Absolute Quantification of Selected microRNAs Expression in Endometrial Cancer by Digital PCR.

MicroRNAs (miRNA) are involved in the process of carcinogenesis, including the development of endometrial cancer (EC). This study aimed to investigate the association between the expression of three miRNAs (miR-21-5p, miR-205-5p, and miR-222-3p) in endometrial cancer tissues. In addition, the stability of expression of SNORD48 and U6, which were initially planned to be used as reference miRNAs for normalization, was investigated. Endometrial tissue was obtained from 111 patients with EC during hysterectomy and from 19 patients undergoing surgery for uterine fibroids or pelvic organ prolapse as a control group without neoplastic changes. Our study was based on calculations made with a digital PCR method (Qiagen, Hilden, Germany) to measure the absolute expression. In the endometrial cancer tissue, miR-205-5p was upregulated, while miR-222-3p and SNORD48 were downregulated compared to the control group. We detected statistically significant correlation of miR-205-5p, U6, and SNORD48 expression with different histological grades; the expression of miR-205-5p increases with the histopathological grade advancement (intraepithelial neoplasia- EIN = 1590, G1 = 3367.2, G2 = 8067 and G3 = 20,360), while U6 and SNORD expression decreases from EIN to G2 and increases again in the G3 grade (U6: EIN = 19,032, G1 = 16,482.4, G2 = 13,642.4, G3 = 133,008; SNORD48: EIN = 97,088, G1 = 59,520, G2 = 43,544, G3 = 227,200). Our study suggests that upregulation of miR-205-5p and downregulation of miR-222-3p and SNORD48 may influence development of endometrial cancer. Moreover, miR-205-5p, U6, and SNORD48 expression changes may be associated with progression of endometrial cancer. The results also indicate that SNORD48 and U6, commonly used as internal references, may influence endometrial cancer development and progression; therefore, they should not be used as references. However, it is important to note that further research is required to understand their role in endometrial cancer.

Evaluation of miRNA Expression in Patients with Gestational Diabetes Mellitus: Investigating Diagnostic Potential and Clinical Implications.

Purpose: Gestational diabetes mellitus (GDM) is common pregnancy complication (8%), characterized by hyperglycemia resulting from pathological homeostatic mechanisms. There's a concerning trend of increasing GDM prevalence. New markers, particularly epigenetic ones, are sought for early detection and enhanced care. miRNA are small non-coding RNA molecules. The main goal was to investigate the potential role of miRNA (miR-16-5p, miR-222-3p, miR-21-5p) in GDM and their association with clinical features. Patients and Methods: The study included 72 pregnant patients, with 42 having GDM and 30 in the control group. miRNA expression was measured using ELISA. Results: There were no significant differences in miR-222-3p expression between GDM patients and the control group. The GDM group exhibited a positive correlation between miR-16-5p expression and miR-21-5p expression as well as between miR-16-5p expression and insulin resistance. In the GDM group, a positive correlation was observed between miR-21-5p expression and fasting glucose levels. Conclusion: Results do not confirm the role of miR-222-3p in GDM pathogenesis or as a diagnostic marker. Additionally, a role for miR-16-5p in GDM pathogenesis was observed. Furthermore, a potential role for miR-21-5p in monitoring GDM treatment is indicated.

Bioinformatic analysis shows the correlation of hsa_circ_0006220-miR-221/222-3p-ESR1/KDR axis with sorafenib resistance of hepatocellular carcinoma.

Sorafenib resistance is a major obstacle influencing its therapeutic efficacy in advanced hepatocellular carcinoma (HCC). The knowledge of circular RNAs (circRNAs), a group of novel endogenous non-coding RNAs, in sorafenib resistance of HCC is still inadequate. In this study, a series of bioinformatic analyses and validation were performed. Firstly, a dataset GSE101850 was included for screening out differentially expressed circRNAs between sorafenib resistant and sensitive HCC, after which the structural patterns and binding microRNAs (miRNAs) of these candidate circRNAs were acquired. By combination of the results from expression, prognosis and diagnosis analysis, miR-221-3p and miR-222-3p were selected as the most potential binding miRNAs of hsa_circ_0006220, which was correlated with sorafenib resistance of HCC. Next, the target genes of miR-221-3p and miR-222-3p were predicted. Subsequently, ESR1 was identified as the top one hub gene among all these target genes. By conducting survival analysis and correlation analysis, ESR1 and KDR were considered as the most potential genes associated with sorafenib resistance of HCC. Collectively, we elucidated a potential hsa_circ_0006220-miR-221/222-3p-ESR1/KDR regulatory axis linked to sorafenib resistance of HCC.

