Expression Levels Of Cyclin D1 Research Articles

Neuromedin S regulates goat ovarian granulosa cell proliferation and steroidogenesis via endoplasmic reticulum Ca2+-YAP1-ATF4-c-Jun pathway.

Neuromedin S (NMS) plays key roles in reproductive regulation, while its function and mechanism in follicular development remain unclear. The current study aims to investigate the specific role and mechanisms of NMS and its receptors in regulating the proliferation and steroidogenesis of ovarian granulosa cells (GCs). Phenotypically, a certain concentration of NMS addition promoted the proliferation and estrogen production of goat GCs, accompanied by an increase in the G1/S cell population and upregulation of the expression levels of cyclin D1, cyclin dependent kinase 6, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450, family 11, subfamily A, polypeptide 1, while the effects of NMS treatment were effectively hindered by knockdown of neuromedin U receptor type 2 (NMUR2). Mechanistically, activation of NMUR2 with NMS maintained endoplasmic reticulum (ER) calcium (Ca2+) homeostasis by triggering the PLCG1-IP3R pathway, which helped preserve ER morphology, sustained an appropriate level of endoplasmic reticulum unfoldedprotein response (UPRer), and suppressed the nuclear translocation of activating transcription factor 4. Moreover, NMS maintained intracellular Ca2+ homeostasis to activate the calmodulin 1-large tumor suppressor kinase 1pathway, ultimately orchestrating the regulation of goat GC proliferation and estrogen production through the Yes1 associated transcriptional regulator-ATF4-c-Jun pathway. Crucially, the effects of NMS were mitigated by concurrent knockdown of the NMUR2 gene. Collectively, these data suggest that activation of NMUR2 by NMS enhances cell proliferation and estrogen production in goat GCs through modulating the ER and intracellular Ca2+ homeostasis, leading to activation of the YAP1-ATF4-c-Jun pathway. These findings offer valuable insights into the regulatory mechanisms involved in follicular growth and development, providing a novel perspective for future research.

Neuromedin S regulates goat ovarian granulosa cell proliferation and steroidogenesis via endoplasmic reticulum Ca2+-YAP1-ATF4-c-Jun pathway.

Neuromedin S (NMS) plays key roles in reproductive regulation, while its function and mechanism in follicular development remain unclear. The current study aims to investigate the specific role and mechanisms of NMS and its receptors in regulating the proliferation and steroidogenesis of ovarian granulosa cells (GCs). Phenotypically, a certain concentration of NMS addition promoted the proliferation and estrogen production of goat GCs, accompanied by an increase in the G1/S cell population and upregulation of the expression levels of cyclin D1, cyclin dependent kinase 6, steroidogenic acute regulatory protein, cytochrome P450, family 11, subfamily A, polypeptide 1, 3beta-hydroxysteroid dehydrogenase, and cytochrome P450, family 11, subfamily A, polypeptide 1, while the effects of NMS treatment were effectively hindered by knockdown of neuromedin U receptor type 2 (NMUR2). Mechanistically, activation of NMUR2 with NMS maintained endoplasmic reticulum (ER) calcium (Ca2+) homeostasis by triggering the PLCG1-IP3R pathway, which helped preserve ER morphology, sustained an appropriate level of endoplasmic reticulum unfoldedprotein response (UPRer), and suppressed the nuclear translocation of activating transcription factor 4. Moreover, NMS maintained intracellular Ca2+ homeostasis to activate the calmodulin 1-large tumor suppressor kinase 1pathway, ultimately orchestrating the regulation of goat GC proliferation and estrogen production through the Yes1 associated transcriptional regulator-ATF4-c-Jun pathway. Crucially, the effects of NMS were mitigated by concurrent knockdown of the NMUR2 gene. Collectively, these data suggest that activation of NMUR2 by NMS enhances cell proliferation and estrogen production in goat GCs through modulating the ER and intracellular Ca2+ homeostasis, leading to activation of the YAP1-ATF4-c-Jun pathway. These findings offer valuable insights into the regulatory mechanisms involved in follicular growth and development, providing a novel perspective for future research.

Effect of neratinib on HER2 mRNA stability via hsa-miR-23a-5p and efficacy of CDK4/6 inhibitors in HER2-low breast cancer.

