Simvastatin inhibits stem cell proliferation in human leiomyoma via TGF‐β3 and Wnt/β‐Catenin pathways

Uterine leiomyoma (UL) is the most common gynaecologic tumour, affecting an estimated 70 to 80% of women. Leiomyomas develop from the transformation of myometrial stem cells into leiomyoma stem (or tumour‐initiating) cells. These cells undergo self‐renewal and differentiation to mature cells, both are necessary for the maintenance of tumour stem cell niche and tumour growth, respectively. Wnt/β‐catenin and TGF‐β/SMAD pathways, both overactive in UL, promote stem cell self‐renewal, crosstalk between stem and mature cells, cellular proliferation, extracellular matrix (ECM) accumulation and drive overall UL growth. Recent evidence suggests that simvastatin, an antihyperlipidemic drug, may have anti‐leiomyoma properties. Herein, we investigated the effects of simvastatin on UL stem cells. We isolated leiomyoma stem cells by flow cytometry using DyeCycle Violet staining and Stro‐1/CD44 surface markers. We found that simvastatin inhibits proliferation and induces apoptosis in UL stem cells. In addition, it also suppressed the expression of the stemness markers Nanog, Oct4 and Sox2. Simvastatin significantly decreased the production of the key ECM proteins, collagen 1 and fibronectin. Finally, it inhibited genes and/or proteins expression of TGF‐β1, 2 and 3, SMAD2, SMAD4, Wnt4, β‐Catenin, LRP6, AXIN2 and Cyclin D1 in UL stem cells, all are key drivers of the TGF‐β3/SMAD2 and Wnt4/β‐Catenin pathways. Thus, we have identified a novel stem cell‐targeting anti‐leiomyoma simvastatin effect. Further studies are needed to replicate these findings in vivo.     

MiR‐26a‐tetrahedral framework nucleic acids mediated osteogenesis of adipose‐derived mesenchymal stem cells

ObjectivesDelivery systems that provide time and space control have a good application prospect in tissue regeneration applications, as they can effectively improve the process of wound healing and tissue repair. In our experiments, we constructed a novel micro‐RNA delivery system by linking framework nucleic acid nanomaterials to micro‐RNAs to promote osteogenic differentiation of mesenchymal stem cells.Materials and MethodsTo verify the successful preparation of tFNAs–miR‐26a, the size of tFNAs–miR‐26a were observed by non‐denaturing polyacrylamide gel electrophoresis and dynamic light scattering techniques. The expression of osteogenic differentiation‐related genes and proteins was investigated by confocal microscope, PCR and western blot to detect the impact of tFNAs–miR‐26a on ADSCs. And finally, Wnt/β‐catenin signaling pathway related proteins and genes were detected by confocal microscope, PCR and western blot to study the relevant mechanism.ResultsBy adding this novel complex, the osteogenic differentiation ability of mesenchymal stem cells was significantly improved, and the expression of alkaline phosphatase (ALP) on the surface of the cell membrane and the formation of calcium nodules in mesenchymal stem cells were significantly increased on days 7 and 14 of induction of osteogenic differentiation, respectively. Gene and protein expression levels of ALP (an early marker associated with osteogenic differentiation), RUNX2 (a metaphase marker), and OPN (a late marker) were significantly increased. We also studied the relevant mechanism of action and found that the novel nucleic acid complex promoted osteogenic differentiation of mesenchymal stem cells by activating the canonical Wnt signaling pathway.ConclusionsThis study may provide a new research direction for the application of novel nucleic acid nanomaterials in bone tissue regeneration.

MiR‐26a‐tetrahedral framework nucleic acids mediated osteogenesis of adipose‐derived mesenchymal stem cells.     

ClC‐c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila

ObjectivesAdult stem cells uphold a delicate balance between quiescent and active states, which is crucial for tissue homeostasis. Whereas many signalling pathways that regulate epithelial stem cells have been reported, many regulators remain unidentified.Materials and MethodsFlies were used to generate tissue‐specific gene knockdown and gene knockout. qRT‐PCR was used to assess the relative mRNA levels. Immunofluorescence was used to determine protein localization and expression patterns. Clonal analyses were used to observe the phenotype. RNA‐seq was used to screen downstream mechanisms.ResultsHere, we report a member of the chloride channel family, ClC‐c, which is specifically expressed in Drosophila intestinal stem/progenitor cells and regulates intestinal stem cell (ISC) proliferation under physiological conditions and upon tissue damage. Mechanistically, we found that the ISC loss induced by the depletion of ClC‐c in intestinal stem/progenitor cells is due to inhibition of the EGFR signalling pathway.ConclusionOur findings reveal an ISC‐specific function of ClC‐c in regulating stem cell maintenance and proliferation, thereby providing new insights into the functional links among the chloride channel family, ISC proliferation and tissue homeostasis.     

