Overexpression Group Research Articles

MiR-4298 and lncKRTAP5-6-3 regulated Cathepsin D expression through ERK-MAPK signaling pathway in chronic UVB-damaged HaCaT cells

ObjectiveMiRNAs and lncRNAs are important regulators in the process of skin photoaging. In this study, we investigated the expression changes and interactions between miR4298 and lncKRTAP5-6-3 in chronically UVB-damaged human keratinocyte cell line (HaCaT) cells and explored miR4298-MAPK/ERK signaling pathway-Cathepsin D-lncKRTAP5-6-3 mechanisms in photoaging cells.MethodsHaCaT cells were irradiated with 12 mJ/cm2 UVB once a day for 7 days. miR-4298 mimics and miR-4298 inhibitors were transfected into HaCaT cells by lipo3000 transfection reagent, and the HaCaT cells were divided into three groups: blank control group; UVB-damaged group; and UVB damage+miR-4298 regulation (overexpression or inhibition) group. The expression levels of miR4298 and lncKRTAP5-6-3 were quantitatively analyzed using RT-PCR, while the expression of Cathepsin D and MAPK/ERK signaling pathway proteins was detected using Western blot.ResultsAfter 7 consecutive days of UVB irradiation, the expression of miR-4298 decreased by 0.64 ± 0.06 (P < 0.001) compared to the un-irradiated HaCaT cells, and the expression of the KRTAP5-6-3 decreased by 0.80 ± 0.13 (P < 0.001) compared to the control group. The expression of p-ERK signaling was increased by 0.9437 ± 0.1186 (P < 0.0001), and Cathepsin D was decreased by 0.6163 ± 0.075 (P < 0.0001). In HaCaT cells transfected with miR-4298 mimics and then irradiated by UVB for 7 days, the expression of lncKRTAP5-6-3 was increased to 0.5114 ± 0.1438 (P < 0.05)-fold, and the phosphorylation level of ERK signaling was decreased by 0.3880 ± 0.1185 (P < 0.01), while Cathepsin D expression was increased by 0.2617 ± 0.0749 (P < 0.0001) compared to the UVB-damaged group. In HaCaT cells transfected with miR-4298 inhibitors and then irradiated by UVB for 7 days, lncKRTAP5-6-3 was decreased by 0.1697 ± 0.1383, the phosphorylation level of ERK signaling was increased by 1.096 ± 0.7836 (P < 0.05), while Cathepsin D expression was decreased by 0.05197 ± 0.24827 compared to the UVB-damaged group.ConclusionThe synergistic effects of miR4298 and lncKRTAP5-6-3 play important roles in chronic UVB-damaged HaCaT cells by regulating the MAPK/ERK signaling pathway and Cathepsin D expression. This study presents novel targets for intervening in chronic ultraviolet damage (photoaging) skin and UV-related dermatoses.

ALKBH5, an m6A demethylase, attenuates tumor growth and inhibits metastasis in papillary thyroid carcinoma

The significance of ALKBH5 in erasing mRNA methylation in mRNA biogenesis, decay, and translation control has emerged as a prominent research focus. Additionally, ALKBH5 is associated with the development of numerous human cancers. However, it remains unclear whether ALKBH5 regulates the growth and metastasis of papillary thyroid carcinoma (PTC). Here, we compared cancer tissues and paracancerous tissues from PTC patients, along with cultured cells expressing ALKBH5 (overexpression, silent gene expression, normal stable expression). Our primary objective was to investigate the impact of ALKBH5 on PTC. Selected 30 cases of PTC tissues and their adjacent noncancerous tissues to compare the protein expression levels of ALKBH5 between the two groups using immunohistochemical analysis. qRT-PCR and western blot were used to detect the expression of ALKBH5 in normal thyroid follicular epithelial cells (Nthy-ori3-1) and 4 PTC cell lines (human PTC cell lines K1, BCPAP, IHH4, and TPC1). Appropriate cell lines were screened for subsequent experiments. Immunofluorescence staining was used to localize the high accumulation of ALKBH5 in cells. Construct the ALKBH5 knockdown vector and ALKBH5 overexpression vector separately, and construct the overexpression ALKBH5-mut vector with m6A domain mutation. The impact of different levels of ALKBH5 in the three cell lines on RNA m6A methylation levels was compared using qRT-PCR and western blot methods. Furthermore, cell viability was assessed using the CCK-8 assay, while the impact on cell proliferation was examined using plate colony formation assay. Cell invasion was evaluated using the Transwell assay. Immunohistochemical staining results showed that the expression of ALKBH5 protein in PTC cancer tissue was significantly lower than in adjacent non-cancerous tissue (P < 0.05). Lymph node metastasis in PTC patients may have been linked to ALKBH5 protein levels in their cancerous tissues (P = 0.034). The expression of ALKBH5 in PTC cell lines BCPAP, IHH4, and TPC1 was significantly lower than Nthy-ori3-1 (P < 0.05). IHH4 and TPC1 cell lines were selected for subsequent experiments. Immunofluorescence single staining results showed a high accumulation of ALKBH5 protein in the cell nucleus. Cell viability results suggested that compared to the overexpression-negative control group, cell proliferation, and invasion were significantly decreased in the ALKBH5 overexpression group (P < 0.05) and the mut-ALKBH5 overexpression group (P < 0.05). Additionally, compared to the ALKBH5 overexpression group, cell proliferation and invasion were significantly more decreased in the mut-ALKBH5 overexpression group (P < 0.05). However, compared to the interference-negative control group, cell proliferation and invasion were significantly increased in the ALKBH5 interference group (P < 0.05). The presented findings suggested that m6A demethylase ALKBH5 inhibits tumor growth and metastasis in PTC. Moreover, effective inhibition of m6A modification of ALKBH5 might constitute a potential treatment strategy for PTC.