e12550 Background: Traditional HER2-negative breast cancer sometimes includes HER2-low components, and CDK4/6 inhibitors are the first- and second-line treatments for advanced HER2-low breast cancer. Nonetheless, little is understood about how the stability of HER2 mRNA in HER2-low breast cancer affects the effectiveness of CDK4/6 inhibitor therapy and the associated intervention techniques. Methods: In order to investigate the impact of HER2-low on the effectiveness of CDK4/6 inhibitor in treating breast cancer, we first used HER2-0 and HER2-low breast cancer cells/mice as the study object and assessed the anti-proliferation effect of CDK4/6 inhibitor using CCK8 technique and monoclonal formation experiment. Second, we increased the treatment of anti-HER2 inhibitor neratinib and assessed its impact on the efficacy of HER2 low breast cancer CDK4/6 inhibitors. By reducing the expression of HER2 in HER2 low breast cancer cells, we analyzed and determined whether HER2 is the primary pathway that influences the poor efficacy of HER2 low breast cancer CDK4/6 inhibitors; At last, actinomycin D was used to detect the mRNA stability of HER2 in the treatment of HER2-low breast cancer by neratinib against CDK4/6 inhibitors, and then microRNA sequencing and luciferase reporter gene detection technology were used. To investigate the specific mechanism of action of neratinib on the mRNA stability of HER2 in the treatment of HER2-low breast cancer by CDK4/6 inhibitors. Results: Our study found that treatment with CDK4/6 inhibitors was significantly less effective against HER2-low breast cancer compared to HER2-0 subtype (P=0.005). Notably, in HER2-low subtype cells, HER2 knockdown significantly inhibited cell proliferation (P<0.001), G1 phase cell cycle arrest (P=0.001), and downregulated cyclin D1 expression and CDK4 protein levels (P=0.007, P=0.003). Second, it was found that in the cell and animal models of HER2-low breast cancer, the addition of the CDK4/6 inhibitor drug neratinib effectively reduced the stability of HER2 mRNA (P=0.001), inhibited the cell cycle of G1 phase (P=0.005). In addition, we observed that lowering the dose of neratinib to 1/2 (20 mg/kg) remained effective and well tolerated (P=0.002). Finally, we found that neratinib can reduce the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3'UTR region of HER2 (P<0.001), thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. Conclusions: We found that low expression of HER2 reduced the efficacy of CDK4/6 inhibitors in HER2-low breast cancer; Neratinib reduces the mRNA stability of HER2 by binding hsa-miR-23a-5p to the 3 'UTR region of HER2, thereby improving the efficacy of CDK4/6 inhibitors in HER2-low breast cancer. These findings provide a tolerable and effective two-drug combination regimen for patients with clinical HER2-low breast cancer.

PL-hMSC and CH-hMSC derived soluble factors inhibit proliferation but improve hGBM cell migration by activating TGF-β and inhibiting Wnt signaling.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. GBM resists most chemotherapeutic agents, resulting in a high mortality rate in patients. Human mesenchymal stem cells (hMSCs), which are parts of the cancer stroma, have been shown to be involved in the development and progression of GBM. However, different sources of hMSCs might affect GBM cells differently. In the present study, we established hMSCs from placenta (PL-hMSC) and chorion (CH-hMSC) to study the effects of their released soluble factors on the proliferation, migration, invasion, gene expression, and survival of human GBM cells, U251. We found that the soluble factors derived from CH-hMSCs and PL-hMSCs suppressed the proliferation of U251 cells in a dose-dependent manner. In contrast, soluble factors derived from both hMSC sources increased U251 migration without affecting their invasive property. The soluble factors derived from these hMSCs decreased the expression levels of CyclinD1, E2Fs and MYC genes that promote GBM cell proliferation but increased the expression level of TWIST gene, which promotes EMT and GBM cell migration. The functional study suggests that both hMSCs might exert their effects, at least in part, by activating TGF-β and suppressing Wnt/β-catenin signaling in U251 cells. Our study provides a better understanding of the interaction between GBM cells and gestational tissue-derived hMSCs. This knowledge might be used to develop safer and more effective stem cell therapy that improves the survival and quality of life of patients with GBM by manipulating the interaction between hMSCs and GBM cells.

Mechanism of Si Ni San Combined with Astragalus in Treating Hepatic Fibrosis: A Network Pharmacology and Molecular Docking Study.

Si Ni San combined with Astragalus (SNSQ) has demonstrated significant efficacy in the treatment of hepatic fibrosis (HF), as confirmed by clinical practice. However, its pharmacological mechanism remains unclear. This study employs network pharmacology to identify key targets and proteins for molecular docking. Additionally, animal experiments were conducted to validate the network pharmacology results, providing further insights into the mechanism of SNSQ in treating HF. Effective compounds of SNSQ were screened from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Encyclopedia of Traditional Chinese Medicine (ETCM) databases. Molecular formula structures of these effective compounds were obtained from the PubChem database. Partial target proteins with a probability greater than 0.6 were sourced from the SWISS database. Uniprot IDs corresponding to these target proteins were retrieved from the SUPERPRED database. The remaining target proteins of the compounds were obtained from the Uniprot database based on the Uniprot IDs. The drug target proteins were then summarized. Target points related to HF were selected from the GeneCards and OMIM databases. Common target points were identified in the Venn diagram and imported into Cytoscape 3.9.1 software to construct the "SNSQ-effective compound-target pathway-HF" network. AutoDock software was used for molecular docking of compounds and target proteins with high-degree values. The common target points underwent GO function enrichment and KEGG pathway enrichment analysis using the DAVID database. An HF rat model was established, and serum AST and ALT activities were measured. The Hyp assay kit was utilized to detect the Hyp content in liver tissue. To the transcription levels of pro-inflammatory factors (IL-1β, TNF-α, IL-6) and anti-inflammatory factors (IL-10, TGF-β1, IL-4) in rat serum and liver.IL-1β, TNF-α, IL-10, and TGF-β1 were chosen for validation through ELISA. Western blotting and qRT-PCR were used to assess the expression of related proteins, namely NFKB1, NF-κBp65, NF-κBp50, α-SMA, and Col-1 in liver tissue. qRT-PCR was also employed to study the expression of ECM synthesis and proliferation-related genes, including Cyclin D1, TIMP1, COL1A1 in HSC-T6 cells and rat liver tissue, as well as the inhibition of the ECM-related gene MMP13 in HSC-T6 cells and rat liver tissue. A total of 16 valid compounds were predicted, with kaempferol, sitosterol, and isorhamnetin exhibiting high-degree values. KEGG enrichment analysis revealed that the target genes of SNSQ were enriched in multiple pathological pathways, with the NF-Kappa B signaling pathway being predominant. Molecular docking simulations indicated strong affinities between SNSQ's primary components-kaempferol, sitosterol, isorhamnetin-and NFKB1. Experimental results demonstrated significant reductions in AST, ALT, and Hyp levels in the SNSQ group. Pro-inflammatory factors (IL-1β, TNF-ɑ) were markedly reduced, while anti-inflammatory factors (IL-10, TGF-β1) were substantially increased. The protein expression and transcription levels of α-SMA and Col-1 were significantly decreased, whereas those of NFKB1, NF-κBp65, and NF-κBp50 were notably elevated. mRNA expression levels of Cyclin D1, TIMP1, COL1A1 in HSC-T6 cells and rat liver tissue were significantly decreased, whereas MMP13 mRNA expression level was significantly increased. Treatment of HF with SNSQ involves multiple targets and pathways, with a close association with the overexpression of NFKB1 and activation of the NF-Kappa B signaling pathway. Its mechanism is closely linked to the activation of inflammatory responses, HSC activation, and proliferation.

