Objective: To investigate the mechanism of Yishen Tongluo Formula in ameliorating renal pyroptosis in diabetic nephropathy mice by regulating the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. Methods: Sixty C57BL/6 male mice were randomly divided into control (10 mice) and intervention groups (50 mice) using random number table method. The diabetes nephropathy model was established by intraperitoneally injecting streptozotocin(50 mg/kg). After modeling, the intervention group was further divided into model, semaglutide (40 μg/kg), and high-, medium-, and low-dose Yishen Tongluo Formula groups (15.6, 7.8, and 3.9 g/kg, respectively) using random number table method. The high-, medium-, and low-dose Yishen Tongluo Formula groups were administered corresponding doses of medication by gavage, the semaglutide group received a subcutaneous injection of semaglutide injection, and the control group and model groups were administered distilled water by gavage for 12 consecutive weeks. Random blood glucose levels of mice in each group were monitored, and the 24-h urinary protein content was measured using biochemical method every 4 weeks; after treatment, the serum creatinine and urea nitrogen levels were measured using biochemical method. The weight of the kidneys was measured, and the renal index was calculated. Hematoxylin and eosin, periodic acid-Schiff, periodic Schiff-methenamine, and Masson staining were used to observe the pathological changes in renal tissue. An enzyme-linked immunosorbent assay was used to detect urinary β2-microglobulin (β2-MG), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) levels. Western blotting and real-time fluorescence PCR were used to detect the relative protein and mRNA expression levels of nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3 (NLRP3), Caspase-1, gasdermin D (GSDMD), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in renal tissue. Immunohistochemistry was used to detect the proportion of protein staining area of the TLR4, MyD88, and NF-κB in renal tissue. Results: Compared with the control group, the random blood glucose, 24-h urinary protein, serum creatinine, urea nitrogen, and renal index of the model group increased, and the urine β2-MG, NGAL, and KIM-1 levels increased. The relative protein and mRNA expression levels of NLRP3, Caspase-1, GSDMD, IL-1β, and IL-18 in renal tissue increased, and the proportion of TLR4, MyD88, and NF-κB protein positive staining areas increased (P<0.05). Pathological changes such as glomerular hypertrophy were observed in the renal tissue of the model group. Compared with the model group, the Yishen Tongluo Formula high-dose group showed a decrease in random blood glucose after 12 weeks of treatment (P<0.05). The Yishen Tongluo Formula high- and medium-dose groups showed a decrease in 24-h urinary protein, creatinine, urea nitrogen, and renal index, as well as decreased β2-MG, NGAL, and KIM-1 levels. NLRP3, Caspase-1, GSDMD, IL-1 β, and IL-18 relative protein and mRNA expression levels were also reduced, and the proportion of TLR4, MyD88, and NF-κB protein positive staining areas was reduced (P<0.05). Pathological damage to renal tissue was ameliorated. Conclusion Yishen Tongluo Formula may exert protective renal effects by inhibiting renal pyroptosis and alleviating tubular interstitial injury in diabetic nephropathy mice by regulating the TLR4/MyD88/NF-κB signaling pathway.