ARC protects cochlear hair cells from neomycin-induced ototoxicity via the Ras/JNK signaling pathway.

The present study was designed to investigate the role and mechanism of the Apoptosis repressor with caspase recruitment domain (ARC) in protecting the neomycin-induced hair cell damage. HEI-OC1 cells and basilar membrane culture were applied to determine the effect of ARC. Plasmid transfection was used to regulate the ARC or Ras expression. We have found the ARC overexpression in HEI-OC1 cells can increase the cell viability and decrease cell apoptosis after neomycin injury. The cleaved caspase 3 was reduced in ARC overexpression group after neomycin treatment. The p-CREB expression was increased in ARC overexpression group, while the p-c-Jun expression was decreased after neomycin incubation. In HEI-OC1 cells and basilar membranes, JNK and Ras inhibitions both can reduce ARC expression, and Ras overexpression can increase the ARC expression. This study indicates that ARC can protect the hair cells from neomycin-induced apoptosis through Ras/JNK signaling pathway. Our findings provide new insights in preventing cochlear HC death after drug-induced ototoxicity.

A Mechanism Study on the Antioxidant Pathway of Keap1-Nrf2-ARE Inhibiting Ferroptosis in Dopaminergic Neurons.

The pathology of Parkinson's disease (PD) indicates that iron deposition exists in dopaminergic neurons, which may be related to the death of cellular lipid iron peroxide. The extracellular autophagy adaptor SQSTM1(p62) of dopamine (DA) neurons can activate the intracellular Keap1-Nrf2-ARE signaling pathway to inhibit ferroptosis, which has a protective effect on DA neurons. The objective of this study was to investigate the protective mechanism of the Keap1- Nrf2-ARE antioxidant pathway against iron death in dopaminergic neurons. The experiment was divided into a control group (Control group), 1-methyl-4- phenylpyridiniumion control group (MPP+ Control group), p62 overexpression group (MPP+OVp62), and p62 overexpression no-load group (MPP+ OV-P62-NC). The inhibitors brusatol and ZnPP inhibited the activation of NF-E2-related factor 2(Nrf2) and Heme oxygenase-1(HO-1), respectively, and were divided into brusatol group (MPP+OV-p62+brusatol) and ZnPP group (MPP+OV-p62+ZnPP). RT-qPCR was used to detect transfection efficiency, and Cell Counting Kit-8 (CCK8) was used to detect cell activity. FerroOrange, 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), and Liperfluo probes were used to detect intracellular iron, reactive oxygen species (ROS), and lipid peroxidation (LPO) levels. Western Blotting detected the levels of Nrf2, HO-1, Kelch-like ECH-associated protein1 (Keap1), and their downstream Glutathione peroxidase 4(GPX4) and Acyl-CoA synthetase long-chain family member 4(ACSL4). The levels of LGlutathione (GSH) and Malondialdehyde (MDA) were detected by GSH and MDA kits, and the activation of Keap1-Nrf2-ARE pathway was verified at the cellular level to have an antioxidant protective effect on iron death in dopaminergic neurons. (1) The results of RT-qPCR showed that compared with the control group, the expression of the p62 gene was significantly increased in the MPP+OV-p62 groups (P = 0.039), and the p62 gene was significantly increased in the brusatol and ZnPP groups, indicating successful transfection (P =0.002; P=0.008). (2) The immunofluorescence probe flow results showed that compared to the normal control group, the contents of three kinds of probes in MPP+ model group were significantly increased (P =0.001; P ＜0.001; P＜0.001), and the contents of three kinds of probes in MPP+OV-p62 group were decreased compared to the MPP+ model group (P =0.004). The results indicated that the levels of iron, ROS, and LPO were increased in the MPP+ group and decreased in the MPP+OV-p62 group. (3) Compared with the control group, the expressions of Nrf2, HO-1, and GPX4 in the MPP+OV-p62 group were increased (P =0.007; P =0.004; P=0.010), and the expressions of Keap1 and ACSL4 in MPP+p62 overexpression group were decreased (P =0.017; P =0.005). Compared with the MPP+ control group, Nrf2 and GPX4 were increased in the MPP+OV-p62 group, and ACSL4 was decreased in the MPP+OVp62 group (P =0.041; P ＜0.001; P ＜0.001). The results of the GSH and MDA kit showed that compared with the normal control group, the content of GSH in the MPP+ control group was decreased (P < 0.01), and the content of MDA was increased (P < 0.01). Compared with the MPP+ model group, GSH content was increased (P = 0.003), and MDA content was decreased in the MPP+OV-p62 group (P < 0.001). Nrf2, HO-1, and GPX4 increased in the MPP+p62 overexpression group but decreased in the brusatol group and ZnPP group (P < 0.001). Based on the transfection of P62 plasmid, it was found that P62 plasmid can inhibit the lipid peroxidation of iron death in dopaminergic nerve cells by activating the Nrf2 signaling pathway, thus playing a protective role in dopaminergic nerve cells.

