Figure 2. (B) Representative TRAP gel from the experiment. In this set let-7g* had slightly higher activity than control which resulted in bigger variance (see A). Therefore, we also provided let-7g* from another set together with appropriate control. (C) Jurkat cells were treated with miRNA inhibitors and cells were harvested for TRAP 48 hours after electroporation. Telomerase activity was evaluated by the intensity of the telomerase product ladder as described above.

Figure 4. Downregulation of luciferase activity of the TCF7, MSI1, and PAX5 3′UTR reporters.

The results are expressed as percent of reporter activity in cells transfected with a scrambled control (SC). Differences between the luciferase activity in SC and transfection agent treated cells were not statistically significant, demonstrating that transfection of miRNA did not induce a nonspecific effect (data not shown). Means and standard errors were calculated from 4 independent experiments. Statistically significant differences relative to the SC control are indicated as in Figure 2. Number of miRNA sites in respective 3′UTR is displayed. let-7g* has no predicted binding sites in the 3′UTRs of these three genes.

Figure 5. miRNAs targeting TERT regulate genes in the Wnt pathway.

Western blot analysis of endogenously expressed TCF7 (38–80 kDa) and MSI1 (39 kDa and 43 kDa) proteins in DLD-1 cells transfected with scrambled control (SC), and MIX1, MIX2, and MIX3 as described in Figure 3 or with transfection reagent alone (TR). The mixtures of miRNAs as well as SC were at a concentration of 60 nM. Single transfected miRNAs gave more modest differences than mixtures. Molecular weight markers are shown in the left margin. β-actin served as a loading control. The quantification of Western blot analysis (Table) is shown on the right side. The signals of TCF7 or MSI1 were normalized to β-actin and expressed as a percentage of SC control. The ratio of 43 to 38 kDa MSI1 forms is shown on the picture under MSI1 bands.

Figure 6. miRNAs targeting hTERT decrease cell proliferation in the DLD-1 and MCF-7 cell lines.

The cells were transfected by miRNA mimics and cell numbers were determined 48 hours post-transfection. An increase in cell death was not detected (data not shown). Means and standard errors were calculated from 5 (single miRNAs) and 3 independent experiments (mixtures of miRNAs). Statistically significant differences relative to the negative control is expressed as in Figure 2. Differences between the SC and transfection agent treated cells were not statistically significant (data not shown). (a) Proliferation of DLD-1 cell transfected with miRNA mimic molecules are expressed as the percent of number of cells transfected with a scrambled control (SC). (b) Proliferation of DLD-1 and MCF-7 cells transfected with mixtures of miRNA mimics molecules expressed as a percent of the number of cells transfected with a scrambled control (SC).