Receptor C(k)-dependent signaling regulates hTERT gene transcription

BACKGROUND: Available evidence suggests that the regulation of telomerase activity primarily depends on the transcriptional control of the human telomerase reverse transcriptase (hTERT) gene. Although several activators and repressors of hTERT gene transcription have been identified, the exact mechanism by which hTERT transcription is repressed in normal cells and activated in cancer cells remains largely unknown. In an attempt to identify possible novel mechanisms involved in the regulation of hTERT transcription, the present study examined the role of Receptor C(k), a cell surface receptor specific for cholesterol, in the transcription of hTERT gene in normal human peripheral blood mononuclear cells. RESULTS: Activated Receptor C(k )was found to down-regulate hTERT mRNA expression by repressing the transcription of c-myc gene. Receptor C(k)-dependent signaling was also found to down-regulate the mRNA expression of the gene coding for the ligand inducible transcription factor, peroxisome proliferator-activated receptor γ (PPARγ). The ligand activation of PPARγ resulted in the down-regulation of c-myc and hTERT mRNA expression. By using specific activator and inhibitor of protein kinase C (PKC), it was demonstrated that Receptor C(k )dependent down-regulation of hTERT gene transcription involved inhibition of PKC. In addition, 25-hydroxycholesterol was found to contribute to the transcriptional regulation of hTERT gene. CONCLUSION: Taken together, the findings of this study present evidence for a molecular link between cholesterol-activated Receptor C(k )and hTERT transcription, and provide new insights into the regulation of hTERT expression in normal human peripheral blood mononuclear cells

Intrinsic expression of host genes and intronic miRNAs in prostate carcinoma cells

Abstract Background Recent data show aberrant and altered expression of regulatory noncoding micro (mi) RNAs in prostate cancer (PCa). A large number of miRNAs are encoded in organized intronic clusters within many protein coding genes. While expression profiling studies of miRNAs are common place, little is known about the host gene and their resident miRNAs coordinated expression in PCa cells. Furthermore, whether expression of a subset of miRNAs is distinct in androgen-responsive and androgen-independent cells is not clear. Here we have examined the expression of mature miRNAs of miR 17–92, miR 106b-25 and miR 23b-24 clusters along with their host genes C13orf25, MCM7 and AMPO respectively in PCa cell lines. Results The expression profiling of miRNAs and host genes was performed in androgen-sensitive MDA PCa 2b and LNCaP as well as in androgen-refractory PC-3 and DU 145 cell culture models of PCa. No significant correlation between the miRNA expression and the intrinsic hormone-responsive property of PCa cells was observed. Androgen-sensitive MDA PCa 2b cells exhibited the highest level of expression of most miRNAs studied in this report. We found significant expression variations between host genes and their resident miRNAs. The expressions of C13orf25 and miR 17–92 cluster as well as MCM7 and miR 106b-25 cluster did not reveal statistically significant correlation, thus suggesting that host genes and resident miRNAs may be expressed independent of each other. Conclusion Our results suggest that miRNA expression profiles may not predict intrinsic hormone-sensitive environment of PCa cells. More importantly, our data indicate the possibility of additional novel mechanisms for intronic miRNA processing in PCa cells.</p

Functionally important structural elements of U12 snRNA

U12 snRNA is analogous to U2 snRNA of the U2-dependent spliceosome and is essential for the splicing of U12-dependent introns in metazoan cells. The essential region of U12 snRNA, which base pairs to the branch site of minor class introns is well characterized. However, other regions which are outside of the branch site base pairing region are not yet characterized and the requirement of these structures in U12-dependent splicing is not clear. U12 snRNA is predicted to form an intricate secondary structure containing several stem–loops and single-stranded regions. Using a previously characterized branch site genetic suppression assay, we generated second-site mutations in the suppressor U12 snRNA to investigate the in vivo requirement of structural elements in U12-dependent splicing. Our results show that stem–loop IIa is essential and required for in vivo splicing. Interestingly, an evolutionarily conserved stem–loop IIb is dispensable for splicing. We also show that stem–loop III, which binds to a p65 RNA binding protein of the U11-U12 di.snRNP complex, is essential for in vivo splicing. The data validate the existence of proposed stem–loops of U12 snRNA and provide experimental support for individual secondary structures

Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a.

Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments

Conservation of miR-644a target site.

<p>Panels A and B show alignments of GAPDH and β-actin 3′ UTR sequences containing miR-644a binding site in 7 mammalian species. miR-644a target site sequence is shown in gray box and seed binding region is shown in bold. Stars indicate conserved nucleotides in the target sequence in at least 5 out of 7 species.</p

β-actin is a direct target of miR-644a.