A phase II trial of larotrectinib in tumors with NTRK fusions or extremes of NTRK mRNA overexpression identified by comprehensive genomic profiling

Abstract Background TRK-inhibitors have demonstrated efficacy across several cancers with NTRK fusions. Their activity in cancers with NTRK overexpression remains unclear. Methods This trial enrolled patients with advanced cancers harboring NTRK fusions or extreme mRNA overexpression, defined as NTRK1/2/3 expression by RNA profiling &amp;gt;5 SDs for a given cancer type. The primary endpoint was objective response rate (ORR), with secondary endpoints including time-to-progression (TTP) ratio [TTP on study to TTP on previous systemic therapy (TTP1)], progression-free survival (PFS), and overall survival (OS). Initially planned for 2 non-comparator groups: primary central nervous system (CNS) and non-CNS tumours with NTRK fusions, the protocol was amended to permit NTRK overexpression. Results Seventeen patients were treated with larotrectinib: one glioblastoma with a SPECC1L::NTRK2 fusion (group 1), and a peripheral nerve sheath tumor with a TPM3::NTRK1 fusion and 15 patients with overexpression (group 2). The ORR was 6%. An additional 3 of 12 (25%) TTP1-evaluable patients achieved a TTP ratio ≥1.3 and 2 of 5 without an evaluable TTP1 had a PFS &amp;gt;6 months. Median PFS and OS were 3.5 (95% CI, 1.4-6.0) and 15.9 months (95% CI, 6.4-NR), respectively. Conclusion Unlike its efficacy in NTRK-fusion positive cancers, larotrectinib did not demonstrate a signal of efficacy among tumors with NTRK overexpression.

NEDD4 enhances bone‑tendon healing in rotator cuff tears by reducing fatty infiltration.

Rotator cuff tears (RCT) can cause shoulder pain, weakness and stiffness, significantly affecting daily life. Analysis of the GSE103266 dataset revealed significant changes in the mTOR/PI3K/Akt signaling pathway and lipid metabolism‑related pathways, suggesting that fatty infiltration may affect RCT. The analysis indicated that the ubiquitin ligase NEDD4 plays a critical role in RCT. NEDD4 was found to be highly associated with the mTOR/PI3K/Akt signaling pathway. An RCT model in Sprague‑Dawley (SD) rats was established to study the role of NEDD4 in regulating the mTOR pathway and investigate its effects on fatty infiltration. SD rats were divided into NEDD4 overexpression and knockout groups. Tissue recovery, apoptosis and fat deposition were measured through histological staining, reverse transcription‑quantitative PCR and western blotting. Additionally, cell culture of fibro‑adipogenic progenitors and lentiviral transfection were conducted to investigate the effect of NEDD4 on adipocyte differentiation. NEDD4 overexpression significantly reduced lipid accumulation, whereas NEDD4 knockdown enhanced lipid accumulation. NEDD4 was found to regulate the mTOR pathway and the expression of adipogenesis‑related genes, promoting fat metabolism and inhibiting adipocyte differentiation. Histological analysis indicated that NEDD4 overexpression improved tissue recovery and reduced apoptosis. Targeting NEDD4 offers a potential therapeutic strategy to improve the clinical outcomes of patients with RCT by modulating the mTOR pathway and fat metabolism.

Hepatocyte HIF-2α aggravates NAFLD by inducing ferroptosis through increasing extracellular iron.

Recent research has illuminated the pivotal role of the hypoxia-inducible factor-2α (HIF-2α)/peroxisome proliferator-activated receptor alpha (PPARα) pathway in the progression of nonalcoholic fatty liver disease (NAFLD). Meanwhile, it has been reported that HIF-2α is involved in iron regulation, and that aberrant iron distribution leads to liver lipogenesis. Therefore, we hypothesize that HIF-2α exacerbates fatty liver by affecting iron distribution. To substantiate this hypothesis, we utilized liver-specific HIF-2α knockout mice and the LO2 cell line with overexpressed HIF-2α. HIF-2α overexpression (OE) was induced via lentiviral infection, followed by exposure to free fatty acids (FFAs) and deferoxamine (DFO). In animal experiments, hepatic HIF-2α knockout resulted in lower liver lipid levels, lower liver weight, and higher serum iron levels. Enrichment in autophagy, ferroptosis, and the PI3K-AKT pathway was demonstrated through Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in the liver of mice. In vitro experiments showed that HIF-2α increased supernatant iron. In the HIF-2α OE group, the addition of FFA led to decreased levels of reduced glutathione (GSH) and glutathione peroxidase 4 (GPX4) protein, along with increased lipid peroxidation (LPO), cellular lipid droplets, and triglyceride content. Impressively, DFO intervention decreased supernatant iron, reversed these changes by increasing GSH and GPX4 levels, and simultaneously reduced LPO levels, cellular lipid droplets, and triglyceride content. In addition, the expression of proteins related to β-oxidation increased, and lipid deposition in hepatocytes improved, which may be associated with the PI3K/AKT pathway. In summary, our findings suggest that HIF-2α-mediated iron flux enhances NAFLD cell susceptibility to ferroptosis, thereby impacting lipid metabolism-related genes and contributing to lipid accumulation.NEW & NOTEWORTHY The experiment demonstrated that HIF-2α increased extracellular iron. In LO2 cells overexpressing HIF-2α, FFAs not only increased cellular lipid and triglyceride levels but also induced key features of ferroptosis, such as reduced GSH and GPX4 levels and increased LPO, despite the absence of cellular iron overload. These effects were reversed by lowering extracellular iron with DFO. Furthermore, DFO treatment increased β-oxidation protein expression and improved lipid deposition in hepatocytes, potentially through the PI3K/AKT pathway.

