Zinc-finger protein CXXC5 promotes breast carcinogenesis by regulating the TSC1/mTOR signaling pathway.

CXXC5, a member of the CXXC family of zinc-finger proteins, is associated with numerous pathological processes. However, the pathophysiological function of CXXC5 has not been clearly established. Herein, we found that CXXC5 interacts with the CRL4B and NuRD complexes. Screening of transcriptional targets downstream of the CXXC5-CRL4B-NuRD complex by next-generation sequencing (chromatin immunoprecipitation sequencing) revealed that the complex regulates the transcriptional repression process of a cohort of genes, including TSC1 (tuberous sclerosis complex subunit 1), which play important roles in cell growth and mammalian target of rapamycin signaling pathway regulation, and whose abnormal regulation results in the activation of programmed cell death-ligand protein 1 (PD-L1). Intriguingly, CXXC5 expression increased after stimulation with vitamin B2 but decreased after vitamin D treatment. We also found that the CXXC5-CRL4B-NuRD complex promotes the proliferation of tumor cells in vitro and accelerates the growth of breast cancer in vivo. The expression of CXXC5, CUL4B, and MTA1 increased during the occurrence and development of breast cancer, and correspondingly, TSC1 expression decreased. Meanwhile, a high expression of CXXC5 was positively correlated with the histological grade of high malignancy and poor survival of patients. In conclusion, our study revealed that CXXC5-mediated TSC1 suppression activates the mammalian target of rapamycin pathway, reduces autophagic cell death, induces PD-L1-mediated immune suppression, and results in tumor development, shedding light on the mechanism of the pathophysiological function of CXXC5.

FAT10 induces immune suppression by upregulating PD-L1 expression in hepatocellular carcinoma.

The upregulation of programmed death ligand 1 (PD-L1) plays a crucial role in facilitating cancer cells to evade immune surveillance through immunosuppression. However, the precise regulatory mechanisms of PD-L1 in hepatocellular carcinoma (HCC) remain undefined. The correlation between PD-L1 and ubiquitin-like molecules (UBLs) was studied using sequencing data from 20 HCC patients in our center, combined with TCGA data. Specifically, the association between FAT10 and PD-L1 was further validated at both the protein and mRNA levels in HCC tissues from our center. Subsequently, the effect of FAT10 on tumor progression and immune suppression was examined through both in vivo and in vitro experiments. Utilizing sequencing data, qPCR, and Western blotting assays, we confirmed that FAT10 was highly expressed in HCC tissues and positively correlated with PD-L1 expression. Additionally, in vitro experiments demonstrated that the overexpression of FAT10 fostered the proliferation, migration, and invasion of HCC cells. Furthermore, the overexpression of FAT10 in HCC cells led to an increase in PD-L1 expression, resulting in the inhibition of T cell proliferation and the enhancement of HCC cell resistance to T cell-mediated cytotoxicity. Moreover, in vivo experiments utilizing the C57BL/6 mouse model revealed that overexpression of FAT10 effectively suppressed the infiltration of CD8 + GZMB + and CD8 + Ki67 + T cells, as well as reduced serum levels of TNF-α and IFN-γ. Mechanistically, we further identified that FAT10 upregulates PD-L1 expression via activating the PI3K/AKT/mTOR pathway, but not in a ubiquitin-like modification. In conclusion, our findings indicate that FAT10 promotes immune evasion of HCC via upregulating PD-L1 expression, suggesting its potential as a novel target to enhance the efficiency of immunotherapy in HCC.

The Host Protein CAD Regulates the Replication of FMDV through the Function of Pyrimidines' De Novo Synthesis.