ObjectiveTo investigate the effect and mechanism of Yishen Tongluo prescription in protecting mice from oxidative stress injury in diabetic kidney disease （DKD） via the nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1）/NAD（P）H quinone oxidoreductase 1 （NQO1） signaling pathway. MethodsSpecific pathogen-free （SPF） male C57BL/6 mice were assigned into a control group （n=10） and a modeling group （n=50）. The DKD model was established by intraperitoneal injection of streptozotocin. The mice in the modeling group were randomized into a model group， a semaglutide （40 μg·kg^-1） group， and high-， medium-， and low-dose （18.2， 9.1， 4.55 g·kg^-1， respectively） Yishen Tongluo prescription groups， with 10 mice in each group. The treatment lasted for 12 weeks. Blood glucose and 24-h urine protein levels were measured， and the kidney index （KI） was calculated. Serum levels of creatinine （SCr）， blood urea nitrogen （BUN）， alanine aminotransferase （ALT）， and aspartate aminotransferase （AST） were assessed. The pathological changes in the renal tissue were evaluated by hematoxylin-eosin， periodic acid-Schiff， periodic acid-silver methenamine， and Masson’s trichrome staining. Enzyme-linked immunosorbent assay kits were used to measure the levels of β2-microglobulin （β2-MG）， neutrophil gelatinase-associated lipocalin （NGAL）， kidney injury molecule-1 （KIM-1）， liver fatty acid-binding protein （L-FABP）， nitric oxide synthase （NOS）， glutathione （GSH）， total antioxidant capacity （T-AOC）， and 8-hydroxy-2'-deoxyguanosine （8-OHdG）. Immunohistochemical staining was performed to examine the expression of Kelch-like ECH-associated protein 1 （Keap1） and malondialdehyde （MDA）. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR） and Western blot were employed to determine the mRNA and protein levels， respectively， of factors in the Nrf2/HO-1/NQO1 signaling pathway. ResultsCompared with the control group， the DKD model group showed rises in blood glucose， 24-h urine protein， KI， SCr， BUN， and ALT levels， along with glomerular hypertrophy， renal tubular dilation， thickened basement membrane， mesangial expansion， and collagen deposition. Additionally， the model group showed elevated levels of β2-MG， NGAL， KIM-1， L-FABP， NOS， and 8-OHdG， lowered levels of GSH and T-AOC， up-regulated expression of MDA and Keap1， and down-regulated expression of Nrf2， HO-1， NQO1， and glutamate-cysteine ligase catalytic subunit （GCLC） （P<0.05）. Compared with the model group， the semaglutide group and the medium- and high-dose Yishen Tongluo prescription groups showed reductions in blood glucose， 24-h urine protein， KI， SCr， BUN， and ALT levels， along with alleviated pathological injuries in the renal tissue. In addition， the three groups showed lowered levels of β2-MG， NGAL， KIM-1， L-FABP， NOS， and 8-OHdG， elevated levels of GSH and T-AOC， down-regulated expression of MDA and Keap1， and up-regulated expression of Nrf2， HO-1， NQO1， and GCLC （P<0.05）. ConclusionYishen Tongluo prescription exerts renoprotective effects in the mouse model of DKD by modulating the Nrf2/HO-1/NQO1 signaling pathway， mitigating oxidative stress， and reducing renal tubular injuries.

OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na⁺, K⁺, and especially Ca²⁺. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies. METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage Tübingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group. RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity. CONCLUSIONS We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.
Animals ; Zebrafish/genetics* ; Receptors, Purinergic P2X2/deficiency* ; CRISPR-Cas Systems/genetics* ; Gene Knockout Techniques ; Phenotype ; Zebrafish Proteins/genetics* ; Disease Models, Animal

Objective To explore the clinical characteristics and prognostic analysis of acute corpus callosum in-farction.Methods A total of 1 466 patients with acute cerebral infarction admitted to the Neurology Department of Jiangbin Hospital in Guangxi from January 2019 to October 2023 and Neurology Department of Fuwai Hospital,Chinese Academy of Medical Sciences from January 2022 to December 2022.Among them,21 patients with acute corpus callosum infarction confirmed by MRI(observation group)and 25 patients with isolated subcortical infarction at the same period(control group)were selected.By comparing clinical data and follow-up information between two groups,we summarized the clinical characteristics of acute corpus callosum infarction,and analyzed the relevant factors affecting the prognosis of the corpus callosum infarction group.Results Acute corpus callosum infarction accounted for 1.43％(21/1 466)of patients with acute cerebral infarction in the same period.The main clinical manifestations include limb paralysis,cognitive decline,and speech impairment.Compared with the control group,the group with corpus callosum infarction had a longer onset visit time(P<0.05),more cognitive impair-ment(P<0.05),a higher proportion of mild stroke(P<0.05),more worsening symptoms during the course of the disease(P<0.05),a higher proportion of cardioembolic type(P<0.05),a lower proportion of small artery occlu?sion type(P<0.05)and a higher proportion of good prognosis(mRS score≤2 points)(P<0.05).The poor prog?nosis of corpus callosum infarction was found to be related to factors such as large lesions,multiple high?risk factors(≥3),concomitant lobar infarction and worsening of symptoms during the course of the disease.Conclusions Acute corpus callosum infarction often leads to cognitive impairment and mild limb paralysis,which frequently de?lays treatment.The poor prognosis is mainly related to factors such as lobar infarction,major lesions,and worsening of symptoms during the process.

