Bone Marrow Mesenchymal Stem Cells (BMSCs) with a Disintegrin and Metalloprotease 17 (ADAM17) Overexpression Increase Cervical Cancer Cell Proliferation and Migration Through Epidermal Growth Factor Receptor (EGFR)/Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase b (AKT) Signaling

ADAM-17 is a membrane-bound protease and highly expressed in multiple tumors. BMSCs carrying target genes are delivered to damaged sites. This study aimed to investigate the mechanism underlying BMSCs with ADAM-17 in cervical cancer (CC). BMSCs were transfected with ADAM-17 mimics and co-cultured with CC cells followed by analysis of cell proliferation and migration by MTT assay and scratch assay, ADAM-17 and target genes (LAMB3, Robol) level by Western blot and RT-qPCR. As the effectiveness of ADAM-17 transfection was confirmed by its increased level, the presence of empty vector rarely affected ADAM-17 expression and biological activities of CC cells compared to control group (p > 0.05). BMSCs with ADAM-17 overexpression increased CC cell proliferation and enhanced scratch healing rate (p < 0.05), accompanied with upregulated LAMB3 and Robol. The difference in LAMB3 and Robol expression between empty vector group and control group did not reach a significance. In conclsuion, this study elucidates that BMSCs with ADAM-17 overexpression promotes CC cell progression through up-regulation of LAMB3 and Robol and activation EGFR/PI3K/Akt signaling, providing a novel BMSC-based targeted therapy.

Time-Resolved Gene Expression Analysis Monitors the Regulation of Inflammatory Mediators and Attenuation of Adaptive Immune Response by Vitamin D

Peripheral blood mononuclear cells (PBMCs) belong to the innate and adaptive immune system and are highly sensitive and responsive to changes in their systemic environment. In this study, we focused on the time course of transcriptional changes in freshly isolated human PBMCs 4, 8, 24 and 48 h after onset of stimulation with the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Taking all four time points together, 662 target genes were identified and segregated either by time of differential gene expression into 179 primary and 483 secondary targets or by driver of expression change into 293 direct and 369 indirect targets. The latter classification revealed that more than 50% of target genes were primarily driven by the cells' response to ex vivo exposure than by the nuclear hormone and largely explained its down-regulatory effect. Functional analysis indicated vitamin D’s role in the suppression of the inflammatory and adaptive immune response by down-regulating ten major histocompatibility complex class II genes, five alarmins of the S100 calcium binding protein A family and by affecting six chemokines of the C-X-C motif ligand family. Taken together, studying time-resolved responses allows to better contextualize the effects of vitamin D on the immune system.

An RNA Interference Tool to Silence Genes in Sarcoptes scabiei Eggs

In a quest for new interventions against scabies—a highly significant skin disease of mammals, caused by a parasitic mite Sarcoptes scabiei—we are focusing on finding new intervention targets. RNA interference (RNAi) could be an efficient functional genomics approach to identify such targets. The RNAi pathway is present in S. scabiei and operational in the female adult mite, but other developmental stages have not been assessed. Identifying potential intervention targets in the egg stage is particularly important because current treatments do not kill this latter stage. Here, we established an RNAi tool to silence single-copy genes in S. scabiei eggs. Using sodium hypochlorite pre-treatment, we succeeded in rendering the eggshell permeable to dsRNA without affecting larval hatching. We optimised the treatment of eggs with gene-specific dsRNAs to three single-copy target genes (designated Ss-Cof, Ss-Ddp, and Ss-Nan) which significantly and repeatedly suppressed transcription by ~66.6%, 74.3%, and 84.1%, respectively. Although no phenotypic alterations were detected in dsRNA-treated eggs for Ss-Cof and Ss-Nan, the silencing of Ss-Ddp resulted in a 38% reduction of larval hatching. This RNAi method is expected to provide a useful tool for larger-scale functional genomic investigations for the identification of essential genes as potential drug targets.

HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30–60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

S ilencing of SCD and SREBP1 Genes by Short Hairpin RNAs in Chicken Hepatic Cells in Vitro

