HIF-2α mediates a marked increase in migration and stemness characteristics in a subset of glioma cells under hypoxia by activating an Oct-4/Sox-2-Mena (INV) axis.

Hypoxia is a salient feature of most solid tumors and plays a central role in tumor progression owing to its multiple contributions to therapeutic resistance, metastasis, angiogenesis and stemness properties. Reports exist in literature about hypoxia increasing stemness characteristics and invasiveness potential of malignant cells. In order to delineate molecular crosstalk among factors driving glioma progression, we used knockdown and overexpression strategies. We have demonstrated that U87MG and A172 glioma cells inherently have a subset of cells with high migratory potential due to migration-inducing Mena transcripts. These cells also have elevated stemness markers (Sox-2 and Oct-4). There was a significant increase of number in this subset of migratory cells on exposure to hypoxia with corresponding elevation (over 1000 fold) in migration-inducing Mena transcripts. We were able to demonstrate that a HIF-2α-Sox-2/Oct-4-Mena (INV) axis that is strongly activated in hypoxia and markedly increases the migratory potential of the cells. Such cells also formed tumor spheres with greater efficiency. We have correlated our in-vitro results with human glioblastoma samples and found that hypoxia, invasiveness and stemness markers correlated well in native tumor samples. This study identifies a novel signaling mechanism mediated by HIF-2α in regulating invasiveness and stemness characteristics, suggesting that under hypoxic conditions, some tumor cells acquire more migratory potential by increased Pan Mena and Mena INV expression as a consequence of this HIF-2α mediated increase in Oct-4 and Sox-2. These properties would help the cells to form a new nidus after local invasion or metastasis.

Gene Silencing and Activation of Human Papillomavirus 18 Is Modulated by Sense Promoter Associated RNA in Bidirectionally Transcribed Long Control Region.

BACKGROUND: Recently various studies have demonstrated the role of promoter associated non-coding RNAs (pRNA) in dsRNA induced transcriptional gene silencing and activation. However the exact mechanistic details of these processes with respect to the orientation of pRNAs are poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: We have identified novel sense and antisense long control region (LCR) associated RNAs (pRNAs) in HPV18 positive cervical cancer cell lines HeLa, C-4 I and C-4 II. Using dsRNAs against these pRNAs, we were able to achieve upregulation or downregulation of the sense and antisense pRNAs and the downstream E6 and E7 oncogenes. We present evidence that knockdown of the sense pRNA is associated with reduction in E6 and E7 oncogenes and an upregulation of antisense pRNA. Conversely upregulation of sense pRNA is accompanied by an induction of the oncogenes and a concomitant reduction in antisense pRNA. Moreover, the exact role of sense and antisense pRNAs in dsRNA mediated gene modulation was confirmed by their selective degradation using antisense phosphorothioate oligodeoxynucleotides (ODN). Degradation of sense pRNA with antisense ODN led to loss of dsRNA induced silencing and activation, suggesting that dsRNA mediated gene modulation requires sense pRNA. Both processes were accompanied with congruent changes in the methylation pattern of activating and repressive histones. CONCLUSION/SIGNIFICANCE: Thus this data identifies and demonstrates the role of previously unknown important regulatory transcripts in HPV18 gene expression which can prove valuable targets in cervical cancer therapeutics. This mode of gene regulation by bidirectional transcription could be operational in other promoters as well and serve as a mechanism of regulating gene expression.

Long-term suppression of HIV-1C virus production in human peripheral blood mononuclear cells by LTR heterochromatization with a short double-stranded RNA.

OBJECTIVES: A region in the conserved 5' long terminal repeat (LTR) promoter of the integrated HIV-1C provirus was identified for effective targeting by a short double-stranded RNA (dsRNA) to cause heterochromatization leading to a long-lasting decrease in viral transcription, replication and subsequent productive infection in human host cells. METHODS: Small interfering RNAs (siRNAs) were transfected into siHa cells containing integrated LTR-luciferase reporter constructs and screened for efficiency of inducing transcriptional gene silencing (TGS). TGS was assessed by a dual luciferase assay and real-time PCR. Chromatin modification at the targeted region was also studied. The efficacy of potent siRNA was then checked for effectiveness in TZM-bl cells and human peripheral blood mononuclear cells (PBMCs) infected with HIV-1C virus. Viral Gag-p24 antigen levels were determined by ELISA. RESULTS: One HIV-1C LTR-specific siRNA significantly decreased luciferase activity and its mRNA expression with no such effect on HIV-1B LTR. This siRNA-mediated TGS was induced by histone methylation, which leads to heterochromatization of the targeted LTR region. The same siRNA also substantially suppressed viral replication in TZM-bl cells and human PBMCs infected with various HIV-1C clinical isolates for ≥3 weeks after a single transfection, even of a strain that had a mismatch in the target region. CONCLUSIONS: We have identified a potent dsRNA that causes long-term suppression of HIV-1C virus production in vitro and ex vivo by heritable epigenetic modification at the targeted C-LTR region. This dsRNA has promising therapeutic potential in HIV-1C infection, the clade responsible for more than half of AIDS cases worldwide.

