PTEN mRNA Expression Research Articles

Regulation of Curcumin on Follicle Initiation Rate in Diminished Ovarian Reserve.

To study the mechanism by which curcumin regulates ovarian primordial follicle initiation in rats with triptolide-induced diminished ovarian reserve (DOR). An in vitro gelatin sponge culture was performed on 3-day-old rat ovaries. After the establishment of the DOR model with triptolide, curcumin was administered for 3 days. Histological analysis and follicle counts were performed using H&E staining. ELISA detection of ovarian hormones in the culture medium (E2, FSH and LH), western blotting and Q-PCR for protein and mRNA expression (LTCONS-00011173, TGF-β1, Smad1, AMH, PTEN and GDF-9). Ovarian primordial and growing follicles increased significantly after curcumin intervention (p < 0.05), FSH/LH and E2 levels were increased significantly (p < 0.05). Curcumin also significantly decreased the expression of LTCONS-00011173. Meanwhile, curcumin increased the expression of TGF-β, AMH, and GDF-9 (p < 0.05). In addition, curcumin increased Smad1 gene expression and protein phosphorylation in the ovary on the one hand (p < 0.05), but inhibited Smad1 and p-Smad1 protein expression on the other hand (p < 0.05). Moreover, curcumin decreased PTEN protein and mRNA expression (p < 0.05). Curcumin activates primordial follicles in DOR model rats through TGF-β1 and downstream AMH signaling pathways and may limit follicle exhaustion through LncRNA.

Abstract A060: Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly malignancy. Cancer associated fibroblasts (CAFs) play a complex role in the PDAC tumor microenvironment (TME), with a subset contributing significantly to tumor progression. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and its loss is associated with increased tumor aggressiveness. Our group previously demonstrated PTEN loss in SMA+ positive CAFS correlated with worsened outcomes in patients and in mouse PDAC models. Our group previously demonstrated the loss of PTEN in CAFS led to activation of Stat3, resulting in an immunosuppressive microenvironment (Lefler et al, 2022 PMID: 35803738). However, little is known of the signals produced by tumor cells or other cell types in the TME that trigger loss of PTEN in CAFs. This study aims to to elucidate the molecular mechanisms underlying PTEN loss in CAFs in PDAC. To address this gap, we employed single cell RNA sequencing of tumors from a mouse PDAC model and identified a (PDRGFRa+Lys6+Stat3+Pten-low) CAF subpopulation. Comparing normal pancreas epithelial cells to tumor cells in this data set, we identified the IL6 family of cytokines, (IL6; Oncostatin-M, OSM; and Leukemia Inhibitory Factor, LIF) and Transforming Growth Factor beta 1 (TGFb1) as potential ligands that regulated PTEN, while myeloid cells were a source of the IL6 family member Oncostatin M (OSM). Treatment of isolated CAFs with these factors revealed no significant difference in PTEN mRNA expression. Thus, we developed an in vitro reporter system in which a PTEN-GFP fusion protein is expressed in CAFs. Treating these CAFs with IL6, OSM and TGFb1 (but not LIF) led to a significant decrease in GFP fluorescence. Analysis of endogenous PTEN by western blot confirmed these results. We posited that expression of PTEN E3 ligases might be increased by treatment with these factors, leading to destruction of PTEN. In support of this hypothesis, the expression of E3 ligases WWP1 and WWP2 was increased by treatment with IL6 and OSM. Additionally, preliminary data has revealed that PTEN is oxidized in isolated CAFs, which causes loss of PTEN activity and potentially increased protein turnover. Current experiments are aimed at testing these potential mechanisms. To conclude, PTEN is dysregulated post-transcriptionally in CAFs. The mechanism of PTEN dysregulation could be via increased expression of E3 ligases that target PTEN and/or PTEN oxidation. Future efforts will be aimed at testing whether inhibition of these pathways can restore PTEN expression in mouse PDAC models. These pathways and interactions could be further exploited for therapeutic benefit in curbing PDAC progression. Citation Format: Ivo N. Woogeng, Lu Han, Samaneh Saberi, Cameron Bumbleburg, Joseph Beaudet, Sudarshana Sharma, Michael Ostrowski. Dysregulation of PTEN expression in a subset of Cancer Associated Fibroblasts by Tumor Secreted Factors in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A060.

