Quality and Safety Considerations for Therapeutic Products Based on Extracellular Vesicles.

Extracellular vesicles (EVs) serve as an intrinsic system for delivering functional molecules within our body, playing significant roles in diverse physiological phenomena and diseases. Both native and engineered EVs are currently the subject of extensive research as promising therapeutics and drug delivery systems, primarily due to their remarkable attributes, such as targeting capabilities, biocompatibility, and low immunogenicity and mutagenicity. Nevertheless, their clinical application is still a long way off owing to multiple limitations. In this context, the Science Board of the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan has conducted a comprehensive assessment to identify the current issues related to the quality and safety of EV-based therapeutic products. Furthermore, we have presented several examples of the state-of-the-art methodologies employed in EV manufacturing, along with guidelines for critical processes, such as production, purification, characterization, quality evaluation and control, safety assessment, and clinical development and evaluation of EV-based therapeutics. These endeavors aim to facilitate the clinical application of EVs and pave the way for their transformative impact in healthcare.

Case report: Rare heterozygous variant in the NR5A1 gene causing 46,XY complete gonadal dysgenesis with a non-communicating rudimentary uterus.

The nuclear receptor subfamily 5 group A member 1 (NR5A1) gene encodes NR5A1, also known as steroidogenic factor 1, a crucial transcriptional factor regulating adrenal and gonadal development and function. Although pathogenic variants in NR5A1 are known to cause a spectrum of disorders of sex development (DSD), individuals with 46,XY DSD with fully female internal and external genitalia are relatively rare. Herein, we present the case of a patient with 46,XY complete gonadal dysgenesis (CGD) who had a non-communicating rudimentary uterus due to a c.132_134del (p.Asn44del) heterozygous in-frame-deletion in NR5A1 that was diagnosed while treating a pelvic mass in which gynecological malignancy could not be disregarded. Unlike two previous cases with the p.Asn44del variant, this case presented with CGD, a severe DSD phenotype, and we found that the oligogenic inheritance of DSD-causative genes such as SRY, DHX37, SLC26A8, and CFTR may have affected the severity of the clinical phenotype.

Novel AI Combining CNN and SVM to Predict Colorectal Cancer Prognosis and Mutational Signatures from HE Images.

Reducing recurrence following radical resection of colon cancer without over- or under-treatment remains a challenge. Postoperative adjuvant chemotherapy (Adj) is currently administered based solely on pathological tumor, node, and metastasis (TNM) stage. However, prognosis can vary significantly among patients with the same disease stage. Therefore, novel classification systems in addition to the TNM are necessary to inform decision-making regarding postoperative treatment strategies, especially stage II and III disease, and to minimize overtreatment and undertreatment with Adj. We developed a prognostic prediction system for colorectal cancer by using a combined convolutional neural network (CNN) and support vector machine (SVM) approach to extract features from hematoxyling and eosin staining (HE) images. We combined the TNM and our AI-based classification system into a TNM-AI (mTNM-AI) classification system with high discriminative power for recurrence-free survival (RFS). Furthermore, the cancer cell population recognized by this system as low risk of recurrence exhibited the mutational signature SBS87 as a genetic phenotype. The novel AI-based classification system developed here is expected to play an important role in prognostic prediction and personalized treatment selection in oncology.

Repression of SMAD3 by STAT3 and c-Ski induces conventional dendritic cell differentiation.

A pleiotropic immunoregulatory cytokine, TGF-ß, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.

Analysis of Carcinogenic Involvement of MicroRNA Pattern in Peripheral Non-Cancerous Tissues and Chronic Viral Liver Injury.

