Induction of oxidative- and endoplasmic-reticulum-stress dependent apoptosis in pancreatic cancer cell lines by DDOST knockdown.

The dolichyl-diphosphooligosaccharide-protein glycosyltransferase non-catalytic subunit (DDOST) is a key component of the oligosaccharyltransferase complex catalyzing N-linked glycosylation in the endoplasmic reticulum lumen. DDOST is associated with several cancers and congenital disorders of glycosylation. However, its role in pancreatic cancer remains elusive, despite its enriched pancreatic expression. Using quantitative mass spectrometry, we identify 30 differentially expressed proteins and phosphopeptides (DEPs) after DDOST knockdown in the pancreatic ductal adenocarcinoma (PDAC) cell line PA-TU-8988T. We evaluated DDOST / DEP protein-protein interaction networks using STRING database, correlation of mRNA levels in pancreatic cancer TCGA data, and biological processes annotated to DEPs in Gene Ontology database. The inferred DDOST regulated phenotypes were experimentally verified in two PDAC cell lines, PA-TU-8988T and BXPC-3. We found decreased proliferation and cell viability after DDOST knockdown, whereas ER-stress, ROS-formation and apoptosis were increased. In conclusion, our results support an oncogenic role of DDOST in PDAC by intercepting cell stress events and thereby reducing apoptosis. As such, DDOST might be a potential biomarker and therapeutic target for PDAC.

The role of cilia in the development, survival, and regeneration of hair cells.

Mutations impacting cilia genes lead to a class of human diseases known as ciliopathies. This is due to the role of cilia in the development, survival, and regeneration of many cell types. We investigated the extent to which disrupting cilia impacted these processes in lateral line hair cells of zebrafish. We found that mutations in two intraflagellar transport (IFT) genes, ift88 and dync2h1, which lead to the loss of kinocilia, caused increased hair cell apoptosis. IFT gene mutants also have a decreased mitochondrial membrane potential, and blocking the mitochondrial uniporter causes a loss of hair cells in wild-type zebrafish but not mutants, suggesting mitochondria dysfunction may contribute to the apoptosis seen in these mutants. These mutants also showed decreased proliferation during hair cell regeneration but did not show consistent changes in support cell number or proliferation during hair cell development. These results show that the loss of hair cells seen following disruption of cilia through either mutations in anterograde or retrograde IFT genes appears to be due to impacts on hair cell survival but not necessarily development in the zebrafish lateral line.

HSF1 is required for cellular adaptation to daily temperature fluctuations.

The heat shock response (HSR) is a universal mechanism of cellular adaptation to elevated temperatures and is regulated by heat shock transcription factor 1 (HSF1) or HSF3 in vertebrate endotherms, such as humans, mice, and chickens. We here showed that HSF1 and HSF3 from egg-laying mammals (monotremes), with a low homeothermic capacity, equally possess a potential to maximally induce the HSR, whereas either HSF1 or HSF3 from birds have this potential. Therefore, we focused on cellular adaptation to daily temperature fluctuations and found that HSF1 was required for the proliferation and survival of human cells under daily temperature fluctuations. The ectopic expression of vertebrate HSF1 proteins, but not HSF3 proteins, restored the resistance in HSF1-null cells, regardless of the induction of heat shock proteins. This function was associated with the up-regulation of specific HSF1-target genes. These results indicate the distinct role of HSF1 in adaptation to thermally fluctuating environments and suggest association of homeothermic capacity with functional diversification of vertebrate HSF genes.

TRIM44, a Novel Prognostic Marker, Supports the Survival of Proteasome-Resistant Multiple Myeloma Cells.