Calcitonin gene‑related peptide alleviates hyperoxia‑induced human alveolar cell injury via the CGRPR/TRPV1/Ca2+ axis.

Although exogenous calcitonin gene‑related peptide (CGRP) protects against hyperoxia‑induced lung injury (HILI), the underlying mechanisms remain unclear. The present study attempted to elucidate the molecular mechanism by which CGRP protects against hyperoxia‑induced alveolar cell injury. Human alveolar A549 cells were treated with 95% hyperoxia to establish a hyperoxic cell injury model. ELISA was performed to detect the CGRP secretion. Immunofluorescence, quantitative (q)PCR, and western blotting were used to detect the expression and localization of CGRP receptor (CGRPR) and transient receptor potential vanilloid 1 (TRPV1). Cell counting kit‑8 and flow cytometry were used to examine the proliferation and apoptosis of treated cells. Digital calcium imaging and patch clamp were used to analyze the changes in intracellular Ca2+ signaling and membrane currents induced by CGRP in A549 cells. The mRNA and protein expression levels of Cyclin D1, proliferating cell nuclear antigen (PCNA), Bcl‑2 and Bax were detected by qPCR and western blotting. The expression levels of CGRPR and TRPV1 in A549 cells were significantly downregulated by hyperoxic treatment, but there was no significant difference in CGRP release between cells cultured under normal air and hyperoxic conditions. CGRP promoted cell proliferation and inhibited apoptosis in hyperoxia, but selective inhibitors of CGRPR and TRPV1 channels could effectively attenuate these effects; TRPV1 knockdown also attenuated this effect. CGRP induced Ca2+ entry via the TRPV1 channels and enhanced the membrane non‑selective currents through TRPV1 channels. The CGRP‑induced increase in intracellular Ca2+ was reduced by inhibiting the phospholipase C (PLC)/protein kinase C (PKC) pathway. Moreover, PLC and PKC inhibitors attenuated the effects of CGRP in promoting cell proliferation and inhibiting apoptosis. In conclusion, exogenous CGRP acted by inversely regulating the function of TRPV1 channels in alveolar cells. Importantly, CGRP protected alveolar cells from hyperoxia‑induced injury via the CGRPR/TRPV1/Ca2+ axis, which may be a potential target for the prevention and treatment of the HILI.

ASPSCR1::TFE3 Drives Alveolar Soft Part Sarcoma by Inducing Targetable Transcriptional Programs.

Alveolar soft part sarcoma (ASPS) is a rare mesenchymal malignancy driven by the ASPSCR1::TFE3 fusion. A better understanding of the mechanisms by which this oncogenic transcriptional regulator drives cancer growth is needed to help identify potential therapeutic targets. Here, we characterized the transcriptional and chromatin landscapes of ASPS tumors and preclinical models, identifying the essential role of ASPSCR1::TFE3 in tumor cell viability by regulating core transcriptional programs involved in cell proliferation, angiogenesis, and mitochondrial biology. ASPSCR1::TFE3 directly interacted with key epigenetic regulators at enhancers and promoters to support ASPS-associated transcription. Among the effector programs driven by ASPSCR1::TFE3, cell proliferation was driven by high levels of cyclin D1 expression. Disruption of cyclin D1/CDK4 signaling led to loss of ASPS proliferative capacity, and combined inhibition of CDK4/6 and angiogenesis halted tumor growth in xenografts. These results define the ASPS oncogenic program, reveal mechanisms by which ASPSCR1::TFE3 controls tumor biology, and identify a strategy for therapeutically targeting tumor cell-intrinsic vulnerabilities.

Knockdown of circXPO1 inhibits the development of oral squamous cell carcinoma cells.

