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Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.

DNA methylation is a class of epigenetic modification manner, which is responsible for the inactivation of various tumor suppressors. Recently, long non-coding RNAs (lncRNAs) were revealed to be implicated in a variety of malignancies, including non-small cell lung cancer (NSCLC). However, the contributions of lncRNAs to DNA-methylation-induced oncogenic effects in NSCLC remain largely unknown. In this study, we identified a DNA-methylation-repressed lncRNA DIO3 opposite strand upstream RNA (DIO3OS) in NSCLC. DIO3OS is downregulated in NSCLC, and its low expression is related to poor prognosis. Ectopic expression of DIO3OS repressed NSCLC cell growth and motility and promoted NSCLC cell apoptosis in vitro. DIO3OS also repressed NSCLC tumorigenesis and metastasis in vivo. DIO3OS knockdown exhibited opposite biological effects. DIO3OS competitively bound heterogeneous nuclear ribonucleoprotein K (hnRNPK), repressed the binding of hnRNPK to MYC DNA and MYC mRNA, reduced the promoting roles of hnRNPK on MYC transcription and translation, led to the repression of MYC transcription and translation, and therefore remarkably decreased the expression of MYC and CDC25A, a downstream target of MYC. Additionally, depletion of hnRNPK blocked the tumor-suppressive roles of DIO3OS in NSCLC. In conclusion, these findings identified DIO3OS as an important protective factor against NSCLC via modulating hnRNPK-MYC-CDC25A axis., Graphical abstract, DIO3OS is downregulated by DNA methylation in NSCLC. DIO3OS repressed NSCLC progression via competitively binding hnRNPK, repressing the binding of hnRNPK to MYC DNA and MYC mRNA, reducing the promoting roles of hnRNPK on MYC transcription and translation, and therefore decreasing the expression of MYC.

It has been reported that many receptors and proteases are required for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Although angiotensin-converting enzyme 2 (ACE2) is the most important of these receptors, little is known about the contribution of other genes. In this study, we examined the roles of neuropilin-1, basigin, transmembrane serine proteases (TMPRSSs), and cathepsins (CTSs) in SARS-CoV-2 infection using the CRISPR interference system and ACE2-expressing human induced pluripotent stem (iPS) cells. Double knockdown of TMPRSS2 and cathepsin B (CTSB) reduced the viral load to 0.036% ± 0.021%. Consistently, the combination of the CTPB inhibitor CA-074 methyl ester and the TMPRSS2 inhibitor camostat reduced the viral load to 0.0078% ± 0.0057%. This result was confirmed using four SARS-CoV-2 variants (B.1.3, B.1.1.7, B.1.351, and B.1.1.248). The simultaneous use of these two drugs reduced viral load to less than 0.01% in both female and male iPS cells. These findings suggest that compounds targeting TMPRSS2 and CTSB exhibit highly efficient antiviral effects independent of gender and SARS-CoV-2 variant., Graphical abstract, Hashimoto et al. used iPS cells and the CRISPRi system to compare the functions of receptors and proteases involved in SARS-CoV-2 infection. Dual inhibition of TMPRSS2 and CTSB prevented SARS-CoV-2 infection. The combination of TMPRSS2 and CTSB inhibitor treatment exhibited highly efficient antiviral effects independent of gender and SARS-CoV-2 variant.

The progression of breast cancer is closely related to obstructive sleep apnea-hypopnea syndrome (OSAHS). Low concentrations of cannabinoids promote tumor proliferation. However, the role of cannabinoid receptors (CBs) in chronic intermittent hypoxia (CIH)-induced breast cancer has not been reported. The migration and invasion of breast cancer cell lines (MCF-7 and T47D) were measured by scratch assay and transwell assay. Gene and protein expressions were analyzed by qPCR and western blotting. Tumor xenograft mice model were established to evaluate the function of CBs. We observed that chronic hypoxia (CH) and CIH increased CBs expression and promoted migration and invasion in breast cancer. Mice grafted with MCF-7 exhibited obvious tumor growth, angiogenesis, and lung metastasis in CIH compared with CH and control. In addition, CIH induced CBs expression, which subsequently activated insulin-like growth factor-1 receptor (IGF-1R)/AKT/glycogen synthase kinase-3β (GSK-3β) axis. Knockdown of CBs alleviated CIH-induced migration and invasion of breast cancer in vitro. Furthermore, CIH exaggerated the malignancy of breast cancer and silencing of CBs suppressed tumor growth and metastasis in vivo. Our study contributed to understanding the role of CIH in breast cancer development modulation., Graphical abstract, The role of CBs in CIH-induced breast cancer remains unknown. In this study, we found that CIH induced CBs expression via activating IGF-1R/AKT/GSK-3β signaling. CBs knockdown inhibited CIH-induced progression of breast cancer. Our study revealed that CBs might be a new therapeutic strategy for breast cancer.

Based on our initial bioinformatics finding of the upregulated expression of sentrin-specific protease 1 (SENP1) and cyclin E1 (CCNE1) in Wilms tumor, this study aimed to illustrate the molecular mechanism of SENP1 in Wilms tumor, which involved the hypoxia-inducible factor 1α (HIF-1α)/stanniocalcin-1 (STC1)/CCNE1 axis. Wilms tumor and adjacent normal tissues were clinically collected. Gain- and loss-of-function assays were performed to evaluate the effects of the regulatory axis on malignant phenotypes of Wilms tumor cells. A mouse model of Wilms tumor xenografts was further established for in vivo substantiation. Overexpression of CCNE1 and SENP1 occurred in Wilms tumor tissues and cells. Silencing SENP1 inhibited viability and enhanced cell-cycle arrest of Wilms tumor cells. SENP1 promoted STC1 expression and upregulated CCNE1 by driving the small ubiquitin-like modifier (SUMO)ylation of HIF-1α, which ultimately promoted the malignant phenotypes of Wilms tumor cells. It was further confirmed that silencing SENP1 downregulated the expression of CCNE1 and restricted tumorigenicity of Wilms tumor cells in vivo. Taken together, SENP1 elevated STC1 expression by driving the SUMOylation of HIF-1α, thereby upregulating the expression of CCNE1 and ultimately promoting the development of Wilms tumor., Graphical abstract, Wilms tumor is a frequently occurring embryonal tumor in children. This study elucidates a novel mechanism by which sentrin-specific protease 1 (SENP1) triggers the development of Wilms tumor through regulating the HIF-1α/STC1/CCNE1 axis. Herein, this study provides potential therapeutic targets for the treatment of Wilms tumor.

Breast cancer (BC) is one of the most common malignancies among women worldwide with a high incidence of recurrence and metastasis. In this study, we demonstrate that hsa_circ_0068631, a circRNA generated from the transferrin receptor (TFRC), is upregulated in BC tissues and cell lines. Knockdown of hsa_circ_0068631 inhibited the proliferation and migration of BC cells in vitro and in vivo. Mechanistically, an RNA pull-down assay and RNA immunoprecipitation assay revealed that eukaryotic translation initiation factor 4A3 (EIF4A3) could bind to hsa_circ_0068631 and c-Myc mRNA. Additionally, the expression of hsa_circ_0068631 was positively correlated with c-Myc, and the upregulation of hsa_circ_0068631 was a crucial factor for the dysregulation of c-Myc. Through an actinomycin D assay, we confirmed that the mRNA stability of c-Myc was influenced by hsa_circ_0068631 and EIF4A3. Furthermore, hsa_circ_0068631 could recruit EIF4A3 to increase c-Myc mRNA stability. Rescue assays manifesting depletion of c-Myc rescued the promotive effect of hsa_circ_0068631 overexpression on biological activities in BC. In conclusion, to our knowledge, this study is the first to unveil the role of hsa_circ_0068631 and the hsa_circ_0068631/EIF4A3/c-Myc axis in BC, providing a new target for BC treatment., Graphical abstract, circRNA hsa_circ_0068631 was upregulated in BC tissues and cell lines. hsa_circ_0068631 promoted the proliferation and migration of BC cells in vitro and in vivo. Mechanistically, hsa_circ_0068631 could recruit EIF4A3 to increase c-Myc mRNA stability, unveiling the role of the hsa_circ_0068631/EIF4A3/c-Myc axis in BC.