Circulating mRNA Expression of VEGF, PTEN, and SOCS1 as Potential Prognostic Predictor for Nasopharyngeal Carcinoma Progression.

The molecular mechanisms underlying nasopharyngeal carcinoma (NPC) progression remain poorly understood. In particular, the roles of circulating mRNAs encoding key regulatory proteins have yet to be explored. This study aimed to identify NPC-associated expression signatures of circulating VEGF, PTEN, and SOCS1 mRNAs and their potential as biomarkers. A case-control study was conducted comprising 160 nasopharyngeal carcinoma (NPC) patients and 80 controls, from whom peripheral blood samples. Total RNA was extracted and the levels of VEGF, PTEN, and SOCS1 mRNAs were quantified using reverse transcription quantitative PCR (RT-qPCR). Relative expression was calculated using the 2-ΔΔCt method. Bioinformatic analyses, including GeneMANIA, Gene Ontology (GO), and KEGG pathway analysis, were performed to predict the functional roles and interactions of these mRNAs. We identified significantly increased circulating VEGF mRNA in lymph node metastases (1.66-fold, p<0.05) and elevated SOCS1 mRNA in late-stage NPC (20-fold, p<0.05). PTEN mRNA was reduced 4.26-fold in NPC patients. These data suggest that circulating VEGF, PTEN, and SOCS1 mRNAs represent signatures of NPC progression and can potentially be biomarkers. Network analyses implicate these mRNAs in mechanisms enabling NPC pathogenesis. Our study reveals NPC-associated expression changes of circulating VEGF, PTEN, and SOCS1 mRNAs. These molecular signatures may serve as biomarkers during NPC progression and provide insights into underlying mechanisms. Further validation of their utility as prognostic indicators of NPC is warranted.

The interplay of PTEN and AKT nexus in breast cancer: a molecular perspective.

Breast cancer is a highly prevalent and life-threatening ailment that is commonly detected among the females. The downregulation of PTEN in breast cancer is associated with a poor prognosis, aggressive tumor type, and metastasis to lymph nodes, as it activates the pro-survival pathway PI3K/AKT, which is considered the ultimate proliferative pathway. The mRNA expression of PTEN and AKT genes was investigated using RT-qPCR and TaqMan primer probe chemistry. Moreover DNA was also isolated from the same tissue samples and exonic regions of both genes were amplified for mutational analysis. The proteins expression of PTEN and AKT from seven human breast cancer cell lines was checked through western blot experiments. The study revealed a decrease in PTEN expression in 73.3% of the samples, whereas an increase in AKT expression in 40% of samples was observed when compared to the distant normal breast tissue. Conversely, the remaining 60% of samples exhibited a decrease in AKT mRNA expression. There was no observed alteration in the genetic sequence of AKT and PTEN within the targeted amplified regions ofbreast cancer samples. The high levels of PTEN protein in T-47D and MDA-MB-453 resulted in a lower p-AKT. Two cell lines ZR-75-1 and MDA-MB-468 appeared to be PTEN negative on western blot but mRNA was detected on RT-qPCR. In breast cancer the status/expression of PTEN & AKT at mRNA and protein level might be obliging in forecasting the path of disease progression, treatment and prognosis.

Simultaneous and Sensitive Sensing of Intracellular MicroRNA and mRNA for the Detection of the PI3K/AKT Signaling Pathway in Live Cells.

Simultaneous detection of the concentration variations of microRNA-221 (miRNA-221) and PTEN mRNA molecules in the PI3K/AKT signaling pathway is of significance to elucidate cancer cell migration and invasion, which is useful for cancer diagnosis and therapy. In this work, we show the biodegradable MnO2 nanosheet-assisted and target-triggered DNAzyme recycling signal amplification cascaded approach for the specific detection of the PI3K/AKT signaling pathway in live cells via simultaneous and sensitive monitoring of the variation of intracellular miRNA-221 and PTEN mRNA. Our nanoprobes enable highly sensitive and multiplexed sensing of miRNA-221 and PTEN mRNA with low detection limits of 23.6 and 0.59 pM in vitro, respectively, due to the signal amplification cascades. Importantly, the nanoprobes can be readily delivered into cancer cells and the MnO2 nanosheets can be degraded by intracellular glutathione to release the Mn2+ cofactors to trigger multiple DNAzyme recycling cycles to show highly enhanced fluorescence at different wavelengths to realize sensitive and multiplexed imaging of PTEN mRNA and miRNA-221 for detecting the PI3K/AKT signaling pathway. Moreover, the regulation of PTEN mRNA expression by miRNA-221 upon stimulation by various drugs can also be verified by our method, indicating its promising potentials for both disease diagnosis and drug screening.

