Coexpression of TP53, BIM, and PTEN Enhances the Therapeutic Efficacy of Non-Small-Cell Lung Cancer.

For non-small-cell lung cancer (NSCLC), the ubiquitous occurrence of concurrent multiple genomic alterations poses challenges to single-gene therapy. To increase therapeutic efficacy, we used the branch-PCR method to develop a multigene nanovector, NP-TP53-BIM-PTEN, that carried three therapeutic gene expression cassettes for coexpression. NP-TP53-BIM-PTEN exhibited a uniform size of 104.8 ± 24.2 nm and high serum stability. In cell transfection tests, NP-TP53-BIM-PTEN could coexpress TP53, BIM, and PTEN in NCI-H1299 cells and induce cell apoptosis with a ratio of up to 94.9%. Furthermore, NP-TP53-BIM-PTEN also inhibited cell proliferation with a ratio of up to 42%. In a mouse model bearing an NCI-H1299 xenograft tumor, NP-TP53-BIM-PTEN exhibited a stronger inhibitory effect on the NCI-H1299 xenograft tumor than the other test vectors without any detectable side effects. These results exhibited the potential of NP-TP53-BIM-PTEN as an effective and safe multigene nanovector to enhance NSCLC therapy efficacy, which will provide a framework for genome therapy with multigene combinations.

Zinc Inhibits Lead-Induced Oxidative Stress and Apoptosis of ST Cells Through ROS/PTEN/PI3K/AKT Axis.

Lead (Pb) is a widely distributed toxic heavy metal element known to have strong male reproductive toxicity, which can result in issues such as abnormal count and morphology of sperm. Zinc (Zn) is an essential trace element for the human body that can antagonize the activity of Pb in some physiological environments, and it also possesses antioxidant and anti-inflammatory effects. However, the specific mechanism of Zn's antagonism against Pb remains largely unclear. In our study, we conducted research using swine testis cells (ST cells) and confirmed that the half maximal inhibitory concentration of Pb on ST cells was 994.4 µM, and the optimal antagonistic concentration of Zn was 10 µM. Based on this information, we treated ST cells with Pb and Zn and detected related indices such as apoptosis, oxidative stress, and the PTEN/PI3K/AKT pathway using flow cytometry, DCFH-DA staining, RT-PCR, and Western blot. Our results demonstrated that Pb exposure can generate excessive reactive oxygen species (ROS), disrupt the antioxidant system, upregulate PTEN expression, and inhibit the PI3K/AKT pathway in ST cells. In contrast, Zn significantly inhibited the overproduction of ROS, improved oxidative stress, and decreased PTEN expression, thus protecting the PI3K/AKT pathway compared to Pb-exposed ST cells. Furthermore, we found that Pb exposure exacerbated the expression of genes related to the apoptosis pathway and reduced the expression of anti-apoptotic genes. Furthermore, this situation was significantly improved when co-cultured with Pb and Zn. In summary, our study demonstrated that Zn alleviated Pb-induced oxidative stress and apoptosis through the ROS/PTEN/PI3K/AKT axis in ST cells.

Impacts of delta 9-tetrahydrocannabinol against myocardial ischemia/reperfusion injury in diabetic rats: Role of PTEN/PI3K/Akt signaling pathway.

Despite the current optimal therapy, patients with myocardial ischemia/reperfusion (IR) injury still experience a high mortality rate, especially when diabetes mellitus is present as a comorbidity. Investigating potential treatments aimed at improving the outcomes of myocardial IR injury in diabetic patients is necessary. Our objective was to ascertain the cardioprotective effect of delta 9-tetrahydrocannabinol (THC) against myocardial IR injury in diabetic rats and examine the role of phosphatase and tensin homolog (PTEN)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in mediating this effect. Diabetes was induced in male Wistar rats (8-10 weeks old, 200-250 g; n = 60) by a single injection of streptozotocin. The duration of the diabetic period was 10 weeks. During the last 4 weeks of diabetic period, rats were treated with THC (1.5 mg/kg/day; intraperitoneally), either alone or in combination with LY294002, and then underwent IR intervention. After 24 h of reperfusion, infarct size, cardiac function, lactate dehydrogenase (LDH) and cardiac-specific isoform of troponin-I (cTn-I) levels, myocardial apoptosis, oxidative stress markers, and expression of PTEN, PI3K, and Akt proteins were evaluated. THC pretreatment resulted in significant improvements in infarct size and cardiac function and decreases in LDH and cTn-I levels (P < 0.05). It also reduced myocardial apoptosis and oxidative stress, accompanied by the downregulation of PTEN expression and activation of the PI3K/Akt signaling pathway (P < 0.05). LY294002 pretreatment abolished the cardioprotective action of THC. This study revealed the cardioprotective effects of THC against IR-induced myocardial injury in diabetic rats and also suggested that the mechanism may be associated with enhanced activity of the PI3K/Akt signaling pathway through the reduction of PTEN phosphorylation.