Objective To investigate the effects of exosomal microRNA-1234-3p(miR-1234-3p)of lung cancer cells on the malignant behaviors of lung cancer cells,the polarization of tumor-associat-ed macrophages(TAMs),and the functions of T cells,as well as its potential molecular mecha-nisms.Methods Exosomes were extracted from lung cancer A549 cells,and their morphology was observed using transmission electron microscopy.The expression of exosomal marker proteins[tumor susceptibility gene 101(TSG101),CD63 and CD9]was detected by western blot.The relative ex-pression level of miR-1234-3p was measured using real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).The expression of exosomal miR-1234-3p was inhibited by transfecting,the proliferation,migration,and invasion abilities of A549 cells were detected using the CCK-8 method,wound healing assay,and Transwell invasion assay,respectively.The exosomes were co-cultured with macrophages and T lymphocytes separately.The expression of TAM polarization markers CD206 and CD163 was detected by western blot.The secretion levels of cytokines[interleukin(IL)-6,IL-10,and transforming growth factor-β(TGF-β)]were measured using an enzyme-linked immu-nosorbent assay(ELISA).The proliferation and apoptosis abilities of T lymphocytes,as well as the expression of functional molecules[γ-interferon(IFN-γ)and programmed death receptor 1(PD-1)]were detected through cell experiments.The expression levels of proteins related to the phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(AKT)signaling pathway were detected by western blot.Results The isolated exosomes were round double-layer vesicles with a diameter of 50 to 200 nm and expressed TSG101,CD63 and CD9 proteins.The expression level of miR-1234-3p in A549 cells was higher than that in human normal lung epithelial cells BEAS-2B,and the expression level of miR-1234-3p in A549 cell exosomes was higher than that in parental A549 cells,with statis-tically significant differences(P＜0.001).After inhibiting exosomal miR-1234-3p,the prolifera-tion,migration,and invasion abilities of A549 cells decreased,with statistically significantdifferenc-es(P＜0.001).After inhibiting exosomal miR-1234-3p,the levels of M2-type polarization marker proteins CD206 and CD163 in macrophages decreased,the levels of cytokines IL-10 and TGF-β de-creased,and the level of IL-6 increased,with statistically significant differences(P＜0.001),indi-cating that the polarization of macrophages toward the M2 type was inhibited.In T lymphocytes,af-ter inhibiting exosomal miR-1234-3p,cell viability increased,the apoptosis rate decreased,the ex-pression level of the functional molecule IFN-γ increased,and the expression level of PD-1 de-creased,with statistically significant differences(P＜0.01 orP＜0.001).After inhibiting exosom-al miR-1234-3p,the protein levels of phosphorylated(p)-PI3K and p-AKT in A549 cells de-creased,with statistically significant differences(P＜0.001).Conclusion Exosomal miR-1234-3p of lung cancer cells may promote the malignant behaviors of lung cancer cells,the polarization of TAMs toward the M2 type,and the dysfunction of T lymphocytes by activating the PI3K/AKT signa-ling pathway,thereby regulating the immunosuppressive microenvironment and promoting the pro-gression of lung cancer.

Objective To investigate the predictive value of National Institutes of Health Stroke Scale(NIHSS)score,serum brain-derived neurotrophic factor(BDNF)and interleukin-6(IL-6)in post-stroke depression(PSD).Methods A total of 180 patients with stroke were selected and divided into the PSD group(n=80,HAMD≥8 points)and the non-PSD(NPSD)group(n=100,HAMD＜8 points),according to HAMD score at 3 months after stroke.The basic information,NIHSS score,serum BDNF and IL-6 were compared between the two groups.The influencing factors of PSD were analyzed by Logistic regression method.Receiver operating characteristic(ROC)curves were plotted to evaluate the predictive value of NIHSS score,serum BDNF and IL-6 for PSD.Results Compared with the NPSD grope,low density lipoprotein cholesterol(LDL-C)and serum BDNF level were significantly lower,NIHSS score and serum IL-6 level were significantly increased in the PSD group(P＜0.05).Results of Logistic regression analysis showed that increased NIHSS score and serum IL-6 were risk factors for PSD,and increased serum BDNF was the protective factor in patents with PSD(P＜0.05).The area under curve(AUC)of NIHSS score,serum BDNF and IL-6 and their combination prediction of PSD were 0.762,0.746,0.796 and 0.839,respectively.The sensitivity of the combined prediction was 86.0%and a specificity was 95.0%.Conclusion Compared with NPSD patients,the NIHSS score and serum IL-6 level are increased,and the serum BDNF level is decreased in patients with PSD.The combination of all three has a high predictive value for patients of PSD.