TRIM44, a tripartite motif (TRIM) family member, is pivotal in linking the ubiquitin-proteasome system (UPS) to autophagy in multiple myeloma (MM). However, its prognostic impact and therapeutic potential remain underexplored. Here, we report that TRIM44 overexpression is associated with poor prognosis in a Multiple Myeloma Research Foundation (MMRF) cohort of 858 patients, persisting across primary and recurrent MM cases. TRIM44 expression notably increases in advanced MM stages, indicating its potential role in disease progression. Single-cell RNA sequencing across MM stages showed significant TRIM44 upregulation in smoldering MM (SMM) and MM compared to normal bone marrow, especially in patients with t(4;14) cytogenetic abnormalities. This analysis further identified high TRIM44 expression as predictive of lower responsiveness to proteasome inhibitor (PI) treatments, underscoring its critical function in the unfolded protein response (UPR) in TRIM44-high MM cells. Our findings also demonstrate that TRIM44 facilitates SQSTM1 oligomerization under oxidative stress, essential for its phosphorylation and subsequent autophagic degradation. This process supports the survival of PI-resistant MM cells by activating the NRF2 pathway, which is crucial for oxidative stress response and, potentially, other chemotherapy-induced stressors. Additionally, TRIM44 counters the TRIM21-mediated suppression of the antioxidant response, enhancing MM cell survival under oxidative stress. Collectively, our discoveries highlight TRIM44's significant role in MM progression and resistance to therapy, suggesting its potential value as a therapeutic target.

USP36 regulates the proliferation, survival, and differentiation of hFOB1.19 osteoblast.

BACKGROUND: Effective bone formation relies on osteoblast differentiation, a process subject to intricate post-translational regulation. Ubiquitin-specific proteases (USPs) repress protein degradation mediated by the ubiquitin-proteasome pathway. Several USPs have been documented to regulate osteoblast differentiation, but whether other USPs are involved in this process remains elusive. METHODS: In this study, we conducted a comparative analysis of 48 USPs in differentiated and undifferentiated hFOB1.19 osteoblasts, identifying significantly upregulated USPs. Subsequently, we generated USP knockdown hFOB1.19 cells and evaluated their osteogenic differentiation using Alizarin red staining. We also assessed cell viability, cell cycle progression, and apoptosis through MTT, 7-aminoactinomycin D staining, and Annexin V/PI staining assays, respectively. Quantitative PCR and Western blotting were employed to measure the expression levels of osteogenic differentiation markers. Additionally, we investigated the interaction between the USP and its target protein using co-immunoprecipitation (co-IP). Furthermore, we depleted the USP in hFOB1.19 cells to examine its effect on the ubiquitination and stability of the target protein using immunoprecipitation (IP) and Western blotting. Finally, we overexpressed the target protein in USP-deficient hFOB1.19 cells and evaluated its impact on their osteogenic differentiation using Alizarin red staining. RESULTS: USP36 is the most markedly upregulated USP in differentiated hFOB1.19 osteoblasts. Knockdown of USP36 leads to reduced viability, cell cycle arrest, heightened apoptosis, and impaired osteogenic differentiation in hFOB1.19 cells. USP36 interacts with WD repeat-containing protein 5 (WDR5), and the knockdown of USP36 causes an increased level of WDR5 ubiquitination and accelerated degradation of WDR5. Excessive WDR5 improved the impaired osteogenic differentiation of USP36-deficient hFOB1.19 cells. CONCLUSIONS: These observations suggested that USP36 may function as a key regulator of osteoblast differentiation, and its regulatory mechanism may be related to the stabilization of WDR5.

Effects of TET2-mediated methylation reconstruction on A2058 melanoma cell sensitivity to matrix stiffness in a 3D culture system.