Triptolide Reduces MDA-MB-231 Cell Metastasis by Attenuating Epithelial-Mesenchymal Transition through the ROCK/PTEN/Akt Axis.

Triple-negative breast cancer (TNBC) is a highly heterogeneous and invasive subtype of breast cancer. The prognosis of TNBC is poor because of its high distant metastasis rate. Triptolide is a type of diterpene trioxide natural compound with potential anti-tumor activities. This study explored the metastatic inhibitory effect of triptolide on MDA-MB-231 cells and its underlying mechanism. Triptolide suppressed cell proliferation and induced cell apoptosis in a time- and dose-dependent manner. Low doses of triptolide (0-8 nM) reduced the migration and invasion capabilities of MDA-MB-231 cells. Triptolide decreased ROCK1, p-Akt, N-cadherin, vimentin and MMP-9 expressions, but increased PTEN and E-cadherin expressions on protein and mRNA levels. Furthermore, the down-regulation of ROCK1 expression in MDA-MB-231 cells after being treated by triptolide could be rescued by ROCK1 specific inhibitor Y27632. Molecular docking showed that triptolide and Y27632 shared the same active center of ROCK1 protein. This article's findings taken together showed that ROCK1 is the primary target of triptolide, which can cause cell apoptosis and inhibit the epithelial-mesenchymal transition of MDA-MB-231 cells.

ELK4 Promotes Cell Cycle Progression and Stem Cell-like Characteristics in HPV-associated Cervical Cancer by Regulating the FBXO22/PTEN Axis

Background: Cervical cancer (CC) is a prevalent gynecological carcinoma, and patients infected with human papillomavirus (HPV) have a higher morbidity rate. Aims: To explore the effects of ETS-like transcription factor 4 (ELK4) in patients with HPV+ CC. Study design: In vitro cell lines and human-sample study. Methods: The ELK4 levels in human tissue (65 HPV+ CC tissue and 25 HPV− normal cervical tissue) and cell lines (human cervical epithelial immortalized cell line H8 and CC cell lines HeLa [HPV18], CaSki [HPV16], and SiHa [HPV−]) were quantified using qRT-PCR and western blot assay. ELK4 knockdown transfection was effective and confirmed by western blotting. The MTT and EDU assays were used to evaluate cell viability and proliferation, respectively. Flow cytometry was used to detect the CC cell cycle stage. Stem cell markers, such as cluster of differentiation 133 (CD133), CD44, and aldehyde dehydrogenase 1, and the cervicospheres formed were measured. ChIP-qPCR and luciferase activity experiments were used to assess the bond between ELK4 and F-box protein 22 (FBXO22). Results: ELK4 was highly expressed in the HPV+ CC tissue. CC cells with ELK4 knockdown had lower viability and proliferation than the control cells. ELK4 knockdown blocked the progression of the cell cycle from G1 to S phase. ELK4 knockdown suppressed the stem cell-like characteristics of the HPV+ CC cells. ELK4 bonded with the FBXO22 promoter, inhibiting the levels of phosphatase and tensin homolog (PTEN). Conclusion: ELK4 facilitated cell cycle progression and stem cell-like characteristics by regulating the FBXO22/PTEN axis. Thus, ELK4 could be a potential therapeutic target to arrest the progress of HPV-associated CC.

Rapamycin improves social and stereotypic behavior abnormalities induced by pre-mitotic neuronal subset specific Pten deletion.