Foot-and-mouth disease virus (FMDV) is a single-stranded picornavirus that causes economically devastating disease in even-hooved animals. There has been little research on the function of host cells during FMDV infection. We aimed to shed light on key host factors associated with FMDV replication during acute infection. We found that HDAC1 overexpression in host cells induced upregulation of FMDV RNA and protein levels. Activation of the AKT-mammalian target of rapamycin (mTOR) signaling pathway using bpV(HOpic) or SC79 also promoted FMDV replication. Furthermore, short hairpin RNA (shRNA)-induced suppression of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), a transcription factor downstream of the AKT-mTOR signaling pathway, resulted in downregulation of FMDV RNA and protein levels. Coimmunoprecipitation assays showed that the ACTase domain of CAD could interact with the FMDV 2C protein, suggesting that the ACTase domain of CAD may be critical in FMDV replication. CAD proteins participate in de novo pyrimidine synthesis. Inhibition of FMDV replication by deletion of the ACTase domain of CAD in host cells could be reversed by supplementation with uracil. These results revealed that the contribution of the CAD ACTase domain to FMDV replication is dependent on de novo pyrimidine synthesis. Our research shows that HDAC1 promotes FMDV replication by regulating de novo pyrimidine synthesis from CAD via the AKT-mTOR signaling pathway. IMPORTANCE Foot-and-mouth disease virus is an animal virus of the Picornaviridae family that seriously harms the development of animal husbandry and foreign trade of related products, and there is still a lack of effective means to control its harm. Replication complexes would generate during FMDV replication to ensure efficient replication cycles. 2C is a common viral protein in the replication complex of Picornaviridae virus, which is thought to be an essential component of membrane rearrangement and viral replication complex formation. The host protein CAD is a key protein in the pyrimidines de novo synthesis. In our research, the interaction of CAD and FMDV 2C was demonstrated in FMDV-infected BHK-21 cells, and it colocalized with 2C in the replication complex. The inhibition of the expression of FMDV 3D protein through interference with CAD and supplementation with exogenous pyrimidines reversed this inhibition, suggesting that FMDV might recruit CAD through the 2C protein to ensure pyrimidine supply during replication. In addition, we also found that FMDV infection decreased the expression of the host protein HDAC1 and ultimately inhibited CAD activity through the AKT-mTOR signaling pathway. These results revealed a unique means of counteracting the virus in BHK-21 cells lacking the interferon (IFN) signaling pathway. In conclusion, our study provides some potential targets for the development of drugs against FMDV.

TRIM26 inhibits clear cell renal cell carcinoma progression through destabilizing ETK and thus inactivation of AKT/mTOR signaling.

BACKGROUND: Tripartite motif-containing 26 (TRIM26), a member of the TRIM protein family, exerts dual function in several types of cancer. Nevertheless, the precise role of TRIM26 in clear cell renal cell carcinoma (ccRCC) has not been investigated. METHODS: The expression of TRIM26 in ccRCC tissues and cell lines were examined through the use of public resources and experimental validation. The impacts of TRIM26 on cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process were determined via CCK-8, colony formation, EdU incorporation, wound healing, Transwell invasion, Western blot, and Immunofluorescence assays. RNA-seq followed by bioinformatic analyses were used to identify the downstream pathway of TRIM26. The interaction between TRIM26 and ETK was assessed by co-immunoprecipitation, qRT-PCR, Western blot, cycloheximide (CHX) chase, and in vivo ubiquitination assays. RESULTS: We have shown that TRIM26 exhibits a downregulation in both ccRCC tissues and cell lines. Furthermore, this decreased expression of TRIM26 is closely linked to unfavorable overall survival and diseases-free survival outcomes among ccRCC patients. Gain- and loss-of-function experiments demonstrated that increasing the expression of TRIM26 suppressed the proliferation, migration, invasion, and EMT process of ccRCC cells. Conversely, reducing the expression of TRIM26 had the opposite effects. RNA sequencing, coupled with bioinformatic analysis, revealed a significant enrichment of the mTOR signaling pathway in the control group compared to the group with TRIM26 overexpression. This finding was then confirmed by a western blot assay. Subsequent examination revealed that TRMI26 had a direct interaction with ETK, a non-receptor tyrosine kinase. This interaction facilitated the ubiquitination and degradation of ETK, resulting in the deactivation of the AKT/mTOR signaling pathway in ccRCC. ETK overexpression counteracted the inhibitory effects of TRIM26 overexpression on cell proliferation, migration, and invasion. CONCLUSION: Our results have shown a novel mechanism by which TRIM26 hinders the advancement of ccRCC by binding to and destabilizing ETK, thus leading to the deactivation of AKT/mTOR signaling. TRIM26 shows promise as both a therapeutic target and prognostic biomarker for ccRCC patients.

Chondrocyte autophagy mediated by T-2 toxin via AKT/TSC/Rheb/mTOR signaling pathway and protective effect of CSA-SeNP.