5‐Fluorouracil efficacy requires anti‐tumor immunity triggered by cancer‐cell‐intrinsic STING

5‐Fluorouracil (5‐FU) is a widely used chemotherapeutic drug, but the mechanisms underlying 5‐FU efficacy in immunocompetent hosts in vivo remain largely elusive. Through modeling 5‐FU response of murine colon and melanoma tumors, we report that effective reduction of tumor burden by 5‐FU is dependent on anti‐tumor immunity triggered by the activation of cancer‐cell‐intrinsic STING. While the loss of STING does not induce 5‐FU resistance in vitro, effective 5‐FU responsiveness in vivo requires cancer‐cell‐intrinsic cGAS, STING, and subsequent type I interferon (IFN) production, as well as IFN‐sensing by bone‐marrow‐derived cells. In the absence of cancer‐cell‐intrinsic STING, a much higher dose of 5‐FU is needed to reduce tumor burden. 5‐FU treatment leads to increased intratumoral T cells, and T‐cell depletion significantly reduces the efficacy of 5‐FU in vivo. In human colorectal specimens, higher STING expression is associated with better survival and responsiveness to chemotherapy. Our results support a model in which 5‐FU triggers cancer‐cell‐initiated anti‐tumor immunity to reduce tumor burden, and our findings could be harnessed to improve therapeutic effectiveness and toxicity for colon and other cancers.     

Gastric stem cells promote inflammation and gland remodeling in response to Helicobacter pylori via Rspo3‐Lgr4 axis

Helicobacter pylori is a pathogen that colonizes the stomach and causes chronic gastritis. Helicobacter pylori can colonize deep inside gastric glands, triggering increased R‐spondin 3 (Rspo3) signaling. This causes an expansion of the “gland base module,” which consists of self‐renewing stem cells and antimicrobial secretory cells and results in gland hyperplasia. The contribution of Rspo3 receptors Lgr4 and Lgr5 is not well explored. Here, we identified that Lgr4 regulates Lgr5 expression and is required for H. pylori‐induced hyperplasia and inflammation, while Lgr5 alone is not. Using conditional knockout mice, we reveal that R‐spondin signaling via Lgr4 drives proliferation of stem cells and also induces NF‐κB activity in the proliferative stem cells. Upon exposure to H. pylori, the Lgr4‐driven NF‐κB activation is responsible for the expansion of the gland base module and simultaneously enables chemokine expression in stem cells, resulting in gland hyperplasia and neutrophil recruitment. This demonstrates a connection between R‐spondin‐Lgr and NF‐κB signaling that links epithelial stem cell behavior and inflammatory responses to gland‐invading H. pylori.     

Cancer‐secreted miRNAs regulate amino‐acid‐induced mTORC1 signaling and fibroblast protein synthesis

Metabolic reprogramming of non‐cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer‐derived metabolic and non‐metabolic factors, is an emerging aspect of cancer–host interaction. We show that in normal and cancer‐associated fibroblasts, breast cancer‐secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer‐derived miR‐105 and miR‐204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re‐feeding, ¹³C‐arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer‐derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR‐105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re‐prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma‐produced proteins during periodic nutrient fluctuations.     

