Long non-coding RNAs HERH-1 and HERH-4 facilitate cyclin A2 expression and accelerate cell cycle progression in advanced hepatocellular carcinoma

Background The advanced hepatocellular carcinoma (HCC), such as the recurrent tumor after liver transplantation (LT), is an obstacle of HCC treatment. The aim of this study was to discover the underlying mechanism of HCC progression caused by non-coding RNAs (ncRNAs). Methods To this end, we investigated the selected patient cohort of matching primary and recurrent HCC after receiving LT. The recurrent tumors after LT were regarded as clinical models of the advanced HCC. Microarrays were used to profile lncRNA and mRNA expression in HCC recurrent and primary tissue samples. The mRNA profile characteristics were analyzed by bioinformatics. Two cell lines, HepG2 and QGY-7703, were used as HCC cell models. The protein-coding potential, length, and subcellular location of the interested lncRNAs were examined by bioinformatics, Northern blot, fluorescent in situ hybridization (FISH), and quantitative RT-PCR (qRT-PCR) assays. HCC cell proliferation was detected by CCK-8, doubling time and proliferation marker gene quantitation assays. DNA replication during the cell cycle was measured by EdU/PI staining and flow cytometry analyses. Promoter activity was measured using a luciferase reporter assay. Interactions between DNA, RNA, and protein were examined by immunoprecipitation and pull-down assays. The miRNA-target regulation was validated by a fluorescent reporter assay. Results Both lncRNA and mRNA profiles exhibited characteristic alterations in the recurrent tumor cells compared with the primary HCC. The mRNA profile in the HCC recurrent tissues, which served as model of advanced HCC, showed an aberrant cell cycle regulation. Two lncRNAs, the highly expressed lncRNA in recurrent HCC (HERH)-1 and HERH-4, were upregulated in the advanced HCC cells. HERH-1/4 enhanced proliferation and promoted DNA replication and G1-S transition during the cell cycle in HCC cells. HERH-1 interacted with the transcription factor CREB1. CREB1 enhanced cyclin A2 (CCNA2) transcription, depending on HERH-1-CREB1 interaction. HERH-4 acted as an miR-29b/c sponge to facilitate CCNA2 protein translation through a competing endogenous RNA (ceRNA) pathway. Conclusions The oncogenic lncRNA HERH-1/4 promoted CCNA2 expression at the transcriptional and post-transcriptional levels and accelerated cell cycle progression in HCC cells. The HERH-1-CREB1-CCNA2 and HERH-4-miR-29b/c-CCNA2 axes served as molecular stimuli for HCC advance.

Profile of Podocyte Translatome During Development of Type 2 and Type 1 Diabetic Nephropathy Using Podocyte-Specific TRAP mRNA RNA-seq

Podocyte injury is important in development of diabetic nephropathy (DN). Although several studies have reported single cell-based RNA-seq of podocytes in type 1 DN (T1DN), the podocyte translating mRNA profile in type 2 DN (T2DN) <u>has not been previously compared</u> to that of T1DN. <u>We</u> analyzed the podocyte translatome in T2DN in podocin-Cre; Rosa26^fsTRAP; eNOS-/-; <i>db/db </i>mice and compared it to streptozotocin-induced T1DN in podocin-Cre; Rosa26^fsTRAP; eNOS-/- mice utilizing Translating Ribosome Affinity Purification (TRAP) and RNA-seq. Over 125 genes were highly enriched in the podocyte ribosome. More podocyte TRAP genes were differentially expressed in T2DN compared to T1DN. TGF-β signaling pathway genes were upregulated while MAPK pathway genes were downregulated only in T2DN while ATP binding and cAMP-mediated signaling genes were downregulated only in T1DN. Genes regulating actin filament organization and apoptosis increased while genes regulating VEGFR signaling and glomerular basement membrane components decreased in both type 1 and type 2 diabetic podocytes. A number diabetes-induced genes not previously been linked to podocyte injury <u>were confirmed in both</u> <u>mouse and human DN</u>. Differences and similarities in the podocyte translatome in T2DN and T1DN can identify factors underlying the pathophysiology of DN and novel therapeutic targets to treat diabetes-induced podocyte injury.

Profile of Podocyte Translatome During Development of Type 2 and Type 1 Diabetic Nephropathy Using Podocyte-Specific TRAP mRNA RNA-seq

Podocyte injury is important in development of diabetic nephropathy (DN). Although several studies have reported single cell-based RNA-seq of podocytes in type 1 DN (T1DN), the podocyte translating mRNA profile in type 2 DN (T2DN) <u>has not been previously compared</u> to that of T1DN. <u>We</u> analyzed the podocyte translatome in T2DN in podocin-Cre; Rosa26^fsTRAP; eNOS-/-; <i>db/db </i>mice and compared it to streptozotocin-induced T1DN in podocin-Cre; Rosa26^fsTRAP; eNOS-/- mice utilizing Translating Ribosome Affinity Purification (TRAP) and RNA-seq. Over 125 genes were highly enriched in the podocyte ribosome. More podocyte TRAP genes were differentially expressed in T2DN compared to T1DN. TGF-β signaling pathway genes were upregulated while MAPK pathway genes were downregulated only in T2DN while ATP binding and cAMP-mediated signaling genes were downregulated only in T1DN. Genes regulating actin filament organization and apoptosis increased while genes regulating VEGFR signaling and glomerular basement membrane components decreased in both type 1 and type 2 diabetic podocytes. A number diabetes-induced genes not previously been linked to podocyte injury <u>were confirmed in both</u> <u>mouse and human DN</u>. Differences and similarities in the podocyte translatome in T2DN and T1DN can identify factors underlying the pathophysiology of DN and novel therapeutic targets to treat diabetes-induced podocyte injury.

