miR-429 regulates the transition between Hypoxia-Inducible Factor (HIF)1A and HIF3A expression in human endothelial cells.

Hypoxia-inducible factors (HIF) are heterodimeric transcription factors that allow cells to adapt and survive during hypoxia. Regulation of HIF1A and HIF2A mRNA is well characterized, whereas HIF3A mRNA regulation and function are less clear. Using RNA-Seq analysis of primary human umbilical vein endothelial cells, we found two isoforms of HIF3A were expressed, HIF3A2 and HIF3A3. Comparing HIF3A expression profiles to HIF1A mRNA during 48 hours of hypoxia revealed that HIF1A message peaked at 4 hours, whereas HIF3A expression increased while HIF1A was decreasing. Given that HIF1A mRNA is regulated by miR-429, we tested miR-429 effects on both HIF3A isoforms and found that they too were regulated by miR-429. Analysis of a HIF-3 target, DNA-damage-inducible transcript 4, a key survival gene, indicated that DDIT4 mRNA is induced by HIF-3 and negatively regulated by miR-429 through miR-429's actions on HIF3A message. This provides a compelling model for how hypoxia-induced miR-429 regulates the switch between HIF-1 adaptive responses to HIF-3 survival responses by rapidly decreasing HIF1A levels while simultaneously slowing the progression of HIF3A expression until the miR-429 levels drop below normoxic levels. Since HIF-1 drives HIF3A and miR-429 expression, this establishes a regulatory network in which miR-429 plays a pivotal role.

Pennogenyl Saponins from Paris quadrifolia L. Induce Extrinsic and Intrinsic Pathway of Apoptosis in Human Cervical Cancer HeLa Cells.

Pennogenyl saponins are the active compounds of large number of plant species and consequently many polyherbal formulations. Hence, great interest has been shown in their characterization and in the investigation of their pharmacological and biological properties, especially anticancer. This present study reports on the evaluation of cytotoxic effects and explanation of the molecular mechanisms of action of the two pennogenyl saponins (PS 1 and PS 2) isolated from Paris quadrifolia L. rhizomes on human cervical adenocarcinoma cell line HeLa. To determine the viability of the cells treated with the compounds we used real-time cell proliferation analysis and found that the pennogenyl saponins PS 1 and PS 2 strongly inhibited the tumor cells growth with IC50 values of 1.11 ± 0.04 µg/ml and 0.87 ± 0.05 µg/ml, respectively. The flow cytometry analysis indicated that the two compounds induced apoptosis in a dose-dependent manner and decreased mitochondrial membrane potential in HeLa cells in the early stage of apoptosis. Quantitative PCR and Western Blot analysis showed that the two saponins significantly increased mRNA expression of FADD and BID as well as induced caspase-8 via increased of procaspase-8 processing in the treated cells. The results of this study suggest that both the extrinsic death receptor and intrinsic mitochondrial pathways are involved in the programmed cell death.

Concurrent DNA Copy-Number Alterations and Mutations in Genes Related to Maintenance of Genome Stability in Uninvolved Mammary Glandular Tissue from Breast Cancer Patients.

Somatic mosaicism for DNA copy-number alterations (SMC-CNAs) is defined as gain or loss of chromosomal segments in somatic cells within a single organism. As cells harboring SMC-CNAs can undergo clonal expansion, it has been proposed that SMC-CNAs may contribute to the predisposition of these cells to genetic disease including cancer. Herein, the gross genomic alterations (>500 kbp) were characterized in uninvolved mammary glandular tissue from 59 breast cancer patients and matched samples of primary tumors and lymph node metastases. Array-based comparative genomic hybridization showed 10% (6/59) of patients harbored one to 359 large SMC-CNAs (mean: 1,328 kbp; median: 961 kbp) in a substantial portion of glandular tissue cells, distal from the primary tumor site. SMC-CNAs were partially recurrent in tumors, albeit with considerable contribution of stochastic SMC-CNAs indicating genomic destabilization. Targeted resequencing of 301 known predisposition and somatic driver loci revealed mutations and rare variants in genes related to maintenance of genomic integrity: BRCA1 (p.Gln1756Profs*74, p.Arg504Cys), BRCA2 (p.Asn3124Ile), NCOR1 (p.Pro1570Glnfs*45), PALB2 (p.Ser500Pro), and TP53 (p.Arg306*). Co-occurrence of gross SMC-CNAs along with point mutations or rare variants in genes responsible for safeguarding genomic integrity highlights the temporal and spatial neoplastic potential of uninvolved glandular tissue in breast cancer patients.