The mechanistic target of rapamycin (mTOR) pathway is a signaling system integral to neural growth and migration. In both patients and rodent models, mutations to the phosphatase and tensin homolog gene (PTEN) on chromosome 10 results in hyperactivation of the mTOR pathway, as well as seizures, intellectual disabilities and autistic behaviors. Rapamycin, an inhibitor of mTOR, can reverse the epileptic phenotype of neural subset specific Pten knockout (NS-Pten KO) mice, but its impact on behavior is not known. To determine the behavioral effects of rapamycin, male and female NS-Pten KO and wildtype (WT) mice were assigned as controls or administered 10 mg/kg of rapamycin for 2 weeks followed by behavioral testing. Rapamycin improved social behavior in both genotypes and stereotypic behaviors in NS-Pten KO mice. Rapamycin treatment resulted in a reduction of several measures of activity in the open field test in both genotypes. Rapamycin did not reverse the reduced anxiety behavior in KO mice. These data show the potential clinical use of mTOR inhibitors by showing its administration can reduce the production of autistic-like behaviors in NS-Pten KO mice.

LINC00470 represses cell autophagy and cisplatin sensitivity of glioma via suppressing PTEN expression.

INTRODUCTION: Glioma is one of primary brain tumours which has the worst clinical prognoses of patients. As an alternative chemotherapeutic drug for malignant glioma, the therapeutic effect of cisplatin (CDDP) is devastatingly affected due to resistance in patients. In this study, we investigated the effect of LINC00470/PTEN on the CDDP sensitivity of glioma cells. MATERIAL AND METHODS: Differentially expressed lncRNAs and the downstream regulators in glioma tissue were obtained via bioinformatics analysis. LINC00470 and PTEN mRNA expression levels were detected using qRT-PCR. IC50 values of glioma cells were examined using Cell Counting Kit-8 (CCK-8). Cell apoptosis was revealed by flow cytometry. The expression level of autophagy-related protein was detected by western blot. Intracellular autophagosome formation was detected by immunofluorescence staining, and the methylation level of PTEN promoter was detected via methylation-specific PCR (MSP). RESULTS: Through the above steps, we found that LINC00470 was highly expressed in glioma cells, and the survival rate of patients was reduced in the presence of high expression of LINC00470. Silenced LINC00470 promoted LC3 II expression and autophagosome formation, and facilitated cell apoptosis to inhibit resistance to CDDP. While silenced PTEN could successfully reverse the previous effects on glioma cells. CONCLUSIONS: Based on the above, LINC00470 repressed cell autophagy by constraining PTEN, thereby enhancing CDDP resistance of glioma cells.

MiR-328-3p promotes TGF-ß1-induced proliferation, migration, and inflammation of airway smooth muscle cells by regulating the PTEN/Akt pathway.

BACKGROUND: Recent studies have shown that the up-regulation of microRNA miR-328-3p expression increases seasonal allergy and asthma symptoms in children, but the specific mechanism remains unclear. Therefore, the aim of this study was to explore the role and mechanism of -miR-328-3p in transforming growth factor (TGF)-ß1-induced airway smooth muscle cells (ASMCs). METHODS: The effect of TGF-ß1 on the expression of miR-328-3p in ASMCs was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cells proliferation, migration, and inflammatory factors in TGF-ß1-induced ASMCs were measured by cell counting kit-8 (CCK-8), transwell, and enzyme-linked immunosorbent assay (ELISA), respectively. Besides, TargetScan was used to predict phosphatase and tensin homolog (PTEN), the downstream target of miR-328-3p; double-luciferase reporter assay, western blot, and qRT-PCR were used to verify the targeting relationship between miR-328-3p and PTEN; western blot was also used to examine the effects of PTEN and miR-328-3p knockdown on the expression levels of PTEN, Akt, and p-Akt proteins. RESULTS: The expression of miR-328-3p was up-regulated in TGF-ß1-induced ASMCs. Knockdown of miR-328-3p significantly inhibited proliferation, migration, and inflammation of ASMCs induced by TGF-ß1 and decreased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß. The dual--luciferase reporter assay results confirmed that PTEN was a target gene of miR-328-3p. Moreover, inhibition of PTEN expression reversed the inhibitory effect of low miR-328-3p expression on -TGF-ß1-induced ASMC's proliferation, migration, and inflammation. In comparison to the knockdown of miR-328-3p alone, the simultaneous knockdown of miR-328-3p with PTEN decreased PTEN protein expression levels and increased p-Akt/Akt ratio in TGF-ß1-induced ASMCs. CONCLUSION: Through regulating the expression of PTEN and the activity of Akt signaling pathway, miR-328-3p promotes TGF-ß1-induced proliferation, migration, and inflammation of ASMCs.

