OBJECTIVES: Oxaliplatin (OXA) and 5-fluorouracil (5-FU) are 2 commonly used chemotherapeutic agents for colorectal cancer (CRC). MicroRNAs (miRNAs, miRs) play crucial roles in the development of chemoresistance in various cancers. However, the role and mechanism of miR-224-5p in regulating CRC chemoresistance remain unclear. This study aims to investigate the function of miR-224-5p in chemoresistant CRC cells and the underlying mechanisms. METHODS: CRC datasets GSE28702 and GSE69657 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed miRNAs between drug-sensitive and resistant groups (OXA or 5-FU) were analyzed, and miR-224-5p was identified as the target miRNA. Chemoresistant cell lines HCT15-OXR, HCT15-5-FU, SW480-OXR, and SW480-5-FU were established. Transient transfections were performed using miR-224-5p mimics, inhibitors, and their respective negative controls (control mimic, control inhibitor) in these cell lines. Cells were treated with different concentrations of OXA or 5-FU post-transfection, and the half-maximal inhibitory concentration (IC₅₀) was determined using the cell counting kit-8 (CCK-8) assay. Cell proliferation was assessed by CCK-8 and colony formation assays. The expression levels of miR-224-5p, LC3, and P62 were measured by real-time polymerase chain reaction (real-time PCR) and/or Western blotting. Autophagic flux was assessed using a tandem fluorescent-tagged LC3 reporter assay. TargetScan 8.0, miRTarBase, miRPathDB, and HADb were used to predict B-cell lymphoma-2 (Bcl-2) as a potential miR-244-5p target, which was further validated by dual-luciferase reporter assays. RESULTS: Chemoresistant CRC cells exhibited down-regulated miR-224-5p expression, whereas up-regulation of miR-224-5p enhanced chemotherapy sensitivity. Exposure to OXA or 5-FU significantly increased autophagic activity in chemoresistant CRC cells, which was reversed by miR-224-5p overexpression. Dual-luciferase assays verified Bcl-2 as a direct target of miR-224-5p. CONCLUSIONS MiR-224-5p regulates chemoresistance in CRC by modulating autophagy through direct targeting of Bcl-2.
Humans ; MicroRNAs/physiology* ; Colorectal Neoplasms/drug therapy* ; Drug Resistance, Neoplasm/genetics* ; Autophagy/drug effects* ; Fluorouracil/pharmacology* ; Oxaliplatin ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Gene Expression Regulation, Neoplastic

OBJECTIVES: The core pathology of osteoporosis lies in bone resorption exceeding bone formation; thus, promoting osteogenesis is a key therapeutic strategy. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) forms the biological basis of bone formation. Astragaloside IV (A-IV), a major active component of Astragalus membranaceus, is known to enhance osteogenesis, but its precise molecular mechanisms remain unclear. This study aims to investigate the effects of A-IV on the proliferation and osteogenic differentiation of BMSCs from osteoporotic rats and to elucidate its molecular mechanism through the regulation of microRNA-21 (miR-21) and Notch2 expression. METHODS: After 1 week of adaptive feeding, mature female SD rats were randomly divided into a sham-operated (Sham) group (n=4) and an ovariectomized (OVX) group (n=8) to establish an osteoporosis model. Twelve weeks after surgery, BMSCs were isolated from femoral bone marrow and cultured. Cells were divided into a S-BMSCs group (from Sham), an O-BMSCs group (from OVX), and an A-BMSCs group (from OVX-derived BMSCs treated with A-IV). S-BMSCs and O-BMSCs were induced for osteogenic differentiation using osteogenic induction medium, whereas A-BMSCs were treated with A-IV before induction. Flow cytometry was used to identify mesenchymal stem cell surface markers (CD29) and hematopoietic stem cell marker (CD34) to confirm BMSC characteristics. Cell proliferation was assessed using the methyl thiazolyl tetrazolium (MTT) assay. Alizarin red staining was performed to quantify calcium nodule formation, and alkaline phosphatase (ALP) activity assays were used to evaluate osteogenic differentiation. Real-time reverse transcription PCR (real-time RT-PCR) was used to detect changes in osteogenic-related genes, runt-related transcription factor 2 (Runx2) and osteopontin (OPN), as well as miR-21 expression. Western blotting was performed to assess Runx2, OPN, and Notch2 protein expression. RESULTS: Flow cytometry confirmed that O-BMSCs retained the phenotypic characteristics of mesenchymal stem cells. A-IV significantly enhanced the proliferation of BMSCs from osteoporotic rats (P<0.05), increased ALP activity, and upregulated the mRNA and protein expression of Runx2 and OPN (P<0.05). Bioinformatic and experimental analyses demonstrated that miR-21 directly targeted Notch2. A-IV treatment increased miR-21 expression while suppressing Notch2 protein expression and inhibiting activation of the Notch signaling pathway (P<0.05). CONCLUSIONS Astragaloside IV promotes the osteogenic differentiation of BMSCs derived from osteoporotic rats by upregulating miR-21 expression and inhibiting the key Notch signaling protein Notch2, thereby relieving the Notch2-mediated suppression of osteogenesis.
Animals ; Triterpenes/pharmacology* ; Saponins/pharmacology* ; Osteogenesis/drug effects* ; MicroRNAs/metabolism* ; Rats, Sprague-Dawley ; Female ; Cell Differentiation/drug effects* ; Mesenchymal Stem Cells/drug effects* ; Signal Transduction/drug effects* ; Osteoporosis/pathology* ; Rats ; Cells, Cultured ; Receptor, Notch2/metabolism* ; Receptors, Notch/metabolism* ; Ovariectomy ; Cell Proliferation/drug effects*