The hypoxia-inducible miR-429 regulates hypoxia-inducible factor-1α expression in human endothelial cells through a negative feedback loop.

Hypoxia-inducible factors (HIFs) 1 and 2 are dimeric α/ß transcription factors that regulate cellular responses to low oxygen. HIF-1 is induced first, whereas HIF-2 is associated with chronic hypoxia. To determine how HIF1A mRNA, the inducible subunit of HIF-1, is regulated during hypoxia, we followed HIF1A mRNA levels in primary HUVECs over 24 hours using quantitative PCR. HIF1A and VEGF A (VEGFA) mRNA, a transcriptional target of HIF-1, increased ∼ 2.5- and 8-fold at 2-4 hours, respectively. To determine how the mRNAs were regulated, we identified a microRNA (miRNA), miR-429, that destabilized HIF1A message and decreased VEGFA mRNA by inhibiting HIF1A. Target protector analysis, which interferes with miRNA-mRNA complex formation, confirmed that miR-429 targeted HIF1A message. Desferoxamine treatment, which inhibits the hydroxylases that promote HIF-1α protein degradation, stabilized HIF-1 activity during normoxic conditions and elevated miR-429 levels, demonstrating that HIF-1 promotes miR-429 expression. RNA-sequencing-based transcriptome analysis indicated that inhibition of miRNA-429 in HUVECs up-regulated 209 mRNAs, a number of which regulate angiogenesis. The results demonstrate that HIF-1 is in a negative regulatory loop with miR-429, that miR-429 attenuates HIF-1 activity by decreasing HIF1A message during the early stages of hypoxia before HIF-2 is activated, and this regulatory network helps explain the HIF-1 transition to HIF-2 during chronic hypoxia in endothelial cells.

Mangiferin has an additive effect on the apoptotic properties of hesperidin in Cyclopia sp. tea extracts.

A variety of biological pro-health activities have been reported for mangiferin and hesperidin, two major phenolic compounds of Honeybush (Cyclopia sp.) tea extracts. Given their increasing popularity, there is a need for understanding the mechanisms underlying the biological effects of these compounds. In this study, we used real-time cytotoxicity cellular analysis of the Cyclopia sp. extracts on HeLa cells and found that the higher hesperidin content in non-fermented "green" extracts correlated with their higher cytotoxicity compared to the fermented extracts. We also found that mangiferin had a modulatory effect on the apoptotic effects of hesperidin. Quantitative PCR analysis of hesperidin-induced changes in apoptotic gene expression profile indicated that two death receptor pathway members, TRADD and TRAMP, were up regulated. The results of this study suggest that hesperidin mediates apoptosis in HeLa cells through extrinsic pathway for programmed cell death.

Regulation of the unfolded protein response by microRNAs.

The unfolded protein response (UPR) is an adaptive response to the stress that is caused by an accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). It is an important component of cellular homeostasis. During ER stress, the UPR increases the protein-folding capacity of the endoplasmic reticulum to relieve the stress. Failure to recover leads to apoptosis. Specific cellular mechanisms are required for the cellular recovery phase after UPR activation. Using bioinformatics tools, we identified a number of microRNAs that are predicted to decrease the mRNA expression levels for a number of critical components of the UPR. In this review, we discuss the potential role of microRNAs as key regulators of this pathway and describe how microRNAs may play an essential role in turning off the UPR after the stress has subsided.