OBJECTIVE: Kashin-Beck disease (KBD) is an endemic, degenerative, and cartilage-damaging disease for which low selenium and T-2 toxins are considered environmental pathogenic factors. This study aimed to investigate the molecular mechanisms of autophagy in cartilage damage caused by T-2 toxin and the protective effect of chondroitin sulfate A nano-elemental selenium (CSA-SeNP) on the cartilage. METHODS: KBD chondrocytes and C28/I2 human chondrocyte cell lines were used. T-2 toxin, AKT inhibitor, and CSA-SeNP treatment experiments were conducted separately, with a treatment time of 24 h. Autophagy was monitored using MDC staining, and mRFP-GFP-LC3 adenovirus, respectively. RT-qPCR and western blotting were used to detect the expression of the relevant genes and proteins. RESULTS: The suppression of autophagy observed in KBD chondrocytes was replicated by applying 10 ng/mL T-2 toxin to C28/I2 chondrocytes for 24 h. The AKT/TSCR/Rheb/mTOR signaling pathway was activated by T-2 toxin, which inhibits autophagy. The supplementation with CSA-SeNP alleviated the inhibition of autophagy by T-2 toxin through the AKT/TSCR/Rheb/mTOR signaling pathway. CONCLUSIONS: Loss of autophagy regulated by the AKT/TSCR/Rheb/mTOR signaling pathway plays an important role in cartilage damage caused by T-2 toxin. CSA-SeNP supplementation attenuated inhibition of autophagy in chondrocytes by T-2 toxin by modulating this signaling pathway. These findings provide promising new targets for the prevention and treatment of cartilage disease.

The lncRNA LINC01605 promotes the progression of pancreatic ductal adenocarcinoma by activating the mTOR signaling pathway.

BACKGROUND: This study investigated the molecular mechanism of long intergenic non-protein coding RNA 1605 (LINC01605) in the process of tumor growth and liver metastasis of pancreatic ductal adenocarcinoma (PDAC). METHODS: LINC01605 was filtered out with specificity through TCGA datasets (related to DFS) and our RNA-sequencing data of PDAC tissue samples from Renji Hospital. The expression level and clinical relevance of LINC01605 were then verified in clinical cohorts and samples by immunohistochemical staining assay and survival analysis. Loss- and gain-of-function experiments were performed to estimate the regulatory effects of LINC01605 in vitro. RNA-seq of LINC01605-knockdown PDAC cells and subsequent inhibitor-based cellular function, western blotting, immunofluorescence and rescue experiments were conducted to explore the mechanisms by which LINC01605 regulates the behaviors of PDAC tumor cells. Subcutaneous xenograft models and intrasplenic liver metastasis models were employed to study its role in PDAC tumor growth and liver metastasis in vivo. RESULTS: LINC01605 expression is upregulated in both PDAC primary tumor and liver metastasis tissues and correlates with poor clinical prognosis. Loss and gain of function experiments in cells demonstrated that LINC01605 promotes the proliferation and migration of PDAC cells in vitro. In subsequent verification experiments, we found that LINC01605 contributes to PDAC progression through cholesterol metabolism regulation in a LIN28B-interacting manner by activating the mTOR signaling pathway. Furthermore, the animal models showed that LINC01605 facilitates the proliferation and metastatic invasion of PDAC cells in vivo. CONCLUSIONS: Our results indicate that the upregulated lncRNA LINC01605 promotes PDAC tumor cell proliferation and migration by regulating cholesterol metabolism via activation of the mTOR signaling pathway in a LIN28B-interacting manner. These findings provide new insight into the role of LINC01605 in PDAC tumor growth and liver metastasis as well as its value for clinical approaches as a metabolic therapeutic target in PDAC.

Effect of berberine combined with metformin on autophagy in polycystic ovary syndrome by regulating AMPK/AKT/mTOR pathway.

The pathologic mechanism of polycystic ovary syndrome (PCOS) is related to increased autophagy of granulosa cells. Both berberine and metformin have been shown to improve PCOS, but whether the combination of berberine and metformin can better improve PCOS by inhibiting autophagy remains unclear. PCOS models were constructed by injecting dehydroepiandrosterone into rats, and berberine, metformin or berberine combined with metformin was administered to rats after modeling. Rats' body weight and ovarian weight were measured before and after modeling. Histopathological examination of ovarian tissue and estrous cycle analysis of rats were performed. Insulin resistance, hormone levels, oxidative stress, and lipid metabolism in PCOS rats were assessed. Expression of the AMPK/AKT/mTOR pathway and autophagy-related proteins was analyzed by Western blot assays. Granulosa cells were isolated from rat ovarian tissue and identified by immunofluorescence staining followed by transmission electron microscopy analysis. Berberine combined with metformin reduced the body weight and ovarian weight of PCOS rats, increased the number of primordial and primary follicles, decreased the number of secondary and atretic follicles, normalized the estrous cycle, and improved insulin resistance, androgen biosynthesis, oxidative stress and lipid metabolism disorders, and increased estrogen production. In addition, berberine combined with metformin reduced the number of autophagosomes in granulosa cells, which may be related to AMPK/AKT/mTOR pathway activation, decreased Beclin1 and LC3II/LC3I levels, and increased p62 expression. Berberine combined with metformin could inhibit autophagy by activating the AMPK/AKT/mTOR pathway in PCOS, indicating that berberine combined with metformin is a potential treatment strategy for PCOS.