Inhibition of PTEN promotes osteointegration of titanium implants in type 2 diabetes by enhancing anti-inflammation and osteogenic capacity of adipose-derived stem cells.

Background: The low osteogenic differentiation potential and attenuated anti-inflammatory effect of adipose-derived stem cells (ADSCs) from animals with type 2 diabetes mellitus (T2DM) limits osseointegration of the implant. However, the underlying mechanisms are not fully understood. Methods: Western blotting and qRT-PCR analyses were performed to investigate the effects of PTEN on the osteogenic capacity of ADSCs of T2DM rats (TADSCs). We conducted animal experiments in T2DM-Sprague Dawley (SD) rats to evaluate the osteogenic capacity of modified TADSC sheets in vivo. New bone formation was assessed by micro-CT and histological analyses. Results: In this study, adipose-derived stem cells of T2DM rats exhibited an impaired osteogenic capacity. RNA-seq analysis showed that PTEN mRNA expression was upregulated in TADSCs, which attenuated the osteogenic capacity of TADSCs by inhibiting the AKT/mTOR/HIF-1α signaling pathway. miR-140-3p, which inhibits PTEN, was suppressed in TADSCs. Overexpression or inhibition of PTEN could correspondingly reduce or enhance the osteogenic ability of TADSCs by regulating the AKT/mTOR/HIF-1α signaling pathway. TADSCs transfected with PTEN siRNA resulted in higher and lower expressions of genes encoded in M2 macrophages (Arg1) and M1 macrophages (iNOS), respectively. In the T2DM rat model, PTEN inhibition in TADSC sheets promoted macrophage polarization toward the M2 phenotype, attenuated inflammation, and enhanced osseointegration around implants. Conclusion: Upregulation of PTEN, which was partially due to the inhibition of miR-140-3p, is important for the attenuated osteogenesis by TADSCs owing to the inhibition of the AKT/mTOR/HIF-1α signaling pathway. Inhibition of PTEN significantly improves the anti-inflammatory effect and osteogenic capacity of TADSCs, thus promoting peri-implant bone formation in T2DM rats. Our findings offer a potential therapeutic approach for modifying stem cells derived from patients with T2DM to enhance osseointegration.

Resveratrol Inhibits T-acute Lymphoblastic Leukemia in Mice by Regulating Notch1 Signaling Pathway

To observe the effect of resveratrol (Res) on T-acute lymphoblastic leukemia (T-ALL) mice, and further explore its mechanism on Notch1 signaling pathway. Twenty-five 6-8 weeks old female C57BL/6 mice were randomly divided into control group, T-ALL group and Res group. Res group was further divided into low-Res, middle-Res and high-Res group. The percentage of leukemia cells in peripheral blood and spleen cell suspension were detected by flow cytometry and Wright-Giemsa staining, pathological morphology of spleen and bone marrow tissues were observed by HE staining, the expression levels of Notch1, Hes-1, c-Myc, miR-19b and PTEN mRNA in spleen tissue were detected by RT-qPCR, and the protein levels of Notch1, Hes-1, c-Myc, p-PTEN and PTEN were detected by Western blot. Compared with control group, the leukemia cells in peripheral blood of mice in T-ALL group were markedly increased, accompanied by diffuse infiltration of leukemia cells in spleen and bone marrow tissues, the mRNA levels of Notch1, Hes-1, c-Myc, miR-19b and the protein levels of Notch1, Hes-1, c-Myc were increased (P <0.01), while the expression of PTEN mRNA and protein were significantly decreased in the spleen tissue of T-ALL mice (P <0.01). The above indicators in the H-Res group were reversed compared with T-ALL group after administration of resveratrol. Resveratrol may play a role in anti T-ALL by inhibiting Notch1 signaling pathway in mice.

Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma.