MiR-221-3p/222-3p Cluster Expression in Human Adipose Tissue Is Related to Obesity and Type 2 Diabetes.

The progression of obesity and type 2 diabetes (T2D) is intricately linked with adipose tissue (AT) angiogenesis. Despite an established network of microRNAs (miRNAs) regulating AT function, the specific role of angiogenic miRNAs remains less understood. The miR-221/222 cluster has recently emerged as being associated with antiangiogenic activity. However, no studies have explored its role in human AT amidst the concurrent development of obesity and T2D. Therefore, this study aims to investigate the association between the miR-221-3p/222-3p cluster in human AT and its regulatory network with obesity and T2D. MiR-221-3p/222-3p and their target gene (TG) expression levels were quantified through qPCR in visceral (VAT) and subcutaneous (SAT) AT from patients (n = 33) categorized based on BMI as normoweight (NW) and obese (OB) and by glycemic status as normoglycemic (NG) and type 2 diabetic (T2D) subjects. In silico analyses of miR-221-3p/222-3p and their TGs were conducted to identify pertinent signaling pathways. The results of a multivariate analysis, considering the simultaneous expression of miR-221-3p and miR-222-3p as dependent variables, revealed statistically significant distinctions when accounting for variables such as tissue depot, obesity, sex, and T2D as independent factors. Furthermore, both miRNAs and their TGs exhibited differential expression patterns based on obesity severity, glycemic status, sex, and type of AT depot. Our in silico analysis indicated that miR-221-3p/222-3p cluster TGs predominantly participate in angiogenesis, WNT signaling, and apoptosis pathways. In conclusion, these findings underscore a promising avenue for future research, emphasizing the miR-221-3p/222-3p cluster and its associated regulatory networks as potential targets for addressing obesity and related metabolic disorders.

Identification and comprehensive analysis of epithelial-mesenchymal transition related target genes of miR-222-3p in breast cancer.

Background: Epithelial-mesenchymal transition (EMT) is a crucial mechanism that microRNA-222-3p (miR-222-3p) promotes breast cancer (BC) progression. Our study aimed to identify EMT-associated target genes (ETGs) of miR-222-3p for further analysis of their roles in BC based on bioinformatics tools. Methods: Based on bioinformatics analysis, we identified 10 core ETGs of miR-222-3p. Then, we performed a comprehensive analysis of 10 ETGs and miR-222-3p, including pathway enrichment analysis of ETGs, differential expression, clinical significance, correlation with immune cell infiltration, immune checkpoint genes (ICGs) expression, tumor mutational burden (TMB), microsatellite instability (MSI), stemness, drug sensitivity, and genetic alteration. Results: The expression of miR222-3p in basal-like BC was significantly higher than in other subtypes of BC and the normal adjacent tissue. Pathway analysis suggested that the ETGs might regulate the EMT process via the PI3K-Akt and HIF-1 signaling pathway. Six of the 10 core ETGs of miR-222-3p identified were down-expressed in BC, which were EGFR, IL6, NRP1, NTRK2, LAMC2, and PIK3R1, and SERPINE1, MUC1, MMP11, and BIRC5 were up-expressed in BC, which also showed potential diagnostic values in BC. Prognosis analysis revealed that higher NTRK2 and PIK3R1 expressions were related to a better prognosis, and higher BIRC5 and miR-222-3p expressions were related to a worse prognosis. Most ETGs and miR-222-3p were positively correlated with various infiltration of various immune cells and ICGs expression. Lower TMB scores were correlated with higher expression of MUC1 and NTRK2, and higher BIRC5 was related to a higher TMB score. Lower expression of MUC1, NTRK2, and PIK3R1 were associated with higher MSI scores. Higher expression of ETGs was associated with lower mRNAsi scores, except BIRC5 and miR-222-3p conversely. Most ETGs and miR-222-3p expression were negatively correlated with the drug IC50 values. The analysis of the genetic alteration of the ETGs suggested that amplification was the main genetic alteration of eight ETGs except for NTRK2 and PIK3R1. Conclusion: MiR-222-3p might be a specific biomarker of basal-like BC. We successfully identify 10 core ETGs of miR-222-3p, some might be useful diagnostic and prognostic biomarkers. The comprehensive analysis of 10 ETGs and miR-222-3p indicated that they might be involved in the development of BC, which might be novel therapeutic targets for the treatment of BC.

Protumorigenic role of the atypical cadherin FAT1 by the suppression of PDCD10 via RelA/miR221-3p/222-3p axis in glioblastoma.