Mechanism of WAVE1 regulation of lipopolysaccharide-induced mitochondrial metabolic abnormalities and inflammatory responses in macrophages

To explore the mechanism by which Wiskott-Aldrich syndrome protein family verprolin-homologous protein 1 (WAVE1) regulates lipopolysaccharide (LPS)-induced mitochondrial metabolic abnormalities and inflammatory responses in macrophages. Macrophage cell lines with overexpressed WAVE1 (mouse BMDM and human THP1 cells) were prepared. The macrophages were treated with LPS (500 ng/mL) to simulate sepsis-induced inflammatory responses. The experiment consisted of two parts. The first part included control, LPS, vector (LPS+oe-NC), WAVE1 overexpression (LPS+oe-WAVE1) groups. The second part included LPS, LPS+oe-NC, LPS+oe-WAVE1 and exogenous high mobility group box-1 (HMGB1) intervention (LPS+oe-WAVE1+HMGB1) groups. RT-PCR was used to measure mitochondrial DNA content, and RT-qPCR was used to detect the mRNA expression levels of WAVE1, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. Western blot was performed to measure the protein expression of WAVE1, hexokinase 2, and pyruvate kinase M2. ELISA was utilized to detect the levels of TNF-α, IL-1β, IL-6, and HMGB1. JC-1 staining was used to assess mitochondrial membrane potential. Seahorse XP96 was used to evaluate oxygen consumption rate and extracellular acidification rate. MitoSOX probe was employed to measure mitochondrial reactive oxygen species levels, and 2-NBDG method was used to assess glucose uptake. Kits were used to measure pyruvate kinase activity, lactate, adenosine triphosphate (ATP), and HMGB1 levels. Compared with the control group, the LPS group showed lower levels of WAVE1 protein and mRNA expression, mitochondrial membrane potential, oxygen consumption rate, and mitochondrial DNA content (P<0.05), while TNF-α, IL-1β, IL-6 levels and mRNA expression, mitochondrial reactive oxygen species, glucose uptake, lactate, ATP, hexokinase 2, and pyruvate kinase M2 protein expression levels as well as extracellular acidification rate, pyruvate kinase activity, and HMGB1 release were significantly increased (P<0.05). Compared with the LPS+oe-NC group, the LPS+oe-WAVE1 group showed increased WAVE1 protein and mRNA expression, mitochondrial membrane potential, oxygen consumption rate, and mitochondrial DNA content (P<0.05), while TNF-α, IL-1β, IL-6 levels and mRNA expression, mitochondrial reactive oxygen species, glucose uptake, lactate, ATP, hexokinase 2, and pyruvate kinase M2 protein expressions, as well as extracellular acidification rate, pyruvate kinase activity, and HMGB1 release were decreased (P<0.05). Compared with the LPS+oe-WAVE1 group, the LPS+oe-WAVE1+HMGB1 group exhibited increased glucose uptake, lactate, ATP levels, and extracellular acidification rate (P<0.05). WAVE1 participates in the regulation of LPS-induced inflammatory responses in macrophages by modulating the release of inflammatory factors, mitochondrial metabolism, and HMGB1 release.

Effect of WW-domain transcription regulator 1 on aging regulation of human dental pulp stem cells