Vitamin D Relieves Epilepsy Symptoms and Neuroinflammation in Juvenile Mice by Activating the mTOR Signaling Pathway via RAF1: Insights from Network Pharmacology and Molecular Docking Studies.

Epilepsy is a common neurological disorder, and the exploration of potential therapeutic drugs for its treatment is still ongoing. Vitamin D has emerged as a promising treatment due to its potential neuroprotective effects and anti-epileptic properties. This study aimed to investigate the effects of vitamin D on epilepsy and neuroinflammation in juvenile mice using network pharmacology and molecular docking, with a focus on the mammalian target of rapamycin (mTOR) signaling pathway. Experimental mouse models of epilepsy were established through intraperitoneal injection of pilocarpine, and in vitro injury models of hippocampal neurons were induced by glutamate (Glu) stimulation. The anti-epileptic effects of vitamin D were evaluated both in vivo and in vitro. Network pharmacology and molecular docking analysis were used to identify potential targets and regulatory pathways of vitamin D in epilepsy. The involvement of the mTOR signaling pathway in the regulation of mouse epilepsy by vitamin D was validated using rapamycin (RAPA). The levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) were assessed by enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining was used to analyze the apoptosis of hippocampal neurons. In in vivo experiments, vitamin D reduced the Racine scores of epileptic mice, prolonged the latency of epilepsy, and inhibited the production of TNF-α, IL-1ß, and IL-6 in the hippocampus. Furthermore, network pharmacology analysis identified RAF1 as a potential target of vitamin D in epilepsy, which was further confirmed by molecular docking analysis. Additionally, the mTOR signaling pathway was found to be involved in the regulation of mouse epilepsy by vitamin D. In in vitro experiments, Glu stimulation upregulated the expressions of RAF1 and LC3II/LC3I, inhibited mTOR phosphorylation, and induced neuronal apoptosis. Mechanistically, vitamin D activated the mTOR signaling pathway and alleviated mouse epilepsy via RAF1, while the use of the pathway inhibitor RAPA reversed this effect. Vitamin D alleviated epilepsy symptoms and neuroinflammation in juvenile mice by activating the mTOR signaling pathway via RAF1. These findings provided new insights into the molecular mechanisms underlying the anti-epileptic effects of vitamin D and further supported its use as an adjunctive therapy for existing anti-epileptic drugs.

The pathogenicity of vancomycin-resistant Enterococcus faecalis to colon cancer cells.

BACKGROUND: The aim of this study was to investigate the pathogenicity of vancomycin-resistant Enterococcus faecalis (VREs) to human colon cells in vitro. METHODS: Three E. faecalis isolates (2 VREs and E. faecalis ATCC 29212) were cocultured with NCM460, HT-29 and HCT116 cells. Changes in cell morphology and bacterial adhesion were assessed at different time points. Interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGFA) expression were measured via RT-qPCR and enzyme-linked immunosorbent assay (ELISA), respectively. Cell migration and human umbilical vein endothelial cells (HUVECs) tube formation assays were used for angiogenesis studies. The activity of PI3K/AKT/mTOR signaling pathway was measured by Western blotting. RESULTS: The growth and adhesion of E. faecalis at a multiplicity of infection (MOI) of 1:1 were greater than those at a MOI of 100:1(p < 0.05). Compared to E. faecalis ATCC 29212, VREs showed less invasive effect on NCM460 and HT-29 cells. E. faecalis promoted angiogenesis by secreting IL-8 and VEGFA in colon cells, and the cells infected with VREs produced more than those infected with the standard strain (p < 0.05). Additionally, the PI3K/AKT/mTOR signaling pathway was activated in E. faecalis infected cells, with VREs demonstrating a greater activation compared to E. faecalis ATCC 29212 (p < 0.05). CONCLUSION: VREs contribute to the occurrence and development of CRC by promoting angiogenesis and activating the PI3K/AKT/mTOR signaling pathway.

Capsaicin attenuates Porphyromonas gingivalis-suppressed osteogenesis of periodontal ligament stem cells via regulating mitochondrial function and activating PI3K/AKT/mTOR pathway.