High-grade gliomas are extremely fatal tumors, marked by severe hypoxia and therapeutic resistance. Autophagy is a cellular degradative process that can be activated by hypoxia, ultimately resulting in tumor advancement and chemo-resistance. Our study aimed to examine the link between autophagy markers' expression in low-grade gliomas (LGGs) and high-grade gliomas (HGGs). In 39 glioma cases, we assessed the protein expression of autophagy markers LC3B, SQSTM1/p62, and DRAM by immunohistochemistry (IHC) and the mRNA expression of the autophagy genes PTEN, PI3K, AKT, mTOR, ULK1, ULK2, UVRAG, Beclin 1, and VPS34 using RT-qPCR. LC3B, SQSTM1/p62, and DRAM expression were positive in 64.1%, 51.3%, and 28.2% of glioma cases, respectively. The expression of LC3B and SQSTM1/p62 was notably higher in HGGs compared to LGGs. VPS34 exhibited a significant differential expression, displaying increased fold change in HGGs compared to LGGs. Additionally, it exhibited robust positive associations with Beclin1 (rs = 0.768), UVRAG (rs = 0.802), and ULK2 (rs = 0.786) in HGGs. This underscores a potential association between autophagy and the progression of gliomas. We provide preliminary data for the functional analysis of autophagy using a cell culture model and to identify potential targets for therapeutic interventions.

Colquhounia root tablet improves diabetic kidney disease by regulating epithelial-mesenchymal transition via the PTEN/PI3K/AKT pathway.

Diabetic kidney disease (DKD) is a severe microvascular complication of diabetes mellitus that can lead to end-stage renal disease. Colquhounia root tablet (CRT) has shown therapeutic potential in treating DKD, but its efficacy and underlying mechanisms remain to be elucidated. A randomized controlled clinical trial was conducted on 61 DKD patients. The treatment group received CRT in addition to standard therapy, while the control group received standard therapy alone. Treatment efficacy and adverse events were evaluated after 3months. Additionally, in vitro experiments using human renal tubular epithelial cells (HK-2) were performed to investigate the effect of CRT on high glucose (HG)-induced epithelial-mesenchymal transition (EMT) and the involvement of the PTEN/PI3K/AKT signaling pathway. CRT treatment significantly improved proteinuria and increased the effective treatment rate in DKD patients compared to the control group, with no significant difference in adverse events. Moreover, CRT reversed HG-induced EMT in HK-2 cells, as evidenced by the downregulation of α-SMA and upregulation of E-cadherin at both mRNA and protein levels. Mechanistically, CRT increased PTEN expression and inhibited the PI3K/AKT pathway, similar to the effects of the PI3K inhibitor LY29400. The combination of CRT and LY29400 further enhanced PTEN mRNA expression under HG conditions. CRT effectively improves proteinuria in DKD patients and ameliorates HG-induced EMT in HK-2 cells. The underlying mechanism may involve the upregulation of PTEN and subsequent inhibition of the PI3K/AKT signaling pathway. These findings provide new insights into the therapeutic potential of CRT for DKD treatment.

Selenium bioavailability modulates the sensitivity of thyroid cells to iodide excess

IntroductionIodide is an essential micronutrient for the synthesis of thyroid hormones and its imbalance is involved in the origin of different thyroid pathological processes. Selenium (Se) is another essential trace element that contributes to thyroid preservation through the control of the redox homeostasis. Different studies have demonstrated that sodium-iodide-symporter (NIS) is downregulated in the presence of iodide excess and Se supplementation reverses this effect. We also demonstrated that NOX4-derived ROS are involved in NIS repression induced by iodide excess. The aim of this study was to investigate how Se bioavailability is decisive in the sensitivity to iodide excess on a differentiated rat thyroid cell line (FRTL-5). ResultsWe demonstrated that siRNA-mediated silencing of Nox4 suppressed AKT phosphorylation induced by iodide excess. Iodide increases TGF-β1 mRNA expression, AKT phosphorylation, ROS levels and decreases GPX1 and TXRND1 mRNAs expression while Se reversed these effects. Furthermore, iodide induced Nrf2 transcriptional activity only in Se-supplemented cultures, suggesting that Se positively influences Nrf2 activation and selenoenzyme response in FRTL-5. Se, also inhibited NF-κB phosphorylation induced by iodide excess. In addition, we found that iodide excess decreased total phosphatase activity and PTP1B and PTEN mRNA expression. Se supply restored only PTEN mRNA expression. Finally, we studied the 2-α-iodohexadecanal (2-IHD) effects since it has been proposed as intermediary of iodide action on thyroid autoregulation. 2-IHD stimulated PI3K/AKT activity and reduced NIS expression by a ROS-independent mechanism. Also, we found that 2-IHD increased TGF-β1 mRNA and TGF-β inhibitor (SB431542) reverses the 2-IHD inhibitory effect on NIS mRNA expression, suggesting that TGF-β1 signaling pathway could be involved. Although Se reduced 2-IHD-induced TGFB1 levels, it could not reverse its inhibitory effect on NIS expression. ConclusionOur study suggests that Se bioavailability may improve the expression of antioxidant genes through the activation of Nrf2, interfere in PI3K/AKT signaling and NIS expression by redox modulation.