[Effect and Mechanism of Atorvastatin on Reversing Drug Resistance in Leukemia by Regulating Glycolysis through PTEN/mTOR Pathway].

OBJECTIVE: To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM. METHODS: HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected. RESULTS: CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 µmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 µmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 µmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance. CONCLUSION: Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.

Fisetin alleviates cellular senescence through PTEN mediated inhibition of PKCδ-NOX1 pathway in vascular smooth muscle cells.

Reactive oxygen species (ROS) are a key risk factor of cellular senescence and age-related diseases, and protein kinase C (PKC) has been shown to activate NADPH oxidases (NOXs), which generate ROS. Although PKC activation induces oxidative stress, leading to the cellular dysfunction in various cell types, the correlation between PKC and senescence has not been reported in vascular smooth muscle cell (VSMC). Several studies have indicated cellular senescence is accompanied by phosphatase and tensin homolog (PTEN) loss and that an interaction exists between PTEN and PKC. Therefore, we aimed to determine whether PTEN and PKC are associated with VSMC senescence and to investigate the mechanism involved. We found hydrogen peroxide (H2O2) decreased PTEN expression and increased PKCδ phosphorylation. Moreover, H2O2 upregulated the NOX1 subunits, p22phox and p47phox, and induced VSMC senescence via p53-p21 signaling pathway. We identified PKCδ activation contributed to VSMC senescence through activation of NOX1 and ROS production. However, fisetin inhibited cellular senescence induced by the PTEN-PKCδ-NOX1-ROS signaling pathway, and this anti-aging effect was attributed to reduced ROS production caused by suppressing NOX1 activation. These results suggest that the PTEN-PCKδ signaling pathway is directly related to senescence via NOX1 activation and that the downregulation of PKCδ by flavonoids provides a potential means of treating age-associated diseases.

ELANE Promotes M2 Macrophage Polarization by Down-Regulating PTEN and Participates in the Lung Cancer Progression.

BACKGROUND: Macrophages are one of the most important immunoinflammatory cell populations in the tumor microenvironment (TME). In this study, we preliminarily investigated the upstream pathway of M2 macrophage polarization affecting lung cancer progression. METHODS: Bioinformatics analysis was used to evaluate genes closely associated with lung adenocarcinoma and their relationship with immune cells. THP-1 monocytes were induced into M2 macrophages. The expression of markers in M2 macrophages was detected by quantitative reverse transcription-PCR (qRT-PCR), enzyme linked immunosorbent assay (ELISA), and flow cytometry. The effects of neutrophil elastase (ELANE)-mediated M2 macrophages on lung cancer cell proliferation, migration and invasion and tumor growth were investigated by in vitro and in vivo experiments after co-culture of macrophage conditioned medium (CM) and lung cancer cell lines A549 and H1299. The PTEN protein expression was detected by Western blotting. RESULTS: ELANE was significantly positively correlated with M2 macrophages. ELANE up-regulated the expression of the M2 macrophage markers CD206, CCL22, IL-10 and CCL18 and increased the proportion of CD206+ macrophages. Compared with M0-CM, M2-CM promoted cell proliferation, migration, and invasion, and (M2+ELANE)-CM further enhanced this effect. In vivo, ELANE promoted M2 macrophage-induced tumor growth in lung cancer mice model. In vitro experiments showed that ELANE can down-regulate the expression of PTEN and promote the polarization of M2 macrophages. CONCLUSION: ELANE promotes the polarization of M2 macrophages by down-regulating PTEN, thus promoting cell proliferation, migration, and invasion in vitro and growth of lung cancer cells in vivo.