Taurine represses age‐associated gut hyperplasia in Drosophila via counteracting endoplasmic reticulum stress

As they age, adult stem cells become more prone to functional decline, which is responsible for aging‐associated tissue degeneration and diseases. One goal of aging research is to identify drugs that can repair age‐associated tissue degeneration. Multiple organ development‐related signaling pathways have recently been demonstrated to have functions in tissue homeostasis and aging process. Therefore, in this study, we tested several chemicals that are essential for organ development to assess their ability to delay intestinal stem cell (ISC) aging and promote gut function in adult Drosophila. We found that taurine, a free amino acid that supports neurological development and tissue metabolism in humans, represses ISC hyperproliferation and restrains the intestinal functional decline seen in aged animals. We found that taurine represses age‐associated ISC hyperproliferation through a mechanism that eliminated endoplasmic reticulum (ER) stress by upregulation of the target genes of unfolded protein response in the ER (UPR^ER) and inhibiting the c‐Jun N‐terminal kinase (JNK) signaling. Our findings show that taurine plays a critical role in delaying the aging process in stem cells and suggest that it may be used as a natural compound for the treatment of age‐associated, or damage‐induced intestinal dysfunction in humans.     

B‐cell capacity for differentiation changes with age

BackgroundAge‐related immune deficiencies are thought to be responsible for increased susceptibility to infection in older adults, with alterations in lymphocyte populations becoming more prevalent over time. The loss of humoral immunity in ageing was attributed to the diminished numbers of B cells and the reduced ability to generate immunoglobulin.AimsTo compare the intrinsic B‐cell capacity for differentiation into mature plasma cells (PCs), between young and old donors, using in vitro assays, providing either effective T‐cell help or activation via TLR engagement.MethodsB cells were isolated from healthy individuals, in younger (30–38 years) and older (60–64 years) donors. An in vitro model system of B‐cell differentiation was used, analysing 5 differentiation markers by flow cytometry, under T‐dependent (TD: CD40/BCR stimulation) or T‐independent (TI: TLR7/BCR activation) conditions. Antibody secretion was measured by ELISA and gene expression using qPCR.ResultsTI and TD differentiation resulted in effective proliferation of B cells followed by their differentiation into PC. B‐cell‐executed TI differentiation was faster, all differentiation marker and genes being expressed earlier than under TD differentiation (day 6), although generating less viable cells and lower antibody levels (day 13). Age‐related differences in B‐cell capacity for differentiation were minimal in TD differentiation. In contrast, in TI differentiation age significantly affected proliferation, viability, differentiation, antibody secretion and gene expression, older donors being more efficient.ConclusionAltogether, B‐cell differentiation into PC appeared similar between age groups when provided with T‐cell help, in contrast to TI differentiation, where multiple age‐related changes suggest better capacities in older donors. These new findings may help explain the emergence of autoantibodies in ageing.     

Tnfaip2/exoc3‐driven lipid metabolism is essential for stem cell differentiation and organ homeostasis

Lipid metabolism influences stem cell maintenance and differentiation but genetic factors that control these processes remain to be delineated. Here, we identify Tnfaip2 as an inhibitor of reprogramming of mouse fibroblasts into induced pluripotent stem cells. Tnfaip2 knockout impairs differentiation of embryonic stem cells (ESCs), and knockdown of the planarian para‐ortholog, Smed‐exoc3, abrogates in vivo tissue homeostasis and regeneration—processes that are driven by somatic stem cells. When stimulated to differentiate, Tnfaip2‐deficient ESCs fail to induce synthesis of cellular triacylglycerol (TAG) and lipid droplets (LD) coinciding with reduced expression of vimentin (Vim)—a known inducer of LD formation. Smed‐exoc3 depletion also causes a strong reduction of TAGs in planarians. The study shows that Tnfaip2 acts epistatically with and upstream of Vim in impairing cellular reprogramming. Supplementing palmitic acid (PA) and palmitoyl‐L‐carnitine (the mobilized form of PA) restores the differentiation capacity of Tnfaip2‐deficient ESCs and organ maintenance in Smed‐exoc3‐depleted planarians. Together, these results identify a novel role of Tnfaip2 and exoc3 in controlling lipid metabolism, which is essential for ESC differentiation and planarian organ maintenance.     