Risk factors for hepatocarcinogenesis include chronic inflammation due to viral infection, liver fibrosis, and aging. In this study, we separated carcinogenic and non-carcinogenic cases due to hepatitis C virus (HCV) infection, aiming to comprehensively analyze miRNA expression in liver tissues by age, and identify factors that contribute to carcinogenesis. Total RNA was extracted from 360 chronic hepatitis C (CH), 43 HCV infected hepatocellular carcinoma (HCC), and surrounding non-tumor (SNT) tissues. MicroRNA (miRNA) expression patterns were analyzed using microarray. Using machine learning, we extracted characteristic miRNA expression patterns for each disease and age. There were no age-dependent changes in miRNA expression in the disease-specific comparisons; however, miRNA expression differed among the age groups of 50, 60, and 70 years of age between CH and SNT. The expression of miRNA was different between SNT and HCC only in patients in their 70s. Of the 55 miRNAs with significant differences in expression between CH and SNT, 34 miRNAs showed significant differences in expression even in the degree of liver fibrosis. The observation that miRNAs involved in hepatocarcinogenesis differ at different ages suggests that the mechanisms of carcinogenesis differ by age group as well. We also found that many miRNAs whose expression did not affect liver fibrosis were involved in carcinogenesis. These findings are expected to define biomarkers for detection of HCC at early stage, and develop novel therapeutic targets for HCC.

Aberrant regulation of serine metabolism drives extracellular vesicle release and cancer progression.

Cancer cells secrete extracellular vesicles (EVs) to regulate cells in the tumor microenvironment to benefit their own growth and survive in the patient's body. Although emerging evidence has demonstrated the molecular mechanisms of EV release, regulating cancer-specific EV secretion remains challenging. In this study, we applied a microRNA library to reveal the universal mechanisms of EV secretion from cancer cells. Here, we identified miR-891b and its direct target gene, phosphoserine aminotransferase 1 (PSAT1), which promotes EV secretion through the serine-ceramide synthesis pathway. Inhibition of PSAT1 affected EV secretion in multiple types of cancer, suggesting that the miR-891b/PSAT1 axis shares a common mechanism of EV secretion from cancer cells. Interestingly, aberrant PSAT1 expression also regulated cancer metastasis via EV secretion. Our data link the PSAT1-controlled EV secretion mechanism and cancer metastasis and show the potential of this mechanism as a therapeutic target in multiple types of cancer.

Liver-specific DICER1 syndrome model mice develop cystic liver tumors with defective primary cilia.

DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome. © 2024 The Pathological Society of Great Britain and Ireland.

Preoperative evaluation of visceral pleural invasion in peripheral lung cancer utilizing deep learning technology.

PURPOSE: This study aimed to assess the efficiency of artificial intelligence (AI) in the detection of visceral pleural invasion (VPI) of lung cancer using high-resolution computed tomography (HRCT) images, which is challenging for experts because of its significance in T-classification and lymph node metastasis prediction. METHODS: This retrospective analysis was conducted on preoperative HRCT images of 472 patients with stage I non-small cell lung cancer (NSCLC), focusing on lesions adjacent to the pleura to predict VPI. YOLOv4.0 was utilized for tumor localization, and EfficientNetv2 was applied for VPI prediction with HRCT images meticulously annotated for AI model training and validation. RESULTS: Of the 472 lung cancer cases (500 CT images) studied, the AI algorithm successfully identified tumors, with YOLOv4.0 accurately localizing tumors in 98% of the test images. In the EfficientNet v2-M analysis, the receiver operating characteristic curve exhibited an area under the curve of 0.78. It demonstrated powerful diagnostic performance with a sensitivity, specificity, and precision of 76.4% in VPI prediction. CONCLUSION: AI is a promising tool for improving the diagnostic accuracy of VPI for NSCLC. Furthermore, incorporating AI into the diagnostic workflow is advocated because of its potential to improve the accuracy of preoperative diagnosis and patient outcomes in NSCLC.

A Cerebral Embolism Caused by a Malignant Peripheral Nerve Sheath Tumor in a Patient with Neurofibromatosis Type 1.

