Methylation of KSHV vCyclin by PRMT5 contributes to cell cycle progression and cell proliferation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus that encodes numerous cellular homologs, including cyclin D, G protein-coupled protein, interleukin-6, and macrophage inflammatory proteins 1 and 2. KSHV vCyclin encoded by ORF72, is the homolog of cellular cyclinD2. KSHV vCyclin can regulate virus replication and cell proliferation by constitutively activating cellular cyclin-dependent kinase 6 (CDK6). However, the regulatory mechanism of KSHV vCyclin has not been fully elucidated. In the present study, we identified a host protein named protein arginine methyltransferase 5 (PRMT5) that interacts with KSHV vCyclin. We further demonstrated that PRMT5 is upregulated by latency-associated nuclear antigen (LANA) through transcriptional activation. Remarkably, knockdown or pharmaceutical inhibition (using EPZ015666) of PRMT5 inhibited the cell cycle progression and cell proliferation of KSHV latently infected tumor cells. Mechanistically, PRMT5 methylates vCyclin symmetrically at arginine 128 and stabilizes vCyclin in a methyltransferase activity-dependent manner. We also show that the methylation of vCyclin by PRMT5 positively regulates the phosphorylate retinoblastoma protein (pRB) pathway. Taken together, our findings reveal an important regulatory effect of PRMT5 on vCyclin that facilitates cell cycle progression and proliferation, which provides a potential therapeutic target for KSHV-associated malignancies.

Role and Regulatory Mechanism of circRNA_14820 in the Proliferation and Differentiation of Goat Skeletal Muscle Satellite Cells.

Skeletal muscle satellite cells (SMSCs), a type of myogenic stem cell, play a pivotal role in postnatal muscle regeneration and repair in animals. Circular RNAs (circRNAs) are a distinct class of non-coding RNA molecules capable of regulating muscle development by modulating gene expression, acting as microRNAs, or serving as protein decoys. In this study, we identified circ_14820, an exonic transcript derived from adenosine triphosphatase family protein 2 (ATAD2), through initial RNA-Seq analysis. Importantly, overexpression of circ_14820 markedly enhanced the proliferation of goat SMSCs while concomitantly suppressing their differentiation. Moreover, circ_14820 exhibited predominant localization in the cytoplasm of SMSCs. Subsequent small RNA and mRNA sequencing of circ_14820-overexpressing SMSCs systematically elucidated the molecular regulatory mechanisms associated with circ_14820. Our preliminary findings suggest that the circ_14820-miR-206-CCND2 regulatory axis may govern the development of goat SMSCs. These discoveries contribute to a deeper understanding of circRNA-mediated mechanisms in regulating skeletal muscle development, thereby advancing our knowledge of muscle biology.

Doxycycline-Mediated Control of Cyclin D2 Overexpression in Human-Induced Pluripotent Stem Cells.

Previous studies have demonstrated that when the cyclin D2 (CCND2), a cell-cycle regulatory protein, is overexpressed in human-induced pluripotent stem cells (hiPSCs), cardiomyocytes (CMs) differentiated from these CCND2-overexpressing hiPSCs can proliferate after transplantation into infarcted hearts, which significantly improves the cells' potency for myocardial regeneration. However, persistent CM proliferation could lead to tumor growth or the development of arrhythmogenic complications; thus, the goal of the current study was to generate a line of hiPSCs in which CCND2 overexpression could be tightly controlled. First, we transfected hiPSCs with vectors coding for a doxycycline-inducible Tet-On transactivator and S. pyogenes dCas9 fused to the VPR activation domain; then, the same hiPSCs were engineered to express guide RNAs targeting the CCND2 promotor. Thus, treatment with doxycycline (dox) activated dCas9-VPR expression, and the guide RNAs directed dCas9-VPR to the CCND2 promoter, which activated CCND2 expression. Subsequent experiments confirmed that CCND2 expression was dox-dependent in this newly engineered line of hiPSCs (doxCCND2-hiPSCs): CCND2 protein was abundantly expressed after 48 h of treatment with dox and declined to near baseline level ~96 h after dox treatment was discontinued.