Exosomal MicroRNA-223, MicroRNA-146, and MicroRNA-21 Profiles and Biochemical Changes in Laryngeal Cancer.

Laryngeal squamous cell carcinoma (LSCC) is one of the most aggressive cancers, and its early diagnosis is urgent. Exosomes are believed to have diagnostic significance in cancer. However, the role of serum exosomal microRNAs, miR-223, miR-146, and miR-21, and phosphatase and tensin homologue (PTEN) and hemoglobin subunit delta (HBD) mRNAs in LSCC is unclear. Exosomes were isolated from the blood serum of 10 LSCC patients and 10 healthy controls to perform scanning electron microscopy and liquid chromatography quadrupole time-of-flight mass spectrometry analyses to characterize them and to undergo reverse transcription polymerase chain reaction to identify miR-223, miR-146, miR-21, and PTEN and HBD mRNA expression phenotypes. Biochemical parameters, including serum C-reactive protein (CRP) and vitamin B12, were also obtained. Serum exosomes of 10-140 nm were isolated from LSCC and controls. Serum exosomal miR-223, miR-146, and PTEN were found to be significantly decreased (p < 0.05), in contrast to serum exosomal miRNA-21 (p < 0.01), and serum vitamin B12 and CRP (p < 0.05) were found to be significantly increased, in LSCC vs controls. Our novel data show that the combination of reduced serum exosomal miR-223, miR-146, and miR-21 profiles and biochemical alterations in CRP and vitamin B12 levels may be useful indicators of LSCC that could be validated by large studies. Our findings also suggest a possible negative regulatory effect of miR-21 on PTEN in LSCC, encouraging a more extensive investigation of its role.

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

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. 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. 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). 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.

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

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. 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). 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. Based on the above, LINC00470 repressed cell autophagy by constraining PTEN, thereby enhancing CDDP resistance of glioma cells.

Otizmli Hastalarda PTEN Ve POGZ Genlerinin Ekspresyon Profilleri

Aim: Autism spectrum disorder (ASD), a group of heterogeneous neurodevelopmental disorders, is characterized by complex behavioral phenotypes. Despite extensive studies over many years, the causes of ASD are still unknown. PTEN and POGZ genes are studied as candidate genes that may be responsible for the ASD phenotype. We aimed to investigate the expression levels of PTEN and POGZ genes in autistic patients.Methods: Gene expressions of PTEN and POGZ were investigated in 50 ASD patients and 50 age and gender matched healthy controls. This study was conducted in the Erciyes University Genome and Stem Cell Center (GENKOK).Results: POGZ gene expression was increased in patients compared to controls. According to gender, the expression results of the autistic male patients were significant. PTEN mRNA expression was not statistically significant but found to be lower in patients than in controls. The relationship between the expression of these genes and cognitive deficits was not significant.Conclusion: We recommend investigating other possible candidate genes in larger cohorts and comparing the results with different additional cilinical findings in ASD.

Melatonin Mitigates Cisplatin-Induced Ovarian Dysfunction via Altering Steroidogenesis, Inflammation, Apoptosis, Oxidative Stress, and PTEN/PI3K/Akt/mTOR/AMPK Signaling Pathway in Female Rats.