We have previously reported an important role of PR55α, a regulatory subunit of PP2A Ser/Thr phosphatase, in the support of critical oncogenic pathways required for oncogenesis and the malignant phenotype of pancreatic cancer. The studies in this report reveal a novel mechanism by which the p53 tumor suppressor inhibits the protein-stability of PR55α via FBXL20, a p53-target gene that serves as a substrate recognition component of the SCF (Skp1_Cullin1_F-box) E3 ubiquitin ligase complex that promotes proteasomal degradation of its targeted proteins. Our studies show that inactivation of p53 by siRNA-knockdown, gene-deletion, HPV-E6-mediated degradation, or expression of the loss-of-function mutant p53R175H results in increased PR55α protein stability, which is accompanied by reduced protein expression of FBXL20 and decreased ubiquitination of PR55α. Subsequent studies demonstrate that knockdown of FBXL20 by siRNA mimics p53 deficiency, reducing PR55α ubiquitination and increasing PR55α protein stability. Functional tests indicate that ectopic p53R175H or PR55α expression results in an increase of c-Myc protein stability with concomitant dephosphorylation of c-Myc-T58, which is a PR55α substrate, whose phosphorylation otherwise promotes c-Myc degradation. A significant increase in anchorage-independent proliferation is also observed in normal human pancreatic cells expressing p53R175H or, to a greater extent, overexpressing PR55α. Consistent with the common loss of p53 function in pancreatic cancer, FBXL20 mRNA expression is significantly lower in pancreatic cancer tissues compared to pancreatic normal tissues and low FBXL20 levels correlate with poor patient survival. Collectively, these studies delineate a novel mechanism by which the p53/FBXL20 axis negatively regulates PR55α protein stability.

Highlights • CLIC2 is highly expressed in benign, less invasive and less metastatic tumors. • Forced expression of CLIC2 prevents metastasis and invasion in animal tumor models. • CLIC2 is associated with decreased vascular permeability in tumor masses. • CLIC2, a secretable soluble protein, can bind to and inhibit MMP14. • Extracellular CLIC2 can suppress malignant cell invasion., The abilities to invade surrounding tissues and metastasize to distant organs are the most outstanding features that distinguish malignant from benign tumors. However, the mechanisms preventing the invasion and metastasis of benign tumor cells remain unclear. By using our own rat distant metastasis model, gene expression of cells in primary tumors was compared with that in metastasized tumors. Among many distinct gene expressions, we have focused on chloride intracellular channel protein 2 (CLIC2), an ion channel protein of as-yet unknown function, which was predominantly expressed in the primary tumors. We created CLIC2 overexpressing rat glioma cell line and utilized benign human meningioma cells with naturally high CLIC2 expression. CLIC2 was expressed at higher levels in benign human brain tumors than in their malignant counterparts. Moreover, its high expression was associated with prolonged survival in the rat metastasis and brain tumor models as well as with progression-free survival in patients with brain tumors. CLIC2 was also correlated with the decreased blood vessel permeability likely by increased contents of cell adhesion molecules. We found that CLIC2 was secreted extracellularly, and bound to matrix metalloproteinase (MMP) 14. Furthermore, CLIC2 prevented the localization of MMP14 in the plasma membrane, and inhibited its enzymatic activity. Indeed, overexpressing CLIC2 and recombinant CLIC2 protein effectively suppressed malignant cell invasion, whereas CLIC2 knockdown reversed these effects. Thus, CLIC2 suppress invasion and metastasis of benign tumors at least partly by inhibiting MMP14 activity., Graphical abstract Image, graphical abstract

Background Kruppel like factor 10 (KLF10), which is also known as TGF-β Inducible Early Gene-1 (TIEG1), plays a crucial role in regulating cell proliferation, cell apoptosis and inflammatory reaction in human carcinoma cells. Moreover, KLF10 knockout in mice leads to severe defects associated with muscle, skeleton and heart etc. However, the function of KLF10 in intervertebral disc degeneration (IVDD) has not been reported yet. Methods The relationship between KLF10 and IVDD were investigated in nucleus pulposus (NP) tissues from human and rats. The role of KLF10 in NP cells was explored via loss or gain of function experiments. IVDD rat models were constructed through needle puncture and the effects of KLF10 in IVDD model of rats were investigated via intradiscal injection of KLF10. Results We first found that KLF10 was lowly expressed in degenerative NP tissues and the level of KLF10 showed negative correlation with the disc grades of IVDD patients. Loss or gain of function experiments demonstrated that KLF10 could inhibit apoptosis and enhance migration and proliferation of IL-1β induced NP cells. And KLF10 overexpression reduced extracellular matrix (ECM) degeneration and enhanced ECM synthesis, whereas knockdown of KLF10 resulted in adverse effects. These positive effects of KLF10 could be reversed by the inhibition of TGF-β signaling pathway. In vivo, KLF10 overexpression alleviated IVDD. Conclusions This is the first study to reveal that KLF10 was dysregulated in IVDD and overexpressed KLF10 could alleviate IVDD by regulating TGF-β signaling pathway both in vitro and in vivo, which were involved in prohibiting apoptosis, promoting proliferation and migration of NP cells. The translational potential of this article: Overexpression of KLF10 might be an effective therapeutic strategy in the treatment of IVDD.

Long non-coding RNAs (lncRNAs) have been shown to impact important biological functions such as proliferation, survival, and genomic stability. To analyze radiation-induced lncRNA expression in human tumors, we irradiated prostate cancer cells with a single dose of 10 Gy or a multifractionated radiotherapeutic regimen of 10 fractions with a dose of 1 Gy (10 × 1 Gy) during 5 days. We found a stable upregulation of the lncRNA LINC00261 and LINC00665 at 2 months after radiotherapy that was more pronounced after single-dose irradiation. Analysis of the The Cancer Genome Atlas (TCGA) and The Atlas of Non-coding RNAs in Cancer (TANRIC) databases showed that high expression of these two lncRNAs may be a potential negative prognostic marker for overall survival of prostate cancer patients. Knockdown of LINC00261 and LINC00665 in long-term survivors decreased survival after re-irradiation and affected DNA double-strand break repair. Mechanistically, both lncRNAs showed an interdependent expression and regulated expression of the DNA repair proteins CtIP (RBBP8) and XPC as well as the microRNA miR-329. Identifying long-term tumor adaptation mechanisms can lead to the discovery of new molecular targets, in effect opening up new research directions and improving multimodal radiation oncologic treatment., Graphical Abstract, Irradiation can induce long-term effects on the expression of long non-coding RNAs in cancer cells, resulting in tumor adaptation, enhanced tumor cell survival, and radiation resistance, which can be targeted to increase radiosensitivity and treatment efficacy.

Osteosarcoma is a bone tumor frequently diagnosed in children and young adults. Despite advances in chemotherapy and surgical resection, tumors metastasize in 30% of osteosarcoma patients. In addition, side effects caused by chemotherapeutic drugs, as well as the development of chemoresistance, highlight the need to identify the molecular mechanisms involved in the pathogenesis of osteosarcoma. We compared 65 osteosarcoma samples to their adjacent normal tissues, as well as commercially obtained osteosarcoma cell lines with normal osteoblast cell lines, and identified a role for the microRNA (miR)-140/ubiquitin-specific protease 22 (USP22)/lysine-specific demethylase 1 (LSD1)/p21 axis in the development of osteosarcoma. Osteosarcoma tissues and cells exhibited poor miR-140 and p21 expression, whereas the expression of USP22 and LSD1 was increased. Overexpression of miR-140 inhibited cell proliferation, migration, and invasion and promoted cell apoptosis by directly targeting USP22, resulting in its decreased expression. Overexpression of USP22 reversed the effects of miR-140 overexpression in osteosarcoma cells. Overexpression of miR-140 or USP22 knockdown led to the ubiquitination and degradation of LSD1. miR-140 overexpression also suppressed tumorigenesis in vivo. This study revealed a role for miR-140 in the restriction of osteosarcoma development and identified miR-140 as a potential target for therapeutic intervention., Graphical Abstract, This study reveals a role for miR-140 in the restriction of osteosarcoma development and identifies miR-140 as a potential target for therapeutic intervention.