ErbB3 is required for hyperaminoacidemia-induced pancreatic α cell hyperplasia.

Blood amino acid levels are maintained in a narrow physiological range. The pancreatic α cells have emerged as the primary aminoacidemia regulator through glucagon secretion to promote hepatic amino acid catabolism. Interruption of glucagon signaling disrupts the liver-α cells axis leading to hyperaminoacidemia, which triggers a compensatory rise in glucagon secretion and α cell hyperplasia. The mechanisms of hyperaminoacidemia-induced α cell hyperplasia remain incompletely understood. Using a mouse α cell line and in vivo studies in zebrafish and mice, we found that hyperaminoacidemia-induced α cell hyperplasia requires ErbB3 signaling. In addition to mechanistic target of rapamycin complex 1, another ErbB3 downstream effector signal transducer and activator of transcription 3 also plays a role in α cell hyperplasia. Mechanistically, ErbB3 may partner with ErbB2 to stimulate cyclin D2 and suppress p27 via mechanistic target of rapamycin complex 1 and signal transducer and activator of transcription 3. Our study identifies ErbB3 as a new regulator for hyperaminoacidemia-induced α cell proliferation and a critical component of the liver-α cells axis that regulates aminoacidemia.

Trans-Activation of the Coactivator-Associated Arginine Methyltransferase 1 (Carm1) Gene by the Oncogene Product Tax of Human T-Cell Leukemia Virus Type 1.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.

[Bioinformatic analysis of CCND2 expression in papillary thyroid carcinoma and its impact on immune infiltration].

OBJECTIVE: To investigate cyclin D2 (CCND2) expression in papillary thyroid carcinoma (PTC) and its association with the clinicopathological features. METHODS: The public databases TCGA, TIMER 2.0 and UALCAN were used to explore CCND2 expression level in PTC and adjacent tissues, and its diagnostic value for PTC was analyzed using ROC curves. GO enrichment analysis of CCND2-related differentially expressed genes (DEGs) in PTC was performed, and tumor immune infiltration of CCND2 in thyroid cancer was analyzed using TIMER database and CIBERSORT data source. RT-qPCR and Western blot were used to detect CCND2 expression in normal human thyroid cell line Nthy-ori-3-1 and human PTC cell lines TPC-1 and BCPAP. CCND2 expression was also detected in clinical specimens of PTC and adjacent tissues by immunohistochemistry, and its correlation with clinicopathological features of the patients were analyzed. RESULTS: Informatic analysis revealed significantly higher CCND2 mRNA expression in thyroid cancer than in the adjacent tissues (P < 0.001) in close correlation with tumor stage, gender, age, pathological subtype, and lymph node involvement (P < 0.05). ROC curve analysis showed that at the cutoff value of 4.983, the diagnostic sensitivity, specificity, and accuracy of CCND2 expression for PTC was 83.6%, 94.9%, and 78.5%, respectively. CCND2 expression was positively correlated with B cells, CD4+ T cells, and macrophages (P < 0.001) and negatively with CD8+ T cells (P < 0.01), and also correlated with memory B-cell infiltration, CD4+ T-cell memory activation, M2 macrophages, resting mast cells, and mast cell activation (P < 0.05). RT-qPCR, Western blot and immunohistochemistry showed significantly higher CCND2 expression in the PTC cells than in Nthy-ori-3-1 cells (P < 0.01) and also in clinical PTC tissues than in the adjacent tissues (P < 0.05) in correlation with tumor size, lymph node metastasis and TNM stage (P < 0.05). CONCLUSION: CCND2 overexpression is closely correlated with tumor progression and immune cell infiltration in PTC patients..

Circ_0002331 Interacts with ELAVL1 to Improve ox-LDL-Induced Vascular Endothelial Cell Dysfunction via Regulating CCND2 mRNA Stability.