Ovarian damage and fertility impairment are major side effects of chemotherapy in pre-menopausal cancer patients. Cisplatin is a widely used chemotherapeutic drug. The present study was designed to assess the ameliorative effects of melatonin as an adjuvant for fertility preservation. Thirty-two adult female Wistar rats were divided randomly into four equal groups: Control, Melatonin, Cisplatin (CP) treated, and CP + Melatonin treated. The cisplatin-treated group showed decreased body and ovarian weights, decreased serum E2 and AMH, increased serum LH and FSH, reduced ovarian levels of SOD, CAT, GSH, and TAC, and increased ovarian MDA. The histopathological examination of the cisplatin-treated group showed deleterious changes within ovarian tissue in the form of damaged follicles and corpus luteum, hemorrhage, and inflammatory infiltrates with faint PAS reaction in zona pellucida, increased ovarian collagen deposition, and marked expression of caspase-3 immune reaction in granulosa and theca cells, stroma, and oocytes. Alongside, there was a significant downregulation in the mRNA expression of steroidogenic enzymes, IL10, AMPK, PI3K, AKT, mTOR, and PTEN, while TGF-β1, IL1β, IL6, TNF-α, NF-Kβ, P53, p38-MAPK, JNK, and FOXO3 mRNA expressions were upregulated in cisplatin-treated rats' ovarian tissue. Coadministration of cisplatin-treated rats with melatonin reversed these changes significantly. In conclusion, melatonin's antioxidant, anti-inflammatory, and anti-apoptotic activities could modulate ovarian disturbances induced by cisplatin and preserve fertility.

Rheum officinale and Salvia miltiorrhiza inhibit renal fibrosis via miR-21/PTEN/Akt signaling pathway in vitro and in vivo

Ethnopharmacological relevanceAs one of the main components of many famous Chinese herbal formulas, Rheum palmatum L. and Salvia miltiorhiza Bunge (RS) are extensively used to treat chronic kidney disease (CKD). RS has been proved to improve renal function and relieve renal fibrosis (RF), but the potential mechanism remains a mystery. Aim of the studyThe purpose of this study is to determine whether microRNA-21 (miR-21) is associated with RF progression, as well as whether RS protects against RF through miR-21/PTEN/AKT signaling. Materials and methods(1) The rat model of RF was established using unilateral ureteral obstruction (UUO). After UUO surgery, miR-21 levels in plasma were detected by RT-PCR and RF scores were assessed by Masson's trichrome stain at days 3, 7, 14 and 21. The correlation analysis of the above two indexes was carried out by Spearman correlation analysis. (2) Human proximal tubular epithelial cells (HK-2) was transfected with miR-21 mimic and inhibitor, and then the levels of phosphatase and tensin homolog (PTEN) protein and mRNA were measured with Western blotting and RT-PCR, respectively. (3) TGF-β (10 ng/mL) was added into HK-2 cells to induce fibrosis, followed by the intervention of RS-containing rat serum. PTEN and protein kinase-B (Akt) phosphorylation, as well as the expression of PTEN protein in HK-2 cells, were assessed by RT-PCR, Western blotting and immunofluorescence. (4) The rat models of RF were prepared by UUO and treated with RS. Serum creatinine and urea nitrogen levels were measured. RF score was determined by Masson's trichrome stain. RT-PCR was used to determine the expression of miR-21, PTEN, and Akt mRNA. Western blotting was used to determine the expression of PTEN and Akt proteins. ResultsA positive correlation was found between plasma miR-21 levels and RF scores of rats after UUO surgery at Days 3, 7, 14 and 21. It was confirmed that miR-21 targeted PTEN. RS drug-containing serum could rise the expression of PTEN and reduce Akt phosphorylation of HK-2 cells induced by TGF-β. Moreover, RS drug-containing serum could increase PTEN expression and reduce Akt phosphorylation induced by miR-21 mimic in HK-2 cells. The rats treated with RS had significantly decreased serum creatinine and urea nitrogen levels and a lower RF score. RS also decreased miR-21 and Akt expressions, increased PTEN expression of UUO rats. ConclusionThere was a positive correlation between plasma miR-21 levels and RF scores. The inhibitory effect of RS on RF might be mediated by miR-21/PTEN/AKT signaling.

Protective effect of total saponins of ginseng stems and leaves (GSLS) on chlorpyrifos-induced brain toxicity in mice through the PTEN/PI3K/AKT axis.