Circular RNAs (circRNAs) have emerged as pivotal regulators of cellular processes in human malignancies, including oral squamous cell carcinoma (OSCC). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect RNA expression levels of circXPO1, miR-524-5p and cyclin D1 (CCND1). Colony formation assay and 5-Ethynyl-2'-deoxyuridine (EdU) assay were performed to analyze cell proliferation, while transwell assay was carried out to investigate the cell migration and invasion. Cell apoptosis was assessed by flow cytometry. Protein expression analysis was implemented by Western blot assay. Additionally, lactate production and glucose consumption were investigated using a lactate assay kit and glucose assay kit, respectively. The invivo tumorigenic potential of circXPO1 was evaluated using a xenograft mouse model assay. Elevated levels of circXPO1 and CCND1, alongside reduced miR-524-5p expression were decreased in OSCC tissues and cells. Knockdown of circXPO1 in OSCC cells inhibited their proliferative, migratory and invasive capacities, as well as glycolysis, prompting apoptosis. Moreover, circXPO1 silencing hindered tumor growth invivo. MiR-524-5p could be sequestered by circXPO1, and its inhibition could counteract the beneficial effects of circXPO1 knockdown on OSCC progression. Knockdown of circXPO1 inhibited OSCC progression by up-regulating miR-524-5p and down-regulating CCND1 expression, which might provide potential targets for OSCC treatment.

CircDDX17 targets miR-223-3p / RIP3 to regulate the proliferation and apoptosis of non-small cell lung cancer cells

Objective: To explore the molecular mechanism of circDDX17 regulating the proliferation and apoptosis of non-small cell lung cancer cells by targeting the miR-223-3p/RIP3 molecular axis. Methods: The expression levels of circDDX17, miR-223-3p, and RIP3 in human normal lung epithelial cell lines BEAS-2B and non-small cell lung cancer cells H1299, A549, and H446 were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The plasmids of pcDNA, pcDNA-circDDX17, anti-miR-con, anti-miR-223-3p, pcDNA-circDDX17 and miR-con, pcDNA-circDDX17 and miR-223-3p mimics were transfected into H1299 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay was used to detect the cell proliferation. Flow cytometry was used to detect the cell cycle and cell apoptosis. Plate cloning experiment was used to detect cell proliferation ability. The dual luciferase report experiment was applied to verify the targeting relationship between miR-223-3p with circDDX17 and RIP3. Western blot was used to detect the protein expression of cyclinD1, CDK2, cleaved caspase-3 and Bax. Results: The expression levels of circDDX17 and RIP3 mRNA in H1299, A549, and H446 cells were significantly reduced (P<0.05), the expression level of miR-223-3p mRNA was significantly increased (P<0.05) compared with BEAS-2B. The cell viability [(69.46±4.68)%], the number of cell clones (83.49±7.86), the proportion of cells in S phase [(22.52±1.41) %], the protein expression levels of cyclinD1 and CDK2 in PCDNa-CircDDX17 group were lower than those in pcDNA group [(97.54±7.72)%, 205.03±13.37, (28.69±1.49)%, respectively, P<0.05], while the percentage of G0/G1 phase cells [(64.45±3.56)%], apoptosis rate [(18.36±1.63)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17 group were higher than those of pcDNA group [(51.33±2.76) % and (5.21±0.54) %, respectively, P<0.05]. The viability [(72.64±5.44)%], the number of cell clones (78.16±8.23), the proportion of S-stage cells [(21.34±1.59) %], the protein expression levels of CyclinD1 and CDK2 in anti-miR-223-3p group were lower than those in anti-miR-con group [(103.47±6.25)%, 169.32±14.53, (28.43±1.26)%, respectively, P<0.05]. Percentage of G0/G1 phase cells [(62.86±3.28)%], apoptosis rate [(14.64±1.67)%], the protein expression levels of cleaved caspase-3 and Bax in the anti-miR-223-3p group were higher than those of anti-miR-con group [(51.33±2.71)% and (4.83±0.39)%, respectively, P<0.05]. MiR-223-3p has complementary sites with circDDX17 or RIP3. The viability [(135.45±9.28)%], the number of cell clones (174.64±10.68), the proportion of S-phase cells [(26.39±2.25)%], the protein expression levels of cyclinD1 and CDK2 in pcDNA-circDDX17+miR-223-3p group were higher than those in pcDNA-circDDX17+miR-con group [(101.56±6.68)%, 107.65±7.62, (21.64±1.72)%, P<0.05]. Percentage of G0/G1 phase cells [(56.64±2.76)%], apoptosis rate [(8.34±0.76)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17+miR-223-3p group were lower than those of pcDNA-circDDX17+miR-con group [(64.03±3.48)% and (15.21±1.18)%, respectively, P<0.05]. Conclusion: circDDX17 could inhibit the proliferation and induce apoptosis of non-small cell lung cancer cells via targeting the miR-223-3p / RIP3 molecular axis.