Matrix stiffness is a crucial factor in the tumor microenvironment, impacting tumor progression and development. TET2 is vital for epigenetic regulation in melanoma and is significantly reduced in advanced melanomas compared with nevi and thin melanomas. However, it is unclear how TET2 mediates the effect of matrix stiffness on melanoma cells. This study utilized A2058 cell lines and prepared different stiffness collagen hydrogels to evaluate TET2 overexpression (TET2OE) and mutant (TET2M) melanoma cells' activity, proliferation, and invasion. A2058 melanoma cells' viability and invasion decreased with increased matrix stiffness, with TET2OE cells experiencing a more significant impact than TET2M cells. Methylation analysis revealed that TET2 determines gene methylation levels, influencing cell-ECM interactions. Transcriptome analysis confirmed that TET2 promotes matrix stiffness's effect on melanoma cell fate. This research provides promising directions and opportunities for melanoma treatment.

Experimental Study: The Development of a Novel Treatment for Chemotherapy-Resistant Tongue Cancer with the Inhibition of the Pathological Periostin Splicing Variant 1-2 with Exon 21.

Tongue squamous cell carcinoma (TSCC) occurs frequently in the oral cavity, and because of its high proliferative and metastatic potential, it is necessary to develop a novel treatment for it. We have reported the importance of the inhibition of the periostin (POSTN) pathological splicing variant, including exon 21 (PN1-2), in various malignancies, but its influence is unclear in tongue cancer. In this study, we investigated the potential of POSTN exon 21-specific neutralizing antibody (PN21-Ab) as a novel treatment for TSCC. Human PN2 was transfected into the human TSCC (HSC-3) and cultured under stress, and PN2 was found to increase cell viability. PN2 induced chemotherapy resistance in HSC-3 via the phosphorylation of the cell survival signal Akt. In tissues from human TSCC and primary tumors of an HSC-3 xenograft model, PN1-2 was expressed in the tumor stroma, mainly from fibroblasts. The intensity of PN1-2 mRNA expression was positively correlated with malignancy. In the HSC-3 xenograft model, CDDP and PN21-Ab promoted CDPP's inhibition of tumor growth. These results suggest that POSTN exon 21 may be a biomarker for tongue cancer and that PN21-Ab may be a novel treatment for chemotherapy-resistant tongue cancer. The treatment points towards important innovations for TSCC, but many more studies are needed to extrapolate the results.

Circular RNA circMRPS35 represses malignant progression in osteosarcoma cells via targeting miR-105-5p/FOXO1.

Osteosarcoma is a highly metastatic, aggressive bone cancer that occurs in children and young adults worldwide. Circular RNAs (circRNAs) are crucial molecules for osteosarcoma progression. In this study, we aimed to investigate the impact of circMRPS35 overexpression and its interaction with FOXO1 via evaluating apoptosis, cell cycle, and bioinformatic analyses on the malignant development of osteosarcoma in MG63 and MNNG/HOS cells. We found that circMRPS35 overexpression reduced osteosarcoma cell viability and inhibited tumor growth in vivo. It increased the apoptosis rate and induced cell cycle arrest in osteosarcoma cells. We identified a potential interaction between circMRPS35 and FOXO1 with miR-105-5p using bioinformatics analysis. Overexpression of circMRPS35 decreased miR-105-5p expression, whereas miR-105-5p mimic treatment increased its expression. This mimic also suppressed the luciferase activity of circMRPS35 and FOXO1 and reduced FOXO1 expression. Overexpression of circMRPS35 elevated FOXO1 protein levels, but this effect was reversed by co-treatment with the miR-105-5p mimic. We demonstrated that inhibiting miR-105-5p decreased viability and induced apoptosis. Overexpression of FOXO1 or treatment with a miR-105-5p inhibitor could counteract the effects of circMRPS35 on viability and apoptosis in osteosarcoma cells. Therefore, we concluded that circMRPS35 suppressed the malignant progression of osteosarcoma via targeting the miR-105-5p/FOXO1 axis.

Genetic switch selectively kills hepatocellular carcinoma cell based on microRNA and tissue-specific promoter.