Identification of SRY‐box 30 as an age‐related essential gatekeeper for male germ‐cell meiosis and differentiation

Although important factors governing the meiosis have been reported in the embryonic ovary, meiosis in postnatal testis remains poorly understood. Herein, we first report that SRY‐box 30 (Sox30) is an age‐related and essential regulator of meiosis in the postnatal testis. Sox30‐null mice exhibited uniquely impaired testis, presenting the abnormal arrest of germ‐cell differentiation and irregular Leydig cell proliferation. In aged Sox30‐null mice, the observed testicular impairments were more severe. Furthermore, the germ‐cell arrest occurred at the stage of meiotic zygotene spermatocytes, which is strongly associated with critical regulators of meiosis (such as Cyp26b1, Stra8 and Rec8) and sex differentiation (such as Rspo1, Foxl2, Sox9, Wnt4 and Ctnnb1). Mechanistically, Sox30 can activate Stra8 and Rec8, and inhibit Cyp26b1 and Ctnnb1 by direct binding to their promoters. A different Sox30 domain required for regulating the activity of these gene promoters, providing a “fail‐safe” mechanism for Sox30 to facilitate germ‐cell differentiation. Indeed, retinoic acid levels were reduced owing to increased degradation following the elevation of Cyp26b1 in Sox30‐null testes. Re‐expression of Sox30 in Sox30‐null mice successfully restored germ‐cell meiosis, differentiation and Leydig cell proliferation. Moreover, the restoration of actual fertility appeared to improve over time. Consistently, Rec8 and Stra8 were reactivated, and Cyp26b1 and Ctnnb1 were reinhibited in the restored testes. In summary, Sox30 is necessary, sufficient and age‐associated for germ‐cell meiosis and differentiation in testes by direct regulating critical regulators. This study advances our understanding of the regulation of germ‐cell meiosis and differentiation in the postnatal testis.     

Effect of MiR‐100‐5p on proliferation and apoptosis of goat endometrial stromal cell in vitro and embryo implantation in vivo

The growth of endometrial stromal cells (ESCs) at implantation sites may be a potential factor affecting the success rate of embryo implantation. Incremental proofs demonstrated that ncRNAs (e.g. miRNAs, lncRNAs and circRNAs) were involved in various biological procedures, including proliferation and apoptosis. In this study, the role of miR‐100‐5p on proliferation and apoptosis of goat ESCs in vitro and embryo implantation in vivo was determined. The mRNA expression of miR‐100‐5p was significantly inhibited in the receptive phase (RE) rather than in the pre‐receptive phase (PE). Overexpression of miR‐100‐5p suppressed ESCs proliferation and induced apoptosis. The molecular target of MiR‐100‐5p, HOXA1, was confirmed by 3′‐UTR assays. Meanwhile, the product of HOXA1 mRNA RT‐PCR increased in the RE more than that in the PE. The HOXA1‐siRNA exerted significant negative effects on growth arrest. Instead, incubation of ESCs with miR‐100‐5p inhibitor or overexpressed HOXA1 promoted the cell proliferation. In addition, Circ‐9110 which acted as a sponge for miR‐100‐5p reversed the relevant biological effects of miR‐100‐5p. The intrinsic apoptosis pathway was suppressed in ESCs, revealing a crosstalk between Circ‐9110/miR‐100‐5p/HOXA1 axis, PI3K/AKT/mTOR, and ERK1/2 pathways. To further evaluate the progress in study on embryo implantation regulating mechanism of miR‐100‐5p in vivo, the pinopodes of two phases were observed and analysed, suggesting that, as similar as in situ, miR‐100‐5p was involved in significantly regulating embryo implantation in vivo. Mechanistically, miR‐100‐5p performed its embryo implantation function through regulation of PI3K/AKT/mTOR and ERK1/2 pathways by targeting Circ‐9110/miR‐100‐5p/HOXA1 axis in vivo.     

UBE2C triggers HIF‐1α‐glycolytic flux in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the most common malignancy in Taiwan. Therefore, refining the diagnostic sensitivity of biomarkers for early‐stage tumours and identifying therapeutic targets are critical for improving the survival rate of HNSCC patients. Metabolic reprogramming contributes to cancer development and progression. Metabolic pathways, specifically, play a crucial role in these diverse biological and pathological processes, which include cell proliferation, differentiation, apoptosis and carcinogenesis. Here, we investigated the role and potential prognostic value of the ubiquitin‐conjugating enzyme E2 (UBE2) family in HNSCC. Gene expression database analysis followed by tumour comparison with non‐tumour tissue showed that UBE2C was upregulated in tumours and was associated with lymph node metastasis in HNSCC patients. Knockdown of UBE2C significantly reduced the invasion/migration abilities of SAS and CAL27 cells. UBE2C modulates glycolysis pathway activation and HIF‐1α expression in SAS and CAL27 cells. CoCl₂ (HIF‐1α inducer) treatment restored the expression of glycolytic enzymes and the migration/invasion abilities of UBE2C knockdown cells. Based on our findings, UBE2C expression mediates HIF‐1α activation, increasing glycolysis pathway activation and the invasion/migration abilities of cancer cells. UBE2C may be an independent prognostic factor and a therapeutic target in HNSCC.     