Objective:This study explored the effect of nanoparticle-encapsulated curcumin on the highly expressed multidrug resistance gene 1 （MDR1） in a human low-differentiated nasopharyngeal carcinoma cell line （CNE2）. Methods:Curcumin/chitosan deoxycholic acid nanoparticles were prepared, and the cells were subjected to different treatments: radiotherapy, empty carriers, curcumin, and curcumin-loaded nanoparticles. Cell survival was analyzed using the clonogenic assay, and assessments of apoptosis, MDR1 levels, and miR593 levels were conducted. Results:The cell survival fractions in the curcumin group and the curcumin-loaded nanoparticles group were significantly reduced. Notably, higher apoptosis rates were observed in cells treated with curcumin or curcumin-loaded nanoparticles compared to those that received only radiotherapy. Moreover, a decreased MDR1 level was noted in both the curcumin group and the curcumin-loaded nanoparticles group, with further reduction in MDR1 expression observed in the nanoparticle group （P<0.05）. Enhanced expression of miR593 was found in the curcumin group and the curcumin-loaded nanoparticles group, with a relatively higher level in the nanoparticle group （P<0.05）. Curcumin encapsulated in nanoparticles exhibited a stronger radiosensitizing effect. The combination of curcumin and radiotherapy effectively inhibited nasopharyngeal carcinoma （NPC） tumor growth, suppressed MDR1 expression, and enhanced miR593 levels. After inhibiting miR593, MDR1 expression increased. The radiosensitizing effect of curcumin-loaded nanoparticles was regulated by miR593 rather than being triggered by MDR1. Conclusion:Curcumin-loaded nanoparticles mediated enhanced expression of miR593, which in turn inhibited the transcription and translation of the MDR1 gene, thereby reducing the radioresistance of NPC and effectively restraining its growth.
Humans ; Curcumin/pharmacology* ; Nasopharyngeal Neoplasms/pathology* ; Nasopharyngeal Carcinoma ; Nanoparticles ; Cell Line, Tumor ; Apoptosis/drug effects* ; MicroRNAs ; ATP Binding Cassette Transporter, Subfamily B ; ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism* ; Cell Survival

OBJECTIVES: To explore the mechanism of Qingda Granules (QDG) for alleviating brain damage in spontaneously hypertensive rats (SHRs). METHODS: Twelve 5-week-old SHRs were randomized into SHR control group and SHR+QDG group treated with QDG by gavage at the daily dose of 0.9 g/kg for 12 weeks. The control rats, along with 6 age-matched WKY rats, were treated with saline only. Blood pressure changes of the rats were monitored, and pathologies and neuronal apoptosis in the cerebral cortex were examined with HE staining and TUNEL staining. Cerebral cortical expressions of miR-124 and STAT3 mRNA were detected using RT-qPCR, and the protein expressions of NeuN, STAT3, Bcl-2, Bax, and cleaved caspase-3 were detected with immunohistochemistry and Western blotting. In a HT22 cell model of oxygen and glucose deprivation/reoxygenation (OGD/R), the effects of QDG on cell viability and apoptosis, expressions of miR-124 and STAT3 mRNA, and protein expressions of STAT3, Bcl-2, Bax, and cleaved caspase-3 were evaluated using CCK8 assay, Hoechst 33342 staining, RT-qPCR, and Western blotting. RESULTS: Compared with WKY rats, SHRs had significantly elevated systolic blood pressure, diastolic blood pressure and mean arterial pressure with significantly increased neuronal apoptosis in the cerebral cortex, reduced expressions of NeuN, miR-124 and Bcl-2, and enhanced expressions of STAT3, Bax and cleaved caspase-3 (P<0.05). All these changes in the SHRs were significantly ameliorated by treatment with QDG (P<0.05). In the HT22 cell model, QDG treatment obviously reduced OGD/R-induced cell apoptosis, increased the expressions of miR-124 and Bcl-2, and suppressed the elevation of protein expressions of STAT3, Bax and cleaved caspase-3. CONCLUSIONS QDG inhibits cerebral cortical neuronal apoptosis and thereby attenuates brain damage in SHR rats by modulating the miR-124/STAT3 signaling axis.
Animals ; Rats, Inbred SHR ; MicroRNAs/metabolism* ; STAT3 Transcription Factor/metabolism* ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Rats ; Apoptosis/drug effects* ; Rats, Inbred WKY ; Male ; Hypertension