The clinical treatment of hepatocellular carcinoma (HCC) is still a heavy burden worldwide. Intracellular microRNAs (miRNAs) commonly express abnormally in cancers, thus they are potential therapeutic targets for cancer treatment. miR-21 is upregulated in HCC whereas miR-122 is enriched in normal hepatocyte but downregulated in HCC. In our study, we first generated a reporter genetic switch compromising of miR-21 and miR-122 sponges as sensor, green fluorescent protein (GFP) as reporter gene and L7Ae:K-turn as regulatory element. The reporter expression was turned up in miR-21 enriched environment while turned down in miR-122 enriched environment, indicating that the reporter switch is able to respond distinctly to different miRNA environment. Furthermore, an AAT promoter, which is hepatocyte-specific, is applied to increase the specificity to hepatocyte. A killing switch with AAT promoter and an apoptosis-inducing element, Bax, in addition to miR-21 and miR-122 significantly inhibited cell viability in Huh-7 by 70 % and in HepG2 by 60 %. By contrast, cell viability was not affected in five non-HCC cells. Thus, we provide a novel feasible strategy to improve the safety of miRNA-based therapeutic agent to cancer.

Up-regulation of miR-126 via DNA methylation in hypoxia-preconditioned endothelial cells may contribute to hypoxic tolerance of neuronal cells.

BACKGROUND: Endothelial cells (ECs) can confer neuroprotection by secreting molecules. This study aimed to investigate whether DNA methylation contributes to the neuroprotective gene expression induced by hypoxia preconditioning (HPC) in ECs and to clarify that the secretion of molecules from HPC ECs may be one of the molecular mechanisms of neuroprotection. METHODS: Human microvascular endothelial cell-1 (HMEC-1) was cultured under normal conditions (C), hypoxia(H), and hypoxia preconditioning (HPC), followed by the isolation of culture medium (CM). SY5Y cell incubated with the isolated CM from HMEC-1 was exposed to oxygen-glucose deprivation (OGD). The DNA methyltransferases (DNMTs), global methylation level, miR-126 and its promotor DNA methylation level in HMEC-1 were measured. The cell viability and cell injury in SY5Y were detected. RESULTS: HPC decreased DNMTs level and global methylation level as well as increased miR-126 expression in HMEC-1. CM from HPC treated HMEC-1 also relieved SY5Y cell damage, while CM from HMEC-1 which over-expression of miR-126 can reduce injury in SY5Y under OGD condition. CONCLUSIONS: These findings indicate EC may secrete molecules, such as miR-126, to execute neuroprotection induced by HPC through regulating the expression of DNMTs.

Latrophilins as Downstream Effectors of Androgen Receptors including a Splice Variant, AR-V7, Induce Prostate Cancer Progression.

Latrophilins (LPHNs), a group of the G-protein-coupled receptor to which a spider venom latrotoxin (LTX) is known to bind, remain largely uncharacterized in neoplastic diseases. In the present study, we aimed to determine the role of LPHNs in the progression of prostate cancer. We assessed the actions of LPHNs, including LPHN1, LPHN2, and LPHN3, in human prostate cancer lines via their ligand (e.g., α-LTX, FLRT3) treatment or shRNA infection, as well as in surgical specimens. In androgen receptor (AR)-positive LNCaP/C4-2/22Rv1 cells, dihydrotestosterone considerably increased the expression levels of LPHNs, while chromatin immunoprecipitation assay revealed the binding of endogenous ARs, including AR-V7, to the promoter region of each LPHN. Treatment with α-LTX or FLRT3 resulted in induction in the cell viability and migration of both AR-positive and AR-negative lines. α-LTX and FLRT3 also enhanced the expression of Bcl-2 and phosphorylated forms of JAK2 and STAT3. Meanwhile, the knockdown of each LPHN showed opposite effects on all of those mediated by ligand treatment. Immunohistochemistry in radical prostatectomy specimens further showed the significantly elevated expression of each LPHN in prostate cancer, compared with adjacent normal-appearing prostate, which was associated with a significantly higher risk of postoperative biochemical recurrence in both univariate and multivariable settings. These findings indicate that LPHNs function as downstream effectors of ARs and promote the growth of androgen-sensitive, castration-resistant, or even AR-negative prostate cancer.