Centrosome linker protein C‐Nap1 maintains stem cells in mouse testes

The centrosome linker component C‐Nap1 (encoded by CEP250) anchors filaments to centrioles that provide centrosome cohesion by connecting the two centrosomes of an interphase cell into a single microtubule organizing unit. The role of the centrosome linker during development of an animal remains enigmatic. Here, we show that male CEP250 ^−/− mice are sterile because sperm production is abolished. Premature centrosome separation means that germ stem cells in CEP250 ^−/− mice fail to establish an E‐cadherin polarity mark and are unable to maintain the older mother centrosome on the basal site of the seminiferous tubules. This failure prompts premature stem cell differentiation in expense of germ stem cell expansion. The concomitant induction of apoptosis triggers the complete depletion of germ stem cells and consequently infertility. Our study reveals a role for centrosome cohesion in asymmetric cell division, stem cell maintenance, and fertility.     

Genome‐wide selective detection of Mile red‐bone goat using next‐generation sequencing technology

The ecotype population of goats (Capra hircus) was created by long‐term artificial selection and natural adaptation. Mile red‐bone goat is an indigenous breed with visible red bones, and its special bone structure has received extensive attention. This study aimed to identify genetic variants and candidate genes associated with specific bone phenotypes using next‐generation sequencing technology (NGS). The results revealed that 31,828,206 single nucleotide polymorphisms (SNPs) were obtained from 72 goats (20 Mile red‐bone goats and 52 common goats) by NGS. A total of 100 candidate genes were identified on the basis top 1% window interaction from nucleotide diversity (π), π ratio (π _A/π _B), and pairwise fixation index (F _ST). Exactly 77 known signaling pathways were enriched. Specifically, three coding genes (NMNAT2, LOC102172983, and PNLIP) were annotated in the vitamin metabolism signaling pathways, and NCF2 was annotated to the osteoclast (OC) differentiation pathway. Furthermore, 5862 reliable copy number variations (CNVs) were obtained, and 14 and 24 genes were annotated with the top 1‰ CNV based on F _ST (>0.490) and V _ST (>0.527), respectively. Several pathways related to bone development and metabolism of exogenous substances in vivo, including calcium signaling pathway, OC differentiation, and glycerophospholipid metabolism, were annotated. Specifically, six genes from 19 candidate CNVs, which were obtained by interaction of the top 1‰ CNVs with F _ST and V _ST, were annotated to mucin‐type O‐glycan biosynthesis and metabolic pathways. Briefly, the results implied that pseudopurpurin and specific genetic variants work together to contribute to the red‐bone color and specific bone structure of Mile red‐bone goat. This study is helpful to understanding the genetic basis of the unique bone phenotype of Mile red‐bone goats.     

DJ‐1 depletion prevents immunoaging in T‐cell compartments

Decline in immune function during aging increases susceptibility to different aging‐related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naïve/memory T‐cell subpopulations, still remain largely elusive. Here, we show that loss of DJ‐1 encoded by PARK7/DJ‐1, causing early‐onset familial Parkinson’s disease (PD), unexpectedly diminished signs of immunoaging in T‐cell compartments of both human and mice. Compared with two gender‐matched unaffected siblings of similar ages, the index PD patient with DJ‐1 deficiency showed a decline in many critical immunoaging features, including almost doubled non‐senescent T cells. The observation was further consolidated by the results in 45‐week‐old DJ‐1 knockout mice. Our data demonstrated that DJ‐1 regulates several immunoaging features via hematopoietic‐intrinsic and naïve‐CD8‐intrinsic mechanisms. Mechanistically, DJ‐1 depletion reduced oxidative phosphorylation (OXPHOS) and impaired TCR sensitivity in naïve CD8 T cells at a young age, accumulatively leading to a reduced aging process in T‐cell compartments in older mice. Our finding suggests an unrecognized critical role of DJ‐1 in regulating immunoaging, discovering a potent target to interfere with immunoaging‐ and aging‐associated diseases.