The atypical cadherin FAT1 function either as a pro or antitumorigenic in tumors of different tissue origins. Our group previously demonstrated the protumorigenic nature of FAT1 signaling in glioblastoma (GBM). In this study, we investigated how FAT1 influences the expression of clustered oncomiRs (miR-221-3p/miR-222-3p) and their downstream effects in GBM. Through several experiments involving the measurement of specific gene/microRNA expression, gene knockdowns, protein and cellular assays, we have demonstrated a novel oncogenic signaling pathway mediated by FAT1 in glioma. These results have been verified using antimiRs and miR-mimic assays. Initially, in glioma-derived cell lines (U87MG and LN229), we observed FAT1 as a novel up-regulator of the transcription factor NFκB-RelA. RelA then promotes the expression of the clustered-oncomiRs, miR-221-3p/miR-222-3p, which in turn suppresses the expression of the tumor suppressor gene (TSG), PDCD10 (Programmed cell death protein10). The suppression of PDCD10, and other known TSG targets (PTEN/PUMA), by miR-221-3p/miR-222-3p, leads to increased clonogenicity, migration, and invasion of glioma cells. Consistent with our in-vitro findings, we observed a positive expression correlation of FAT1 and miR-221-3p, and an inverse correlation of FAT1 and the miR-targets (PDCD10/PTEN/PUMA), in GBM tissue-samples. These findings were also supported by publicly available GBM databases (The Cancer Genome Atlas [TCGA] and The Repository of Molecular Brain Neoplasia Data [Rembrandt]). Patients with tumors displaying high levels of FAT1 and miR-221-3p expression (50% and 65% respectively) experienced shorter overall survival. Similar results were observed in the TCGA-GBM database. Thus, our findings show a novel FAT1/RelA/miR-221/miR-222 oncogenic-effector pathway that downregulates the TSG, PDCD10, in GBM, which could be targeted therapeutically in a specific manner.

Role of Increased miR-222-3p Expression in Peripheral Blood and Wound Marginal Tissues of Type 2 Diabetes Mellitus Patients with Diabetic Foot Ulcer.

Purpose: To study the correlations of miR-222-3p expression in the peripheral blood and wound marginal tissues of type 2 diabetes mellitus (T2DM) patients with the onset of diabetic foot ulcer (DFU), as well as explore the clinical value possessed by miR-222-3p in the diagnosis and treatment outcomes of DFU. Methods: The study included 70 T2DM patients who did not suffer foot ulcers (T2DM group), 146 T2DM patients who suffered foot ulcers (DFU group), as well as 70 normal controls (NC group). Quantitative real-time PCR determined the MiR-222-3p relative expression. Clinical features and risk factors regarding DFU were assessed. Multiple stepwise logistic regression analysis assisted in confirming whether miR-222-3p expression could serve for independently predicting the risk factors for DFU. ROC curve analysis evaluated the diagnostic value exhibited by miR-222-3p level against DFU. Results: T2DM group exhibited an obviously higher MiR-222-3p expression relative to NC group [1.98 (0.98, 3.62) vs 0.92 (0.61, 1.87)] (P < 0.01), but DFU group exhibited an obviously higher miR-222-3p expression relative to T2DM group [5.61 (1.98, 10.24) vs 1.98 (0.98, 3.62)] (P < 0.01). Besides, miR-222-3p expression presented a negative correlation with DFU healing rate (P < 0.05). According to Kaplan-Meier survival curve analysis, the group with high miR-222-3p expression showed higher unhealed DFU cumulative rate relative to the group with low expression (log-rank, P = 0.011, 0.001, respectively). Multivariate logistic regression analysis confirmed that high miR-222-3p expressions could independently predict DFU risk (OR=3.85, 95% CI 1.18~12.37, P = 0.008). According to the ROC curve analysis, the AUC of miR-222-3p specific to DFU diagnosis reached 0.803, with the best sensitivity of 95.93% and best specificity of 96.27%. Conclusion: The increased expression of miR-222-3p in the peripheral blood of T2DM patients is closely related to the occurrence of DFU. MiR-222-3p is a biomarker with potential clinical value in diagnosing and evaluating the prognosis of DFU.

MiR-222-3p suppresses C2C12 myoblast proliferation and differentiation via the inhibition of IRS-1/PI3K/Akt pathway.