Colorectal cancer (CRC) is one of the most common and deadly cancers, and the incidence of CRC is on the rise. Due to the lack of early diagnosis method and high metastasis of the disease, the prognosis of CRC remains very poor. Exploring the underlying molecular mechanisms of CRC is very necessary for effective therapy. In this study, we investigated the function of circBANP in CRC. The results showed that circBANP was elevated in both CRC tissues and cells and its level positively correlated with the stage of CRC. Knockdown of circBANP greatly suppressed the epithelial-mesenchymal transition (EMT) process and CRC cell proliferation, migration, and invasion. In addition, knockdown of circBANP inhibited CRC tumor growth and metastasis in vivo. Further, circBANP directly bound to let-7d-5p and regulated CRC development via acting as a let-7d-5p sponge. Let-7d-5p directly targeted HMGA1 and thus circBANP/let-7d-5p regulated Wnt/β-catenin signaling via HMGA1. Collectively, circBANP promotes CRC development and metastasis via acting as a let-7d-5p sponge to regulate HMGA1/Wnt/β-catenin signaling, providing a potential biomarker and therapeutic target for the management of CRC., Graphical abstract, This study reveals that circBANP promotes colorectal cancer development and metastasis via acting as a let-7d-5p sponge to regulate HMGA1/Wnt/β-catenin signaling, providing a potential biomarker and therapeutic target for the management of colorectal cancer.

Non-small cell lung cancer (NSCLC) is one of the major causes of morbidity and mortality worldwide. We aimed to investigate the role of N6-methyladenosine (m6A) methyltransferase-like 3 (METTL3) regulating microRNA-1246 (miR-1246) in the progression of NSCLC by targeting paternally expressed gene 3 (PEG3). METTL3, miR-1246, and PEG3 expression in tissues was assessed, and the predictive role of METTL3 in prognosis of patients with NSCLC was detected. NSCLC cells were relatively treated with altered expression of METTL3, miR-1246, or PEG3 to measure their roles in the proliferation, migration, invasion, apoptosis, and in vivo growth of the NSCLC cells. The RNA m6A level was determined, and the targeting relationship between miR-1246 and PEG3 was confirmed. Our results revealed that METTL3 and miR-1246 were upregulated, whereas PEG3 was downregulated in NSCLC tissues. METTL3 knockdown or PEG3 overexpression in NSCLC cells suppressed malignant behaviors of NSCLC cells. METTL3 affected the m6A modification of miR-1246, thus upregulating miR-1246 and miR-1246-targeted PEG3. The elevation of PEG3 reversed the effects of miR-1246 upregulation on NSCLC cells. This study revealed that m6A methyltransferase METTL3 affects the m6A modification of miR-1246, thus upregulating miR-1246 to promote NSCLC progression by inhibiting PEG3., Graphical Abstract, Huang et al. revealed that m6A methyltransferase METTL3 affects the m6A modification of miR-1246, thus upregulating miR-1246 to promote non-small cell lung cancer progression by inhibiting PEG3. The study may contribute to the exploration on therapeutic strategies for non-small cell lung cancer.

Long non-coding RNAs (lncRNAs), microRNAs (miRNAs or miRs), and genes are emerging players in cancer progression. In the present study, we explored the roles and interactions of oncogenic lncRNA small nucleolar RNA host gene 1 (SNHG1), miR-376, forkhead box protein K1 (FOXK1), and Snail in hepatocellular carcinoma (HCC). Expression of SNHG1, miR-376, and FOXK1 in HCC was characterized in clinical HCC tissues of 75 patients with HCC. The interactions between SNHG1 and miR-376 and between miR-376 and FOXK1 were predicted and confirmed by dual-luciferase reporter gene and RNA immunoprecipitation assays. Overexpression and knockdown experiments were performed in HCC cells to examine the effects of the SNHG1/miR-376/FOXK1/Snail axis on viability, apoptosis, invasiveness, and migrating abilities. Their effects on tumor growth and metastasis were validated in nude mouse models. SNHG1 and FOXK1 were upregulated, and miR-376a was downregulated in HCC. SNHG1 knockdown contributed to suppression of HCC cell viability, invasion, and migration properties and promotion of apoptosis. SNHG1 could competitively bind to miR-376a to upregulate its target gene FOXK1, which upregulated Snail. SNHG1 knockdown delayed cancer progression both in vitro and in vivo by upregulating miR-376a and downregulating FOXK1 and Snail. SNHG1 knockdown exerts anti-tumor activity in HCC, suggesting a therapeutic target., Graphical Abstract, SNHG1 is highly expressed in HCC. SNHG1 competitively binds to miR-376a and inhibits the expression of miR-376a, thereby promoting FOXK1 and Snail expression, leading to reduction of HCC cell proliferative, migrating, and invasive properties, and promotes cell apoptosis and promotion of tumor growth and metastasis in HCC.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression in vivo. Collectively, we have demonstrated that upregulation of TRAIL and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL., Graphical abstract, The corresponding author and colleagues show that metformin is capable of increasing the levels of a death ligand TRAIL in some breast and lung cancer cells, thereby activating death-receptor-mediated cell death. They discover a novel mechanism of action of metformin exhibiting antitumor activity via induction of internal TRAIL.

Summary Maintenance and self-renewal of the spermatogonial stem cell (SSC) population is the cornerstone of male fertility. Here, we have identified a key role for the nucleosome remodeling protein CHD4 in regulating SSC function. Gene expression analyses revealed that CHD4 expression is highly enriched in the SSC population in the mouse testis. Using spermatogonial transplantation techniques it was established that loss of Chd4 expression significantly impairs SSC regenerative capacity, causing a ∼50% reduction in colonization of recipient testes. An scRNA-seq comparison revealed reduced expression of “self-renewal” genes following Chd4 knockdown, along with increased expression of signature progenitor genes. Co-immunoprecipitation analyses demonstrated that CHD4 regulates gene expression in spermatogonia not only through its traditional association with the remodeling complex NuRD, but also via interaction with the GDNF-responsive transcription factor SALL4. Cumulatively, the results of this study depict a previously unappreciated role for CHD4 in controlling fate decisions in the spermatogonial pool., Highlights • CHD4 is highly expressed in spermatogonial stem cells in the mouse testis • CHD4 expression is required for spermatogonial stem cell maintenance • CHD4 interacts with SALL4 and NuRD to activate expression of “self-renewal” genes, In this article, Lord and colleagues show that the remodeling protein CHD4 is highly expressed in spermatogonial stem cells (SSCs), and is functionally required for SSC maintenance. The authors demonstrate that CHD4 interacts with the NuRD complex and SALL4 to activate expression of “self-renewal” genes, such as Gfra1.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths, characterized by highly hypoxic tumor microenvironment. Hypoxia-inducible factor-1α (HIF-1α) is a major regulator involved in cellular response to changes of oxygen levels, supporting the adaptation of tumor cells to hypoxia. Bruceine D (BD) is an isolated natural quassinoid with multiple anti-cancer effects. Here, we identified BD could significantly inhibit the HIF-1α expression and its subsequently mediated HCC cell metabolism. Using biophysical proteomics approaches, we identified inhibitor of β-catenin and T-cell factor (ICAT) as the functional target of BD. By targeting ICAT, BD disrupted the interaction of β-catenin and ICAT, and promoted β-catenin degradation, which in turn induced the decrease of HIF-1α expression. Furthermore, BD could inhibit HCC cells proliferation and tumor growth in vivo, and knockdown of ICAT substantially increased resistance to BD treatment in vitro. Our data highlight the potential of BD as a modulator of β-catenin/HIF-1α axis mediated HCC metabolism., Graphical abstract Bruceine D disrupted the direct interaction between ICAT and β-catenin, inducing β-catenin degradation, which in turn induced the decrease of HIF-1α expression and its subsequently mediated HCC cell metabolism.Image 1

Oncogenic transformation enables cells to behave differently from their neighboring normal cells. Both cancer and normal cells recognize each other, often promoting the extrusion of the former from the epithelial cell layer. Here, we show that RasV12-transformed normal rat kidney 52E (NRK-52E) cells are extruded towards the basal side of the surrounding normal cells, which is concomitant with enhanced motility. The active migration of the basally extruded RasV12 cells is observed when surrounded by normal cells, indicating a non-cell-autonomous mechanism. Furthermore, specific inhibitor treatment and knockdown experiments elucidate the roles of PI3K and myosin IIA in the basal extrusion of Ras cells. Our findings reveal a new aspect of cancer cell invasion mediated by functional interactions with surrounding non-transformed cells.