Chlorpyrifos (CPF) is a class of toxic compounds which has been widely used in agriculture that can cause multi-organ damage to the liver, kidneys, testes, and nervous system. Currently, most studies on ginseng have concentrated on the roots and rhizomes, and less research has been conducted on the above-ground parts. Our laboratory found that ginseng stem and leaf total saponin (GSLS) features strong antioxidant activity. In this experiment, we selected different concentrations of CPF to induce hippocampal neuronal cell injury model in mice, conducted a cell survival screening test, and also selected appropriate concentrations of CPF to induce brain injury model in mice. CCK-8, flow cytometry, Elisa, Hoechst 33258 staining, Annexin V-FITC/PI staining, HE staining, Morris water maze, and qRT-PCR were adopted for detecting the effects of GSLS treatment on CPF-induced cell viability, mitochondrial membrane potential, reactive oxygen species (ROS) levels, Ca2+ concentration and GSLS treatment on CPF-induced brain injury and related signaling in mice, respectively. The effects of GSLS treatment on CPF-induced brain injury and the related signaling pathways in mice were examined. The results showed that GSLS at 60 μg/ml and 125 μg/ml concentrations elevated the viability of CPF-induced HT22 cells, increased mitochondrial membrane potential, depleted ROS, decreased Ca2+ concentration, and decreased apoptosis rate. Meanwhile, GSLS treatment significantly reduced CPF-induced escape latency in mice, elevated the number of entries into the plateau and effective area, increased the effective area and target quadrant residence time, as well as improved the pathological damage of mouse hippocampal neurons. The results of mouse brain sections demonstrated that GSLS treatment significantly increased SOD and CAT activities and lowered MDA accumulation in CPF-induced mice. qRT-PCR revealed that PTEN mRNA expression was significantly decreased with PI3K and AKT expression being significantly increased in GSLS-treated CPF-induced mice. Thus, the obtained results indicate that GSLS can effectively antagonize CPF-induced brain toxicity in mice through regulating PTEN/PI3K/AKT pathway.

LncRNA MEG3 promotes cisplatin sensitivity of cervical cancer cells by regulating the miR-21/PTEN axis

BackgroundCervical cancer (CC) is a common gynecological malignancy worldwide. Some patients perform serious resistance after chemotherapy, and long-stranded non-coding RNA MEG3 is reported to be involved in the regulation of chemoresistance in many solid tumors. However, its involvement in cervical adenocarcinoma has not been reported.MethodsHela cell lines, cisplatin-resistant cell lines (Hela-CR) and nude mice were used in this study. After MEG3 was overexpressed or knocked down in cells by the lentivirus vector, cell growth was detected by the CCK-8 assay, and cell migration was evaluated using Transwell assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of MEG3, miR-21 and PTEN mRNA. Apoptosis was detected by flow cytometry. The targeting relationship between mRNAs was predicted and verified using dual-luciferase reporter gene experiments. Western blot was executed to examine Bax, cleaved-caspase 3, Bcl-2, PTEN and GAPDH expression. Cells were injected into the mice to form xenograft tumors to compare tumorigenesis capacity.ResultsWe demonstrated that MEG3 was down-regulated in cervical cancer by analyzing the TCGA database. Moreover, knockdown of MEG3 promoted CC cell proliferation, migration and inhibited the apoptosis. These changes of CC cells were more pronounced under cisplatin treatment. Further studies showed that the MEG3/miR-21/PTEN axis affected cisplatin sensitivity in cervical cancer cells, and these results of recue assay were used to confirm this conclusion.ConclusionsMEG3 performing as ceRNA promotes cisplatin sensitivity in CC cells through the miR-21/PTEN axis.

RF22 | PMON08 The effects of Bisphenol A on miR-21 expression and miR-21 mediated apoptosis in bovine granulosa cells.