Circular RNAs (circRNAs) have been discovered to serve as vital regulators in atherosclerosis (AS). However, the role and mechanism of circ_0002331 in AS process are still unclear. Human umbilical vein endothelial cells (HUVECs) were treated with ox-LDL to establish an in vitro model for AS. The expression levels of circ_0002331, Cyclin D2 (CCND2) and ELAVL1 were analyzed by quantitative real-time PCR. Cell proliferation, apoptosis, migration, invasion and angiogenesis were assessed by EdU assay, flow cytometry, transwell assay and tube formation assay. The protein levels of CCND2, ELAVL1, and autophagy-related markers were detected using western blot analysis. IL-8 level was analyzed by ELISA. The relationship between ELAVL1 and circ_0002331 or CCND2 was analyzed by RIP assay and RNA pull-down assay. Moreover, FISH assay was used to analyze the co-localization of ELAVL1 and CCND2 in HUVECs. Our data showed that circ_0002331 was obviously downregulated in AS patients and ox-LDL-induced HUVECs. Overexpression of circ_0002331 could promote proliferation, migration, invasion and angiogenesis, while inhibit apoptosis, autophagy and inflammation in ox-LDL-induced HUVECs. Furthermore, CCND2 was positively regulated by circ_0002331, and circ_0002331 could bind with ELAVL1 to promote CCND2 mRNA stability. Besides, CCND2 overexpression suppressed ox-LDL-induced HUVECs dysfunction, and its knockdown also reversed the regulation of circ_0002331 on ox-LDL-induced HUVECs dysfunction. In conclusion, circ_0002331 might be a potential target for AS treatment, which could improve ox-LDL-induced dysfunction of HUVECs via regulating CCND2 by binding with ELAVL1.

Up-regulation of lncRNA WT1-AS ameliorates Aß-stimulated neuronal injury through modulation of miR-186-5p/CCND2 axis in Alzheimer's disease.

As a common neurodegenerative disorder, Alzheimer's disease (AD) seriously threatens human life. Long non-coding RNAs (lncRNAs) exhibit essential functions in AD development. Nevertheless, the detailed effects and possible mechanisms of lncRNA Wilms tumor 1 Antisense RNA (WT1-AS) in AD are largely unknown. In our studies, a total of 30 serum samples from AD patients were collected, and WT1-AS expressions were detected through qRT-PCR analysis. Additionally, an in vitro AD model was constructed by treating Aß1-42 in human neuroblastoma cells. Functional assays were implemented to assess the impacts of WT1-AS on Aß1-42-stimulated human neuroblastoma cell proliferation together with apoptosis. Moreover, relationship of WT1-AS, microRNA (miR)-186-5p as well as cyclin D2 (CCND2) could be predicted through bioinformatics tools as well as proved via dual-luciferase reporter experiments. Our results showed that WT1-AS together with CCND2 were low-expressed, while miR-186-5p presented high expression in AD serum samples together with Aß1-42-stimulated human neuroblastoma cells. WT1-AS over-expression or miR-186-5p depletion notably promoted the proliferation, reduced the apoptosis, and decreased the p-Tau protein expressions of human neuroblastoma cells induced with Aß1-42. Moreover, miR-186-5p combined with WT1-AS, and CCND2 was modulated by miR-186-5p. Furthermore, CCND2 elevation partially offsets the impacts of miR-186-5p elevation on Aß1-42-stimulated cell proliferation as well as apoptosis mediated with WT1-AS up-regulation. Our results indicated that up-regulation of lncRNA WT1-AS ameliorated Aß-stimulated neuronal damage through modulating miR-186-5p/CCND2 axis, offering a novel direction for AD therapy.

GABA regulates the proliferation and apoptosis of head and neck squamous cell carcinoma cells by promoting the expression of CCND2 and BCL2L1.