KIT in oocytes: a key factor for oocyte survival and reproductive lifespan.

BACKGROUND: The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage. METHODS: We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes. FINDINGS: Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells. INTERPRETATION: These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan. FUNDING: This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).

Transcriptional coactivation of NRF2 signaling in cardiac fibroblasts promotes resistance to oxidative stress.

We recently discovered that steroid receptor coactivators (SRCs) SRCs-1, 2 and 3, are abundantly expressed in cardiac fibroblasts (CFs) and their activation with the SRC small molecule stimulator MCB-613 improves cardiac function and dramatically lowers pro-fibrotic signaling in CFs post-myocardial infarction. These findings suggest that CF-derived SRC activation could be beneficial in the mitigation of chronic heart failure after ischemic insult. However, the cardioprotective mechanisms by which CFs contribute to cardiac pathological remodeling are unclear. Here we present studies designed to identify the molecular and cellular circuitry that governs the anti-fibrotic effects of an MCB-613 derivative, MCB-613-10-1, in CFs. We performed cytokine profiling and whole transcriptome and proteome analyses of CF-derived signals in response to MCB-613-10-1. We identified the NRF2 pathway as a direct MCB-613-10-1 therapeutic target for promoting resistance to oxidative stress in CFs. We show that MCB-613-10-1 promotes cell survival of anti-fibrotic CFs exposed to oxidative stress by suppressing apoptosis. We demonstrate that an increase in HMOX1 expression contributes to CF resistance to oxidative stress-mediated apoptosis via a mechanism involving SRC co-activation of NRF2, hence reducing inflammation and fibrosis. We provide evidence that MCB-613-10-1 acts as a protectant against oxidative stress-induced mitochondrial damage. Our data reveal that SRC stimulation of the NRF2 transcriptional network promotes resistance to oxidative stress and highlights a mechanistic approach toward addressing pathologic cardiac remodeling.

Small Cell Lung Carcinoma Cells Depend on KIF11 for Survival.

Few efficacious treatment options are available for patients with small cell lung carcinoma (SCLC), indicating the need to develop novel therapeutic approaches. In this study, we explored kinesin family member 11 (KIF11), a potential therapeutic target in SCLC. An analysis of publicly available data suggested that KIF11 mRNA expression levels are significantly higher in SCLC tissues than in normal lung tissues. When KIF11 was targeted by RNA interference or a small-molecule inhibitor (SB743921) in two SCLC cell lines, Lu-135 and NCI-H69, cell cycle progression was arrested at the G2/M phase with complete growth suppression. Further work suggested that the two cell lines were more significantly affected when both KIF11 and BCL2L1, an anti-apoptotic BCL2 family member, were inhibited. This dual inhibition resulted in markedly decreased cell viability. These findings collectively indicate that SCLC cells are critically dependent on KIF11 activity for survival and/or proliferation, as well as that KIF11 inhibition could be a new strategy for SCLC treatment.

Clinical Significance and Potential Mechanism of Circ_00008842 in Acute Myocardial Infarction.