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib, in combination with gemcitabine, has been shown to be a promising therapy in the treatment of pancreatic cancer. Our previous study showed that DJ-1 promotes invasion and metastasis of pancreatic cancer cells by activating SRC/extracellular signal-regulated kinase (ERK)/uPA. The aim of this study was to evaluate whether knockdown of DJ-1 expression can sensitize pancreatic cancer cells to erlotinib treatment. Knockdown of DJ-1 expression accelerated erlotinib-induced cell apoptosis and improved the inhibitory effect of erlotinib on pancreatic cancer cell proliferation (for the BxPC-3, PANC-1, and MiaPACa-2 cell lines, regardless of KRAS mutation status) in vitro and in xenograft tumor growth in vivo. Knockdown of DJ-1 decreased K-RAS expression, membrane translocation, and activity in BxPC-3 cells. Knockdown of DJ-1 also decreased K-RAS, H-RAS, and N-RAS expression in PANC-1 and MiaPACa-2 cells. Knockdown of DJ-1 synergistically inhibited AKT and ERK1/2 phosphorylation with erlotinib in pancreatic cancer cells. These findings indicate that DJ-1 may activate the RAS pathway, reinforcing erlotinib drug resistance. Therefore, blocking DJ-1 in combination with the EGFR tyrosine kinase inhibitor erlotinib may be an attractive therapeutic target in pancreatic cancer.

An increasing number of studies have clarified the functional roles of RNA-binding proteins (RBPs) in driving post-transcriptional mechanisms of cancer progression. In this study, we integrated data from the RBP database and Gene Expression Omnibus (GEO) data with RNA sequencing (RNA-seq) data from 10 ovarian cancer tissues and 8 normal ovarian tissues and identified an RBP, CUGBP- and ETR-3-like family 2 (CELF2). We found that CELF2 expression was downregulated in ovarian cancer and positively correlated with the overall survival (OS) and progression-free survival (PFS) of patients with ovarian cancer. Altered CELF2 expression led to changes in the proliferation, migration, and invasion of ovarian cancer cells in vitro and in vivo. CELF2 expression increased the stability of its target, FAM198B, by binding to AU/U-rich elements (AREs) in the 3′ untranslated region (3′ UTR). FAM198B knockdown restored the CELF2-mediated suppression of proliferation and migration. We also found that CELF2/FAM198B may repress ovarian cancer progression by inhibiting the mitogen-activated protein kinase/extracellular-regulated protein kinase (MAPK/ERK) signaling pathway. Finally, a curcumin-induced increase in CELF2 expression resulted in increased ovarian cancer cell sensitivity to cisplatin. Our study elucidated a novel mechanism by which the CELF2/FAM198B axis regulates proliferation and metastasis in ovarian cancer, providing novel, potential therapeutic targets for ovarian cancer., Graphical Abstract, This study identified a key prognosis-related RBP, named CELF2, in ovarian cancer, which functions as a tumor suppressor. CELF2 stabilizes the FAM198B mRNA by binding to AU/U-rich elements within the 3′ untranslated region. Moreover, CELF2/FAM198B may suppress ovarian cancer progression via the MAPK/ERK signaling pathway.

N6-methyladenosine (m6A) methylation modification is the most prevalent and abundant internal modification of eukaryotic mRNAs. Increasing evidence has shown that mRNA m6A plays important roles in the development of stem cells. However, to the best of our knowledge, no reports about the roles of mRNA m6A in mouse female germline stem cells (mFGSCs) have been published. In this study, we compared the genome-wide profiles of mRNA m6A methylation and DNA methylation between FGSCs and sandosinbred mice (SIM) embryo-derived thioguanine and ouabain-resistant (STO) cells. qRT-PCR revealed that the expression levels of mRNA m6A-related genes (Mettl3, Alkbh5, Ythdf1, Ythdf2, Ythdc1, and Ythdc2) in FGSCs were significantly higher than those in STO cells. m6A RNA immunoprecipitation sequencing (MeRIP-seq) data further showed that the unique m6A-methylated mRNAs in FGSCs and STO cells were related to cell population proliferation and somatic development, respectively. Additionally, knockdown of Ythdf1 inhibited FGSC self-renewal. Comparison of methylated DNA immunoprecipitation sequencing (MeDIP-seq) results between FGSCs and STO cells identified that DNA methylation contributed to FGSC proliferation by suppressing the somatic program. These results suggested that m6A regulated FGSC self-renewal possibly through m6A binding protein YTHDF1, and DNA methylation repressed somatic programs in FGSCs to maintain FGSC characteristics., Graphical Abstract, Ji Wu’s research team found that the genome-wide levels of mRNA m6A methylation and DNA methylation were higher in FGSCs compared with STO cells. Furthermore, they found that knockdown of Ythdf1 inhibited FGSC self-renewal, and DNA methylation repressed the somatic program in FGSCs to maintain FGSC characteristics.

Circular RNA (circRNA) is a novel subclass of noncoding-RNA molecules that participate in development and progression of a variety of human diseases via sponging microRNAs (miRNAs). Until now, the contributions of circRNAs in chemoresistance of hepatocellular carcinoma (HCC) remain largely unknown. In the present study, we aimed to investigate the role of circRNA in cisplatin resistance of HCC. We investigated the expression of circRNAs in 5 paired cisplatin-sensitive and cisplatin-resistant HCC tissues by microarray analysis. The qRT-PCR analysis was to investigate the expression pattern of circARNT2 in HCC patient tissues and cell lines. Then, the effects of circARNT2 on cisplatin resistance, cell proliferation, and apoptosis were assessed in HCC in vitro and in vivo. circARNT2 was significantly upregulated in HCC tissues and cell lines. Overexpression of circARNT2 in HCC was significantly correlated with aggressive characteristics and served as an independent risk factor for overall survival in patients with HCC. In vitro experiments showed that knockdown of circARNT2 inhibited cell proliferation and enhances the cisplatin sensitivity of HCC cells. Furthermore, circARNT2 facilitates HCC progression in vivo. We demonstrated that circARNT2 acts as a sponge for miR-155-5p and verified that PDK1 is a novel target of miR-155-5p. In summary, our study demonstrated that circARNT2 modulates cisplatin resistance through miR-155-5p/PDK1 pathway. Our findings indicated that circARNT2 may serve as a promising therapeutic target for overcoming cisplatin resistance for HCC., Graphical Abstract, Li et al. found that circARNT2 modulates cisplatin resistance through miR-155-5p/PDK1 pathway. Our findings indicated that circARNT2 may serve as a promising therapeutic target for overcoming cisplatin resistance for HCC.