PTEN regulates proliferation and osteogenesis of dental pulp cells and adipogenesis of human adipose-derived stem cells.

OBJECTIVE: To investigate the role of phosphatase and tensin homolog (PTEN) in dental pulp cells (hDPs) and adipose-derived mesenchymal stem cells (hADSCs). MATERIALS AND METHODS: Genetic variant was identified with exome sequencing. The hDPs isolated from a patient with Cowden syndrome were investigated for their proliferation, osteogenesis, adipogenesis, and gene expression compared with controls. The normal hDPs and hADSCs were treated with the PTEN inhibitor, VO-OHpic trihydrate (VOT), to investigate the effect of PTEN inhibition. RESULTS: A heterozygous nonsense PTEN variant, c.289C>T (p.Gln97*), was identified in the Cowden patient's blood and intraoral lipomas. The mutated hDPs showed significantly decreased proliferation, but significantly upregulated RUNX2 and OSX expression and mineralization, indicating enhanced osteogenic ability in mutated cells. The normal hDPs treated with VOT showed the decreases in proliferation, colony formation, osteogenic marker genes, alkaline phosphatase activity, and mineral deposition, suggesting that PTEN inhibition diminishes proliferation and osteogenic potential of hDPs. Regarding adipogenesis, the VOT-treated hADSCs showed a reduced number of cells containing lipid droplets, suggesting that PTEN inhibition might compromise adipogenic ability of hADSCs. CONCLUSIONS: PTEN regulates proliferation, enhances osteogenesis of hDPs, and induces adipogenesis of hADSCs. The gain-of-function PTEN variant, p.Gln97*, enhances osteogenic ability of PTEN in hDPs.

The mRNA Expression of PTEN, LEF1, JAK3, LC3 and p62/SQSTM1 Genes in Patients with Chronic Myeloid Leukemia.

INTRODUCTION: Chronic myeloid leukemia (CML) is a progressive myeloproliferative disorder resulting from forming a chimeric BCR-ABL gene. The proteins derived from this gene can affect some genes from various signaling pathways such as PI3K/AKT/Wnt/catenin/JAK/Stat involved in proliferation, differentiation, cell death, and genes related to autophagy. Imatinib is the first-line treatment for CML patients, with durable and proper responses in Iranian children and adult CML patients. Hence, we aimed to evaluate the mRNA expression of some selected key genes from those pathways in patients with CML before and under treatment. METHODS: In the case-control study, the mRNA expression of PTEN, LEF1, JAK3, LC3 and p62 genes were measured in 51 CML patients (6 patients before treatment and 45 patients under treatment with imatinib mesylate) and 40 healthy controls using the Real-time PCR method. RESULTS: The mRNA expression of PTEN and P62 were significantly higher in newly diagnosed patients than in controls (P<0.0001 and P = 0.0183, respectively), while the expression of the LC3 gene was significantly lower in the untreated newly diagnosed group than in control subjects (P = 0.0191). The expression level of PTEN, LEF1, JAK3 and P62 genes were significantly decreased in patients under treatment than in the group before treatment (P = 0.0172, P = 0.0002, P = 0.0047 and P = 0.0038, respectively). A positive correlation was seen between the gene expression of P62 and BCR-ABL in the patients under treatment (r 0529, P = 0.016). CONCLUSION: Our findings showed that the changes in expression of these genes were related to the patient's treatment. Due to the key role of these genes in proliferation, differentiation and tumor suppression, it is proposed that these genes may be helpful for follow-up of treatment in CML patients.