<p>(A) Schematic representation of firefly luciferase reporter construct containing β-actin 3′ UTR with either wild type (WT) or mutant (MUT) miR-644a target site. The italicized and underlined sequence in β-actin 3′ UTR represents the miR-644a target site. In the MUT-3′ UTR construct, 2 nucleotides (1562–1563) in the seed binding region of the target site were mutated to their complementary nucleotides (shown in bold) in order to disrupt miR-644a binding. (B) Luciferase reporter assay in CHO-K1 cells cotransfected with WT-3′ UTR or MUT-3′ UTR construct and miR-644a mimic (2 nM) or negative control (NC) mimic (2 nM) as indicated. Renilla luciferase reporter plasmid was cotransfected in all cases as a control for transfection efficiency. Luciferase activity is plotted as a ratio of firefly to renilla luciferase activity. Each bar represents mean ± SE of three independent experiments.</p

GAPDH is a direct target of miR-644a.

<p>(A) Schematic representation of firefly luciferase reporter construct containing GAPDH 3′ UTR with either wild type (WT) or mutant (MUT) miR-644a target site. The miR-644a target site in GAPDH 3′ UTR is italicized and underlined. In the MUT-3′ UTR construct, 5 nucleotides (1183–1187) in the seed binding region of the target site were mutated to their complementary nucleotides (shown in bold) in order to disrupt miR-644a binding. (B) Luciferase reporter assay in CHO-K1 cells cotransfected with WT-3′ UTR or MUT-3′ UTR construct and miR-644a mimic (2 nM) or negative control (NC) mimic (2 nM) as indicated. Renilla luciferase reporter plasmid was cotransfected in all cases as a control for transfection efficiency. Luciferase activity is plotted as a ratio of firefly to renilla luciferase activity. Each bar represents mean ± SE of three independent experiments.</p

miR-644a downregulates GAPDH and β-actin protein expression.

<p>(A, B and C) Representative western blots showing the expression of GAPDH, β-actin and STAT2 in LNCaP, 293T and HeLa cells treated with indicated amounts of miR-644a mimic or negative control (NC) mimic for 48 hours. STAT2 expression was used as a loading control. (D, E and F) Quantitation of GAPDH protein expression in the respective lanes as shown in A, B and C. (G, H and I) Quantitation of β-actin protein expression in the respective lanes as shown in A, B and C. Three independent western blots were used for the quantification of protein expression. The signal intensities of bands were measured using ImageJ software. The GAPDH or β-actin expression in each lane was determined by normalizing GAPDH or β-actin band intensity to STAT2 band intensity. Data are plotted as mean ± SE of three independent experiments.</p

miR-644a downregulates GAPDH and β-actin mRNA expression.

<p>(A and B) Quantitative real-time PCR analysis of GAPDH and β-actin mRNA expression in LNCaP, 293T and HeLa cells transfected with miR-644a mimic or negative control (NC) mimic. (C) In order to demonstrate that the repression of GAPDH and β-actin mRNA expression is a consequence of specific targeting by miR-644a, the effect of miR-644a was checked on a computationally predicted non-target gene, STAT2. STAT2 mRNA expression was determined by quantitative real-time PCR analysis in LNCaP, 293T and HeLa cells transfected with miR-644a mimic or NC mimic. GAPDH, β-actin and STAT2 mRNA expression was normalized to 18S rRNA expression. Data are plotted as mean ± SE of three independent experiments.</p

MiR-644a Disrupts Oncogenic Transformation and Warburg Effect by Direct Modulation of Multiple Genes of Tumor-Promoting Pathways

Castration-resistant prostate cancer (CRPC) is defined by tumor microenvironment heterogeneity affecting intrinsic cellular mechanisms including dysregulated androgen signaling, aerobic glycolysis (Warburg effect), and aberrant activation of transcription factors including androgen receptor (AR) and c-Myc. Using in vitro, in vivo, and animal models, we find a direct correlation between miR-644a downregulation and dysregulation of essential cellular processes. MiR-644a downregulated expression of diverse tumor microenvironment drivers including c-Myc, AR coregulators, and antiapoptosis factors Bcl-xl and Bcl2. Moreover, miR-644a modulates epithelial–mesenchymal transition (EMT) by directly targeting EMT-promoting factors ZEB1, cdk6, and Snail. Finally, miR-644a expression suppresses the Warburg effect by direct targeting of c-Myc, Akt, IGF1R, and GAPDH expression. RNA sequencing analysis revealed an analogous downregulation of these factors in animal tumor xenografts. These data demonstrate miR-644a mediated fine-tuning of oncogenesis, stimulating pathways and resultant potentiation of enzalutamide therapy in CRPC patients. Significance: This study demonstrates that miR-644a therapeutically influences the CRPC tumor microenvironment by suppressing androgen signaling and additional genes involved in metabolism, proliferation, Warburg effect, and EMT, to potentiate the enzalutamide therapy