Biogenesis of intronic miRNAs located in clusters by independent transcription and alternative splicing.

miRNAs are generally classified as "intergenic" or "intronic" based upon their genomic location. Intergenic miRNAs are known to be transcribed as independent transcription units, while intronic miRNAs are believed to be processed from the introns of their hosting transcription units and hence share common regulatory mechanisms and expression patterns with its host gene. Recent reports in the literature suggest that some intronic miRNAs, which do not show concordance in expression with their respective host genes, might be transcribed and regulated as independent transcription units. However, there is no direct evidence for the existence of independently transcribed intronic miRNA in humans to date. We have characterized the full-length primary transcripts (pri-miRNAs) of three human intronic miRNAs-miR 106b, miR 93, and miR 24-1-by RNA ligase-mediated RACE and show that human intronic miRNA can indeed be transcribed as independent transcription units. Also, clustered miRNAs are generally believed to arise from a common primary transcript and are expected to have similar expression profiles. However, we have identified several novel alternatively spliced transcripts by RT-PCR, each of which harbors a single pre-miRNA from a cluster of closely located intronic miRNAs. We show that these transcripts represent unique pri-miRNAs for each of these clustered miRNAs. We also report the identification of conserved splice acceptor signals which are responsible for maturation of these novel splice variants. Our results suggest that alternative splicing might play a role in uncoupling the expression of clustered miRNAs from each other, which otherwise are generally believed to be co-transcribed and co-expressed.

CpG hypermethylation of the C-myc promoter by dsRNA results in growth suppression.

Deregulation of the c-myc proto-oncogene plays an important role in carcinogenesis. It is, therefore, commonly found to be overexpressed in various types of tumors. Downregulation of c-myc expression assumes great importance in tumor therapy because of its ability to promote and maintain cancer stem cells. Apart from post-transcriptional gene silencing (PTGS), siRNAs have also been shown to cause transcriptional gene silencing (TGS) through epigenetic modifications of a gene locus. This approach can potentially be used to silence genes for longer periods and at a much lesser dosage than PTGS. In this study, we have examined the effect of transfection of a novel siRNA directed against a CpG island encompassing the CT-I(2) region in the P2 promoter of c-myc in U87MG and other cell lines. Transient transfection with this siRNA resulted in c-myc promoter CpG hypermethylation and decreased expression of c-myc (both mRNA and protein) and its downstream targets. A decrease was also observed in the expression of some stemness markers (oct-4 and nanog). Stable transfection also confirmed the promoter CpG hypermethylation and reduced c-myc expression along with reduced cell proliferation and an increase in apoptosis and senescence. A significant decrease in c-myc levels was also observed in three other cancer cell lines after transient transfection under similar conditions. Thus this novel siRNA has the capability of becoming an effective therapeutic tool in malignancies with overexpression of c-myc and may be of particular use in the eradication of recalcitrant cancer stem cells.

Silencing of integrated human papillomavirus-16 oncogenes by small interfering RNA-mediated heterochromatization.

Double-stranded RNAs or small interfering RNAs (siRNA) targeting the promoters of genes are known to cause gene knockdown by a process known as transcriptional gene silencing (TGS). We screened multiple siRNAs homologous to one of the NF-1 binding sites in the human papillomavirus-16 (HPV-16) enhancer and identified one siRNA which causes specific TGS of the HPV-16 oncogenes E6 and E7 when transfected into two HPV-16-positive cell lines siHa and CaSki. This phenomenon was specific to the HPV-16 enhancer with no effect on the HPV-18 enhancer. TGS was associated with heterochromatization of the targeted region of the enhancer but no DNA methylation was noted during the time period studied. The choice of target in the enhancer was important as siRNAs differing by one or two bases showed no suppression of downstream gene expression. A low copy number enhancer-associated transcript was detected in the cell lines studied and its level decreased significantly after treatment with the siRNA that caused TGS. This supports the RNA:RNA model described previously for TGS. This siRNA which causes simultaneous silencing of E6 as well as E7 oncogenes by an epigenetic mechanism might be useful as a therapeutic modality for HPV-16-positive cervical and other epithelial cancers.