Acute lung injury (ALI), a common complication of sepsis, is characterized by the impairment and injury of pulmonary function. The nuclear factor kappa-B (NF-κB) pathway is activated in ALI. Tripartite motif-containing 37 (TRIM37) can activate the NF-κB pathway and is closely associated with inflammation. The purpose of our study is to reveal the role of TRIM37 in ALI. The present study revealed that TRIM37 presented high levels in lung tissues of ALI mice, and knockdown of TRIM37 alleviated lipopolysaccharide (LPS)-induced lung injury, inflammatory response, and cell apoptosis in vivo. In addition, knockdown of TRIM37 inhibited the inflammatory response, and cell apoptosis of LPS-treated WI-38 cells. Mechanistically, miR-944 was identified to bind with and negatively regulate TRIM37. Furthermore, NEAT1 was indicated to act as a competitive endogenous RNA to promote TRIM37 expression by sequestering miR-944. Detailly, NEAT1 bound with miR-944, negatively modulated miR-944 expression, and positively modulated TRIM37 expression. The rescue assays suggested that overexpression of TRIM37 rescued the influence of NEAT1 knockdown on cell apoptosis and inflammatory response. Overall, NEAT1 facilitated cell apoptosis and inflammatory response of WI-38 cells by the miR-944/TRIM37 axis in sepsis-induced ALI, implying that NEAT1 may provide a novel insight for the treatment of sepsis-induced ALI.

Background & Aims Hepatocellular carcinoma (HCC) is the most common type of hepatic malignancies, with poor prognosis and low survival rate. Paraspeckles, which are unique subnuclear structures, are recently found to be involved in the development of various tumors, including HCC, and are related to induction in chemoresistance of HCC. This study aimed to investigate the possibility of paraspeckle in HCC cells participating in immune escape and its underlying mechanism in vitro and in vivo. Methods Expression of NEAT1_2, the framework of paraspeckle, in HCC cells and tissues was detected by qRT-PCR and RNA-FISH. mRNAs interacted with NEAT1_2 were pull-downed and sequenced in C-terminal S1-aptamer-tagged NEAT1_2 endogenously expressed HCC cells constructed using CRISPR-CAS9 knock-in technology. The effects of paraspeckle on HCC sensitivity to T-cell-mediated cytolysis were detected by T-cell mediated tumor cell killing assay. The roles of NEAT1_2 or NONO on IFNGR1 expression and IFN-γ signaling by applying gene function loss analysis in HCC cells were detected by qRT-PCR, RNA immunoprecipitation, Western blotting, and ELISA. The role of paraspeckle during adoptive T-cell transfer therapy for HCC in vivo was performed with a subcutaneous xenograft mouse. Results Paraspeckle in HCC cells is negatively related to T-cell-mediated cytolysis. Destruction of paraspeckle in HCC cells by knockdown of NEAT1_2 or NONO significantly improved the sensibility of resistant HCC cells to T-cell killing effects. Furthermore, IFNGR1 mRNA, which is sequestered by NEAT1_2 and NONO, is abundant in paraspeckle of T-cell killing-resistant HCC cells. Incapable IFN-γ-IFNGR1 signaling accounts for paraspeckle mediated-adoptive T-cell therapy resistance. Moreover, NEAT1_2 expression negatively correlates with IFNGR1 expression in clinical HCC tissues. Conclusions Paraspeckle in HCC cells helps tumor cells escape from immunosurveillance through sequestering IFNGR1 mRNA to inhibiting IFN-γ-IFNGR1 signaling, thereby avoiding T-cell killing effects. Collectively, our results hint that NEAT1_2 highly expressed HCC patient is more resistant to T-cell therapy in clinic, and NEAT1_2 may be potential target for HCC immunotherapy., Graphical abstract

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas. There is considerable genetic and biological evidence of a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, controlling the biology of these lesions. In addition, activated signal transducer and activator of transcription 3 is associated with clinical progression, although the alterations responsible for this have not been fully elucidated. Here, we studied PLCγ1-dependent mechanisms that can mediate STAT3 activation and control tumor growth and progression. Downstream of PLCγ1, the pharmacological inhibition and genetic knockdown of protein kinase C theta (PKCθ) inhibited signal transducer and activator of transcription 3 activation, impaired proliferation, and promoted apoptosis in cutaneous T-cell lymphoma cells. A PKCθ-dependent transcriptome in mycosis fungoides/Sézary syndrome cells revealed potential effector genes controlling cytokine signaling, TP53, and actin cytoskeleton dynamics. Consistently, an in vivo chicken embryo model xenografted with mycosis fungoides cells showed that PKCθ blockage abrogates tumor growth and spread to distant organs. Finally, the expression of a number of PKCθ target genes found in mycosis fungoides cells significantly correlated with that of PRKCQ (PKCθ) in 81 human mycosis fungoides samples. In summary, PKCθ can play a central role in the activation of malignant cutaneous T-cell lymphoma mechanisms via multiple routes, including, but not restricted to, STAT3. These mechanisms may, in turn, serve as targets for specific therapies., This work has been funded by the Instituto de Salud Carlos III (ISCIII)/FEDER (PI16/00156, PI19/00204, and ASOCIACION LUCHAMOS POR LA VIDA to JPV; PI17/0957 to PLOR). NGD has been supported by a predoctoral contract from UC-IDIVAL. BC holds a RyC contract from MICINN (RYC2018-024004). AEC is funded by ISCIII/MINECO/FEDER (PT17/0009/0019).

The dose-related adverse effects of MDM2‒P53 inhibitors have caused significant concern in the development of clinical safe anticancer agents. Herein we report an unprecedented homo-PROTAC strategy for more effective disruption of MDM2‒P53 interaction. The design concept is inspired by the capacity of sub-stoichiometric catalytic PROTACs enabling to degrade an unwanted protein and the dual functions of MDM2 as an E3 ubiquitin ligase and a binding protein with tumor suppressor P53. The new homo-PROTACs are designed to induce self-degradation of MDM2. The results of the investigation have shown that PROTAC 11a efficiently dimerizes MDM2 with highly competitive binding activity and induces proteasome-dependent self-degradation of MDM2 in A549 non-small cell lung cancer cells. Furthermore, markedly, enantiomer 11a-1 exhibits potent in vivo antitumor activity in A549 xenograft nude mouse model, which is the first example of homo-PROTAC with in vivo therapeutic potency. This study demonstrates the potential of the homo-PROTAC as an alternative chemical tool for tumorigenic MDM2 knockdown, which could be developed into a safe therapy for cancer treatment., Graphical abstract “Suicide” cleavage of MDM2: a homo-PROTAC strategy possessing effective in vitro and in vivo antitumor activities is demonstrated for the first time. The approach may offer an opportunity to overcome the bottleneck of the dose-related adverse effects of MDM2‒P53 inhibitors in the development of clinical anticancer agents.Image 1

Mitochondrial dysfunction and chemokine production have been reported to be involved in the pathogenesis of sepsis. Our initial bioinformatics analysis identified differentially expressed TLR2 in sepsis and the upstream regulatory microRNA-410-3p (miR-410-3p). Hence, the current study was performed to characterize the potential mechanism by which miR-410-3p modulates mitochondrial dysfunction and chemokine production in lipopolysaccharide (LPS)-induced mice in vivo and cardiomyocytes in vitro. Next, we identified that miR-410-3p was downregulated, while TLR2 was upregulated in LPS-induced mice and cardiomyocytes. In addition, miR-410-3p was confirmed to target and inhibit the TLR2 expression. Thereafter, gain- or loss-of-function experiments were conducted to investigate the effect of miR-410-3p and TLR2 on mitochondrial function and chemokine production. TLR2 knockdown or miR-410-3p overexpression was found to alleviate mitochondrial membrane damage and mitochondrial swelling, in addition to augmenting the levels of adenosine triphosphate, mitochondrial membrane potential, and the expression levels of CCL7, CCL5, CXCL1, and CXCL9 in vivo and in vitro. In conclusion, miR-410-3p-mediated TLR2 inhibition alleviated mitochondrial dysfunction and reduced chemokine production in LPS-induced experimental sepsis. Therefore, the overexpression of miR-410-3p may represent a potential strategy for the treatment of sepsis-induced myocardial injury., Graphical Abstract, This study reveals a significance and mechanism of miR-410-3p action in preventing mitochondrial dysfunction and chemokine production and offers a potential prognostic marker and a therapeutic target for sepsis.