Gallic acid ameliorates endometrial hyperplasia through the inhibition of the PI3K/AKT pathway and the down-regulation of cyclin D1 expression

BackgroundGallic acid (GA) is an organic compound with phenolic properties that occurs naturally and can be found in Guizhi Fuling capsules, showcasing a wide range of biological functionalities. PurposeThe objective of this study was to examine the influence of GA on endometrial hyperplasia (EH) and elucidate its underlying mechanism. MethodsInitially, the induction of EH was achieved by administering estradiol to mice via continuous subcutaneous injection for a duration of 21 days. Concurrently, GA treatment was administered, and subsequently, the uterine tissue structure was assessed using hematoxylin and eosin (H&E) staining. Following this, the proliferation of human endometrial cells treated by GA was determined utilizing the CCK-8 method. Furthermore, network pharmacology and single-cell-RNA-seq data were employed to identify the target of GA action. In addition, we will employ immunofluorescence (IF), immunohistochemistry (IHC), flow cytometry, western blot and RT-qPCR methodologies to investigate the impact of GA on the expression level of cyclin D1, PI3K, p-PI3K, AKT, p-AKT. ResultsGA treatment ameliorated histopathological alterations in the uterus and suppress proliferation. Estradiol stimulation can activate the PI3K/AKT pathway, leading to up-regulation of cyclin D1 expression, whereas GA treatment results in down-regulation of its expression. ConclusionsThe expression of cyclin D1 is down-regulated by GA through the inhibition of the PI3K/AKT pathway, effectively mitigating estradiol-induced EH in mice.

Mechanisms of vemurafenib-induced anti-tumor effects in ATC FRO cells

BackgroundAnaplastic Thyroid Carcinoma (ATC) is a rare and deadly malignant tumor in humans. It is prone to developing resistance to radiotherapy and chemotherapy. Molecular targeted therapy offers a novel way to treat ATC. The BRAF mutation is closely associated with many cancers, including thyroid carcinoma. Vemurafenib, a small-molecule inhibitor, is specifically designed to target the mutant serine/threonine kinase BRAF. The objective of this study is to elucidate the regulatory mechanisms underlying the effects of vemurafenib on human anaplastic thyroid carcinoma cell line FRO and to assess its potential therapeutic role. MethodsThe effects of vemurafenib on the proliferation of FRO cells were assessed by the CCK-8 method and Colony-forming assay. Transwell chambers and scratch tests were employed to examine the impact of vemurafenib on the invasion and migration of FRO cells. Apoptosis and cycle distribution of FRO cells were analyzed by tunel assay and flow cytometry. The effects of vemurafenib on the expression of BRAF-activated non-protein coding RNA (BANCR), Bax, Bcl2, and E-cadherin were evaluated by qRT-PCR. Furthermore, the effects of vemurafenib on the expression of phosphoinositol-3-kinase (PI3K)/phosphoinositol-3-kinase (AKT) pathway-related proteins, BRAF, CyclinD1, Bcl-2, Bax, and E-cadherin proteins in FRO cells were investigated through the western-blot method. All experiments were conducted in three replicates. ResultsVemurafenib was observed to inhibit proliferation and induce apoptosis in a dose- and time-dependent manner (P < 0.05). The formation of FRO cell colonies, as well as migration and invasion, all showed a dose-dependent reduction (P < 0.05). Flow cytometric analysis indicated G0/G1 cell cycle arrest (P < 0.05). QRT-PCR revealed that vemurafenib could suppress the expression of BANCR and Bcl2 while increasing the expression of Bax and E-cadherin in a dose-dependent manner (P < 0.05). The protein expression levels of Bax and E-cadherin were up-regulated significantly, and the expression levels of BRAF, CyclinD1, Bcl-2, p-PI3K, p-AKT, and p-mTOR were markedly down-regulated with increasing concentrations of vemurafenib (P < 0.05). ConclusionsThe proliferation and metastasis of FRO cells can be suppressed by vemurafenib through the silencing of BRAF and BANCR expression, inhibition of PI3K/AKT signaling pathway activation, induction of apoptosis, and cell cycle arrest.

WNT5A regulatesthe proliferation, apoptosis and stemness of human stem Leydig cells via the β-catenin signaling pathway.

Stem Leydig cells (SLCs) are essential for maintaining normal spermatogenesis as the significant component of testis microenvironment and gonadal aging. Although progress has been achieved in the regulation of male germ cells in mammals and humans, it remains unknown about the genes and signaling pathways of human SLCs. Here we have demonstrated, for the first time, that WNT5A (Wnt family member 5a) mediates the proliferation, apoptosis, and stemness of human SLCs, namely NGFR+ Leydig cells. We revealed that NGFR+ Leydig cells expressed NGFR, PDGFRA, NES, NR2F2, and THY1, hallmarks for SLCs. RNA-sequencing showed that WNT5A was expressed at a higher level in human SLCs than non-SLCs, while immunohistochemistry and Western blots further illustrated that WNT5A was predominantly expressed in human SLCs. Notably, CCK-8, EdU and Western blots displayed that WNT5A enhanced the proliferation and DNA synthesis and retained stemness of human SLCs, whereas flow cytometry and TUNEL analyses demonstrated that WNT5A inhibited the apoptosis of these cells. WNT5A knockdown caused an increase in LC lineage differentiation of human SLCs and reversed the effect of WNT5A overexpression on fate decisions of human SLCs. In addition, WNT5A silencing resulted in the decreases in nuclear translocation of β-catenin and expression levels of c-Myc, CD44, and Cyclin D1. Collectively, these results implicate that WNT5A regulates theproliferation, apoptosis and stemness of human SLCs through the activation of the β-catenin signaling pathway. This study thus provides a novel molecular mechanism underlying the fate determinations of human SLCs, and it offers a new insight into the niche regulation of human testis.