Objective: Investigating the changes of phenotype and moleculars associated with aging with the increase of passage times of human dental pulp stem cells (hDPSC), to explore the role of WW-containing transcriptional regulator 1 (WWTR1) in the aging mechanism. Methods: hDPSCs were cultured by tissue block method, and were divided into 4 groups according to the age, algebra, cell knockdown and overexpression of WWTR1 in hDPSCs. Group Ⅰ: hDPSCs from human teeth were further divided into youth group (15-25 years old) and group middle-aged group (40-50 years old) according to different ages. Group Ⅱ: according to different passage, hDPSCs were divided into young cells group (hDPSCs were transmitted to P3 generation), and old cells group (hDPSCs were transmitted to P10 generation). Group Ⅲ: hDPSCs were knocked down of WWTR1, which were further divided into knockdown group and knockdown carrier group. Group Ⅳ: hDPSCs were overexpressed of WWTR1, which were further divided into overexpression group and overexpression carrier group. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the changes of WWTR1 expression in groups Ⅰ and Ⅱ, and cell counting kit-8 (CCK-8) was used for groups Ⅱ, Ⅲ, and Ⅳ. Cell proliferation capacity was detected by CCK-8 assay. The ability of osteogenic differentiation was detected by alizarin red staining. Cell senescence positive rate was detected by age-related β-galactosidase staining. The expression levels of age-related genes p53 and p21 were detected by RT-qPCR. Results: The proportion of senescent cells increased gradually with continuous culture. The proliferation and osteogenic differentiation of hDPSCs in the old group were significantly lower than those in the young group (P<0.001). The expression levels of senescence related genes p53 (2.09±0.24) and p21 (4.91±0.54) in old cell group were higher than those in young cell group respectively [p53: (1.08±0.09) and p21: (1.09±0.08)] (P<0.01, P<0.001). The WWTR1 expression levels of hDPSCs in middle-aged group and old cells group were both decreased compared with those in young group and young cells group (P<0.01). The proportion of senescent cells in knockdown group (44.50±2.42) was higher than that in knockdown carrier group (22.27±0.56) (P<0.001). After knocking down WWTR1 in hDPSCs, the expression levels of age-related genes p53 and p21 were up-regulated (P<0.001), and the abilities of proliferation and osteogenic differentiation in the knockdown group were lower than those in the knockdown carrier group (P<0.001). The proportion of senescent cells in overexpression empty carrier group (20.40±0.79) was higher than that in overexpression group (10.07±0.61) (P<0.001). After WWTR1 overexpression ins hDPSCs, the expression levels of age-related genes p53 and p21 were down-regulated, and the proliferation and osteogenic differentiation ability in overexpression group were higher than those in overexpression carrier group (P<0.001). Conclusions: WWTR1 can inhibit the expression levels of age-related genes p53 and p21, thus delaying the aging process as well as promoting the proliferation and osteogenic differentiation of hDPSCs.

Exploration of the biological mechanisms of CENPA as an oncogene in glioma: Screening based on cancer functional status

AbstractGlioma is the most common primary tumour in central nervous system, characterized by high invasiveness, a high recurrence rate and extremely poor prognosis. Machine learning based on cancer functional state helps to combine multi‐omics methods to screen for key gene, such as CENPA, that influences the phenotype of glioma and patients' prognosis. Based on 14 CFS, glioma was divided into three subtypes. Bioinformatics and machine learning methods were utilized to develop an enhanced prognostic prediction signature based on three subtypes. We selected CENPA as a hub biomarker and conducted in vitro experiments such as IHC, western blot, Coip, transwell, cck8, flow cytometry, scratch assay, qPCR, AlphaFold, MOE and in vivo experiments. We identified three subtypes of glioma based on the 14 CFS. The C subtype exhibits poor clinical outcomes, increased carbohydrate and nucleotide metabolism, high infiltration of immune cells, high CNV and tumour mutation burden (p &lt; 0.05). The differential expression of gene between three subtypes were used to construct a novel signature with improved performance in prognostic prediction via machine learning. CENPA was selected as the hub gene, in vitro experiments such as ihc, western blot and qPCR showed that CENPA had high expression in tissues and cell lines (p &lt; 0.05). The scratch assay, edu, cck8, flow cytometry and transwell after CENPA knockdown or overexpression had significant effects on the functions of glioma. Meanwhile, CENPA was regulated by EZH2 and influenced downstream wnt pathway, affecting phosphorylation of two sites, Ser675 and Ser552, on β‐catenin. The effect of CENPA knockdown was reversed by drug CHIR‐99021. Animal experiments indicated that the tumour volume of control and overexpression group increased faster, especially the overexpression group, which was significantly faster (p &lt; 0.001). Machine learning based on CFS is beneficial for the selection of key genes and disease assessment. In glioma, CENPA is positively correlated with WHO grading at both the gene and protein levels, and high CENPA affects patients' poor prognosis. Regulating CENPA can affect functions of glioma, and these phenomena may act through the EZH2/CENPA/β‐catenin signalling axis. CENPA knockdown can be reversed by the drug CHIR‐99021. CENPA may become one of the therapeutic targets in glioma.

S100A11 promotes lipid metabolism and activates the apoptosis in polycystic ovary syndrome.

Apoptosis of ovarian granulosa cells (GCs) affects the development and maturation of oocyte in Polycystic ovary syndrome (PCOS).The objective of our study was to examine the impact of S100A11 on granulosa cells in individuals with polycystic ovary syndrome. We found that the S100A11 level was higher in the ovarian granulosa cells of PCOS patients and ovarian tissue of PCOS mouse models. S100A11 overexpression experiments demonstrated a decrease in the cell proliferation, upregulating the apoptosis and blocking the cell cycle. Immunofluorescence staining showed that S100A11 proteins were mainly located in the nucleus. Integrating the RNA-seq and ChIP-seq, this study found that S100A11 mainly regulated the genetic transcription and lipid metabolism. Furthermore, lipid accumulation and oxidative stress increasing were detected in the S100A11 overexpression group. We also revealed that S100A11 upregulated the p-DRP1 Ser616 to induce the mitochondrial fission.In conclusion, S100A11 upregulated the phospho-DRP1 to activate ovarian granulosa cell apoptosis and induced the lipid metabolism and oxidative stress to regulate the follicular reserve in PCOS. The findings present a novel therapeutic concept for polycystic ovary syndrome.