Patients with peritoneal metastasis of gastric cancer have dismal prognosis, mainly because of inefficient systemic delivery of drugs to peritoneal tumors. We aimed to develop an intraperitoneal treatment strategy using amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotides (ASOs) targeting synaptotagmin XIII (SYT13) and to identify the function of SYT13 in gastric cancer cells. We screened 71 candidate oligonucleotide sequences according to SYT13-knockdown efficacy, in vitro activity, and off-target effects. We evaluated the effects of SYT13 knockdown on cellular functions and signaling pathways, as well as the effects of intraperitoneal administration to mice of AmNA-modified anti-SYT13 ASOs. We selected the ASOs (designated hSYT13-4378 and hSYT13-4733) with the highest knockdown efficiencies and lowest off-target effects and determined their abilities to inhibit cellular functions associated with the metastatic potential of gastric cancer cells. We found that SYT13 interfered with focal adhesion kinase (FAK)-mediated intracellular signals. Intraperitoneal administration of hSYT13-4378 and hSYT13-4733 in a mouse xenograft model of metastasis inhibited the formation of peritoneal nodules and significantly increased survival. Reversible, dose- and sequence-dependent liver damage was induced by ASO treatment without causing abnormal morphological and histological changes in the brain. Intra-abdominal administration of AmNA-modified anti-SYT13 ASOs represents a promising strategy for treating peritoneal metastasis of gastric cancer., Graphical Abstract, To propose a novel treatment option for patients with peritoneal metastasis of gastric cancer, we designed amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotides (ASOs) targeting synaptotagmin XIII (SYT13). Intra-abdominal administration of AmNA-modified anti-SYT13 ASOs represents a promising strategy for treating peritoneal metastasis of gastric cancer.

Increasing circular RNAs (circRNAs) have been reported to act as key players in human malignancies. However, the expression, role, and mechanism of circRNAs in HCC are not well elucidated. In this study, some differentially expressed circRNAs (DECs) between hepatocellular carcinoma (HCC) and normal tissues were identified using three circRNA microarrays (Gene Expression Omnibus [GEO]: GSE78520, GSE94508, and GSE97332). Twenty-one DECs were found to be commonly upregulated in all the three datasets. Among the 21 DECs, hsa_circ_0001955 ranked as the top three most upregulated DECs in GEO: GSE78520, GSE94508, and GSE97332. Moreover, hsa_circ_0001955 expression in HCC cells and tissues was significantly higher than that in corresponding normal controls. Functional experiments revealed that knockdown of hsa_circ_0001955 markedly inhibited proliferation, migration, and invasion of HCC, and its overexpression led to the opposite effects. hsa_circ_0001955 was mainly located in the cytoplasm, in which hsa_circ_0001955 could directly bind to miR-145-5p. miR-145-5p was downregulated in HCC, and its expression was negatively linked to hsa_circ_0001955 expression. Furthermore, we identified that NRAS was a downstream direct target of the hsa_circ_0001955/miR-145-5p axis in HCC. Collectively, our findings demonstrate the oncogenic roles of the hsa_circ_0001955/miR-145-5p/NRAS axis in HCC, which may represent a potential therapeutic target for HCC., Graphical Abstract, The findings demonstrate the oncogenic roles of the hsa_circ_0001955/miR-145-5p/NRAS axis in HCC, which may represent a potential therapeutic target for HCC.

MicroRNA 22 (miR-22) was found in diverse cardiovascular diseases to have a role in regulating multiple cellular processes. However, the regulatory role of miR-22 in aortic dissection (AD) was still unclear. The miR-22 expression in human aorta was explored. A series of mimic, inhibitor, or small interfering RNA (siRNA) plasmids were delivered into vascular smooth muscle cells (VSMCs) to explore the effects of miR-22 and p38 mitogen-activated protein kinase α (p38MAPKα) in controlling VSMC apoptosis in vitro. In addition, a mouse AD model was established, and histopathologic analyses were performed to evaluate the regulatory effects of miR-22. Reduced miR-22 and increased apoptosis of VSMCs was seen in human AD aorta. Downregulation of miR-22 increased the apoptosis of VSMCs in vitro. Bioinformatics analyses revealed that p38MAPKα was a target of miR-22. Inhibiting p38MAPKα expression could reverse the apoptosis of VSMCs induced by miR-22 downregulation. Knockdown of miR-22 in the AD mouse model significantly promoted the development of AD. Our data underscore the importance of vascular remodeling and VSMC function in AD. miR-22 may represent a new therapeutic approach for AD by regulating the apoptosis of VSMCs through the MAPK signaling pathway., Graphical Abstract, This study indicates miR-22 may represent a new therapeutic approach for aortic dissection by regulating the apoptosis of VSMCs through the MAPK signaling pathway. The study provided a novel insight into the mechanism of AD and the innovative therapeutic strategies in the form of new targets for small molecule therapies.

The Warburg effect is a significant hallmark of gastric cancer (GC), and increasing evidence emphasizes the crucial role of circular RNAs (circRNAs) in GC tumorigenesis. However, the precise molecular mechanisms by which circRNAs drive the GC Warburg effect are still elusive. The present study was designed to unveil the roles of circRNAs and the corresponding potential mechanism. High-regulated expression of circCUL3 was observed in both GC tissues and cell lines. Clinically, the high expression of circCUL3 was closely correlated with advanced clinical stage and overall survival in GC patients. Functionally, cellular experimental investigations demonstrated that circCUL3 promoted the proliferation, glucose consumption, lactate production, ATP quantity, and extracellular acidification rate (ECAR) of GC cells. In vivo, circCUL3 knockdown repressed tumor growth. Mechanistic analysis demonstrated that circCUL3 promoted signal transducer and activator of transcription (STAT)3 expression through sponging miR-515-5p; moreover, transcription factor STAT3 accelerated the transcriptional level of hexokinase 2 (HK2). In summary, the present findings provide mechanistic insights into circCUL3/miR-515-5p/STAT3/HK2 axis regulation on the GC Warburg effect, providing a novel possibility for an understanding of GC pathogenesis., Graphical Abstract, The Warburg effect is a significant hallmark of gastric cancer, and circular RNAs markedly regulate GC tumorigenesis. Pu et al. provide mechanistic insights into the circCUL3/miR-515-5p/STAT3/HK2 axis on the GC Warburg effect, providing a novel possibility for an understanding of GC pathogenesis.

Marek's disease (MD), a highly cell-associated and contagious disease of chickens caused by Marek's disease virus (MDV) can result in neural lesions, immunosuppression and neoplasia in chicken. The Meq gene is an important oncogene in the MDV genome, and it is expressed highly in MD tumor tissues and MD T-lymphoblastoid cell lines. An experiment was conducted to elucidate the role of Meq in MD tumor transformation. RNA interference technology was used to block its expression, and then analyzed the biological effects of Meq knockdown on the MD tumor cell line MSB1. A small interfering RNA with an interference efficiency of 70% (P0.05). B-cell lymphoma 2 gene (BCL2) was anti-apoptotic and caspase-6 was the effector in the apoptosis pathway. The activity of caspase-6 was upregulated (P0.05) post-Meq knockdown, but the expression of two genes (matrix metalloproteinase 2 (MMP2) and MMP9) that are correlated closely to cell invasion was downregulated (P