Abstract BPA, a widespread endocrine-disrupting compound (EDC), plays a significant role in the decline of female fertility. Granulosa cells are susceptible to EDCs and are important for oocyte competency. This research focuses on epigenetic mechanisms in granulosa cells that are disrupted by BPA, with a specific focus on microRNA-21(miR-21), which is crucial for oocyte maturation and primarily recognized as an antiapoptotic regulator. miR-21 targets the apoptotic genes PDCD4 and PTEN and limits their expression, resulting in an antiapoptotic effect. Previous studies in our laboratory demonstrate that BPA induces apoptosis and simultaneously increases miR-21 expression, leading to our hypothesis that BPA induces apoptosis in a miR-21 independent manner in bovine granulosa cells.To test this hypothesis, in vitro cultured granulosa cells were transfected with miR-21 inhibitor or scramble with or without BPA, followed by knockdown validation and apoptotic measurements. Using flow cytometry, transfection efficiencies were measured in time- and dose-dependent experiments, showing optimal knock-down greater than 95% at 0.5µM for 12 hrs. miR-21 expression measured by qPCR showed a significant decrease within inhibited groups (p=0.0008, n=3). Although PDCD4 and PTEN mRNA expression remained unaffected, PDCD4 protein expression is inhibited cells was significantly increased (p=0.0233, n=4), suggesting a miR-21 role in PDCD4 translation. PTEN protein was unaffected by miR-21 inhibition.In further experiments, transfected cells were exposed to BPA, and apoptosis was examined. Using Annexin V/PI staining, a significant decrease in healthy cells and an increase in early apoptotic cells within inhibited untreated groups was shown (p=0.0395 and 0.0193, n=3). This finding is likely due to antiapoptotic gene inhibition. BPA exposure resulted in significantly decreased healthy cells, increased late apoptotic cells (p=0.0047 and 0.0145, n=3), with no changes in necrotic cells. Furthermore, no differences were found between inhibited and non-inhibited groups, suggesting that BPA-induced apoptosis is miR-21 independent.Additionally, three proapoptotic (BAX, BAD, Casp-9) and two antiapoptotic (Bcl-2 and HSP70) genes were quantified by qPCR. Unexpectedly, BPA increased the expression of all genes at the mRNA level (p= 0.0021, 0.0283, 0.0143, 0.025, 0.0145, n=4). Quantification of protein levels will reveal whether this change is functional. Moreover, no differences were found between inhibited and non-inhibited groups, suggesting these changes are likely miR-21 independent. Lastly, initiator caspase 9 activity was measured after 6, 12, 24, 48, 72 hrs of BPA exposure, as caspases are primary effectors of apoptosis. 6 and 12hrs timepoints showed no changes; however, at 24 and 48 hrs significantly increased caspase activity was detected (p=0.0087 and 0.0014, n=4). At 72hrs, BPA exposure was lethal, resulting in insufficient cell numbers for measurements.Overall, this research focuses on effects of EDCs on apoptotic regulation within granulosa cells and aims to uncover epigenetic consequences of BPA's mechanism of action, ultimately affecting oocyte competency and fertility. Presentation: Sunday, June 12, 2022 12:54 p.m. - 12:59 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Low miR-222 expression in human visceral adipose tissue is associated with insulin resistance and PTEN and p53 mRNA levels.

The aim of this is study was to analyse the expression of miR-193b, miR-378, miR-Let7-d, and miR-222 in human visceral adipose tissue (VAT), as well as their association with obesity, insulin resistance (IR), and their role in the regulation of genes controlling adipose tissue homeostasis, including adipocytokines, the phosphatase and tension homologue (PTEN), and tumour protein 53 (p53). VAT was obtained from normal-weight (NW), overweight, and obese (OW/OB) subjects with and without IR. Stem-loop RT-qPCR was used to evaluate miRNA expression levels. miRTarBase 4.0, miRWalk, and DIANA-TarBase v8 were used for prediction of validated target gene of the miRNA analysed. A qPCR was used to evaluate PTEN, p53, leptin (LEP), and adiponectin (ADIPOQ) mRNA. miR-222 was lower in IR subjects, and miR-222 and miR-378 negatively correlated with HOMA-IR. PTEN and p53 are miR-222 direct targets according to databases. mRNA expression of PTEN and p53 was lower in OW/OB subjects with and without IR, compared to NW group and its levels positively associated with miR-222. Additionally, p53 and PTEN are positively associated with serum leptin levels. On the other hand, miR-193b and miR-378 negatively correlated with serum leptin but not with mRNA levels. Moreover, miR-Let-7d negatively correlated with serum adiponectin but not with adiponectin mRNA levels. Lower miR-222 levels are associated with IR, and PTEN and p53 expression; the implication of these genes in adipose tissue homeostasis needs more research.