Rapamycin inhibits B16 melanoma cell viability invitro and invivo by inducing autophagy and inhibiting the mTOR/p70‑S6k pathway.

Rapamycin is an immunosuppressant that has been shown to prevent tumor growth following organ transplantation. However, its exact mode of antitumor action remains unknown. The present study used the B16-F10 (B16) murine melanoma model to explore the antitumor mechanism of rapamycin, and it was revealed that rapamycin reduced B16 cell viability in vitro and in vivo. In addition, in vitro and in vivo, the results of western blotting showed that rapamycin reduced Bcl2 expression, and enhanced the protein expression levels of cleaved caspase 3 and Bax, indicating that it can induce the apoptosis of B16 melanoma cells. Furthermore, the results of cell cycle analysis and western blotting showed that rapamycin induced B16 cell cycle arrest in the G1 phase, based on the reduction in the protein expression levels of CDK1, cyclin D1 and CDK4, as well as the increase in the percentage of cells in G1 phase. Rapamycin also significantly increased the number of autophagosomes in B16 melanoma cells, as determined by transmission electron microscopy. Furthermore, the results of RT-qPCR and western blotting showed that rapamycin upregulated the protein expression levels of microtubule-associated protein light chain 3 (LC3) and Beclin-1, while downregulating the expression of p62 in vitro and in vivo, thus indicating that rapamycin could trigger cellular autophagy. The present study revealed that rapamycin in combination with chloroquine (CQ) further increased LC3 expression compared with that in the CQ group, suggesting that rapamycin induced an increase in autophagy in B16 cells. Furthermore, the results of western blotting showed that rapamycin blocked the phosphorylation of p70 ribosomal S6 kinase (p70-S6k) and mammalian target of rapamycin (mTOR) proteins in vitro and in vivo, thus suggesting that rapamycin may exert its antitumor effect by inhibiting the phosphorylation of the mTOR/p70-S6k pathway. In conclusion, rapamycin may inhibit tumor growth by inducing cellular G1 phase arrest and apoptosis. In addition, rapamycin may exert its antitumor effects by inducing the autophagy of B16 melanoma cells in vitro and in vivo, and the mTOR/p70-S6k signaling pathway may be involved in this process.

Activation of RhoA/ROCK2 signaling by hypoxia-inducible factor 1α in promoting tumor growth and metastasis in human colon cancer.

Recent evidence strongly suggests the profound role of the tumor microenvironment in cancer development and progression. A hypoxic microenvironment is widely acknowledged to be a typical feature of solid tumors, and altered hypoxia-inducible factor 1α (HIF-1α) expression has been associated with the formation and the progression of many solid tumors; however, the underlying mechanism of this relationship remains obscure. Clinical colorectal cancer tissue samples were collected to detect the differential expression of HIF-1α, Ras homolog family member A (RhoA), and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2). With hypoxic stress, small interfering RNA (siRNA) targeting HIF-1α, lentivirus transfection of RhoA was used to study the mechanism of HIF-1α and RhoA/ROCK2 signaling pathways in the growth and metastasis of colon cancer. According to Cell Counting Kit 8, wound-healing, and Transwell assays, HIF-1α expression activated the RhoA/ROCK2 pathway within colon cancer cell lines, accelerating their growth and expansion. In cells transfected with LV-RhoA, inactivating the RhoA/ROCK2 pathway with the specific inhibitor Y-27632 decreased tumor growth and metastasis under hypoxic conditions, while activating the RhoA/ROCK2 pathway restored these biological properties. The Western blot results showed that the expression levels of pMYPT1, cyclin D1, and MMP2 in the siRNA + LV-RhoA group were also significantly increased compared with those in the siRNA group. For the first time, this study demonstrated that HIF-1α can promote the growth and metastasis of colon cancer via directly affecting RhoA/ROCK2 signaling and thus represents a novel therapeutic target for colon cancer.

An Essential Role of c-Fos in Notch1-mediated Promotion of Proliferation of KSHV-Infected SH-SY5Y Cells.