Gamma-aminobutyric acid (GABA) is a multifunctional molecule that is widely present in the nervous system and nonneuronal tissues. It plays pivotal roles in neurotransmission, regulation of secretion, cell differentiation, proliferation, and tumorigenesis. However, the exact mechanisms of GABA in head and neck squamous cell carcinomas (HNSCCs) are unknown. We took advantage of RNA sequencing in this work and uncovered the potential gene expression profiles of the GABA-treated HNSCC cell line HN4-2. We found that the expression of CCND2 and BCL2L1 was significantly upregulated. Furthermore, GABA treatment inhibited the cell apoptosis induced by cisplatin and regulated the cell cycle after treatment with cisplatin in HN4-2 cells. Moreover, we also found that GABA could upregulate the expression of CCND2 and BCL2L1 after treatment with cisplatin. Our results not only reveal the potential pro-tumorigenic effect of GABA on HNSCCs but also provide a novel therapeutic target for HNSCC treatment.

MiR-188-5p inhibits cell proliferation and migration in ovarian cancer via competing for CCND2 with ELAVL1.

MicroRNAs (miRNAs) were reportedly demonstrated to participate in ovarian cancer (OC) progression. Here, we inquired into the role of miR-188-5punderneath OC cell proliferation and migration. In this respect, our work examined the miR-188-5p expression and demonstrated its expression level in OC by qRT-PCR analysis. Enforced miR-188-5p expression resulted in a serious downfall of cell growth and mobility, and acceleration apoptosis in OC cells. Furthermore, we identified CCND2 as a target gene of miR-188-5p. RIP assay and luciferase reporter assay verified the interaction of miR-188-5p and CCND2 and exhibited that miR-188-5p greatly hindered the expression of CCND2. Besides, HuR stabilized CCND2 mRNA and counteracted the miR-188-5p suppressive effect on CCND2 mRNA. Functionally, rescue experiments also showed that miR-188-5p-mediated suppression on OC cell proliferation and migration was reverted by CCND2 or HuR overexpression. Our study found miR-188-5p was a tumor suppressor in OC via competing for CCND2 with ELAVL1, contributing to coming up with novel clues for OC therapies.

Paternal sleep deprivation induces metabolic perturbations in male offspring via altered LRP5 DNA methylation of pancreatic islets.

Diabetes and metabolic perturbation are global health challenges. Sleep insufficiency may trigger metabolic dysregulation leading to diabetes. However, the intergenerational transmission of this environmental information is not clearly understood. The research objective was to determine the possible effect of paternal sleep deprivation on the metabolic phenotype of the offspring and to investigate the underlying mechanism of epigenetic inheritance. Male offspring of sleep-deprived fathers exhibit glucose intolerance, insulin resistance, and impaired insulin secretion. In these SD-F1 offspring, a reduction in beta cell mass and proliferation of beta cells were observed. Mechanistically, in pancreatic islets of SD-F1 offspring, we identified alterations in DNA methylation at the promoter region of the LRP5 (LDL receptor related protein 5) gene, a coreceptor of Wnt signaling, resulting in downregulation of downstream effectors cyclin D1, cyclin D2, and Ctnnb1. Restoration of Lrp5 in the pancreas of SD-F1 male mice could improve impaired glucose tolerance and expression of cyclin D1, cyclin D2, and Ctnnb1. This study might significantly contribute to our understanding of the effects of sleeplessness on health and metabolic disease risk from the perspective of the heritable epigenome.

Ndufa4 Regulates the Proliferation and Apoptosis of Neurons via miR-145a-5p/Homer1/Ccnd2.