BACKGROUND AND OBJECTIVE: Prevention of periodontal bone resorption triggered by Porphyromonas gingivalis (P. gingivalis) is crucial for dental stability. Capsaicin, known as the pungent ingredient of chili peppers, can activate key signaling molecules involved in osteogenic process. However, the effect of capsaicin on osteogenesis of periodontal ligament stem cells (PDLSCs) under inflammation remains elusive. METHODS: P. gingivalis culture suspension was added to mimic the inflammatory status after capsaicin pretreatment. The effects of capsaicin on the osteogenesis of PDLSCs, as well as mitochondrial morphology, Ca2+ level, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and osteogenesis-regulated protein expression levels were analyzed. Furthermore, a mouse experimental periodontitis model was established to evaluate the effect of capsaicin on alveolar bone resorption and the expression of osteogenesis-related proteins. RESULTS: Under P. gingivalis stimulation, capsaicin increased osteogenesis of PDLSCs. Not surprisingly, capsaicin rescued the damage to mitochondrial morphology, decreased the concentration of intracellular Ca2+ and ROS, enhanced MMP and activated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. The in vivo results showed that capsaicin significantly attenuated alveolar bone loss and augmented the expression of bone associated proteins. CONCLUSION: Capsaicin increases osteogenesis of PDLSCs under inflammation and reduces alveolar bone resorption in mouse experimental periodontitis.

Total flavone of Abelmoschus manihot regulates autophagy through the AMPK/mTOR signaling pathway to treat intestinal fibrosis in Crohn's disease.

BACKGROUND AND AIM: Drug therapy is the treatment of choice for Crohn's disease because it effectively controls or prevents intestinal inflammation. The purpose was to research the molecular mechanism of the total flavones of Abelmoschus manihot (TFA) on intestinal fibrosis in Crohn's disease. METHODS: A 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis model and IGF-1-treated intestinal fibroblasts were established. Then, TFA, 3-MA, and compound C were used treatments. Hematoxylin and eosin, Masson, and Picrosirius red staining were performed to observe the colon tissue. Immunohistochemical staining was used to detect α-SMA expression. Flow cytometry, CCK8, wound healing, and Transwell assays were conducted to determine apoptosis, proliferation, invasion, and migration. Col1a1 and Col3a1 levels were detected using quantitative polymerase chain reaction. Proteins related to autophagy and apoptosis were detected using western blotting. RESULTS: TFA treated intestinal fibrosis in chronic Crohn's disease. Colon length was the shortest in the ethanol + TNBS group, and TFA treatment significantly improved the situation. Intestinal fibrosis and the percentage of collagen area decreased after TFA treatment. TFA reduced fibrosis by enhancing autophagy stimulation, whereas an autophagy inhibitor reversed the TFA effect. TFA also inhibited migration, proliferation, and collagen synthesis in intestinal fibroblasts. Moreover, it enhanced autophagy and apoptosis of intestinal fibroblasts. TFA upregulated p-AMPK expression and decreases p-mTOR levels. Compound C partially rescued the effect of TFA, indicating that TFA affected intestinal fibroblasts via the AMPK/mTOR pathway in vitro and in vivo. TFA also downregulated Col1a1 and Col3a1 expression. CONCLUSION: TFA regulates autophagy through AMPK/mTOR signaling pathway to treat intestinal fibrosis, which may provide a new therapy for Crohn's disease treatment.

Novel protein C6ORF120 promotes liver fibrosis by activating hepatic stellate cells through the PI3K/Akt/mTOR pathway.

BACKGROUND AND AIM: The role of C6ORF120 in promoting CCL4-induced hepatic fibrosis and its possible mechanisms were explored in C6orf120 knockout rats (C6orf120-/-) and LX-2 cells (a type of human hepatic stellate cell line). METHODS: In vivo experiments, wild-type and C6orf120-/- rats were used to investigate the function of C6ORF120. In the in vitro experiments, C6ORF120 recombinant protein (rC6ORF120) at a concentration of 200 ng/mL was used to stimulate LX-2 cells. Sirius Red staining, Masson staining, western blotting, polymerase chain reaction, immunohistochemistry, and immunofluorescence were used to explore fibrosis-associated factors. RESULTS: C6orf120-/- rats showed mild fibrosis and liver injury in the CCL4-induced liver fibrosis model. Furthermore, RNA-seq revealed that C6orf120-/- rats had less extracellular matrix deposition and activated stellate cells. Consistent with the in vivo, the rC6ORF120 induced LX-2 cell activation. Moreover, mechanistic studies revealed that the p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR levels were significantly elevated and LY294002 (a PI3K/Akt/mTOR typical pathway inhibitor) reversed the function of C6ORF120 in activating LX-2 cells. CONCLUSION: C6ORF120 could activate hepatic stellate cells and promote hepatic fibrosis via the PI3K/Akt/mTOR signaling pathway.

Silencing of GDF11 suppresses hepatocyte apoptosis to relieve LPS/D-GalN acute liver failure.