This study aimed to investigate the influence of Notch1 on c-Fos and the effect of c-Fos on the proliferation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected neuronal cells. Real-time PCR and western blotting were used to determine c-Fos expression levels in KSHV-infected (SK-RG) and uninfected SH-SY5Y cells. C-Fos levels were measured again in SK-RG cells with or without Notch1 knockdown. Next, we measured c-Fos and p-c-Fos concentrations after treatment with the Notch1 γ-secretase inhibitor LY-411575 and the Notch1 activator Jagged-1. MTT and Ki-67 staining were used to evaluate the proliferation ability of cells after c-Fos levels downregulation. CyclinD1, CDK6, and CDK4 expression levels and cell cycle were analyzed by western blotting and flow cytometry, respectively. After the c-Fos intervention, the KSHV copy number and gene expression of RTA, LANA and K8.1 were analyzed by real-time TaqMan PCR. C-Fos was up-regulated in KSHV-infected SK-RG cells. However, the siRNA-mediated knockdown of Notch1 resulted in a significant decrease in the levels of c-Fos and p-c-Fos (P <0.01, P <0.001). Additionally, a decrease in Cyclin D1, CDK6, and CDK4 was also detected. The Notch1 inhibitor LY-411575 showed the potential to down-regulate the levels of c-Fos and p-c-Fos, which was consistent with Notch1 knockdown group (P <0.01), whereas the expression and phosphorylation of c-Fos were remarkably up-regulated by treatment of Notch1 activator Jagged-1 (P <0.05). In addition, our data obtained by MTT and Ki-67 staining revealed that the c-Fos down-regulation led to a significant reduction in cell viability and proliferation of the SK-RG cells (P <0.001). Moreover, FACS analysis showed that the cell cycle was arrested in the G0/G1 stage, and the expressions of Cyclin D1, CDK6, and CDK4 were down-regulated in the c-Fos-knockdown SK-RG cells (P <0.05). Reduction in total KSHV copy number and expressions of viral genes (RTA, LANA and K8.1) were also detected in c-Fos down-regulated SK-RG cells (P <0.05). Our findings strongly indicate that c-Fos plays a crucial role in the promotion of cell proliferation through Notch1 signaling in KSHV-infected cells. Furthermore, our results suggest that the inhibition of expression of key viral pathogenic proteins is likely involved in this process.

The effect and mechanism of low-molecular-weight heparin on the decidualization of stromal cells in early pregnancy

Objective This study aimed to analyze the effect of low-molecular-weight heparin (LMWH) on the decidualization of stromal cells in early pregnancy and explore the effect of LMWH on pregnancy outcomes. Methods Recurrent spontaneous abortion (RSA) mouse model (CBA/J × DBA/2) and normal pregnant mouse model (CBA/J × BALB/c) were established. The female mice were checked for a mucus plug twice daily to identify a potential pregnancy. When a mucus plug was found, conception was considered to have occurred 12 h previously. The pregnant mice were divided randomly into a normal pregnancy control group, an RSA model group, and an RSA + LMWH experimental group (n = 10 mice in each group). Halfway through the 12th day of pregnancy, the embryonic loss of the mice was observed; a real-time quantitative polymerase chain reaction was used to detect the messenger ribonucleic acid (mRNA) expressions of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) in the decidua of the mice. Additionally, the decidual tissues of patients with RSA and those of normal women in early pregnancy who required artificial abortion were collected and divided into an RSA group and a control group. Decidual stromal cells were isolated and cultured to compare cell proliferation between the two groups, and cellular migration and invasion were detected by membrane stromal cells. Western blotting was used to detect the protein expressions of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase- (MMP) 2, and MMP-7 in stromal cells treated with LMWH. Results Compared with the RSA group, LMWH significantly reduced the pregnancy loss rate in the RSA mice (p < 0.05). Compared with the RSA group, the LMWH + RSA group had significantly higher expression levels of PRL and IGFBP1 mRNA (p < 0.01). LMWH promoted the proliferation, migration, and invasion of human decidual stromal cells; compared with the control group, the expression levels of MMP-2, MMP-7, cyclin D1, and PCNA proteins in the decidual stromal cells of the LMWH group increased (p < 0.05). Conclusions The use of LMWH can improve pregnancy outcomes by enhancing the proliferation and migration of stromal cells in early pregnancy and the decidualization of stromal cells.

MiR-383-5p inhibits the proliferation and migration of lung adenocarcinoma cells by targeting SHMT2.

Purpose: To explore the effects of miR-383-5p and serine hydroxymethyltransferase 2 (SHMT2) on the proliferation and migration of lung adenocarcinoma cells. Methods: SHMT2 expression in lung adenocarcinoma and normal tissues was investigated using The Cancer Genome Atlas database. Immunohistochemical analysis was performed to confirm SHMT2 expression in lung adenocarcinoma and adjacent normal lung tissues. Bioinformatics analysis and luciferase reporter assays were used to analyze the relationship between miR-383-5p and SHMT2 expression. The protein expression levels of SHMT2, vimentin, N-cadherin, E-cadherin, Bcl-2, and cyclinD1 were analyzed using western blotting. The reverse transcription-quantitative polymerase chain reaction was used to detect SHMT2 knockdown efficiency, miR-383-5p overexpression, and inhibition efficiency. The proliferative ability of cells was detected using the Cell Counting Kit-8 assay. The Transwell assay was used to detect the migration ability of cells. Results: SHMT2 expression was significantly increased in patients with lung adenocarcinoma compared to that in control patients; the higher the SHMT2 expression the worse the outcomes were in patients with lung adenocarcinoma. SHMT2 knockdown inhibited the proliferation, migration, and epithelial-mesenchymal transition of lung adenocarcinoma A549 and H1299 cells. MiR-383-5p directly targeted and downregulated SHMT2 in A549 and H1299 cells. The effects of miRNA-383-5p on the proliferation and migration of these cells differed from those of SHMT2. Exogenous overexpression of SHMT2 reversed the miR-383-5p-induced proliferation and migration inhibition in A549 and H1299 cells. Conclusion: MiR-383-5p inhibits the proliferation and migration of lung adenocarcinoma cells by targeting and downregulating SHMT2.