Aβ1-42 promotes microglial activation and apoptosis in the progression of AD by binding to TLR4

Alzheimer's disease (AD) is one of the most common age-related neurodegenerative diseases and the most devastating form of senile dementia. It has a complex mechanism and no effective treatment. Exploring the pathogenesis of AD and providing ideas for treatment can effectively improve the prognosis of AD. Microglia were incubated with β-amyloid protein 1-42 (Aβ1-42) to construct an AD cell model. After microglia were activated, cell morphology changed, the expression level of inflammatory factors increased, cell apoptosis was promoted, and the expression of microtubule-associated protein (Tau protein) and related proteins increased. By up-regulating and down-regulating Toll-like receptor 4 (TLR4), the cells were divided into TLR4 knockdown negative control group(Lv-NC group), TLR4 knockdown group(Lv-TLR4 group), TLR4 overexpression negative control group(Sh-NC group), and TLR4 overexpression group(Sh-TLR4 group). The expression of inflammatory factors was detected again. It was found that compared with the Lv-NC group, the expression of various inflammatory factors in the Lv-TLR4 group decreased, cell apoptosis was inhibited, and the expression of Tau protein and related proteins decreased. Compared with the Sh-NC group, the expression of inflammatory factors in the Sh-TLR4 group increased, cell apoptosis was promoted, and the expression of Tau protein and related proteins increased. These results indicate that Aβ1-42 may promote microglial activation and apoptosis by binding to TLR4. Reducing the expression of TLR4 can reduce the occurrence of inflammatory response in AD cells and slow down cell apoptosis. Therefore, TLR4 is expected to become a new target for the prevention and treatment of AD.

Exercise-induced interactions between skeletal muscle and bone via myokines and osteokine in mice: Role of FNDC5/irisin, IGF-1, and osteocalcin

Skeletal muscle and bone interact to maintain their structure and function. Physical exercise is the most effective and easily applicable strategy to maintain their functions; however, exercise-induced interactions by soluble factors remained elusive. Our study aimed to identify exercise-induced interactions between muscle and bone by examining (1) the effects of myokine on bone and (2) the effects of osteocalcin (OCN) on skeletal muscle. To understand the effects of exercise-induced myokines on bone, we examined the effects of FNDC5 for aerobic exercise and IGF-1 for resistance exercise using a muscle-specific myokine overexpression model. To examine OCN effects on muscle, mice were intraperitoneally administered OCN-neutralizing antibody during long-term exercise. Our result showed that aerobic exercise tended to increase serum HA-tag protein attached to FNDC5 in muscle-specific overexpression groups. In addition, osteoblastic activation was increased only after aerobic exercise with HA/FNDC5 overexpression. Resistance exercise did not alter circulating HA-tag (muscle-derived IGF-1) and bone metabolism after IGF-1/HA overexpression. In the OCN study, aerobic exercise enhanced endurance capacity by restoring muscle glycogen content; however, OCN neutralization returned these to baseline. After resistance exercise, OCN suppression inhibited muscle hypertrophy and strength gains by preventing protein synthesis. Our results suggest that aerobic exercise following FNDC5 muscle overexpression promotes osteoblast activity, which may be partially caused by muscle-derived FNDC5 secretion. In addition, OCN was necessary for muscle adaptation in both aerobic and resistance exercises.

Proanthocyanidins protects 3-NPA-induced ovarian function decline by activating SESTRIN2-NRF2-mediated oxidative stress in mice

Abnormal apoptosis of ovarian cells caused by oxidative stress is an important cause of premature ovarian failure (POF). Previous studies revealed that proanthocyanidins (PCs) are powerful natural antioxidants that can safely prevent oxidative damage in humans. However, the protective effect and mechanism of PCs on ovarian function during the course of POF remain unknown. In this study, female mice were injected with 3-nitropropionic acid (3-NPA) to establish an ovarian oxidative stress model; at the same time, the mice were treated with PC via gavage. Thereafter, the expression of various apoptosis genes, hormones, and related molecules was assessed. Compared with those in the control group, the ovarian index, follicle count at all levels, expression of MVH, PCNA and BCL2, and estradiol (E2) and progesterone (P) levels were significantly lower in the POF group, but significant recovery was observed in terms of MVH and PCNA expression and E2 and P levels in the POF + PCs group. The apoptosis marker genes BAX and ROS were significantly increased in the POF group but were notably restored in the POF + PCs group. In addition, the expression of Sestrin2, an antiapoptotic protein, was significantly increased in the PCs treatment group, as were the upstream and downstream regulatory factors NRF2 and SOD2, and the indices of the Sestrin2 overexpression group were similar to those of the PCs treatment group. In summary, these findings suggest that PCs have potential as innovative therapeutic agents for preventing and treating POF by activating the protective SESTRIN2-NRF2 pathway against oxidative stress.