In this paper, we generated a short hairpin RNA growth differentiation factor-11 (sh-GDF11) and evaluated the effects of sh-GDF11 on the pathogenesis of acute liver failure (ALF) in vitro and in vivo. Through bioinformatics study, the key gene related to ALF was assayed. Lipopolysaccharide (LPS) and D-galactoamine (D-GalN) were applied to establish the mouse model of LPS/D-GalN-induced liver injury, and TNF-α and D-Gal were used to construct an in vitro cell model, followed by treatment of sh-GDF11 for analysis of liver cell proliferation. Bioinformatics analysis showed that the protective effect of sh-GDF11 on ALF may be mediated by phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. The results of in vitro study found that sh-GDF11 could promote cell proliferation and inhibit death by blocking the PI3K/Akt/mTOR signaling pathway. In vivo animal experiments further confirmed that sh-GDF11 could suppress hepatocyte apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway. sh-GDF11 relieved LPS/D-GalN-induced ALF by blocking the PI3K/Akt/mTOR signaling pathway, emphasizing its critical role in LPS/D-GalN-induced ALF treatment.

ACTL8 Promotes the Progression of Gastric Cancer Through PI3K/AKT/mTOR Signaling Pathway.

BACKGROUND: Actin-like protein 8 (ACTL8) significantly correlates with tumor growth and prognosis across various cancer types. Nevertheless, the potential relationship between ACTL8 and gastric cancer (GC) remains uncertain. OBJECTIVE: This study aimed to elucidate the role of ACTL8 in human GC cells and to explore its mechanism. METHODS: Bioinformatics analysis tools, such as GEPIA2, Kaplan-Meier, and STRING, were utilized for a comprehensive investigation of the characteristics and functional roles of ACTL8 in GC, including differential expression, prognostic value, and related signaling pathways. Subsequently, gene expression analyses, cell function assays, and signaling pathway experiments were conducted to verify key findings. RESULTS: Bioinformatics analysis showed that ACTL8 was significantly elevated in GC and closely associated with poor prognosis. Gene expression experiments confirmed the bioinformatics results. Furthermore, ACTL8 knockdown markedly reduced GC cell proliferation and inhibited migration and invasion. Mechanistically, a significant increase in the phosphorylation levels of signaling proteins was observed in GC cells following ACTL8 overexpression, and PI3K/Akt/mTOR pathway inhibitors could reverse this effect. CONCLUSION: ACTL8 expression is significantly upregulated in GC cells and is closely correlated with poor patient prognosis. Further mechanistic studies revealed that ACTL8 may promote GC cell migration and proliferation through activation of the PI3K/Akt/mTOR signaling pathway. Consequently, ACTL8 emerges as a promising therapeutic target for GC.

Knockdown of SETD5 Inhibits Colorectal Cancer Cell Growth and Stemness by Regulating PI3K/AKT/mTOR Pathway.

SET domain-containing 5 (SETD5), a member of protein lysine methyltransferase family, is expressed in multiple cancers, making it potential therapeutic targets. However, the role of SETD5 in colorectal cancer remains largely unknown. The expression of SETD5 in the 30 pairs colorectal cancer tissues samples and cell lines were determined by qRT-PCR. The functions of SETD5 was detected by knocked-down or overexpression in colorectal cancer cell lines SW480 and HCT116 cells. Cell proliferative activity, cell death, and stemness characteristics were assessed. BEZ235, a PI3K/AKT/mTOR pathway inhibitor, was used to perform rescue experiment to analyze whether SETD5 exerted its effects through activating PI3K/AKT/mTOR pathway. SETD5 was substantially upregulated in colorectal cancer, and correlated to metastasis and clinical stage of patients. Knockdown of SETD5 inhibited SW480 and HCT116 cell growth, as evidenced by the inhibition of cell viability and clone-forming. Moreover, Knockdown of SETD5 suppressed the capability of tumor sphere formation of SW480 and HCT116 cells, and reduced the expression of stemness-related proteins Nanog and Sox2. Further western blot analysis revealed that SETD5 knockdown inhibited the phosphorylation of proteins associated with the PI3K/AKT/mTOR pathway. In contrast, overexpression of SETD5 exerted the opposite effects. Mechanistically, by blocking PI3K/AKT/mTOR pathway with BEZ235, the effects of SETD5 overexpression on cell viability and Nanog and Sox2 protein expression were reversed. Our results substantiated that SETD5 functioned as an oncogene by promoting cell growth and stemness in colorectal cancer cells through activating the PI3K/AKT/mTOR signaling pathway.

SKI-349, a Sphingosine Kinases 1/2 Inhibitor, Suppresses Cell Viability, Invasion, and AKT/mTOR Signaling Pathway, and Shows Synergistic Cytotoxic Effects with Sorafenib in Hepatocellular Carcinoma.