OBJECTIVES: To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT) against high glucose-induced injury in mouse renal podocytes (MPC5 cells) and the possible mechanism. METHODS: Adult Wistar rats were treated with 19, 38, and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose (30 mmol/L) prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic. The changes in miR-21a-5p expressions and the mRNA levels of FoxO1, PINK1, and Parkin in the treated cells were detected with qRT-PCR, and the protein levels of nephrin, podocin, FoxO1, PINK1, and Parkin were detected with Western blotting. Autophagic activity in the cells were evaluated with MDC staining. The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay. RESULTS: MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression, lowered expressions of FoxO1, PINK1, and Parkin1 mRNAs, and reduced levels of FoxO1, PINK1, parkin, nephrin, and podocin proteins and autophagic activity. Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin, inhibited the expression of miR-21a-5p, increased the mRNA and protein expressions of FoxO1, PINK1 and Parkin, and upregulated autophagic activity of the cells. Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells, and these effects were obviously attenuated by treatment with YYHT-medicated sera. CONCLUSIONS YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.
Animals ; MicroRNAs/genetics* ; Podocytes/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Autophagy/drug effects* ; Rats, Wistar ; Protein Kinases/metabolism* ; Rats ; Forkhead Box Protein O1 ; Mice ; Mitochondria/drug effects* ; Ubiquitin-Protein Ligases/metabolism* ; Glucose ; Diabetic Nephropathies ; Male ; Membrane Proteins/metabolism* ; Intracellular Signaling Peptides and Proteins

OBJECTIVES: To investigate the mechanism through which salidroside inhibits proliferation of gastric cancer (GC) cells focusing on glucose metabolic reprogramming pathways. METHODS: High-throughput sequencing combined with bioinformatics analysis was employed to identify the potential targets of salidroside in human GC MGC-803 cells. Liposome-mediated transfection experiments were carried out to validate the functional and mechanistic roles of these targets. CCK-8 and colony formation assays were used to assess the effects of salidroside on GC cell viability and clonogenic ability. qRT-PCR, Western blotting, and biochemical assay kits were used to analyze the regulatory effects of salidroside on the miR-1343-3p-OGDHL/PDHB enzyme complex-pyruvate metabolic pathway in GC cells. RESULTS: Bioinformatics analysis suggested that the tumor-suppressive factor miR-1343-3p negatively regulated the key glycolytic enzyme gene oxoglutarate dehydrogenase-like (OGDHL) in GC cells, and OGDHL and pyruvate dehydrogenase E1 subunit beta (PDHB) were both significantly upregulated in GC tissues, which was close by correlated with reduced survival rates of GC patients. In MGC-803 cells, salidroside treatment significantly enhanced the expression level of miR-1343-3p and downregulated OGDHL expression, resulting in disruption of the stability of PDHB, reduced pyruvate oxidative decarboxylation, and consequently decreased production of acetyl-CoA and ATP. CONCLUSIONS Salidroside inhibits GC cell proliferation possibly by regulating the miR-1343-3p-OGDHL/PDHB enzyme complex-pyruvate metabolic pathway, which provides new insights into its anti-tumor mechanisms and suggests new strategies for targeted therapy for GC.
Humans ; Stomach Neoplasms/pathology* ; MicroRNAs/genetics* ; Cell Proliferation/drug effects* ; Glucosides/pharmacology* ; Phenols/pharmacology* ; Cell Line, Tumor ; Glucose/metabolism* ; Pyruvate Dehydrogenase (Lipoamide)/metabolism*