Effect and Mechanism of Atorvastatin on Reversing Drug Resistance in Leukemia by Regulating Glycolysis through PTEN/mTOR Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the influence and mechanism of atorvastatin on glycolysis of adriamycin resistant acute promyelocytic leukemia (APL) cell line HL-60/ADM.@*METHODS@#HL-60/ADM cells in logarithmic growth phase were treated with different concentrations of atorvastatin, then the cell proliferation activity was measured by CCK-8 assay, the apoptosis was detected by flow cytometry, the glycolytic activity was checked by glucose consumption test, and the protein expressions of PTEN, p-mTOR, PKM2, HK2, P-gp and MRP1 were detected by Western blot. After transfection of PTEN-siRNA into HL-60/ADM cells, the effects of low expression of PTEN on atorvastatin regulating the behaviors of apoptosis and glycolytic metabolism in HL-60/ADM cells were further detected.@*RESULTS@#CCK-8 results showed that atorvastatin could inhibit the proliferation of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.872, r=0.936), and the proliferation activity was inhibited most significantly when treated with 10 μmol/L atorvastatin for 24 h, which was decreased to (32.3±2.18)%. Flow cytometry results showed that atorvastatin induced the apoptosis of HL-60/ADM cells in a concentration-dependent manner (r=0.796), and the apoptosis was induced most notably when treated with 10 μmol/L atorvastatin for 24 h, which reached to (48.78±2.95)%. The results of glucose consumption test showed that atorvastatin significantly inhibited the glycolytic activity of HL-60/ADM cells in a concentration-dependent and time-dependent manner (r=0.915, r=0.748), and this inhibition was most strikingly when treated with 10 μmol/L atorvastatin for 24 h, reducing the relative glucose consumption to (46.53±1.71)%. Western blot indicated that the expressions of p-mTOR, PKM2, HK2, P-gp and MRP1 protein were decreased in a concentration-dependent manner (r=0.737, r=0.695, r=0.829, r=0.781, r=0.632), while the expression of PTEN protein was increased in a concentration-dependent manner (r=0.531), when treated with different concentrations of atorvastatin for 24 h. After PTEN-siRNA transfected into HL-60/ADM cells, it showed that low expression of PTEN had weakened the promoting effect of atorvastatin on apoptosis and inhibitory effect on glycolysis and multidrug resistance.@*CONCLUSION@#Atorvastatin can inhibit the proliferation, glycolysis, and induce apoptosis of HL-60/ADM cells. It may be related to the mechanism of increasing the expression of PTEN, inhibiting mTOR activation, and decreasing the expressions of PKM2 and HK2, thus reverse drug resistance.

Exosomes Derived from Adipose Mesenchymal Stem Cells Carrying miRNA-22-3p Promote Schwann Cells Proliferation and Migration through Downregulation of PTEN.

Peripheral nerve injury (PNI) is often resulting from trauma, which leads to severe and permanently disability. Schwann cells are critical for facilitating the regeneration process after PNI. Adipose-derived mesenchymal stem cells (ADSCs) exosomes have been used as a novel treatment for peripheral nerve injury. However, the underlying mechanism remains unclear. In this study, we isolated ADSCs and extracted exosomes, which were verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot (WB). Cocultured with Dorsal Root Ganglion (DRG) and Schwann cells (SCs) to evaluate the effect of exosomes on the growth of DRG axons by immunofluorescence, and the proliferation and migration of SCs by CCK8 and Transwell assays, respectively. Through exosomal miRNA sequencing and bioinformatic analysis, the related miRNAs and target gene were predicted and identified by dual luciferase assay. Related miRNAs were overexpressed and inhibited, respectively, to clarify their effects; the downstream pathway through the target gene was determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and WB. Results found that ADSC-exosomes could promote the proliferation and migration of SCs and the growth of DRG axons, respectively. Exosomal miRNA-22-3p from ADSCs directly inhibited the expression of Phosphatase and Tensin Homolog deleted on Chromosome 10 (PTEN), activated phosphorylation of the AKT/mTOR axis, and enhanced SCs proliferation and migration. In conclusion, our findings suggest that ADSC-exosomes could promote SCs function through exosomal miRNA-22-3p, which could be used as a therapeutic target for peripheral nerve injury.

MicroRNA-29a-3p Regulates SH-SY5Y Cell Proliferation and Neurite Growth through Interaction with PTEN-PI3K/AKT/mTOR Signaling Pathway.