The Dandy-Walker malformation (DWM) is characterized by neuron dysregulation in embryonic development; however, the regulatory mechanisms associated with it are unclear. This study aimed to investigate the role of NADH dehydrogenase 1 alpha subcomplex 4 (NDUFA4) in regulating downstream signaling cascades and neuronal proliferation and apoptosis. Ndufa4 overexpression promoted the proliferation of neurons and inhibited their apoptosis in vitro, which underwent reverse regulation by the Ndufa4 short hairpin RNAs. Ndufa4-knockout (KO) mice showed abnormal histological alterations in the brain tissue, in addition to impaired spatial learning capacity and exploratory activity. Ndufa4 depletion altered the microRNA expressional profiles of the cerebellum: Ndufa4 inhibited miR-145a-5p expression both in the cerebellum and neurons. miR-145a-5p inhibited the proliferation of neurons and promoted their apoptosis. Ndufa4 promoted and miR-145a-5p inhibited the expression of human homer protein homolog 1 and cyclin D2 in neurons. Thus, Ndufa4 promotes the proliferation of neurons and inhibits their apoptosis by inhibiting miR-145a-5p, which directly targets and inhibits the untranslated regions of Homer1 and Ccnd2 expression.

Cigarette smoke condensate induces centrosome clustering in normal lung epithelial cells.

BACKGROUND: Unlike normal cells, cancer cells frequently have multiple centrosomes that can cluster to form bipolar mitotic spindles and allow for successful cell division. Inhibiting centrosome clustering, therefore, holds therapeutic promise to promote cancer cell-specific cell death. METHODS: We used confocal microscopy, real-time PCR, siRNA knockdown, and western blot to analyze centrosome clustering and declustering using normal lung bronchial epithelial and nonsmall-cell lung cancer (NSCLC) cell lines. Also, we used Ingenuity Pathway Analysis software to identify novel pathways associated with centrosome clustering. RESULTS: In this study, we found that exposure to cigarette smoke condensate induces centrosome amplification and clustering in human lung epithelial cells. We observed a similar increase in centrosome amplification and clustering in unexposed NSCLC cell lines which may suggest a common underlying mechanism for lung carcinogenesis. We identified a cyclin D2-mediated centrosome clustering pathway that involves a sonic hedgehog-forkhead box protein M1 axis which is critical for mitosis. We also observed that cyclin D2 knockdown induced multipolar mitotic spindles that could eventually lead to cell death. CONCLUSIONS: Here we report a novel role of cyclin D2 in the regulation of centrosome clustering, which could allow the identification of tumors sensitive to cyclin D2 inhibitors. Our data reveal a pathway that can be targeted to inhibit centrosome clustering by interfering with the expression of cyclin D2-associated genes.

FBXL6 is dysregulated in keloids and promotes keloid fibroblast growth by inducing c-Myc expression.

C-MYC-mediated keloid fibroblasts proliferation and collagen deposit may contribute to the development of keloids. F-box and leucine-rich repeat protein 6 (FBXL6) is reported to be involved in tumour progression, while the role of FBXL6 in keloid fibroblasts is not deciphered. Normal control skins, hypertrophic scars and keloid tissues were collected and prepared for FBXL6 detection. FBXL6 short hairpin RNAs (shRNAs) or FBXL6 over-expression plasmids were transfected into keloid fibroblasts, and then c-MYC plasmids were further transfected. Cell viability was assayed with a Cell-Counting Kit-8 kit. The relative expression of FBXL6, Cyclin A1, Cyclin D2, Cyclin E1 and Collagen I was detected with real-time PCR and Western blot. Elevated FBXL6 expression could be observed in keloid tissues and hypertrophic scars. FBXL6 shRNAs transfection could inhibit the viability of keloid fibroblasts with diminished c-MYC expression and down-regulated Cyclin A1, Cyclin D2, Cyclin E1 and Collagen I expression. At the same time, overexpressed FBXL6 could promote the proliferation of keloid fibroblasts. Overexpression of c-MYC could promote the proliferation of keloid fibroblasts reduced by FBXL6 shRNAs with up-regulated Cyclin A1 and Collagen I expression. FBXL6 could promote the growth of keloid fibroblasts by inducing c-MYC expression, which could be targeted in keloids treatment.

[LncRNA RPL22P1-201 affects prostate cancer cell proliferation, cell cycle, and sensitivity to docetaxel by regulating miR-216b-5p expression].