Numerous studies have revealed the profound impact of microRNAs on regulating skeletal muscle development and regeneration. However, the biological function and regulation mechanism of miR-222-3p in skeletal muscle remains largely unknown. In this study, miR-222-3p was found to be abundantly expressed in the impaired skeletal muscles, indicating that it might have function in the development and regeneration process of the skeletal muscle. MiR-222-3p overexpression impeded C2C12 myoblast proliferation and myogenic differentiation, whereas inhibition of miR-222-3p got the opposite results. The dual-luciferase reporter assay showed that insulin receptor substrate-1 (IRS-1) was the target gene of miR-222-3p. We next found that knockdown of IRS-1 could obviously suppress C2C12 myoblast proliferation and differentiation. Additionally, miR-222-3p-induced repression of myoblast proliferation and differentiation was verified to be associated with a decrease in phosphoinositide 3-kinase (PI3K)-Akt signaling. Overall, we demonstrated that miR-222-3p inhibited C2C12 cells myogenesis via IRS-1/PI3K/Akt pathway. Therefore, miR-222-3p may be used as a therapeutic target for alleviating muscle loss caused by inherited and nonhereditary diseases.

Circulating miR-222-3p is associated with ankylosing spondylitis development and predicts therapeutic efficacy of nonsteroidal anti-inflammatory drugs.

Ankylosing spondylitis (AS) is a chronic rheumatic disease, and some microRNAs (miRNAs) in AS have been identified. This study aimed to measure miR-222-3p expression in AS patients, investigate the association of miR-222-3p with AS disease activity, and explore the clinical value of miR-222-3p in diagnosing AS and predicting therapeutic efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) on AS patients. This study included 96 patients with AS, 58 patients with rheumatoid arthritis (RA), and 90 healthy controls. miR-222-3p expression was detected by reverse-transcription quantitative polymerase chain reaction (PCR). The ability of miR-222-3p to discriminate between different groups was evaluated by receiver operating characteristic analysis. The predictive value of miR-222-3p on the efficacy of NSAID treatment for AS was assessed by logistic regression analysis. AS patients treated with oral NSAIDs diclofenac sodium were divided into response (n = 76) and no-response (n = 20) groups after 16 weeks of treatment. miR-222-3p in AS patients was higher than that in healthy subjects and RA patients. miR-222-3p had high diagnostic value in distinguishing patients with AS from RA patients and healthy controls. miR-222-3p, increased in active AS patients, had the ability to screen active AS patients from inactive AS patients. miR-222-3p was decreased in the response group, and had high accuracy in predicting the therapeutic efficiency of NSAIDs. The findings indicate that increased miR-222-3p in AS patients may function as a diagnostic biomarker for AS, and predictive biomarker for the therapeutic efficacy of NSAIDs in patients with AS. In addition, miR-222-3p is associated with AS disease activity.

Endothelial progenitor cell-derived exosomes promote anti-inflammatory macrophages via SOCS3/JAK2/STAT3 axis and improve the outcome of spinal cord injury.

BACKGROUND: Macrophage in the spinal cord injury (SCI) area imparts a chronic pro-inflammation effect that challenges the recovery of SCI. Previously, endothelial progenitor cell-produced exosomes (EPC-EXOs) have been noticed to facilitate revascularization and inflammation control after SCI. However, their effects on macrophage polarization remained unclear. This study aimed to investigate the EPC-EXOs' role in macrophage polarization and reveal its underlying mechanism. METHODS: We extracted the macrophages and EPC from the bone marrow suspension of C57BL/L mice by centrifugation. After cell identification, the EPC-EXOs were collected by ultra-high-speed centrifugation and exosome extraction kits and identified by transmission electron microscopy and nanoparticle tracking analysis. Then, macrophages were cultured with EPC-EXOs in different concentrations. We labeled the exosome to confirm its internalization by macrophage and detected the macrophage polarization marker level both in vitro and in vivo. We further estimated EPC-EXOs' protective effects on SCI by mice spinal cord tissue H&E staining and motor behavior evaluation. Finally, we performed RT-qPCR to identify the upregulated miRNA in EPC-EXOs and manipulate its expression to estimate its role in macrophage polarization, SOCS3/JAK2/STAT3 pathway activation, and motor behavior improvement. RESULTS: We found that EPC-EXOs decreased the macrophages' pro-inflammatory marker expression and increased their anti-inflammatory marker expression on the 7 and 14 days after SCI. The spinal cord H&E staining results showed that EPC-EXOs raised the tissue-sparing area rate significantly after 28 days of SCI and the motor behavior evaluation indicated an increased BMS score and motor-evoked potential by EPC-EXOs treatment after SCI. The RT-qPCR assay identified that miR-222-3P upregulated in EPC-EXOs and its miRNA-mimic also decreased the pro-inflammatory macrophages and increased the anti-inflammatory macrophages. Additionally, miR-222-3P mimic activated the SOCS3/JAK2/STAT3 pathway, and SOCS3/JAK2/STAT3 pathway inhibition blocked miR-2223P's effects on macrophage polarization and mouse motor behavior. CONCLUSION: Comprehensively, we discovered that EPC-EXOs-derived miR-222-3p affected macrophage polarization via SOCS3/JAK2/STAT3 pathway and promoted mouse functional repair after SCI, which reveals EPC-EXOs' role in modulation of macrophage phenotype and will provide a novel interventional strategy to induce post-SCI recovery.