The molecular mechanism of PLD2-mediated regulation of apoptosis and cell edema in pancreatic cells via the Nrf2/NF-κB pathway

This study aimed to elucidate the molecular mechanisms by which PLD2 controls apoptosis and edema in pancreatic cells via the Nrf2/NF-κB pathway. AR42J rat pancreatic cells were treated with 10 nM mitomycin to create an in vitro pancreatitis model (model group), with a control group receiving phosphate-buffered saline. Cells were transfected with a PLD2 overexpression plasmid using Lipofectamine 3000, forming the PLD2 overexpression group. PLD2 protein expression was assessed by Western blotting, and TNF-α, IL-6, and IL-10 levels were measured by RT-qPCR. Nrf2/NF-κB protein expressions were also analyzed. Apoptosis and necrosis were evaluated using Annexin V-FITC/PI staining and the LDH release test. Cell edema was assessed by cell volume, ion content, and membrane damage. Western blotting was used to analyze pan-apoptosis-related proteins. PLD2 expression was lower in the model group compared to controls (P < 0.05) but higher in the PLD2 overexpression group (P < 0.05). TNF-α, IL-6, and IL-10 levels were elevated in the model group (P < 0.05) and reduced in the PLD2 overexpression group (P < 0.05). Nrf2 expression decreased in the model group but increased with PLD2 overexpression (P < 0.05). NF-κB expression increased in the model group but decreased with PLD2 overexpression (P < 0.05). Apoptosis and necrosis rates were higher in the model group (P < 0.05) but lower in the PLD2 overexpression group (P < 0.05). Cell volume, Na + content, and LDH release increased in the model group (P < 0.05) but decreased with PLD2 overexpression (P < 0.05). RIPK1 expression decreased in the model group (P < 0.05) but increased with PLD2 overexpression (P < 0.05). CASP8, FADD, and ZBP1 levels were higher in the model group (P < 0.05) and reduced with PLD2 overexpression (P < 0.05). PLD2 exerts a protective effect in acute pancreatitis by activating Nrf2 and inhibiting NF-κB, reducing apoptosis, cell swelling, and membrane damage. This highlights potential therapeutic targets for pancreatic inflammation.

Transcriptome analysis reveals the regulation of miR-19b on inflammation in bovine mammary epithelial cells

MicroRNAs (miRNAs) are involved in various biological processes where they regulate the expression of mRNAs. Bovine mammary epithelial cells (bMECs) are functional cells that mediate mammary inflammatory immunity. Although numerous miRNAs regulate the function of bMECs, the role of miR-19b in bMECs has not been reported. In this study, the transcriptome of miR-19b overexpressed bMECs was analyzed by RNA-seq. Additionally, the differentially expressed genes (DEGs) were analyzed to establish the role of miR-19b in bMECs. The results revealed 269 DEGs between the miR-19b overexpression group and the negative control, including 199 up-regulated and 70 down-regulated genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that the DEGs regulated immune and inflammatory responses through Staphylococcus aureus (S. aureus) infection and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. In addition, the expression of miR-19b was significantly upregulated in lipophosphoric acid (LTA)-induced bMECs, and overexpression of miR-19b negatively regulated the expression of inflammatory cytokines IL-1β and IL-6, thereby alleviating the inflammatory response of LTA-induced bMECs. Based on the above results, we speculate that miR-19b may inhibit in dairy cow mammary inflammation caused by S. aureus, and this process may be mediated through the regulation of relevant gene expression and signaling pathways. The findings from this study provide a new reference for analyzing the molecular regulation of miR-19b in bMECs.

Assessment of the Role of miR-30a-5p on the Proliferation and Apoptosis of Hair Follicle Stem Cells.

To investigate the role of miR-30a-5p on the proliferation and apoptosis of hair follicle stem cells (HFSCs) and whether the Wnt/β-catenin signaling pathway is involved. HFSCs derived from the vibrissa of mammary rats were obtained by enzymatic digestion, and subsequently the obtained HFSCs were treated with Lipofectamine 2000 cell transfection and divided into normal cell culture group (control), miR-30a-5p overexpression group (miR-30a-5p mimic), miR-30a-5p empty vector group (miR-NC), miR-30a-5p inhibitor group (in-miR-30a-5p), and in-miR-30a-5p empty vector group (in-miR-NC). After transfection, the cell proliferation and apoptosis rates were examined separately. In addition, the mRNA expression of β-catenin, proliferating cell nuclear antigen (PCNA) and apoptosis-related genes (Bax and Bcl-2) were examined. The results of cell proliferation ability showed that in-miR-30a-5p group promoted cell proliferation of HFSCs relative to other groups, along with significant upregulation of gene levels of PCNA. Apoptosis analysis indicated that apoptosis rate was reduced in the in-miR-30a-5p group, and the expression of Bax was suppressed, while that of Bcl-2 was promoted. Wnt/β-catenin signaling pathway investigation revealed a significant increase in the levels of β-catenin in HFSCs in the in-miR-30a-5p group. Downregulation of miR-30a-5p levels inhibited HFSCs apoptosis and simultaneously promoted proliferation, furthermore, the increased expression of β-catenin indirectly confirmed the activation of the Wnt/β-catenin signaling pathway.