Graphical abstract The map illustrating mechanisms associated with lncRNA HIF1A-AS2-mediated inflammation in the atherosclerosis. LncRNA HIF1A-AS2 forms a complex with USF1, which is recruited into the ATF2 promoter to elevate ATF2 expression, thus promoting the development of atherosclerotic inflammation. Meanwhile, downregulation of lncRNA HIF1A-AS2 decreases the expression of ATF2 by reducing the binding of USF1 to the ATF2 promoter regions, thereby inhibiting atherosclerotic inflammation, as reflected by decreased inflammatory factors TNF-α, IL-1β and IL-6 in serum and protein levels of adhesion molecules VCAM-1, ICAM-1, and MCP-1, as well as increased cell viability and reduced apoptosis in ox-LDL-induced inflammation in ECs, SMCs, and HCAECs., Introduction In atherosclerotic lesions, extensive inflammation of the vessel wall contributes to plaque instability. Long noncoding RNAs (lncRNAs) play important roles in diverse biological processes in atherosclerosis. Objectives Here, we aim to identify the functional role and regulatory mechanisms of lncRNA hypoxia-inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) in atherosclerotic inflammation. Methods An atherosclerotic mouse model was induced in ApoE-/- mice by high fat diet (HFD). Endothelial cells (ECs), human aortic smooth muscle cells (SMCs) or human coronary artery endothelial cells (HCAECs) were exposed to ox-LDL to develop the in vitro model. The effects of lncRNA HIF1A-AS2 on inflammation were evaluated by determining levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) and levels of adhesion molecules vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and macrophage cationic peptide 1 (MCP-1). Results It was established that lncRNA HIF1A-AS2 and ATF2 were highly expressed in atherosclerotic ApoE-/- mice. Downregulating lncRNA HIF1A-AS2 in ox-LDL-exposed ECs, SMCs and HCAECs inhibited inflammation by reducing levels of pro-inflammatory factors and adhesion molecules. LncRNA HIF1A-AS2 bound to the transcription factor USF1 to elevate ATF2 expression. USF1 overexpression counteracted the suppressive effect of lncRNA HIF1A-AS2 silencing on ox-LDL-induced inflammation. Knockdown of lncRNA HIF1A-AS2 or ATF2 could also attenuate inflammation in atherosclerotic mice. Collectively, the present study demonstrates that downregulation of lncRNA HIF1A-AS2 represses the binding of USF1 to the ATF2 promoter region and then inhibits ATF2 expression, thereby suppressing atherosclerotic inflammation. Conclusion This study suggests lncRNA HIF1A-AS2 as an promising therapeutic target for atherosclerosis.

Identifying effective drugs to delay the progression of aortic aneurysms is a formidable challenge in vascular medicine. Methyltransferase-like 3 (METTL3) plays a key role in catalyzing the formation of N6-methyladenosine (m6A), but despite the functional importance of METTL3 and m6A in various fundamental biological processes, their roles in abdominal aortic aneurysm (AAA) are unknown. Here, we found that METTL3 knockdown in apolipoprotein E-deficient (ApoE−/−) mice treated with angiotensin II suppressed the formation of AAAs, while METTL3 overexpression exerted the opposite effects. Similar results were obtained in a calcium chloride (CaCl2)-induced mouse AAA model. Mechanistically, METTL3-dependent m6A methylation promoted primary microRNA-34a (miR-34a, pri-miR34a) maturation through DGCR8. Moreover, miR-34a overexpression significantly decreased SIRT1 expression and aggravated AAA formation, while miR-34a deficiency produced the opposite effects. In a rescue experiment, miR-34a knockdown or forced expression of SIRT1 partially attenuated the protective effects of METTL3 deficiency against AAA formation. Our studies reveal an important role for METTL3/m6A-mediated miR-34a maturation in AAA formation and provide a novel therapeutic target and diagnostic biomarker for AAA treatment., Graphical Abstract, Despite the functional importance of METTL3 and m6A in various fundamental biological processes, their roles in abdominal aortic aneurysm (AAA) are unknown. Zhong et al. reveal an important role for METTL3/m6A-mediated miR-34a maturation in AAA formation and provide a novel therapeutic target and diagnostic biomarker for AAA treatment.

Novel therapies tailored to the molecular composition mechanism of esophageal squamous cell carcinoma (ESCC) are needed to improve patient survival. miR-20b-5p expression was significantly upregulated in cancerous tissues and associated with lymph node metastasis, clinical stage, and overall survival (OS). An analysis of the methylation status of the miR-20b-5p gene indicated that the hypomethylation of the CpG sites located upstream of the miR-20b-5p gene in the ESCC tissues was more frequent than in the adjacent normal tissues, and the methylation status of miR-20b-5p correlated inversely with its expression levels. Notably, a series of gain- and loss-of-function assays elucidated that miR-20b-5p promoted ESCC cell proliferation, migration, and invasion both in vitro and in vivo. Luciferase reporter assays, western blot, and qRT-PCR revealed that RB1 and TP53INP1 were the direct targets of miR-20b-5p. Moreover, the effects of ectopic miR-20b-5p expression were abrogated by RB1 and TP53INP1 overexpression. In contrast, the effects of miR-20b-5p depletion were impaired by RB1 and TP53INP1 knockdown. Treatment with a miR-20b-5p antagomir dramatically increased tumor growth and inhibited RB1 and TP53INP1 protein expression in nude mice. This work provided novel insights on the molecular mechanism of ESCC and further provided suggestions for therapy development., Graphical Abstract

MYB proto-oncogene-like 1 (MYBL1) has been reported to be a strong activator of transcription and plays an important role in the development of cancer. However, the precise biological function and molecular mechanism of MYBL1 in hepatocellular carcinoma (HCC) cells remain unclear. In the present study, we found that the expression of MYBL1 was markedly overexpressed in HCC cell lines and HCC samples, respectively. Moreover, MYBL1 expression positively correlated with tumor progression and inversely correlated with patient survival in 368 human HCC tissue samples. Overexpression of MYBL1 induced, whereas knockdown of MYBL1 reduced, HCC proliferation and metastasis both in vitro and in vivo. Furthermore, we demonstrated that HCC patients with high MYBL1 expression had significantly shorter overall and poorer disease-free survival than those with low MYBL1 expression. MYBL1 transcriptionally upregulated TWIST1 expression by directly targeting the TWIST1 promoter. More importantly, the in vitro analysis was consistent with the significant correlation between MYBL1 and TWIST1 expression observed in a large cohort of human HCC specimens. Taken together, our results demonstrate that MYBL1 plays an important role in HCC growth and metastasis and reveal a plausible mechanism for upregulation of TWIST1 in HCC., Graphical Abstract, Corresponding authors and colleagues demonstrate that MYBL1 transcriptionally upregulated TWIST1 expression by directly targeting the TWIST1 promoter and subsequently enhances growth and metastasis of hepatocellular carcinoma cells, which establish MYBL1 as a potential diagnosis biomarkers and may serve as a putative target for HCC diagnosis and therapy.

Circular RNAs (circRNAs) regulate gene expression in different malignancies. However, the molecular mechanisms that link circRNAs with the tumorigenesis of prostate cancer (PCa) are not well understood. In the present study, we attempted to provide a novel basis for targeted therapy for PCa from the aspect of circRNA-microRNA (miRNA)-mRNA interaction. We investigated the expression of circRNAs in 5 paired PCa tissues and adjacent non-tumor tissues by microarray analysis. We focused on hsa_circ_0005100, which is located on chromosome 1 and derived from FMN2, and thus we named it circFMN2. The qRT-PCR was used to detect circFMN2 and target miRNA expression in PCa tissues and cell lines. Biological functional experiments were performed to detect the effects of circFMN2 on the biological behavior of PCa cells in vivo and in vitro. Bioinformatic analysis was utilized to predict potential miRNA target sites on circFMN2. High expression of circFMN2 was associated with PCa progression. Function assays revealed that knockdown of circFMN2 significantly reduced PCa cell growth in vitro and in vivo. Finally, we found that circFMN2 acts as a competing endogenous RNA (ceRNA) for miR-1238 to regulate LIM-homeobox gene 2 (LHX2) expression. circFMN2 regulates the miR-1238/LHX2 axis to promote PCa progression., Graphical Abstract, Chen and colleagues found that circular RNA (circRNA) circFMN2 acts as a competing endogenous RNA (ceRNA) for miR-1238 to regulate LIM-homeobox gene 2 (LHX2) expression. circFMN2 regulates the miR-1238/LHX2 axis to promote PCa progression.