Lean adipose tissue macrophage derived exosome confers immunoregulation to improve wound healing in diabetes.

Chronic non-healing wounds, a prevalent complication of diabetes, are associated with increased mortality in diabetic patients. Excessive accumulation of M1 macrophages in diabetic wounds promotes inflammation and results in dysregulated tissue repair. Adipose tissue macrophages (ATMs) derived from healthy lean donors have the ability to improve glucose tolerance and insulin sensitivity, as well as modulate inflammation. MicroRNAs (miRs), which can be packaged into exosomes (Exos) and secreted from cells, serve as essential regulators of macrophage polarization. Here, we revealed that ATMs isolated from lean mice secrete miRs-containing Exos, which modulate macrophage polarization and promote rapid diabetic wound healing when administered to diabetes-prone db/db mice. The miRs sequence of tissue samples from wounds treated with Exos secreted by lean ATMs (ExosLean) revealed that miR-222-3p was up-regulated. Further analyses showed that inhibiting miR-222-3p using a miR inhibitor impaired the macrophage-reprogramming effect of ExosLean. In the excisional skin wound mouse model, locally inhibiting miR-222-3p disrupted healing dynamics and failed to modulate macrophage polarization. Mechanistic studies revealed a connection between miR-222-3p, Bcl2l11/Bim, an inflammatory response effector, macrophage polarization, and diabetic wound healing. In summary, ExosLean act as positive regulators of macrophage polarization by regulating miR levels in wounds and accelerating wound healing, and thus have important implications for wound management in diabetes.

Suppression of microRNA-222-3p ameliorates ulcerative colitis and colitis-associated colorectal cancer to protect against oxidative stress via targeting BRG1 to activate Nrf2/HO-1 signaling pathway.

Oxidative stress is an important pathogenic factor in ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC), further impairing the entire colon. Intestinal epithelial cells (IECs) are crucial components of innate immunity and play an important role in maintaining intestinal barrier function. Recent studies have indicated that microRNA-222-3p (miR-222-3p) is increased in colon of UC and colorectal cancer (CRC) patients, and miR-222-3p is a crucial regulator of oxidative stress. However, whether miR-222-3p influences IEC oxidative stress in UC and CAC remains unknown. This study investigated the effect of miR-222-3p on the regulation of IEC oxidative stress in UC and CAC. An in vitro inflammation model was established in NCM460 colonic cells, mouse UC and CAC models were established in vivo, and IECs were isolated. The biological role and mechanism of miR-222-3p-mediated oxidative stress in UC and CAC were determined. We demonstrated that miR-222-3p expression was notably increased in dextran sulfate sodium (DSS)-induced NCM460 cells and IECs from UC and CAC mice. In vitro, these results showed that the downregulation of miR-222-3p reduced oxidative stress, caspase-3 activity, IL-1ß and TNF-α in DSS-induced NCM460 cells. We further identified BRG1 as the target gene of miR-222-3p, and downregulating miR-222-3p alleviated DSS-induced oxidative injury via promoting BRG1-mediated activation Nrf2/HO-1 signaling in NCM460 cells. The in vivo results demonstrated that inhibiting miR-222-3p in IECs significantly relieved oxidative stress and inflammation in the damaged colons of UC and CAC mice, as evidenced by decreases in ROS, MDA, IL-1ß and TNF-α levels and increases in GSH-Px levels. Our study further demonstrated that inhibiting miR-222-3p in IECs attenuated oxidative damage by targeting BRG1 to activate the Nrf2/HO-1 signaling. In summary, inhibiting miR-222-3p in IECs attenuates oxidative stress by targeting BRG1 to activate the Nrf2/HO-1 signaling, thereby reducing colonic inflammation and tumorigenesis.

Exosomal microRNA-222-3p increases UVB sensitivity of lens epithelium cells by suppressing MGMT.