RNA interference by short hairpin RNAs (shRNAs) is a widely used post transcriptional silencing mechanism for suppressing expression of the target gene. In the current study, five shRNA molecules each against SCD and SREBP1 genes involved in denovo lipid biosynthesis were designed upon considering parameters such as secondary structures of shRNAs, mRNA target regions, GC content and thermodynamic properties (ΔG overall, ΔG duplex and ΔG break-target), synthesized and cloned in pENTR/U6 entry vector to knockdown the expression of SCD and SREBP1 genes. After transfection of these shRNA constructs into the chicken embryonic hepatocytes, expressions of the target genes were monitored by real time PCR. Significant reduction (P<0.05) in the expression of SCD and SREBP1 genes was observed in hepatocytes. The shRNAs against SCD gene showed the knock down efficiency ranged from 20.4% (shRNA5) to 74.2% (shRNA2). In case of SREBP1 gene, the shRNAs showed knock-down efficiency ranging from 26.8% (shRNA4) to 95.85% (shRNA1). The shRNAs against both the genes introduced in chicken hepatocyte cells did not show any significant impact on expression of immune response genes (IFNA and IFNB) in those cells. These results clearly demonstrated the successful down regulation of the expression of SCD and SREBP1 genes by the shRNA molecules against both the target genes under in vitro condition. It is concluded that the shRNA molecules against SCD and SREBP1 genes showed great potential to silence the expression of these genes under in vitro chicken embryonic hepatocyte cells.

Identification and Expression Profile of Chemosensory Receptor Genes in Aromia bungii (Faldermann) Antennal Transcriptome

The red-necked longicorn beetle, Aromia bungii (Faldermann) (Coleoptera: Cerambycidae), is a major destructive, wood-boring pest, which is widespread throughout the world. The sex pheromone of A. bungii was reported earlier; however, the chemosensory mechanism of the beetle remains almost unknown. In this study, 45 AbunORs, 6 AbunGRs and 2 AbunIRs were identified among 42,197 unigenes derived from the antennal transcriptome bioinformatic analysis of A. bungii adults. The sequence of putative Orco (AbunOR25) found in this study is highly conserved with the known Orcos from other Coleoptera species, and these Orco genes might be potentially used as target genes for the future development of novel and effective control strategies. Tissue expression analysis showed that 29 AbunOR genes were highly expressed in antennae, especially in the antennae of females, which was consistent with the idea that females might express more pheromone receptors for sensing pheromones, especially the sex pheromones produced by males. AbunOR5, 29, 31 and 37 were clustered with the pheromone receptors of the cerambycid Megacyllene caryae, suggesting that they might be putative pheromone receptors of A. bungii. All six AbunGRs were highly expressed in the mouthparts, indicating that these GRs may be involved in the taste perception process. Both AbunIRs were shown to be female-mouthparts-biased, suggesting that they might also be related to the tasting processes. Our study provides some basic information towards a deeper understanding of the chemosensing mechanism of A. bungii at a molecular level.

Hypoxia-inducible factor 1α induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/β-catenin transcriptional cofactor BCL9

Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/β-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

Genome-wide investigation of microRNAs and expression profiles during rhizome development in ginger (Zingiber officinale Roscoe)

Background MicroRNAs (miRNAs) are endogenous, non-coding small functional RNAs that govern the post-transcriptional regulatory system of gene expression and control the growth and development of plants. Ginger is an herb that is well-known for its flavor and medicinal properties. The genes involved in ginger rhizome development and secondary metabolism have been discovered, but the genome-wide identification of miRNAs and their overall expression profiles and targets during ginger rhizome development are largely unknown. In this study, we used BGISEQ-500 technology to perform genome-wide identification of miRNAs from the leaf, stem, root, flower, and rhizome of ginger during three development stages. Results In total, 104 novel miRNAs and 160 conserved miRNAs in 28 miRNA families were identified. A total of 181 putative target genes for novel miRNAs and 2772 putative target genes for conserved miRNAs were predicted. Transcriptional factors were the most abundant target genes of miRNAs, and 17, 9, 8, 4, 13, 8, 3 conserved miRNAs and 5, 7, 4, 5, 5, 15, 9 novel miRNAs showed significant tissue-specific expression patterns in leaf, stem, root, flower, and rhizome. Additionally, 53 miRNAs were regarded as rhizome development-associated miRNAs, which mostly participate in metabolism, signal transduction, transport, and catabolism, suggesting that these miRNAs and their target genes play important roles in the rhizome development of ginger. Twelve candidate miRNA target genes were selected, and then, their credibility was confirmed using qRT-PCR. As the result of qRT-PCR analysis, the expression of 12 candidate target genes showed an opposite pattern after comparison with their miRNAs. The rhizome development system of ginger was observed to be governed by miR156, miR319, miR171a_2, miR164, and miR529, which modulated the expression of the SPL, MYB, GRF, SCL, and NAC genes, respectively. Conclusion This is a deep genome-wide investigation of miRNA and identification of miRNAs involved in rhizome development in ginger. We identified 52 rhizome-related miRNAs and 392 target genes, and this provides an important basis for understanding the molecular mechanisms of the miRNA target genes that mediate rhizome development in ginger.