The importance of long noncoding RNA (lncRNA) in tumorigenesis has been supported by increasing evidence in recent years. However, the mechanism linking lncRNA function with cancer progression remains poorly understood. lncRNA LCPAT1 plays a role in lung cancer. However, how it works in breast cancer (BC) is largely unclear. In this study, we found that LCPAT1 was highly expressed in BC tissues and cell lines. High LCPAT1 expression predicted a low survival rate in BC patients. LCPAT1 promoted BC cell proliferation, migration, and invasion while inhibiting apoptosis in vitro. LCPAT1 knockdown suppressed BC growth in vivo and vice versa. LCPAT1 interacted with RBBP4 and recruited it to the MFAP2 (microfibril-associated protein 2) promoter and then activated MFAP2 transcription. Restoration of MFAP2 rescued the effects of LCPAT1 knockdown in BC cells. In sum, LCPAT1 promotes BC progression through recruiting RBBP4 to initiate MFAP2 transcription., Graphical Abstract, Gong et al. demonstrate that LCPAT1 was highly expressed in breast cancer tissues and promoted breast cancer cell proliferation, migration, and invasion while inhibiting apoptosis through recruiting RBBP4 to initiate MFAP2 transcription. Their work illustrates a new mechanism involved in breast cancer progression and suggests that LCPAT1 may be a new therapeutic target.

Graphical abstract, Highlights • PIBF1 levels peaked in the mid-secretory phase of endometrium. • PIBF1 expression decreased in the mid-secretory endometrium of RIF patients. • PIBF1 regulated HESC proliferation and decidualization via IL6/p-STAT3 signaling. • The IL6/p-STAT3, Ki-67, prolactin, and IGFBP1 levels were lower in RIF patients. • Low PIBF1 expression may account for poor endometrial receptivity in RIF patients., Introduction Recurrent implantation failure (RIF) is a challenging problem of assisted reproductive technology that arises mainly due to inadequate endometrial receptivity and its pathogenesis is still unclear. Objectives In this study, we conducted the first investigation of the effect of decreased PIBF1 expression in mid-secretory phase on endometrial receptivity in patients with RIF. Methods Microarray assay, reverse transcriptase-quantitative polymerase chain reaction, western blot, and in-vitro experiments were conducted. Results The results showed that progesterone-induced blocking factor 1 (PIBF1) expression was highest in the mid-secretory endometrium in control subjects, but was significantly lower in RIF patients. In Ishikawa and human endometrial stromal cells (HESCs), rather than human endometrial epithelial cells, PIBF1 knockdown significantly downregulated cell proliferation and the levels of interleukin 6 (IL6) and phosphorylated signal transducer and activator of transcription-3 (p-STAT3). Besides, in HESCs, the levels of IL6, p-STAT3, prolactin and insulin-like growth factor binding-protein-1 (IGFBP1) decreased after PIBF1 knockdown during in-vitro decidualization. All these cellular changes could be notably restored by PIBF1 or IL6 overexpression. Consistent with our findings with PIBF1, the levels of IL6, p-STAT3, ki-67, prolactin, and IGFBP1 in the mid-secretory endometrium were notably lower in patients with RIF compared with controls. Conclusion In summary, in the mid-secretory phase, decreased expression of PIBF1, IL6, and p-STAT3 inhibited HESC proliferation and decidualization, which is of theoretical and clinical importance for future research and clinical-treatment strategies.

Background/Purpose Gingival overgrowth can occur as a result of poor oral hygiene or a side effect of taking certain medications, such as cyclosporine A (CsA). It has been shown that this immunosuppressant drug induces epithelial-to-mesenchymal transition (EMT) in the gingival epithelium but the associated molecular mechanism remains to be elucidated. Methods We first assessed the relative expression of microRNA-200a (miR-200a) in response to the CsA treatment using qRT-PCR. Next, luciferase reporter assay was applied to examine whether miR-200a was able to regulate ZEB2 and Western blot was utilized to measure the expression of ZEB2 in normal human gingival fibroblasts (HGFs). To confirm the significance of miR-200a and ZEB2 in the CsA-induced gingival overgrowth, miR-200a inhibitor and shRNA mediated knockdown of ZEB2 were used and cell proliferation in HGFs was assessed by MTT assay. Results The expression of miR-200a was dose-dependently downregulated following the CsA treatment. Luciferase reporter assay confirmed that ZEB2 was a direct downstream target regulated by miR-200a and ZEB2 was indeed increased after the administration of CsA. We demonstrated that knockdown of ZEB2 hampered the CsA-induced HGFs proliferation and the elevated cell proliferation due to inhibition of miR-200a was reversed by repression of ZEB2. Conclusion Our results showed that insufficient miR-200a in HGFs caused by CsA administration may lead to gingival enlargement mediated by the upregulation of ZEB2. This finding supported that CsA-induced EMT contributed to the adverse effect of using CsA and miR-200a may serve as an upstream target to prevent the overgrowth of the gingiva.

For over a decade RNA interference (RNAi) has been an important molecular tool for functional genomics studies in parasitic flatworms. Despite this, our understanding of RNAi dynamics in many flatworm parasites, such as the temperate liver fluke (Fasciola hepatica), remains rudimentary. The ability to maintain developing juvenile fluke in vitro provides the opportunity to perform functional studies during development of the key pathogenic life stage. Here, we investigate the RNAi competence of developing juvenile liver fluke. Firstly, all life stages examined possess, and express, core candidate RNAi effectors encouraging the hypothesis that all life stages of F. hepatica are RNAi competent. RNAi effector analyses supported growing evidence that parasitic flatworms have evolved a separate clade of RNAi effectors with unknown function. Secondly, we assessed the impact of growth/development during in vitro culture on RNAi in F. hepatica juveniles and found that during the first week post-excystment liver fluke juveniles exhibit quantitatively lower RNAi mediated transcript knockdown when maintained in growth inducing media. This did not appear to occur in older in vitro juveniles, suggesting that rapidly shifting transcript dynamics over the first week following excystment alters RNAi efficacy after a single 24 h exposure to double stranded (ds)RNA. Finally, RNAi efficiency was found to be improved through use of a repeated dsRNA exposure methodology that has facilitated silencing of genes in a range of tissues, thereby increasing the utility of RNAi as a functional genomics tool in F. hepatica., Graphical abstract Image 1, Highlights • All F. hepatica life stages express the RNAi machinery for a functional pathway. • RNAi-mediated knockdown in juvenile F. hepatica is affected by in vitro growth. • Repeated dsRNA exposures ensure longevity of transcript and protein knockdown. • This provides a basis for a robust RNAi platform in F. hepatica juveniles.

Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury, and its prognosis depends on the balance between hepatocyte death and regeneration. Sirtuin 6 (SIRT6) has been reported to protect against oxidative stress-associated DNA damage. But whether SIRT6 regulates APAP-induced hepatotoxicity remains unclear. In this study, the protein expression of nuclear and total SIRT6 was up-regulated in mice liver at 6 and 48 h following APAP treatment, respectively. Sirt6 knockdown in AML12 cells aggravated APAP-induced hepatocyte death and oxidative stress, inhibited cell viability and proliferation, and downregulated CCNA1, CCND1 and CKD4 protein levels. Sirt6 knockdown significantly prevented APAP-induced NRF2 activation, reduced the transcriptional activities of GSTμ and NQO1 and the mRNA levels of Nrf2, Ho-1, Gstα and Gstμ. Furthermore, SIRT6 showed potential protein interaction with NRF2 as evidenced by co-immunoprecipitation (Co-IP) assay. Additionally, the protective effect of P53 against APAP-induced hepatocytes injury was Sirt6-dependent. The Sirt6 mRNA was significantly down-regulated in P53−/− mice. P53 activated the transcriptional activity of SIRT6 and exerted interaction with SIRT6. Our results demonstrate that SIRT6 protects against APAP hepatotoxicity through alleviating oxidative stress and promoting hepatocyte proliferation, and provide new insights in the function of SIRT6 as a crucial docking molecule linking P53 and NRF2., Graphical abstract Image 1Sirtuin 6 as a transcriptional coactivator of P53 and nuclear factor erythroid 2-related factor 2 (NRF2) protects against acetaminophen-induced hepatotoxicity through alleviating oxidative stress and promoting hepatocyte proliferation.