OBJECTIVES: To investigate the mechanism underlying the inhibitory effect of Buyang Huanwu Decoction (BYHWD) on vascular aging. METHODS: Eighteen male SD rats were randomized into young group, intraperitoneal D-galactose injection-induced aging group, and BYHWD gavage group. The changes in pulse wave velocity (PWV), vascular SA-β-gal activity, and expressions of p16, p21 and SA‑β‑gal of the rats were examined. Serum exosomes were isolated from the rats, and after characterization using NTA and TEM and for surface markers and vascular cell markers, were examined for miR-590-5p expression using qRT-PCR. The M1/M2 macrophage ratio and cytokine levels were evaluated using immunofluorescence staining and qRT-PCR. Bioinformatics analysis and dual-luciferase reporter assays were carried out to predict the potential target genes of miR-590-5p and validate its targeting relationship with SLC8A3, whose expressions were detected in the vascular tissues of the rats by Western blotting. RESULTS: Compared with the young rats, the aging rats exhibited significantly increased PWV in the abdominal aorta with elevated vascular expressions of p16, p21 and SA-β-gal, which were all reversed by BYHWD treatment. The isolated serum exosomes were positive for CD63, CD81, CD31 and SM-22, and the exosomes from aging rats showed significantly downregulated expression of miR-590-5p, which was upregulated after BYHWD treatment. The aging rat vessels showed an increased M1/M2 macrophage ratio with elevated M1-specific cytokines and reduced M2-specific cytokines, and BYHWD treatment effectively inhibited M1 polarization of the macrophages. Pearson analysis revealed a negative correlation between exosomal miR-590-5p upregulation and the M1/M2 ratio. Bioinformatics analysis and dual-luciferase assays confirmed that miR-590-5p targets SLC8A3. Western blotting demonstrated increased SLC8A3 expression in aging rat vessels, which was downregulated after BYHWD treatment. CONCLUSIONS BYHWD attenuates vascular aging in rats by modulating macrophage M1 polarization and suppressing vascular inflammation via exosomal miR-590-5p-mediated downregulation of SLC8A3.
Animals ; MicroRNAs/genetics* ; Rats, Sprague-Dawley ; Drugs, Chinese Herbal/pharmacology* ; Male ; Macrophages/drug effects* ; Rats ; Exosomes/metabolism* ; Aging/drug effects* ; Signal Transduction

OBJECTIVES: To study the molecular mechanisms of LDH-loaded si-NEAT1 for regulating paclitaxel resistance and tumor-associated macrophage (TAM) polarization in breast cancer. METHODS: qRT-PCR and Western blotting were used to detect the expression of lncRNA NEAT1, miR-133b, and PD-L1 in breast cancer SKBR3 cells and paclitaxel-resistant SKBR3 cells (SKBR3-PR). The effects of transfection with si-NEAT1 and miR-133b mimics on MRP, MCRP and PD-L1 expressions and cell proliferation, migration and apoptosis were investigated using qRT-PCR, Western blotting, scratch and Transwell assays, and flow cytometry. Rescue experiments were conducted using si-NEAT1 and miR-133b inhibitor. Human THP-1 macrophages were cultured in the presence of conditioned media (CM) derived from SKBR3 and SKBR3-PR cells with or with si-NEAT1 transfection for comparison of IL-4-induced macrophage polarization by detecting the surface markers. LDH@si-NEAT1 nanocarriers were constructed, and their effects on MRP, MCRP and PD-L1 expressions and cell behaviors of the tumor cells were examined. THP-1 cells were treated with the CM from LDH@si-NEAT1-treated tumor cells, and the changes in their polarization were assessed. RESULTS: SKBR3-PR cells showered significantly upregulated NEAT1 and PD-L1 expressions and lowered miR-133b expression as compared with their parental cells. Transfection with si-NEAT1 and miR-133b mimics inhibited viability, promoted apoptosis and enhanced MRP and BCRP expressions in SKBR3-PR cells. NEAT1 knockdown obvious upregulated miR-133b and downregulated PD-L1, MRP and BCRP expressions. The CM from SKBR3-PR cells obviously promoted M2 polarization of THP-1 macrophages, which was significantly inhibited by CM from si-NEAT1-transfected cells. Treatment with LDH@si-NEAT1 effectively inhibited migration and invasion, promoted apoptosis, and reduced MRP, BCRP and PD-L1 expressions in the tumor cells. The CM from LDH@si-NEAT1-treated SKBR3-PR cells significantly downregulated Arg-1, CD163, IL-10, and PD-L1 and upregulated miR-133b expression in THP-1 macrophages. CONCLUSIONS LDH@si-NEAT1 reduces paclitaxel resistance of breast cancer cells and inhibits TAM polarization by targeting the miR-133b/PD-L1 axis.
Humans ; MicroRNAs/genetics* ; RNA, Long Noncoding/genetics* ; Paclitaxel/pharmacology* ; Breast Neoplasms/metabolism* ; Drug Resistance, Neoplasm ; B7-H1 Antigen/metabolism* ; Cell Line, Tumor ; Female ; Tumor-Associated Macrophages ; Apoptosis ; Cell Proliferation ; Macrophages ; Cell Movement