The expression of Cox-2 and Cyclin D1 with clinicopathological characteristics in oral epithelial dysplasia: A retrospective study

Background: Oral mucosal cancers are often preceded by potentially malignant diseases that may exhibit diverse morphological and histological alterations. The expression of COX 2 has been related to tumor vascularization, dissemination, and survival in oral cancer studies. High levels of cyclin D1 expression to precancerous lesions and oral malignancies. This study was designed to evaluate and compare the immunohistochemical expression of the above-mentioned markers. Materials and Methods: 50 retrospective formalin fixed-paraffin embedded histopathologically confirmed blocks of oral epithelial dysplasia. Immunohistochemical staining was performed using monoclonal antibodies against COX-2 and Cyclin D1. Using a standard camera, photomicrographs of the slides were taken. Results: No significant difference in the age distribution of the patients studied, as well as age pivoting at the fifth and sixth decades with mean and standard deviation (53.36 2.63). There was no significant difference in gender among the studied patients. Furthermore, there was a highly significant difference in grade distribution. Conclusions: The COX-2 and Cyclin D1 markers may assist in diagnosing oral epithelial dysplasia based on severity. It's easy to gather image-analytic data on the stained tissue specimens.

OVEREXPRESSION OF CYCLIN D1 IN ORAL CANCER INCREASES TPF CHEMOTHERAPEUTIC DRUGS VIA ACTIVATING THE CASPASE-3 PATHWAY

Previous studies have indicated that induction chemotherapy can limit the possibility of early lesion eradication and lessen the severity of globally advanced tumors. This study aimed to ascertain if cyclin D1 expression in OSCC patients who were qualified for TPF induction chemotherapy might be regarded as a valid prognostic indicator for long-term outcomes. This study aimed to determine whether or not there was a connection between OSCC cell intervention and the production of cyclin D1 and chemosensitivity to TPF medicines through the apoptotic mechanism. The current work used immunohistochemistry to determine the expression of cyclin D1 in various tissues. To ascertain the survival rate of 232 people diagnosed with locally advanced oral squamous cell carcinoma (OSCC), the study utilized the log-rank test and Kaplan-Meier analysis. The participants in this historical clinical trial were observed for a mean duration of five years. Cytotoxicity assays were employed to assess the therapeutic effectiveness of TPF chemotherapy drugs. The activity of caspase-3 and PARP was evaluated in the HB96, CAL27, and HN30 cell lines following cyclin D1 inhibition, respectively. Long-term clinical outcomes were significantly better for patients with OSCC whose cancer cells expressed low amounts of cyclin D1 than those whose cells expressed high protein levels. Time to initial illness occurrence, time to initial local recurrence, time to mortality, and overall survival were among the metrics evaluated. The groups with low and high expression levels of cyclin D1 showed significantly different survival rates for disease-free survival, overall survival (OS), DMFS, local recurrence-free survival, and local recurrence-free survival (p0.001, 0.003, 0.004, and 0.001, respectively). Patients with cN2 oral squamous cell carcinoma (OSCC) with higher cyclin D1 expression had better distant metastasis-free survival (OS, P=0.024; DMFS, P=0.024) and overall survival (OS, P=0.024; DMFS, P=0.024) with TPF induction chemotherapy. However, patients with cN2 OSCC and low cyclin D1 expression did not show this survival advantage (P=0.024). Research has shown that oral squamous cell carcinoma (OSCC) is more sensitive to TPF-based chemotherapeutic drugs using caspase-3-mediated apoptosis that occurs when cyclin D1 expression rises. Based on these results, TPF induction chemotherapy may provide longer-term benefits than traditional treatment approaches for patients with cN2 OSCC and high cyclin D1 expression. Through a caspase-3-dependent mechanism, oral squamous cell carcinoma (OSCC) cell lines become more susceptible to TPF chemotherapeutic drugs when cyclin D1 is overexpressed.

The inhibitory effect of trastuzumab on BT474 triple‑positive breast cancer cell viability is reversed by the combination of progesterone and estradiol.

Breast cancer expressing the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) is known as triple-positive (TPBC). TPBC represents 9-11% of breast cancer cases worldwide and is a heterogeneous subtype. Notably, TPBC presents a therapeutic challenge due to the crosstalk between the hormonal (ER and PR) and HER2 pathways. Patients with TPBC are treated with trastuzumab (TTZ); however, several patients treated with TTZ tend to relapse. The present study aimed to investigate the effect of the PR on inhibitory effect of TTZ on cell viability. BT474 cells (a model of TPBC) and BT474 PR-silenced cells were treated with either TTZ, progesterone (Pg), the PR antagonist mifepristone (RU486) or estradiol (E2) alone or in combination for 144 h (6 days). Cell viability assays and western blotting were subsequently performed. The results showed that Pg and E2 interfered with the inhibitory effect of TTZ on cell viability and this effect was potentiated when both hormones were combined. Pg was revealed to act through the PR, mainly activating the PR isoform B (PR-B) and inducing the protein expression levels of CDK4 and cyclin D1; however, it did not reactivate the HER2/Akt pathway. By contrast, E2 was able to increase PR isoform A (PR-A) expression, which was inhibited by Pg. Notably, in most of the experiments, RU486 did not antagonize the effects of Pg. In conclusion, Pg and E2 may interfere with the inhibitory effect of TTZ on cell viability through PR-B activation and PR-A inactivation.