SLC7A9 suppression increases chemosensitivity by inducing ferroptosis via the inhibition of cystine transport in gastric cancer

SLC7A9 is responsible for the exchange of dibasic amino acids and cystine (influx) for neutral amino acids (efflux). Cystine/cysteine transport is related to ferroptosis. Sanger sequencing detected TP53 status of cancer cells. Transcriptomic sequencing and untargeted metabolome profiling were used to identify differentially expressed genes and metabolites, respectively, upon SLC7A9 overexpression. CCK8, cell clonality, and EdU assays were used to observe cell proliferation. Cystine probes, glutathione (GSH) probes, and lipid ROS probes were used to examine cystine, GSH, and lipid ROS levels. 13C metabolic flow assays were used to monitor cellular cystine and GSH metabolism. Patient-derived organoids (PDO), immunocompetent MFC mice allograft models and patient-derived xenograft (PDX) models were used to evaluate SLC7A9 impact on chemotherapeutic response and to observe therapeutic effect of SLC7A9 knockdown. Elevated SLC7A9 expression levels in gastric cancer cells were attributed to p53 loss. SLC7A9 knockdown suppressed the proliferation and increased the chemotherapy sensitivity of the cells. Chemotherapy was more effective in PDX and immunocompetent mice models upon SLC7A9 knockdown. Differentially expressed genes and metabolites between the SLC7A9 overexpression and control groups were associated with ferroptosis and GSH metabolism. SLC7A9 knockdown reduced cystine transport into cells, hampered intracellular cystine and GSH metabolic flow, decreased GSH synthesis, and increased lipid ROS levels in gastric cancer cells. Erastin was more effective at inducing ferroptosis in PDO and PDX models upon SLC7A9 knockdown. SLC7A9 promotes gastric cancer progression by acting as a suppressor of ferroptosis, independent of SLC7A11, which is negatively regulated by p53. This work was supported by National Natural Science Foundation of China, Innovation Promotion Program of NHC and Shanghai Key Labs SIBPT, and Shanghai Academy of Science & Technology.

Cystatin C alleviates unconjugated bilirubin-induced neurotoxicity by promoting bilirubin clearance from neurocytes via exosomes, dependent on hepatocyte UGT1A1 activity.

There is an urgent need to identify effective drugs for the treatment of nerve injury caused by unconjugated bilirubin (UCB). Our previous research found that cystatin C (CST3) alleviates UCB-induced neurotoxicity by promoting autophagy in nerve cells, but that autophagy inhibitors did not completely inhibit the effects of CST3. This study investigated whether CST3 could alleviate the neurotoxicity of UCB by promoting the secretion and transport of exosomes containing UCB to the liver for metabolism. It demonstrated that hyperbilirubinemia mice treated with CST3 had a higher number of serum exosomes than those in hyperbilirubinemia mice treated with phosphate-buffered saline. CST3-mediated protection against UCB-induced damage was abolished when autophagy and extracellular vesicle inhibitors were used in combination. The number of exosomes in the CST3 overexpression group was higher than that in the control group. Molecular docking experiments showed that UCB and CST3 had high docking score (-8.2). These results suggest that UCB may be excreted from cells by exosomes, and CST3 may promote this process by binding to UCB and entering the exosomes. We demonstrated that the effect of CST3 relied on liver cells with normal UDP-glucuronyl transferase1A1 (UGT1A1) activity in a coculture system of HT22 and L02 cells. CST3 levels were lower in exosomes secreted by L02 cells than in those secreted by human umbilical vein endothelial cells (HUVECs), whereas CST3 levels were higher in the culture supernatants of L02 cells than in the culture supernatants of HUVECs. This suggests that UCB exosomes in L02 cells may be released and internalized by CST3 and that UCB is then processed by UGT1A1 to conjugate UCB, thus reducing its toxicity. These results suggest that CST3 might alleviate UCB-induced neurotoxicity by promoting the clearance of UCB from cells via exosomes and that these effects are dependent on UGT1A1 activity in liver cells.

Exercise preconditioning mitigates brain injury after cerebral ischemia-reperfusion injury in rats by restraining TIMP1.

Cerebral ischemic disease is a common cerebrovascular disease, especially ischemic stroke. Exercise has protective functions on brain tissues following cerebral ischemia-reperfusion injury (CIRI), but its preventive effects and mechanisms in CIRI remain unclear. We aimed to investigate the effects and mechanisms of exercise preconditioning on CIRI. The middle cerebral artery occlusion (MCAO) operation was prepared to establish CIRI rats. All rats were randomized into the MCAO, exercise (exercise preconditioning plus MCAO operation), vector (exercise preconditioning, MCAO operation plus intraventricular injection of empty vector), and tissue inhibitor of metalloprotease 1 overexpression (OE-TIMP1, exercise preconditioning, MCAO operation plus intraventricular injection of OE-TIMP1) groups. The results indicated that exercise preconditioning suppressed approximately 66.67% of neurological deficit scores and 73.79% of TIMP1 mRNA expression in MCAO rats, which were partially offset by OE-TIMP1. The protective effects of exercise against neuron death status and cerebral infarction size in MCAO rats were reversed by OE-TIMP1. It also confirmed that exercise weakened apoptosis and oxidative stress damage, with notable increases of B-cell lymphoma-2, superoxide dismutase, and glutathione peroxidase production, and evident decreases of BCL2-associated X, caspase 3, and malondialdehyde in MCAO rats, while these effects were partially reversed by OE-TIMP1. Additionally, the inhibitory effects of exercise on the protein levels of TIMP1, hypoxia-inducible factor-alpha, vascular endothelial growth factor receptor 2, vascular endothelial growth factor, and neurogenic locus notch homolog protein 1 in MCAO rats were partially reversed by OE-TIMP1. Altogether, exercise preconditioning had protective effects on CIRI by restraining TIMP1, which provided new therapeutic strategies for preventing CIRI.