The effects of microRNA-29a-3p in the proliferation process of nerve cells are unclear. The purpose of this study is to delve into the regulatory role of microRNA-29a-3p, via interaction with phosphatase and tension homolog (PTEN), in the SH-SY5Y cell proliferation process. Different expressions of microRNA-29a-3p in the SH-SY5Y cells were constructed by transfected miRNA-29a-3p mimic and inhibitor. The effects of cell transfection and the mRNA expressions of PTEN, Akt, and mTOR were detected by qPCR. The expressions of PTEN, Akt, and mTOR protein and the phosphorylation levels of Akt and mTOR were examined using Western blotting. Nerve cell proliferation activity and neurite length of each group were measured and examined by the use of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2Htetrazolium bromide (MTT), and morphological examination. We observed that the levels of PTEN mRNA and protein were distinctly decreased in the microRNA-29a-3p mimic group, but the expressions of the phosphorylated Akt and mTOR mRNA and protein were distinctly upregulated. In the transfected miRNA-29a-3p inhibitor SH-SY5Y cells, the expressions of miRNA-29a-3p were significantly suppressed; however, the expressions of PTEN gene and protein were significantly enhanced. The expressions of phosphorylated Akt and mTOR in the downregulated microRNA-29a-3p group distinctly were suppressed. The SH-SY5Y cell proliferation activity and neurite length in the upregulated microRNA-29a-3p group increased significantly. Our findings revealed that microRNA-29a-3p could enhance the proliferation activity of SH-SY5Y cells and promote neurite growth by inhibiting the expression of PTEN and regulating PI3K/Akt/mTOR signaling pathway.

PGE2 inhibits neutrophil phagocytosis through the EP2R-cAMP-PTEN pathway.

Prostaglandin E2 (PGE2) is a potent lipid mediator of inflammation that modulates immune cell function by binding to unique G protein-coupled receptors (EP receptors). PGE2 production increases during microbial infection and inflammation. In this study, we assessed the effect of PGE2 on the phagocytosis of bacteria by neutrophils, which are key players during infection and inflammation. We also looked for specific EP receptor signaling pathways that contributed to the neutrophil phagocytic activity. PGE2 (50-1000 ng/ml) inhibited the phagocytosis of Escherichia coli by HL-60 human neutrophils in a concentration-dependent manner. Inhibition of neutrophil phagocytosis by PGE2 correlated with increased intracellular cyclic adenosine monophosphate (cAMP) production, and forskolin, an adenosyl cyclase agonist, confirmed the inhibitory effect of cAMP stimulation on neutrophil phagocytosis. The expression of EP2 receptors by HL-60 cells was confirmed by western blot analysis, and selective agonism of EP2 receptors mimicked the inhibition of phagocytosis by PGE2. The EP2 receptor antagonist AH-6089 partially blocked the inhibition of neutrophil phagocytosis PGE2. Specific inhibition of phosphatase and tensin homolog (PTEN) enzyme attenuated the inhibition of neutrophil phagocytosis by PGE2, and both PGE2 and increased intracellular cAMP increased neutrophil PTEN activity, which was associated with decreased PTEN phosphorylation. The results support negative regulation of the antimicrobial activity of neutrophils (i.e., phagocytosis), which has important implications for the future management of bacterial infections.

Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway.

OBJECTIVE: To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/ reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2 (PINK1/PARKIN) signaling pathway. METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells; the hypoxia/ reoxygenation (H/R) model was established in tri-gas incubator; 2', 7'-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species (ROS); the changes of mitochondrial membrane potential was determined by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide staining; the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits; flow cytometry was used to detect the ratio of apoptotic cells; transmission electron microscope was used to observe the ultrastructure of H9C2 cells; Western blot was used to detect the protein changes of mitochondrial 20 kDa outer membrane protein (TOM20), translocase of inner mitochondrial membrane 23 (TIM23), presenilins associated rhomboid-like protein (PARL), PINK1, PARKIN and mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), phosphotyrosine independent ligand for the Lck SH2 domain of 62 kDa (P62), microtubule-associated protein 1 light chain 3 beta (LC3B); the mRNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction; immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin. RESULTS: Resveratrol could inhibit the proliferation of H9C2 cells in a time- and concentration- dependent manner; however, pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels, alleviate the loss of mitochondrial membrane potential induced by H/R, inhibit H/R-induced apoptosis of H9C2 cells, and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage. Resveratrol could further increase the levels of p62, PINK1, PARKIN protein, the expression of PINK1, PARKIN mRNA and the ratio of LC3BⅡ/LC3BⅠin H/R-induced H9C2 cells, inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells, and further reduce the expression of TOM20,TIM23, PARL, Mfn1 and Mfn2 protein in H/R-induced H9C2 cells. The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells. CONCLUSIONS: Resveratrol can protect H9C2 cells from H/R injury, which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.