BACKGROUND: Age-related cataract (ARC) is a leading cause of blindness worldwide with multiple pathogenic factors. Oxidative damage of lens epithelium cells (LECs) is one of the well-accepted pathogenesis of ARC which can be regulated by DNA repair genes (DRGs). The present research aimed to clarify the regulatory mechanism of exosomal microRNAs (miRNAs) on DRGs in LECs. METHODS: The LECs oxidative damage model was established by UVB-irradiation on SRA01/04 (human lens epithelium cell line). Exosomes from UVB-irradiated cells (UVB-exo) and exosomes from normal control cells (NC-exo) were collected from the culture medium. To explore the functions of LECs exosomes, SRA01/04 were incubated with UVB-exo/NC-exo. Then, we detected SRA01/04 proliferation, viability and apoptosis respectively using 5'-ethynyl-2'-deoxyuridine (EdU), cell-counting kit-8 (CCK-8) and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. Next, the miRNA expression profiles of UVB-exo and NC-exo were identified by miRNA microarrays. RNA expression in exosomes, cells, and clinical samples was verified by qRT-PCR. The location and expression of MGMT and CD63 proteins were detected by immunofluorescence and western blot. The 3'UTR regulation of miR-222-3p to MGMT was verified by luciferase analyses. RESULTS: MGMT down-regulated while miR-222-3p up-regulated in LECs sub-central anterior capsule from ARC lenses. MGMT and miR-222-3p expressions in central and peripheral LECs from anterior lens capsules were differential. UVB-exo can transport the up-regulated miR-222-3p from oxidative-damaged LECs to normal LECs, which could suppress MGMT expression and increase UVB sensitivity of LECs. CONCLUSIONS: Findings on exosomal miRNA functions provided novel insights into pathogenesis of ARC. Exosomal miR-222-3p can be a potential target for prevention and cure of ARC.

miR­222-3p reduces neuronal cell apoptosis and alleviates spinal cord injury by inhibiting Bbc3 and Bim.

Spinal cord injury (SCI) is a severe traumatic event, but without any established effective treatment because of the irreversible neuronal death. Here, we investigated the role of miR-222-3p in neuronal apoptosis following SCI. Rat SCI models and neuron hypoxia models were accordingly established. The Bbc3, Bim, Bcl-2, Bax, cleaved-caspase 3, cleaved-caspase 9, Cytochrome c, and miR-222-3p expression levels were examined by Western blotting and real-time reverse transcription polymerase chain reaction (RT-qPCR). The possible association between miR-222-3p and Bbc3/Bim was analyzed by dual-luciferase assay. The neuron viability was assessed by Cell Counting Kit-8 assay and Nissl's staining. Live cell staining was performed to detect the mitochondrial membrane potential and neuronal apoptosis. Rat locomotor function was assessed using the Basso-Beattie-Bresnahan scores. Cytochrome c was outflowed from the mitochondria after SCI or hypoxia treatment, and Bbc3, Bim, Bax, cleaved-caspase 9, and cleaved-caspase 3 were significantly upregulated, while Bcl-2 and miR-222-3p were decreased remarkably. Meanwhile, neuronal cell viability was significantly inhibited. Treatment of miR-222-3p significantly suppressed the Cytochrome c efflux and neuronal apoptosis and improved neuronal cell viability and motor function in SCI rats. Moreover, we found that Bbc3 and Bim were the direct targets of miR-222-3p. Overall, our data suggest that miR-222-3p could alleviate the mitochondrial pathway-mediated apoptosis and motor dysfunction in rats after SCI by targeting Bbc3 and Bim.

Dysregulated miR-222-3p in plasma exosomes of preeclampsia patients and its In vitro effect on HTR8/SVneo extravillous trophoblast cells by targeting STMN1.

OBJECTIVE: To investigate the diagnostic efficiency of miR-222-3p in plasma exosomes (Exos) and plasma for preeclampsia (PE) and the effect of miR-222-3p targeting STMN1 in PE. METHODS: MiR-222-3p levels in total plasma and plasma Exos were detected in PE patients and healthy controls. A bioinformatics database and dual-luciferase reporter assay were employed to verify the targeting relationship between miR-222-3p and STMN1. Trophoblast HTR-8/Svneo cells were transfected with miR-222-3p inhibitors with/without STMN1 shRNA, followed by MTT, wound healing and Transwell invasion assays. The mRNA and protein expressions were measured by qRT‒PCR and Western blotting, respectively. RESULTS: MiR-222-3p levels in total plasma and plasma Exos were higher in PE patients than in healthy controls, particularly in severe PE patients. In addition, miR-222-3p levels in total plasma and plasma Exos from PE patients were positively correlated with diastolic and systolic blood pressure. The area under the curve (AUC) of miR-222-3p in total plasma for PE diagnostic efficiency was 0.798, with a sensitivity of 76.67% and specificity of 71.93%, while the AUC of miR-222-3p in plasma Exos was 0.708 (sensitivity: 61.67%; specificity: 78.95%). In vitro, miR-222-3p targeted STMN1 in HTR-8/Svneo cells. Low miR-222-3p expression reversed the inhibitory effect of STMN1 shRNA on the proliferation, invasion and migration of HTR/SVneo cells. CONCLUSION: PE patients had increased miR-222-3p expression in total plasma and plasma Exos, which both have high diagnostic efficiency for PE. MiR-222-3p can target STMN1 to promote the proliferation, invasion and migration of HTR-8/Svneo cells and is a potential therapeutic target of PE.