This study aimed to evaluate the clinical value of circ_0008842 in acute myocardial infarction (AMI) and explore the potential mechanisms.GSE149051 and GSE160717 datasets analyze common differentially expressed circRNAs (coDEcircRNA) in AMI. RT-qPCR analysis of circ_0008842 mRNA levels in patients with AMI. ROC curve assesses the diagnostic value of circ_0008842 in AMI. A cell model of AMI was constructed by hypoxia-reoxygenation (H/R) -induced H9c2. Cell viability and apoptosis were examined by CCK-8 and flow cytometry. Enzyme-linked immunosorbent assay was used to explore myocardial injury markers CK-MB and cTnI secretion. Dual luciferase reporter assays validate circ_0008842 binding to miRNA. PPI network and gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment reveal potential functions and pathways of targets from the miRNA in AMI.circ_0008842 is recognized as coDEcircRNA in AMI-related databases. circ_0008842 was greatly lower and miR-574-5p was significantly higher in patients with AMI than in healthy individuals. miR-574-5p is a target of circ_0008842. The sensitivity and specificity of circ_0008842 for diagnosing patients with AMI were 87.40% and 83.50%, respectively. Overexpression of circ_0008842 inhibited H/R induced apoptosis, increased cell viability, and decreased CK-MB and cTnI levels, which were partially abrogated by overexpression of miR-574-5p. Calmodulin-like protein 4 (CALML4) was the most connected hub gene in the PPI network of miR-574-5p predicted target genes.circ_0008842 is a diagnostic biomarker for AMI and participates in myocardial injury in AMI by regulating miR-574-5p. Our study provides new insights into the diagnosis for AMI.

Octadecaneuropeptide, ODN, Promotes Cell Survival against 6-OHDA-Induced Oxidative Stress and Apoptosis by Modulating the Expression of miR-34b, miR-29a, and miR-21in Cultured Astrocytes.

Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides including octadecaneuropeptide (ODN). We have previously reported that ODN rescues neurons and astrocytes from 6-OHDA-induced oxidative stress and cell death. The purpose of this study was to examine the potential implication of miR-34b, miR-29a, and miR-21 in the protective activity of ODN on 6-OHDA-induced oxidative stress and cell death in cultured rat astrocytes. Flow cytometry analysis showed that 6-OHDA increased the number of early apoptotic and apoptotic dead cells while treatment with the subnanomolar dose of ODN significantly reduced the number of apoptotic cells induced by 6-OHDA. 6-OHDA-treated astrocytes exhibited the over-expression of miR-21 (+118%) associated with a knockdown of miR-34b (-61%) and miR-29a (-49%). Co-treatment of astrocytes with ODN blocked the 6-OHDA-stimulated production of ROS and NO and stimulation of Bax and caspase-3 gene transcription. Concomitantly, ODN down-regulated the expression of miR-34b and miR-29a and rescued the 6-OHDA-associated reduced expression of miR21, indicating that ODN regulates their expression during cell death. Transfection with miR-21-3p inhibitor prevented the effect of 6-OHDA against cell death. In conclusion, our study indicated that (i) the expression of miRNAs miR-34b, miR-29a, and miR-21 is modified in astrocytes under 6-OHDA injury and (ii) that ODN prevents this deregulation to induce its neuroprotective action. The present study identified miR-21 as an emerging candidate and as a promising pharmacological target that opens new neuroprotective therapeutic strategies in neurodegenerative diseases, especially in Parkinson's disease.

Tert-expressing cells contribute to salivary gland homeostasis and tissue regeneration after radiation therapy.

Salivary gland homeostasis and regeneration after radiotherapy depend significantly on progenitor cells. However, the lineage of submandibular gland (SMG) progenitor cells remains less defined compared with other normal organs. Here, using a mouse strain expressing regulated CreERT2 recombinase from the endogenous Tert locus, we identify a distinct telomerase-expressing (TertHigh) cell population located in the ductal region of the adult SMG. These TertHigh cells contribute to ductal cell generation during SMG homeostasis and to both ductal and acinar cell renewal 1 year after radiotherapy. TertHigh cells maintain self-renewal capacity during in vitro culture, exhibit resistance to radiation damage, and demonstrate enhanced proliferative activity after radiation exposure. Similarly, primary human SMG cells with high Tert expression display enhanced cell survival after radiotherapy, and CRISPR-activated Tert in human SMG spheres increases proliferation after radiation. RNA sequencing reveals upregulation of "cell cycling" and "oxidative stress response" pathways in TertHigh cells following radiation. Mechanistically, Tert appears to modulate cell survival through ROS levels in SMG spheres following radiation damage. Our findings highlight the significance of TertHigh cells in salivary gland biology, providing insights into their response to radiotherapy and into their use as a potential target for enhancing salivary gland regeneration after radiotherapy.