OBJECTIVES: To evaluate the protective effect of Lycium barbarum polysaccharides (LBP) against cisplatin-induced ovarian granulosa cell injury and investigate its possible mechanisms. METHODS: Human granulosa-like tumor cell line (KGN) were treated with 2.5 µg/mL cisplatin for 24 h, followed by treatment with 100, 500, and 1000 mg/L LBP, and the changes in cell viability, apoptosis, level of anti-Müllerian hormone (AMH), and cell ultrastructure were detected with CCK-8 assay, flow cytometry, ELISA and transmission electron microscopy. The cellular expressions of Bax, caspase-3, Bcl-2, and the PI3K/AKT pathway proteins were analyzed using Western blotting, and the expression of miR-23a was detected with RT-qPCR. KGN cell models with lentivirus-mediated miR-23a overexpression or knockdown were used to verify the therapeutic mechanism of LBP. RESULTS: Cisplatin treatment significantly inhibited cell viability, induced apoptosis, decreased AMH level, caused ultrastructural abnormalities, increased Bax and caspase-3 expression, and lowered Bcl-2 expression in KGN cells. Cisplatin also suppressed the activation of the PI3K/AKT signaling pathway and upregulated miR-23a expression in the cells. LBP intervention obviously alleviated cisplatin-induced injuries in KGN cells, and in particular, LBP treatment at the medium dose for 24 h significantly improved KGN cell viability, reduced apoptosis, enhanced their endocrine function, and ameliorated ultrastructural abnormalities. Mechanistically, medium-dose LBP obviously activated the PI3K/AKT pathway by downregulating miR-23a in cisplatin-treated cells, subsequently inhibiting Bax and caspase-3 while upregulating Bcl-2. Overexpression of miR-23a weakened while knockdown of miR-23a significantly enhanced the protective effects of LBP. CONCLUSIONS LBP alleviates cisplatin-induced apoptosis in KGN cells by inhibiting miR-23a expression and activating the PI3K/AKT pathway, suggesting a potential therapeutic strategy for ovarian function preservation.
Humans ; Cisplatin/adverse effects* ; MicroRNAs/genetics* ; Female ; Granulosa Cells/cytology* ; Apoptosis/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Down-Regulation ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Cell Line, Tumor ; Cell Survival/drug effects*

The ambiguity of etiology makes temporomandibular joint osteoarthritis (TMJOA) "difficult-to-treat". Emerging evidence underscores the therapeutic promise of exosomes in osteoarthritis management. Nonetheless, challenges such as low yields and insignificant efficacy of current exosome therapies necessitate significant advances. Addressing lower strontium (Sr) levels in arthritic synovial microenvironment, we studied the effect of Sr element on exosomes and miRNA selectively loading in synovial mesenchymal stem cells (SMSCs). Here, we developed an optimized system that boosts the yield of SMSC-derived exosomes (SMSC-EXOs) and improves their miRNA profiles with an elevated proportion of beneficial miRNAs, while reducing harmful ones by pretreating SMSCs with Sr. Compared to untreated SMSC-EXOs, Sr-pretreated SMSC-derived exosomes (Sr-SMSC-EXOs) demonstrated superior therapeutic efficacy by mitigating chondrocyte ferroptosis and reducing osteoclast-mediated joint pain in TMJOA. Our results illustrate Alix's crucial role in Sr-triggered miRNA loading, identifying miR-143-3p as a key anti-TMJOA exosomal component. Interestingly, this system is specifically oriented towards synovium-derived stem cells. The insight into trace element-driven, site-specific miRNA selectively loading in SMSC-EXOs proposes a promising therapeutic enhancement strategy for TMJOA.
MicroRNAs/metabolism* ; Mesenchymal Stem Cells/drug effects* ; Osteoarthritis/drug therapy* ; Exosomes/drug effects* ; Strontium/pharmacology* ; Synovial Membrane/cytology* ; Humans ; Animals ; Temporomandibular Joint Disorders/therapy* ; Temporomandibular Joint