Circulating plasma miR222-3P status and its potential diagnostic performance in prostate cancer.

BACKGROUND: Although studies suggest that miR222-3p is dysregulated in prostate cancer (PC) cells and tissues, the possible changes in the level of miR222-3p in the plasma samples of PC patients remained unclear. The present study aimed to evaluate the diagnostic value of the plasma miR222-3p expression level as a potential biomarker in PC, benign prostatic hyperplasia (BPH) and healthy people. METHODS: Blood samples were collected from 100 adult males (54 patients with PC, 27 patients with BPH and 19 healthy individuals) referred to our affiliated hospital. The expression level of miR222-3p was evaluated using a quantitative reverse transcription-polymerase chain reaction. Receiver operating characteristic curves were used to evaluate miR222-3p diagnostic accuracy for discriminating between the PC, BPH and healthy individuals. RESULTS: The expression level of miR222-3p was significantly higher in PC patients compared to healthy individuals as a fold change of 5.3 (p = 0.009), but not for BPH individuals. The diagnostic value of the plasma miR222-3p for discrimination of the PC patients from healthy individuals was reasonable [cut-off value (fold change relative to miR16-5p) = 1.69, area under the curve = 0.73, sensitivity = 0.75 and specificity = 0.74]. CONCLUSIONS: Circulating plasma miR-222-3p significantly upregulated in PC patients, but not in BPH ones. Besides these preliminary results showed that miR222-3p has the potential to discriminate PC patients from healthy ones. Addittional studies with a larger sample size are required to confirm these data.

Upregulation of miR-222-3p alleviates the symptom of aortic dissection through targeting STAT3.

OBJECTIVE: This study sought to investigate the differentially expressed miRNAs in Aortic dissection (AD) and explore the downstream mechanisms in regulating AD. METHODS: Exosomes of AD patients and healthy people were isolated by differential centrifugation, and the differentially expressed miRNAs were evaluated by RNA sequencing. The downstream target of miR-222-3p was predicted by bioinformatics method and validated by dual-luciferase assay. Angiotensin II and Promethazine were used to establish AD mouse model and platelet-derived growth factor BB (PDGF-BB) was used to induce human vascular smooth muscle cells (HVSMCs) to elucidate the effect of miR-222-3p upregulation on AD in vivo and in vitro. The relative level of miR-222-3p was evaluated by RT-qPCR. The level of several proteins was investigated by Western blot. Immunofluorescence staining was used to detect the stress fiber formation. Cell migration was evaluated by wound healing and Transwell assay. The proliferation, cell cycle and apoptosis of HVSMCs were assessed by CCK-8 and flow cytometry, respectively. RESULTS: MiR-222-3p was downregulated in AD and PDGF-BB induced HVSMCs. The upregulation of miR-222-3p alleviated the symptom of AD in vivo by targeting STAT3, and inhibited stress fiber formation, abnormal migration, proliferation and apoptosis of HVSMCs induced by PDGF-BB by regulating the expression of α-SMA, SM22α, MMP2, MMP9 and p-Smad2. CONCLUSION: The upregulation of miR-222-3p attenuates the progression of AD. Our study provides a theoretical basis for exploring new strategies against AD.

Correlation study on serum miR-222-3p and glucose and lipid metabolism in patients with polycystic ovary syndrome.