OBJECTIVE: Exploring the effects and mechanisms of long non coding RNA (lncRNA) RPL22P1-201 on prostate cancer cell proliferation, cell cycle, and docetaxel sensitivity by regulating miR-216b-5p expression. METHODS: The Cancer LncRNA Census database was used to analyze the differential expression of RPL22P1-201 between prostate cancer tissue and normal tissue. Real time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression level of RPL22P1-201 in prostate cancer cell lines (DU-145, C4-2B, PC3, 22Rv1, LNCaP) and normal prostate epithelial cells (RWPE-1). PC3 cells were divided into si-RPL22P1-201 group (transfected with RPL22P1-201 interference sequence) and si-NC group (transfected with si-NC sequence). Colony formation assay was used to detect the proliferation ability of PC3 cells. Flow cytometry was used to detect the PC3 cell cycle. The CCK-8 method was used to detect the proliferation of PC3 cells in each group after treatment with docetaxel. The dual luciferase reporter gene experiment verifies the binding of RPL22P1-201 to the target gene. qRT-PCR was used to detect the expression level of miR-216b-5p. Western blot was used to detect the expression levels of TrkB, CDK4, cyclin D2, cyclin D3, and CDK6 proteins. RESULTS: The expression level of RPL22P1-201 in prostate cancer tissue was higher than that in normal tissue (P<0.01). The expression level of RPL22P1-201 in prostate cancer cell lines was higher than that in normal prostate epithelial cells (P<0.01). The number of colonies in the si-NC group and si-RPL22P1-201 group was (256.1 ± 28.79) and (78.77 ± 14.52), respectively. The difference was statistically significant (P<0.01). The G0/G1 cell rates in the si-NC group and si-RPL22P1-201 group were (43.18 ± 4.56)% and (68.85 ± 3.40)%, respectively. The S cell rates were (36.84 ± 2.28)% and (24.27 ± 2.74)%, respectively. The G2/M cell rates were (19.98 ± 2.69)% and (6.88 ± 1.57)%, respectively, and the differences were statistically significant (all P<0.05). The cell survival rate of the si-RPL22P1-201 group under the action of docetaxel was lower than that of the si-NC group (all P<0.05). RPL22P1-201 can pair and bind with miR-216b-5p (P<0.01). Compared with the si-NC group, the si-RPL22P1-201 group showed a decrease in miR-216b-5p expression in PC3 cells (P<0.01), and a decrease in TrkB, CDK4, cyclin D2, cyclin D3, and CDK6 protein expression. CONCLUSIONS: RPL22P1-201 is highly expressed in prostate cancer, and silencing RPL22P1-201 inhibits prostate cancer PC3 cell proliferation and cell cycle by increasing miR-216b-5p expression, and enhances PC3 cell sensitivity to docetaxel.

SRSF1 promotes ASMC proliferation in asthma by competitively binding CCND2 with miRNA-135a.

BACKGROUND: Asthma is an inflammatory syndrome characterized by airway hyperresponsiveness, bronchial inflammation, and airway remodeling. Abnormal proliferation of airway smooth muscle cells (ASMCs) is the main pathological feature of asthma. This study investigated the function and mechanism of serine arginine-rich splicing factor 1 (SRSF1) in ASMC proliferation in asthma. METHODS: SRSF1 expressions in the bronchi of ovalbumin-induced asthmatic mice and IgE-treated mouse ASMCs (mASMCs) were evaluated using quantitative real-time PCR and Western blot. The localization and expression of SRSF1 in the bronchi of asthmatic mice were assessed by immunohistochemistry. Functionally, gain- and loss-of-function assays, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were conducted. Mechanistically, RNA degradation assay, RNA immunoprecipitation, RNA pull-down, and dual-luciferase reporter gene assays were carried out. RESULTS: SRSF1 was highly expressed in the bronchi of ovalbumin-induced asthma mice and IgE-treated mASMCs and was mainly located in the nucleus. Experiments on the function of SRSF1 showed that the silencing of SRSF1 induced the cell cycle of mASMC arrest and restrained mASMC proliferation. Investigations into the mechanism of SRSF1 revealed that SRSF1 and miR-135a are competitively bound to the 3'UTR region of Cyclin D2 (CCND2). SRSF1 overexpression repressed the degradation of CCND2 mRNA, and miR-135a negatively regulated CCND2 expression. Furthermore, SRSF1 knockdown inhibited ASMC proliferation in asthma mouse models by regulating the levels of miR-135a and CCND2. CONCLUSION: SRSF1 knockdown repressed ASMC proliferation in asthma by regulating miR-135a/CCND2 levels.