Objective To investigate the application value of apparent diffusion coefficient(ADC)histogram parameters of multi-plexed sensitivity encoding diffusion weighted imaging(MUSE-DWI)in evaluating lymph node metastasis of cervical cancer(CC).Methods A total of 54 patients with CC diagnosed pathologically after extensive hysterectomy and pelvic lymph node dissection were analyzed retrospectively,and 74 lymph nodes were extracted,including 28 metastatic lymph nodes and 46 non-metastatic lymph nodes.All patients underwent routine MRI examination and MUSE-DWI before surgery.Through the T2WI fat suppression images were referenced,the region of interest(ROI)covering the entire lymph nodes were drawn on the b=800 s/mm2 images of MUSE-DWI,and ADC histogram parameters were obtained including minimum,maximum,mean,median,percentiles(10 th,25 th,75 th,90 th),kurtosis,and skewness.The differences of ADC histogram parameters between the metastatic lymph nodes and the non-metastatic lymph nodes were compared.The receiver operating characteristic(ROC)curves were drawn and the area under the curve(AUC)were calculated to evaluate the diagnostic efficacy of different parameters in distinguishing lymph node metastasis.Results The mean,median,25 th and 75 th percentiles of ADC histogram of the metastatic lymph nodes were significantly lower than those of the non-metastatic lymph nodes(P＜0.05).However,there were no statistically significant differences in minimum,maximum,10 th percentile,90 th percentile,kurtosis,and skewness(P＞0.05).In the evaluation of various parameters for distinguishing lymph node metastasis,the mean had the highest diagnostic efficacy(AUC=0.718),and when the threshold was 963.07×10-6 mm2/s,the sensitivity and specificity were 0.643 and 0.717,respectively.Conclusion The ADC histogram parameters based on MUSE-DWI have high diag-nostic value in differentiating CC lymph node metastasis,and the mean has the highest diagnostic efficiency.

OBJECTIVES: Diabetes mellitus is closely associated with sudden sensorineural hearing loss (SSNHL), but no definitive evidence has established a causal relationship between type 1 diabetes mellitus (T1DM) and SSNHL. This study aims to investigate the impact of T1DM on SSNHL from a genetic perspective, providing insights for risk prediction and treatment strategies. METHODS: Genetic data related to exposure (T1DM) and outcome (SSNHL) were obtained from publicly available genome-wide association studies (GWAS). Instrumental variables were selected, and Mendelian randomization (MR) analysis was conducted to explore the causal association between T1DM and SSNHL. Inverse variance weighted (IVW) analysis was used as the primary method, with random-effects IVW serving as the main analytical approach. MR-Egger, weighted median, simple mode, and weighted mode analyses were utilized as supplementary methods. Cochran's Q test was applied to evaluate the heterogeneity of the selected instrumental variables, MR-PRESSO was applied to detect outliers, MR-Egger regression was used to assess horizontal pleiotropy and leave-one-out analysis was conducted to examine the robustness of individual single nucleotide polymorphisms (SNPs) on the overall results. RESULTS: A total of 127 SNPs were selected as instrumental variables for the MR analysis. IVW analysis demonstrated a genetically determined association between T1DM and SSNHL (OR=1.036, 95% CI 1.002 to 1.071, P=0.038). Forest plots and scatter plots indicated a causal relationship, suggesting that T1DM increases the risk of SSNHL. Cochran's Q test demonstrated no significant heterogeneity among SNPs (MR-Egger: Q=126.030, P=0.356; IVW: Q=126.450, P=0.373). The funnel plot appeared symmetrical, indicating that the selected instrumental variables were primarily related to exposure rather than potential confounding factors. The MR-Egger intercept was not significantly different from zero (P=0.527), indicating no evidence of horizontal pleiotropy among the SNPs. MR-PRESSO analysis did not identify any outlier SNPs (P=0.356). Leave-one-out analysis confirmed the robustness of the findings, as the results remained stable after removing individual SNPs. CONCLUSIONS Two-sample MR analysis supports the conclusion that T1DM patients have an increased risk of developing SSNHL.
Humans ; Mendelian Randomization Analysis ; Hearing Loss, Sensorineural/etiology* ; Diabetes Mellitus, Type 1/genetics* ; Genome-Wide Association Study ; Hearing Loss, Sudden/etiology* ; Polymorphism, Single Nucleotide ; Risk Factors ; Genetic Predisposition to Disease