MiR362-3p Alleviates Osteosarcoma by Regulating the IL6ST/JAK2/STAT3 Pathway in Vivo and in Vitro.

Objectives: To investigate the effects and the related signaling pathway of miR-362-3p on OS. Methods: The bioinformatics analysis approaches were employed to investigate the target pathway of miR-362-3p. After the 143B and U2OS cells and nu/nu male mice were randomly divided into blank control (BC) group, normal control (NC) group, and overexpression group (OG), the CCK-8, EdU staining, wound healing assay, Transwell assay, and TUNEL staining were adopted to respectively determine the effects of overexpressed miR-362-3p on the cell viability, proliferation, migration, invasion, and apoptosis of 143B and U2OS cells in vitro, tumor area assay and hematoxylin and eosin staining were employed to respectively determine the effects of overexpressed miR-362-3p on the growth and pathological injury of OS tissue in vivo. The qRT-PCR, Western blot, and immunohistochemical staining were applied to respectively investigate the effects of overexpressed miR-362-3p on the IL6ST/JAK2/STAT3 pathway in OS in vivo and in vitro. Results: The bioinformatics analysis approaches combined qRT-PCR indicated that the IL6ST/JAK2/STAT3 is one of the target pathways of miR-362-3p. Compared with NC, the cell viability, proliferation, migration, and invasion of 143B and U2OS cells were dramatically (P < 0.01) inhibited but the apoptosis was prominently (P <0 .0001) promoted in OG. Compared with NC, the growth of OS tissue was significantly (P < 0.05) suppressed and the pathological injury of OS tissue was substantially aggravated in OG. The gene expression levels of IL6ST, JAK2, and STAT3 and the protein expression levels of IL6ST, JAK2, p-JAK2, STAT3, and p-STAT3 in 143B and U2OS cells were memorably (P < 0.0001) lower in OG than those in NC. In addition, the positively stained areas of proteins of IL6ST, JAK2, p-JAK2, STAT3, and p-STAT3 of OS tissue in OG were markedly (P < 0.01) reduced compared with those in NC. Conclusion: The overexpression of miR362-3p alleviates OS by inhibiting the IL6ST/JAK2/STAT3 pathway in vivo and in vitro.

Caveolin-2 controls preadipocyte survival in the mitotic clonal expansion for adipogenesis.

Here, we report that Caveolin-2 (Cav-2) is a cell cycle regulator in the mitotic clonal expansion (MCE) for adipogenesis. For the G2/M phase transition and re-entry into the G1 phase, dephosphorylated Cav-2 by protein tyrosine phosphatase 1B (PTP1B) controlled epigenetic activation of Ccnb1, Cdk1, and p21 in a lamin A/C-dependent manner, thereby ensuring the survival of preadipocytes. Cav-2, associated with lamin A/C, recruited the repressed promoters of Ccnb1 and Cdk1 for activation, and disengaged the active promoter of p21 from lamin A/C for inactivation through histone H3 modifications at the nuclear periphery. Cav-2 deficiency abrogated the histone H3 modifications and impeded the transactivation of Ccnb1, Cdk1, and p21, leading to a delay in mitotic entry, retardation of re-entry into G1 phase, and the apoptotic cell death of preadipocytes. Re-expression of Cav-2 restored the G2/M phase transition and G1 phase re-entry, preadipocyte survival, and adipogenesis in Cav-2-deficient preadipocytes. Our study uncovers a novel mechanism by which cell cycle transition and apoptotic cell death are controlled for adipocyte hyperplasia.

N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.

The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer's or Parkinson's, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.