VR-10 polypeptide interacts with CD36 to induce cell apoptosis and autophagy in choroid-retinal endothelial cells: Identification of VR-10 as putative novel therapeutic agent for choroid neovascularization (CNV) treatment.

Choroid neovascularization (CNV) is important adverse pathological changes that contributes to the aggravation of hypoxic-ischemic eye diseases, and our preliminary work evidences that the thrombospondin-1 (TSP-1) synthetic polypeptide VR-10 may be the candidate therapeutic agent for the treatment of CNV, but its detailed effects and molecular mechanisms are not fully delineated. In this study, the CNV models in BN rats were established by using the laser photocoagulation method, which were further subjected to VR-10 peptide treatment. The RNA-seq and bioinformatics analysis suggested that VR-10 peptide significantly altered the expression patterns of genes in the rat ocular tissues, and the changed genes were especially enriched in the CD36-associated signal pathways. Next, by performing the Real-Time qPCR and Western Blot analysis, we expectedly found that VR-10 upregulated the anti-angiogenesis biomarker (PEDF) and downregulated pro-angiogenesis biomarkers (VEGF, HIF-1 and IL-17) in rat tissues. In addition, we evidenced that VR-10 downregulated CDK2, CDK4, CDK6, Cyclin D1 and Cyclin D2 to induce cell cycle arrest, upregulated cleaved Caspase-3, Bax and downregulated Bcl-2 to promote cell apoptosis, and increased LC3B-II/I ratio and facilitate p62 degradation to promote cell autophagy in RF/6A cells, which were all reversed by knocking down CD36. Moreover, VR-10 upregulated PEDF, and decreased the expression levels of VEGF, HIF-1 and IL-17 to block angiogenesis of RF/6A cells in a CD36-dependent manner. Taken together, VR-10 peptide interacts with its receptor CD36 to regulate the biological functions of RF/6A cells, and these data suggest that VR-10 peptide may be the putative therapeutic drug for the treatment of CNV in clinic.

BAP18 induces growth of non-small-cell lung carcinoma through upregulating transcriptional level of CCND1/2.

OBJECTIVE: To elucidate the biological function of BAP18 (BPTF-associated protein of 18 kDa) in non-small-cell lung carcinoma (NSCLC) and the molecular mechanism. PATIENTS AND METHODS: Relative levels of BAP18 in NSCLC tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and its influence on pathological characteristics of NSCLC patients was analyzed. Correlation between BAP18 and Ki67 levels in NSCLC was assessed by Pearson correlation test. Furthermore, Kaplan-Meier curves were depicted for revealing survival difference in NSCLC patients expressing high or low level of BAP18. Relative levels of BAP18, CCND1, CCND2 and CCND3 in A549 and H1299 cells transfected with siBAP18 were determined, as well as colony number. In addition, after knockdown of protein level of BAP18 in A549 and H1299 cells by lentivirus transfection, cell cycle progression was examined. Co-regulation of BAP18 and CCND1/2 on cell growth of NSCLC was finally detected. RESULTS: BAP18 was upregulated in NSCLC tissues, especially cases with advanced stage (III-IV) or large tumor size (>5 cm). BAP18 was closely linked to tumor size, TNM staging and lymphatic metastasis in NSCLC. Knockdown of BAP18 reduced transcriptional levels of CCND1 and CCND2 in A549 and H1299 cells. Furthermore, knockdown of BAP18 delayed transition from G1 to S phase, and weakened growth of NSCLC cells. CONCLUSIONS: BAP18 triggers the progression of NSCLC by regulating transcriptional activities of CCND1/2, which may be a potential target for the treatment and diagnosis of NSCLC.