SKI-349 is a novel sphingosine kinases (SPHK) inhibitor with anti-tumor effects. This study aimed to assess the effect of SKI-349 on cell biological behaviors, downstream pathways, and its synergistic effect with sorafenib in hepatocellular carcinoma (HCC). HCC cell lines (Huh7 and Hep3B) were treated with SKI-349 at concentrations of 1, 2, 4, or 8 µM. Then, SPHK1/2 activity, cell viability, proliferation, apoptosis, invasion, and protein expressions of phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR) and mTOR were detected. Combination index values of SKI-349 (0, 1, 2, 4, or 8 µM) and sorafenib (0, 2.5, 5, 10, or 20 µM) were calculated. SKI-349 decreased the relative SPHK1 and SPHK2 activity compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Meanwhile, SKI-349 reduced cell viability, 5-ethynyl-2'-deoxyuridine (EdU) positive cells, and invasive cells, while it increased apoptotic cells compared to blank control in a dose-dependent manner in Huh7 and Hep3B cell lines. Based on the western blot assay, SKI-349 decreased the ratio of p-AKT to AKT and that of p-mTOR to mTOR compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Additionally, SKI-349 combined with sorafenib declined cell viability with concentration gradient effects compared to SKI-349 sole treatment, and they had synergistic cytotoxic effects in Huh7 and Hep3B cell lines. SKI-349 suppresses SPHK1 and SPHK2 activity, cell viability, invasion, and AKT/mTOR signaling pathway, as well as exhibits a synergistic cytotoxic effect with sorafenib in HCC.

The Exploratory Study of the PTEN-AKT/mTOR Signaling Pathway in the Corresponding Dorsal Root Ganglion during Compensatory Repair via Small Gap Amplification in Sciatic Nerve Injury.

BACKGROUND: Peripheral nerve injury is a challenging orthopedic issue in clinical management that often leads to limb dysfunction or even disability in severe cases. A thorough exploration of the repair process of peripheral nerve injury and the underlying mechanism contributes to formulate more effective therapeutic strategies. METHODS: In the present study, we established a sciatic nerve transection injury model in Sprague-Dawley (SD) rats. A 12-week compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis was then performed via sleeve jointing the proximal common peroneal nerve to the distal tibial nerve and common peroneal nerve, with a 2 mm interval. Compensatory repair via small gap amplification was observed via gross observation of nerve specimen, osmic acid staining, and electrophysiological stimulation of sciatic nerve branches of the tibial and common peroneal nerve. Rat limbs were observed, and the functional recovery of effector muscles of the gastrocnemius and tibialis anterior muscles was assessed through weighing the muscle wet weight, Hematoxylin and Eosin (H&E) staining, and muscle strength detection. H&E staining, Masson staining, and toluidine blue staining were performed to observe the morphological changes of the dorsal root ganglion. Positive expressions of key proteins involved in the Phosphatase and tensin homologue deleted on chromosome ten (PTEN)-protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, including PTEN, AKT, mTOR, Toll-like receptor 4 (TLR4), and Caspase9 in the dorsal root ganglion during compensatory repair of sciatic nerve after injury via small gap amplification, were detected by immunohistochemical staining. RESULTS: It is found that the compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis via sleeve jointing effectively restored the continuity, number of myelinated nerve fibers, and nerve conduction velocity. It promoted toe abduction recovery, improved muscle fiber morphology and increased the wet weight and muscle strength of the gastrocnemius muscle and tibialis anterior muscle. Moreover, it increased the number of neurons and nerve fibers, and improved their morphology. Downregulated PTEN, TLR4, and Caspase9 in the dorsal root ganglia and upregulated AKT and mTOR were observed after small gap amplification than those of the transection injury group, which were closer to those of the control group. CONCLUSIONS: Compensatory repair of sciatic nerve transection injury using a chitin cannula for small gap anastomosis via sleeve jointing can restore the morphology and function of the sciatic nerve, effector muscles, and corresponding dorsal root ganglia by activating the PTEN-AKT/mTOR signaling pathway in the dorsal root ganglia. Our findings provide novel therapeutic targets for peripheral nerve injuries.

Eleutheroside B Pretreatment Attenuates Hypobaric Hypoxia-Induced High-Altitude Pulmonary Edema by Regulating Autophagic Flux via the AMPK/mTOR Pathway.