OBJECTIVE: microRNAs (miRNAs) play pivotal roles in polycystic ovary syndrome (PCOS), an endocrine and metabolic disorder that commonly occurs in women of childbearing age. This paper aimed to measure miR-222-3p expression in sera of PCOS patients and to explore its clinical value on PCOS diagnosis and prediction of diabetic and cardiovascular complications. METHODS: Totally 111 PCOS patients and 94 healthy people were recruited and assigned to the overweight (ow) group and non-overweight (non-ow) group, followed by determination of serum miR-222-3p expression. The diagnostic efficiency of miR-222-3p on PCOS ow and non-ow patients was analyzed. Correlations between miR-222-3p and glycolipid metabolic indicators and diabetic and cardiovascular complications in PCOS were analyzed. The downstream target of miR-222-3p was predicted and their binding relationship was verified. The correlation between PGC-1α and miR-222-3p was analyzed. RESULTS: miR-222-3p was highly-expressed in PCOS patients (p < 0.001), in especially PCOS ow patients. The area under the curve (AUC) of miR-222-3p diagnosing PCOS non-ow patients was 0.9474 and cut-off value was 1.290 (89.06% sensitivity, 98.11% specificity), indicating that non-ow people with serum miR-222-3p > 1.290 could basically be diagnosed with PCOS. AUC of miR-222-3p diagnosing PCOS ow patients was 0.9647 and cut-off value was 2.425 (85.11% sensitivity, 100% specificity), suggesting that ow people with serum miR-222-3p > 2.425 could basically be diagnosed with PCOS. miR-222-3p was positively-correlated with fasting plasma glucose (FPG), fasting insulin (FINS), homeostatic model assessment-insulin resistance (HOMA-IR), and low-density lipoprotein cholesterol (LDL-C) and negatively-correlated with high-density lipoprotein cholesterol (HDL-C). miR-222-3p was independently-correlated with diabetic and cardiovascular complications in PCOS (p < 0.05). High expression of miR-222-3p predicted high risks of diabetic and cardiovascular complications in PCOS. miR-222-3p targeted PGC-1α and was negatively-correlated with PGC-1α (r = - 0.2851, p = 0.0224; r = - 0.3151, p = 0.0310). CONCLUSION: High expression of miR-222-3p assisted PCOS diagnosis and predicted increased risks of diabetic and cardiovascular complications. miR-222-3p targeted PGC-1α and was negatively-correlated with PGC-1α.

Panax notoginseng saponin reduces IL-1ß-stimulated apoptosis and endoplasmic reticulum stress of nucleus pulposus cells by suppressing miR-222-3p.

Background: It is well documented that the malignant biological behaviors of nucleus pulposus cells (NPCs) could trigger intervertebral disc degeneration (IDD). Panax notoginseng saponin (PNS) is a traditional Chinese medicine that inhibits osteoclastogenesis. However, its effects on the phenotypes of NPCs in IDD remains largely unknown. This study sought to examine the role of PNS in IDD and its regulatory mechanism. Methods: First, human NPCs (hNPCs) were treated with interleukin-1 beta (IL-1ß) to induce an IDD cell model. Cell proliferation and apoptosis were estimated by Cell Counting Kit-8 (CCK-8) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Western blot was employed to examine the levels of proteins related to apoptosis and endoplasmic reticulum (ER) stress. Enzyme-linked immunosorbent assays (ELISAs) were used to test inflammatory factors levels. Immunofluorescence (IF) assays were used to determine the nuclear translocation of nuclear factor-kappa beta (NF-κB) p65. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)was used to detect miR-222-3p expression. Results: We discovered that PNS enhanced the viability but reduced the apoptosis, inflammation, and ER stress response of IL-1ß-induced hNPCs in a concentration-dependent manner. Additionally, PNS significantly reduced miR-222-3p expression in the IL-1ß-induced hNPCs. Notably, these PNS effects were reversed by the upregulation of miR-222-3p. Conclusions: In summary, PNS appears to facilitate the proliferation and attenuate the apoptosis, inflammatory response, and ER stress response of IL-1ß-induced hNPCs by inhibiting miR-222-3p expression. Our findings provide a theoretical basis for a novel drug application in IDD research.

Mechanisms underlying microRNA-222-3p modulation of methamphetamine-induced conditioned place preference in the nucleus accumbens in mice.

RATIONALE: MicroRNA (miRNA) control of post-transcription gene expression in the nucleus accumbens (NAc) has been implicated in methamphetamine (METH) dependence. Conditioned place preference (CPP) is a classical animal procedure that reflects the rewarding effects of addictive drugs. miR-222-3p has been reported to play a key role in various neurological diseases and is strongly associated with alcohol dependence. Nevertheless, the role of miR-222-3p in METH dependence remains unclear. OBJECTIVE: To explore the molecular mechanisms underlying the role of miR-222-3p in the NAc in METH-induced CPP. METHODS: miR-222-3p expression in the NAc of METH-induced CPP mice was detected by quantitative real-time (qPCR). Following adeno-associated virus (AAV)-mediated overexpression or knockdown of miR-222-3p in the NAc, mice were subjected to CPP to investigate the effects of miR-222-3p on METH-induced CPP. Target genes of mir-222-3p were predicted using bioinformatics analysis. Candidate target genes for METH-induced CPP were validated by qPCR. RESULTS: miR-222-3p expression in the NAc was decreased in CPP mice. Overexpression of miR-222-3p in the NAc blunted METH-induced CPP. Ppp3r1, Cdkn1c, Fmr1, and PPARGC1A were identified as target gene transcripts potentially mediating the effects of miR-222-3p on METH-induced CPP. CONCLUSION: Our results highlight miR-222-3p as a key epigenetic regulator in METH-induced CPP and suggest a potential role for miR-222-3p in the regulation of METH-induced reward-related changes in the brain.