PTEN-Induced Putative Kinase 1 Dysfunction Accelerates Synucleinopathy.

BACKGROUND: Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive Parkinson's disease (PD) and contribute to the risk of sporadic PD. However, the relationship between PD-related PINK1 mutations and alpha-synuclein (α-syn) aggregation-a main pathological component of PD-remains unexplored. OBJECTIVE: To investigate whether α-syn pathology is exacerbated in the absence of PINK1 after α-syn preformed fibril (PFF) injection in a PD mouse model and its effects on neurodegeneration. METHODS: In this study, 10-week-old Pink1 knockout (KO) and wildtype (WT) mice received stereotaxic unilateral striatal injection of recombinant mouse α-syn PFF. Then, α-syn pathology progression, inflammatory responses, and neurodegeneration were analyzed via immunohistochemistry, western blot analysis, and behavioral testing. RESULTS: After PFF injection, the total α-syn levels significantly increased, and pathological α-syn was markedly aggregated in Pink1 KO mice compared with Pink1 WT mice. Then, earlier and more severe neuronal loss and motor deficits occurred. Moreover, compared with WT mice, Pink1 KO mice had evident microglial/astrocytic immunoreactivity and prolonged astrocytic activation, and a higher rate of protein phosphatase 2A phosphorylation, which might explain the greater α-syn aggravation and neuronal death. CONCLUSION: The loss of Pink1 function accelerated α-syn aggregation, accumulation and glial activation, thereby leading to early and significant neurodegeneration and behavioral impairment in the PD mouse model. Therefore, our findings support the notion that PINK1 dysfunction increases the risk of synucleinopathy.

Exosomes derived from hypoxia preconditioned mesenchymal stem cells laden in a silk hydrogel promote cartilage regeneration via the miR-205-5p/PTEN/AKT pathway.

Tissue engineering has promising prospects for cartilage regeneration. However, there remains an urgent need to harvest high quality seed cells. Bone marrow mesenchymal cells (BMSCs), and in particular their exosomes, might promote the function of articular chondrocytes (ACs) via paracrine mechanisms. Furthermore, preconditioned BMSCs could provide an enhanced therapeutic effect. BMSCs naturally exist in a relatively hypoxic environment (1%-5% O2); however, they are usually cultured under higher oxygen concentrations (21% O2). Herein, we hypothesized that hypoxia preconditioned exosomes (H-Exos) could improve the quality of ACs and be more conducive to cartilage repair. In our study, we compared the effects of exosomes derived from BMSCs preconditioned with hypoxia and normoxia (N-Exos) on ACs, demonstrating that H-Exos significantly promoted the proliferation, migration, anabolism and anti-inflammation effects of ACs. Furthermore, we confirmed that hypoxia preconditioning upregulated the expression of miR-205-5p in H-Exos, suggesting that ACs were promoted via the miR-205-5p/PTEN/AKT pathway. Finally, an injectable silk fibroin (SF) hydrogel containing ACs and H-Exos (SF/ACs/H-Exos) was utilized to repair cartilage defects and effectively promote cartilage regeneration in vivo. The application of SF/ACs/H-Exos hydrogel in cartilage regeneration therefore has promising prospects. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering (CTE) has presented a promising prospect. However, the quality of seed cells is an important factor affecting the repair efficiency. Our study demonstrates for the first time that the exosomes derived from hypoxia preconditioned BMSCs (H-Exos) effectively promote the proliferation, migration and anabolism of chondrocytes and inhibit inflammation through miR-205-5p/PTEN/AKT pathway. Furthermore, we fabricated an injectable silk fibrion (SF) hydrogel to preserve and sustained release H-Exos. A complex composed of SF hydrogel, H-Exos and chondrocytes can effectively promote the regeneration of cartilage defects. Therefore, this study demonstrates that hypoxia pretreatment could optimize the therapeutic effects of BMSCs-derived exosomes, and the combination of exosomes and SF hydrogel could be a promising therapeutic method for cartilage regeneration.