High-altitude pulmonary edema (HAPE) is a life-threatening disease, and autophagy deficiency is implicated in the pathogenesis of HAPE. Eleutheroside B (EB), which is the main bioactive component of Acanthopanax senticosus, exhibits various pharmacological activities. Our previous research demonstrated that autophagic structures were widely found in the ultrastructure of lung tissue in HAPE rats. However, whether EB regulates autophagy deficiency in HAPE remains unknown. This study aimed to investigate the protective effects of EB on hypobaric hypoxia-induced HAPE and explore the underlying molecular mechanism of regulating autophagy. The rat model of high-altitude pulmonary edema was replicated using a hypobaric hypoxic chamber. Rats were pretreated with EB or in combination with chloroquine or compound C. The pulmonary edema was assessed by the lung wet/dry ratio, total protein concentration in bronchoalveolar lavage fluid, and histological analysis. Inflammation and oxidative stress were measured using commercial biochemical kits. Autophagy and autophagic flux were evaluated by western blotting, transmission electron microscopy, and adeno-associated virus-mRFP-GFP-labeled tandem fluorescence LC3. The AMPK/mTOR signaling pathway was detected by western blotting. EB alleviated hypobaric hypoxia-induced pulmonary edema, hypoxemia, acid-base imbalance in the blood, inflammation, and oxidative stress in a dose-dependent manner. EB restored impaired autophagic flux by activating the AMPK/mTOR signaling pathway. However, chloroquine or compound C abolished eleutheroside B-mediated autophagy flux restoration. EB has the potential to restore impaired autophagic flux in the lung of hypobaric hypoxia-induced HAPE rats, which could be attributed to the activation of AMPK/mTOR signaling pathway.

Natural products targeting macroautophagy signaling in hepatocellular carcinoma therapy: Recent evidence and perspectives.

Hepatocellular carcinoma (HCC), presently the second leading cause of global cancer-related mortality, continues to pose significant challenges in the realm of medical oncology, impacting both clinical drug selection and mechanistic research. Recent investigations have unveiled autophagy-related signaling as a promising avenue for HCC treatment. A growing body of research has highlighted the pivotal role of autophagy-modulating natural products in inhibiting HCC progression. In this context, we provide a concise overview of the fundamental autophagy mechanism and delineate the involvement of autophagic signaling pathways in HCC development. Additionally, we review pertinent studies demonstrating how natural products regulate autophagy to mitigate HCC. Our findings indicate that natural products exhibit cytotoxic effects through the induction of excessive autophagy, simultaneously impeding HCC cell proliferation by autophagy inhibition, thereby depriving HCC cells of essential energy. These effects have been associated with various signaling pathways, including PI3K/AKT, MAPK, AMPK, Wnt/ß-catenin, Beclin-1, and ferroautophagy. These results underscore the considerable therapeutic potential of natural products in HCC treatment. However, it is important to note that the present study did not establish definitive thresholds for autophagy induction or inhibition by natural products. Further research in this domain is imperative to gain comprehensive insights into the dual role of autophagy, equipping us with a better understanding of this double-edged sword in HCC management.

MiR-150-5p contributes to unexplained recurrent spontaneous abortion by targeting VEGFA and downregulating the PI3K/AKT/mTOR signaling pathway.

PURPOSE: The purpose of this study is to investigate the function of miR-150-5p in URSA. METHOD: Twenty-six chorionic villous tissues were collected to examine the expression of miR-150-5p and VEGFA by using quantitative polymerase chain reaction (qPCR) and western blot assay, respectively. Transwell assay was conducted to assess the migration and invasion ability of trophoblast cells. The dual-luciferase reporter assay was applied to determine the relationship between miR-150-5p and VEGFA in vitro. Relevant signaling pathway protein expression level was measured via western blot assay. Signaling transduction inhibitor LY294002 was used to block PI3K/AKT/mTOR signaling pathway. Finally, in vivo the effect of miR-150-5p on embryonic absorption rate was evaluated in mice. RESULTS: Clinical samples revealed that miR-150-5p expression was significantly elevated in the villous tissues and serum of URSA patients. Moreover, the overexpressing of miR-150-5p could inhibit both HTR-8/SVneo cell and JAR cell migration, invasion, and restrained PI3K/AKT/mTOR signaling pathway by targeting VEGFA in vitro. This inhibitory effect of miR-150-5p could be reversed by overexpressing the gene of vascular epithelial growth factor A (VEGFA). In contrary, inhibition of miR-150-5p significantly enhanced migration, invasion ability of both HTR-8/SVneo and JAR cells, and also could stimulate PI3K/AKT/mTOR signaling pathway. This promoting effect of miR-150-5p could be ameliorated by LY294002 (PI3K inhibitor). Finally, after miR-150-5p overexpression in vivo, the embryo resorption rate in pregnant mice was increased significantly. CONCLUSIONS: Overall, these findings imply that miR-150-5p is among the key factors that regulate the pathogenesis of URSA.