Use of the ß-Glucan-Producing Lactic Acid Bacteria Strains Levilactobacillus brevis and Pediococcus claussenii for Sourdough Fermentation-Chemical Characterization and Chemopreventive Potential of In Situ-Enriched Wheat and Rye Sourdoughs and Breads.

The aim of the present study was to examine ß-glucan production and the potential prebiotic and chemopreventive effects of wheat and rye sourdoughs and breads generated with wild-type and non-ß-glucan-forming isogenic mutant strains of Levilactobacillus brevis and Pediococcus claussenii. Sourdough and bread samples were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) and pellets (FP) were analyzed (pH values, short-chain fatty acids (SCFA), ammonia, bacterial taxa) and the effects of FS on LT97 colon adenoma cell growth, viability, caspase-2 and -3 activity, genotoxic and antigenotoxic effects and on gene and protein expression of p21, cyclin D2, catalase and superoxide dismutase 2 (SOD2) were examined. Concentrations of SCFA were increased and concentrations of ammonia were partly reduced in the FS. The relative abundance of Bifidobacteriaceae was increased in all FPs. Treatment with FS reduced the growth and viability of LT97 cells and significantly increased caspase-2 and -3 activities without exhibiting genotoxic or antigenotoxic effects. The p21 mRNA and protein levels were increased while that of cyclin D2 was reduced. Catalase and SOD2 mRNA and protein expression were marginally induced. The presented results indicate a comparable chemopreventive potential of wheat and rye sourdoughs and breads without an additional effect of the formed ß-glucan.

Downregulation of Kcnq1ot1 attenuates ß-cell proliferation and insulin secretion via the miR-15b-5p/Ccnd1 and Ccnd2 axis.

AIM: To examine the effect of lncRNA Kcnq1ot1 on pancreatic ß cells in the development of diabetes. METHODS: The expression levels of Kcnq1ot1 were detected in the islets of diabetes mouse models and the serum of patients with type 2 diabetes by qRT-PCR. CCK8, Ki67 staining, immunohistochemical analyses, glucose-stimulated insulin secretion and intraperitoneal glucose tolerance test were performed to detect the effect of Kcnq1ot1 on ß-cell proliferation and insulin secretion in vitro and in vivo. The relationship between Kcnq1ot1 and miR-15b-5p was predicted by bioinformatics prediction, which was confirmed by luciferase reporter assay. RESULTS: Kcnq1ot1 was more abundant in the pancreas. The expression of Kcnq1ot1 was decreased in the islets of db/db mice and diet-induced obese mice and in the serum of patients with type 2 diabetes. Silencing Kcnq1ot1 inhibited the ß-cell proliferation concomitant with a reduction in the levels of Ccnd1 and Ccnd2. Insulin synthesis and secretion were impaired, along with the decreased expression of Ins1, Ins2, and insulin-related transcription factors. Moreover, Kcnq1ot1 knockdown in vivo reduced glucose tolerance and decreased insulin secretion, consistent with the reduction in the relative islet area and Ki67-positive ß-cells detected by immunochemistry and immunofluorescence staining, respectively. Mechanistically, Kcnq1ot1 directly targeted miR-15b-5p which regulated ß-cell proliferation and insulin secretion through Ccnd1 and Ccnd2. Notably, the suppression of miR-15b-5p attenuated the inhibition of Min6 proliferation and insulin production induced by Kcnq1ot1 knockdown. CONCLUSION: Kcnq1ot1 regulated ß-cell proliferation and insulin secretion via the miR-15b-5p/Ccnd1 and Ccnd2 axis, which is worthy of further investigation considering its potential in diabetes treatment.