Integrative Analysis of lncRNA-mRNA Profile Reveals Potential Predictors for SAPHO Syndrome

Synovitis, acne, pustulosis, hyperostosis, and osteitis (SAPHO) syndrome is known as a rare disease characterized by inflammatory lesions on bones and skin. Polymorphism of clinical manifestation and lack of molecular biomarkers have both limited its diagnosis. Our study performed RNA sequencing (RNA-seq) and integrative bioinformatics analysis of long noncoding RNA (lncRNA)-messenger RNA (mRNA) profile in patients with SAPHO syndrome and healthy controls. A total of 4,419 differentially expressed (DE) mRNAs and 2,713 lncRNAs were identified (p &lt; 0.05, fold change &gt; 2) and a coexpression network was constructed to further investigate their regulatory interactions. The DE lncRNAs were predicted to interact with mRNAs in both cis and trans manners. Functional prediction found that the lncRNA-targeted genes may function in SAPHO syndrome by participating in biological process such as adipocytokine signaling pathway, ErbB signaling pathway, FoxO signaling pathway, as well as production and function of miRNAs. The expression levels of three pairs of coexpressed lncRNA-mRNAs were validated by qRT-PCR, and their relative expression levels were consistent with the RNA-seq data. The deregulated RNAs GAS7 and lnc-CLLU1.1-1:2 may serve as potential diagnostic biomarkers, and the combined receiver operating characteristic (ROC) curve of the two showed more reliable diagnostic ability with an AUC value of 0.871 in distinguishing SAPHO patients from healthy controls. In conclusion, this study provides a first insight into long noncoding RNA transcriptome profile changes associated with SAPHO syndrome and inspiration for further investigation on clinical biomarkers and molecular regulators of this inadequately understood clinical entity.

Significance of the Wnt canonical pathway in radiotoxicity via oxidative stress of electron beam radiation and its molecular control in mice

Radiation triggers the cell death events through signaling proteins, but the combined mechanism of events is unexplored. We intend to investigate how the combined cascade works throughout the radiation process and its significance over radiotoxicity. Thirty adult mice were irradiated with electron beam radiation, and five were served as a control (Non treated). Mice were sacrificed after post 24 hours and 30 days of irradiation. We assessed the oxidative stress parameter and mRNA profile (Liver, Kidney, spleen, and germ cells), sperm viability, and motility. The mRNA profile study established the network of combined cascade work during radiation to combat oxidative response and cell survival. The quantitative examination of mRNA uncovers unique critical changes in all mRNA levels in all the cases, particularly in germ cells. Recuperation was likewise seen in post 30 days radiation in the liver, spleen, and kidney followed by oxidative stress parameters, however not in germ cells. It proposes reproductive physiology is exceptionally sensitive towards radiation, even at the molecular level. It also suggests the suppression of Lef1/axin2 could be the main reason for permanent failure in the sperm function process. Post irradiation likewise influences the morphology of sperms. The decrease in mRNA level of Lef1, Axin2, survivin, Ku70 suggests radiation inhibits the Wnt canonical pathway and failure in DNA repair mechanism in a coupled manner. Likewise, the increase in BAX and BCL2 suggests apoptosis activation followed by the decreased expression of enzymatic antioxidants. In summary, controlled several interlinked cascades execute when body exposure to radiation may further be used in a study to counterpart and better comprehend medication focus on radiation treatment.

Identification, Characterization, and Targeting of a Rare and Temporal Dendritic Cell State that Facilitates Adaptive Immune Responses

SummaryThe heterogeneity of innate immune cells facilitates efficient antigen presentation and immune activation in the presence of pathogens via cooperativity of various cell subsets and cell states but also obscures the contribution of individual antigen presenting cells (APCs) to overall immune response.1 It has been hypothesized that a small number of APCs, which are more sensitive to the initial pathogen stimulus, are responsible for coordinating neighboring APCs in an effort to share the metabolic strain associated with heightened pathogen sensitivity.2 In this study, we have identified a temporally-controlled state of dendritic cells (DCs) that demonstrate greater sensitivity to toll-like-receptor (TLR) agonists and secrete the majority of paracrine activating cytokines (TNFα, IL-6…ect). We were able to isolate this distinct population of DCs preferentially phagocytosed the majority of fluorescently labeled, TLR agonist conjugated microparticles (MPs).3 We call this population First Responder cells (FRs) due to their ability to first uptake the MPs and activate neighboring APCs via paracrine signaling. We show that FRs exist in this state for <3 hours, cycle through this state on a <24-hour timescale and show a distinct mRNA profile. Furthermore, FRs are necessary for generation of adaptive responses both in vitro and in vivo. We also show that we can improve both IgG titers and CD8 responses in vivo by targeting two highly upregulated receptors on FR cells, DAP12 and PRG2. Given the significance of FR involvement in APC activation, this study has broad immunological value because it offers a critical first evaluation of a new APC cell state but also has important translational value for improving vaccine efficacy via FR targeting.