BACKGROUND/OBJECTIVES: This study examined the relationship between famine exposure in early life and the risk of type 2 diabetes in adulthood during the 1959–1961 Chinese Famine. SUBJECTS/METHODS: A total of 3,418 individuals aged 35–74 years free of diabetes from two studies in 2006 and 2009 were followed up prospectively in 2009 and 2012, respectively. Famine exposure was classified as unexposed (individuals born in 1962–1978), fetal exposed (individuals born in 1959–1961), child exposed (individuals born in 1949–1958), and adolescent/adult exposed (born in 1931–1948). A logistic regression model was used to assess the relationship between famine exposure and diabetes after adjustment for potential covariates. RESULTS: During a three-year follow-up, the age-adjusted incidence rates of type 2 diabetes were 5.7%, 14.5%, 12.7%, and 17.8% in unexposed, fetal-exposed, child-exposed, and adolescent/adult-exposed groups, respectively (P < 0.01). Relative to the unexposed group, the relative risks (95% confidence interval) for diabetes were 2.15 (1.29–3.60), 1.53 (0.93– 2.51), and 1.65 (0.75–3.63) in the fetal-exposed, child-exposed, and adolescent/adult-exposed groups, after controlling for potential covariates. The interactions between famine exposure and obesity, education level, and family history of diabetes were not observed, except for the urbanization type. Individuals living in rural areas with fetal and childhood famine exposure were at a higher risk of type 2 diabetes, with relative risks of 8.79 (1.82–42.54) and 2.33 (1.17–4.65), respectively. CONCLUSIONS These findings indicate that famine exposure in early life is an independent predictor of type 2 diabetes, particularly in women. Early identification and intervention may help prevent diabetes in later life.

Objective:To screen key genes of renal clear cell carcinoma based on bioinformatics methods, identify possible microRNA (miRNA)-mRNA action axis, and explore the expression of related genes in clear cell renal cell carcinoma tissues and cells.Methods:Gene expression profiles of GSE40435 and GSE71302 datasets were obtained from the Gene Expression Omnibus (GEO) database. TCGA-KIRC datasets were obtained from The Cancer Genome Atlas (TCGA) database. R software was used to identify the differentially expressed mRNA and miRNA, and the functional enrichment analysis was performed. STRING database and Cytoscape software were used to perform the protein interaction analysis. The prognosis-related differentially expressed miRNA was evaluated by the Oncomir database. The potential targeted genes regulated by miRNA were determined by using TargetScan and miRDB targeted gene prediction tools. The tissue samples and clinicopathological features of 34 patients with clear cell renal cell carcinoma in the First Hospital of Shanxi Medical University from June to December 2021 were collected, and normal renal cell line 293T and clear cell renal cell carcinoma cell line 786O were selected. The real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), was used to detect the relative expression of genes; Western blotting and immunohistochemical staining were used to detect the expression levels of the targeted proteins. The dual luciferase reporter gene assay was carried out to verify the targeting relationship between genes.Results:A total of 1 351 differentially expressed mRNA and 50 differentially expressed miRNA were screened and identified. The result of functional enrichment analysis suggested that the fatty acid metabolism pathway and xenobiotic metabolism pathway were suppressed in clear cell renal cell carcinoma, while the apoptosis and immune response pathways were activated. Protein interaction analysis suggested that the signal transduction and protein ubiquitination pathways might play a key role in clear cell renal cell carcinoma. The screening results showed that miRNA-224-5p (miR-224-5p) was most closely associated with clear cell renal cell carcinoma progression and was highly expressed in tumor tissues, and its prognosis-related target gene was NEDD4L. The relative expression of NEDD4L mRNA in clear cell renal cell carcinoma tissues and paraneoplastic tissues were 0.138±0.103 and 1.000±0.026 ( t = 46.23, P < 0.05), and the relative expression of miR-224-5p was 1.000±0.043 and 0.129±0.108 ( t = 45.28, P < 0.05). The differences of NEDD4L mRNA and miR-224-5p expressions in different grades and stages of clear cell renal cell carcinoma tissues were statistically significant (all P < 0.05). The expression of NEDD4L protein was decreased in clear cell renal cell carcinoma. The relative expression of NEDD4L gene in 293T and 786O cells were 1.000±0.125 and 0.210±0.044 ( t = 17.52, P < 0.05); the relative expressions of miR-224-5p gene were 0.209±0.049 and 1.000±0.234 ( t = 10.61, P < 0.05). The relative expressions of NEDD4L mRNA in miRNA mimic group and negative control group were 0.236±0.062 and 1.000±0.024, and the difference was statistically significant ( t = 43.56, P < 0.05). NEDD4L protein expression was reduced in the miRNA mimic group. Dual luciferase reporter gene assay suggested that NEDD4L was a direct target gene of miR-224-5p. Conclusions:In clear cell renal cell carcinoma, miR-224-5p targets and regulates NEDD4L expression, and this mechanism may be related to carcinogenesis and progression of clear cell renal cell carcinoma.