MiR-497-3p induces Premature ovarian failure by targeting KLF4 to inactivate Klotho/PI3K/AKT/mTOR signaling pathway.

BACKGROUND: Premature ovarian failure (POF), as a gynecological endocrine disease, features the manifestation of irregular menstruation, amenorrhea, infertility and perimenopausal syndrome. MicroRNAs (miRNAs) have been reported to modulate POF. However, the specific regulatory mechanism of miR-497-3p in POF remain unclear. METHODS: Quantitative reverse transcription-PCR (RT-qPCR) and western blot were implemented to analyze RNA and protein levels, respectively. Comet assay was performed for the detection of DNA damage. Flow cytometry analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to measure apoptosis of CTX-induced KGN cell (POF cell model). Bioinformatics was utilized to screen out the downstream mRNAs potentially regulated by miR-497-3p. Chromatin immunoprecipitation (ChIP) assay, luciferase reporter assay and RNA pulldown assays were performed to demonstrate the interaction between miR-497-3p and Kruppel-like factor 4 (KLF4) or between KLF4 and Klotho (KL). Rescue assays were performed to verify the involvement of Klotho in miR-497-3p-mediated functions of POF cell model. RESULTS: MiR-497-3p was upregulated in CTX-treated KGN cells. Knockdown of miR-497-3p could reverse the promoting effects of CTX on DNA damage and cell apoptosis. MiR-497-3p negatively regulated Klotho expression by directly targeting the transcription activator KLF4. KLF4 activated Klotho transcription. MiR-497-3p inactivated PI3K/AKT/mTOR signaling pathway through KLF4/Klotho axis. Klotho knockdown reversed the effects of MiR-497-3p on the functions of POF cell model. CONCLUSION: MiR-497-3p promotes DNA damage and apoptosis in CTX-treated KGN cells by targeting KLF4 to downregulate Klotho and inactivate the PI3K/AKT/mTOR signaling pathway. This study unveils novel mechanisms associated with cell functional changes in POF and may enrich therapeutic strategy for POF.

CircCCNB1 Knockdown Blocks the Progression of Cervical Cancer by Acting as Competing Endogenous RNA in the miR-370-3p/SOX4 Pathway.

OBJECTIVE: Cervical cancer is one of the leading causes of cancer-related death in women, which has been shown to be associated with the deregulation of circular RNAs (circRNAs). The aim of this study was to determine the role of circRNA cyclin B1 (circCCNB1) in cervical cancer. METHODS: The expression of circCCNB1, microRNA-370-3p (miR-370-3p), and SRY-box transcription factor 4 (SOX4) mRNA was detected by quantitative real-time PCR (qPCR). Functional experiments, including colony formation assay, EdU assay, transwell assay and flow cytometry assay, were performed. Lactate production and glucose uptake were examined to assess glycolysis metabolism. The protein levels of glycolysis-related markers and SOX4 were detected by western blot. The interaction between miR-370-3p and circCCNB1 or SOX4 was verified by dual-luciferase reporter, RIP, and pull-down assay. Xenograft assay was performed to monitor the role of circCCNB1 in animal models. RESULTS: CircCCNB1 was highly expressed in cervical cancer tissues and cells (squamous cell carcinoma and adenocarcinoma cells). The knockdown of circCCNB1 inhibited cell proliferation, migration, invasion and glycolysis metabolism, and induced cell apoptosis. CircCCNB1 functioned as miR-370-3p sponge to suppress miR-370-3p expression and function. Moreover, circCCNB1 inhibited the expression of miR-370-3p to increase the expression of SOX4. MiR-370-3p inhibition reversed the effects of circCCNB1 knockdown and thus promoted cell proliferation, migration, invasion and glycolysis. SOX4 overexpression reversed the effects of miR-370-3p restoration and thus promoted cell proliferation, migration, invasion and glycolysis. CONCLUSION: CircCCNB1 knockdown blocks cervical cancer development by targeting the miR-370-3p/SOX4 pathway.

Integrating Network Pharmacology, Molecular Docking and Pharmacological Evaluation for Exploring the Polyrhachis vicina Rogers in Ameliorating Depression.

Purpose: To investigate the mechanisms of antidepressant action of active fraction of Polyrhachis vicina Rogers (AFPR) through network pharmacology, molecular docking and experimental validation. Methods: GC-MS was used to predict chemical compounds, corresponding databases were used to predict chemical compound targets and depression targets, Cytoscape software was used to construct and analyze the protein interaction network map, DAVID database was used to analyze gene ontology (GO) and KEGG signaling pathway, and AGFR software was used to perform molecular docking. Subsequently, the underlying action mechanisms of AFPR on depression predicted by network pharmacology analyses were experimentally validated in a CORT-induced depression model in vitro and in vivo. Results: A total of 52 potential targets of AFPR on antidepressant were obtained. GO is mainly related to chemical synaptic transmission, signal transduction and others. KEGG signaling pathways are mainly related to cAMP signaling pathway and C-type lectin receptor signaling pathway. The experiment results showed that AFPR significantly increased the expression of PRKACA, CREB and BDNF in mouse brain tissue and PC12 cells. Furthermore, after interfered of cAMP in PC12 cells, the decreased expression of PRKACA, CREB and BDNF was reversed by AFPR. Conclusion: AFPR may exert antidepressant effects through multiple components, targets and pathways. Furthermore, it could improve neuroplasticity via the cAMP signaling pathway to improve depression-like symptoms.

Active Fraction of Polyrhachis Vicina Roger (AFPR) Ameliorate Depression Induced Inflammation Response by FTO/miR-221-3p/SOCS1 Axis.

Purpose: Neuroinflammation is a significant etiological factor in the development of depression. Traditional Chinese medicine (TCM) has demonstrated notable efficacy in the treatment of inflammation. Our previous study surfaces that the active fraction of Polyrhachis vicina Roger (AFPR) has antidepressant and anti-neuroinflammatory effects, but the specific mechanisms remain to be elucidated. The objective of this study was to examine the impact of AFPR on inflammation in depression via the FTO/miR-221-3p/SOCS1 axis. Methods: Chronic unpredictable stress (CUMS)-induced rats and LPS-induced BV2 cells were employed to simulate depression models in vivo and in vitro. The levels of inflammatory factors were detected using the ELISA assay. The expression of genes and proteins was detected using qRT-PCR and Western blot. Gene interactions were detected using the dual luciferase reporter gene. Protein-RNA interactions were investigated using RNA methylation immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP). Neuroinflammation in the brain was examined through H&E staining, while neuronal apoptosis was assessed using TUNEL staining. Results: The results showed that AFPR ameliorated depression induced inflammation by increasing SOCS1 expression. However, SOCS1 was identified as a target of miR-221-3p. Overexpression of miR-221-3p decreased the expression of SOCS1 and increased the levels of NF-κB, IL-7, and IL-6. In addition, we found that miR-221-3p was regulated by FTO-mediated m6A modification through MeRIP and RIP experiments. Interference with miR-221-3p and overexpression of FTO resulted in increased SOCS1 gene expression and decreased levels of NF-κB, IL-7, and IL-6, which were reversed by AFPR. Conclusion: AFPR inhibits the maturation of pri-miR-221-3p through FTO-mediated m6A modification, reduces the production of miR-221-3p, increases the expression of SOCS1, and reduces the level of inflammation, thereby improving depressive symptoms.