A 31-year-old man with neurofibromatosis type 1 (NF-1) had undergone resection of a malignant peripheral nerve sheath tumor (MPNST) on the buttock 3 months previously. He subsequently underwent mechanical thrombectomy for a hyperacute left middle cerebral artery embolism. Histopathologically, the emboli comprised neurofilament-positive pleomorphic tumor cells with geographic necrosis and conspicuous mitosis and were identified as MPNST. The patient died of respiratory failure due to lung MPNST metastasis on day 15 of hospitalization. To our knowledge, this is the first report of a spontaneous cerebral embolism due to MPNST in a NF-1 patient.

Efficacy of interstitial photodynamic therapy using talaporfin sodium and a semiconductor laser for a mouse allograft glioma model.

To investigate the therapeutic potential of photodynamic therapy (PDT) for malignant gliomas arising in unresectable sites, we investigated the effect of tumor tissue damage by interstitial PDT (i-PDT) using talaporfin sodium (TPS) in a mouse glioma model in which C6 glioma cells were implanted subcutaneously. A kinetic study of TPS demonstrated that a dose of 10 mg/kg and 90 min after administration was appropriate dose and timing for i-PDT. Performing i-PDT using a small-diameter plastic optical fiber demonstrated that an irradiation energy density of 100 J/cm2 or higher was required to achieve therapeutic effects over the entire tumor tissue. The tissue damage induced apoptosis in the area close to the light source, whereas vascular effects, such as fibrin thrombus formation occurred in the area slightly distant from the light source. Furthermore, when irradiating at the same energy density, irradiation at a lower power density for a longer period of time was more effective than irradiation at a higher power density for a shorter time. When performing i-PDT, it is important to consider the rate of delivery of the irradiation light into the tumor tissue and to set irradiation conditions that achieve an optimal balance between cytotoxic and vascular effects.

Pediatric hypertrophic cardiomyopathy caused by a novel TNNI3 variant.

TNNI3 is a gene that causes hypertrophic cardiomyopathy (HCM). A 14-year-old girl who was diagnosed with nonobstructive HCM presented with cardiopulmonary arrest due to ventricular fibrillation. Genetic testing revealed a novel de novo heterozygous missense variant in TNNI3, NM_000363.5:c.583A>T (p.Ile195Phe), which was determined to be the pathogenic variant. The patient exhibited progressive myocardial fibrosis, left ventricular remodeling, and life-threatening arrhythmias. Genetic testing within families is useful for risk stratification in pediatric HCM patients.

Plasma extracellular vesicle microRNAs reflecting the therapeutic effect of the CBP/ß-catenin inhibitor PRI-724 in patients with liver cirrhosis.

There is an unmet need for antifibrotic therapies to prevent the progression of liver cirrhosis. Previously, we conducted an exploratory trial to assess the safety and antifibrotic efficacy of PRI-724, a selective CBP/ß-catenin inhibitor, in patients with liver cirrhosis. PRI-724 was well tolerated and exerted a potential antifibrotic effect. Here, we investigated whether the profiles of circulating microRNAs packaged in extracellular vesicles (EV-miRNAs) are associated with responses to liver fibrosis treatments. Eighteen patients who received PRI-724 for 12 weeks in a phase 1/2a study were classified as responders (n = 10) or non-responders (n = 8) based on changes in liver stiffness. Plasma samples were obtained before and after PRI-724 administration and the levels of EV-miRNAs were analyzed. Three miRNAs (miR-6510-5p, miR-6772-5p, and miR-4261) were identified as predictors of response or non-response to PRI-724, and the levels of three other miRNAs (miR-939-3p, miR-887-3p, and miR-7112-5p) correlated with the efficacy of treatment. Expression of miR-887-3p was detected in hepatocytes and was decreased significantly in liver tissue following PRI-724 treatment. In addition, transfection of a miR-887-3p mimic activated hepatic stellate cells. Thus, decreases in the miR-887-3p level in blood may reflect recovery from liver fibroses in patients with liver cirrhosis treated with PRI-724, although further validation studies are warranted to confirm this.

miR­146a­5p and miR­191­5p as novel diagnostic marker candidates for ovarian clear cell carcinoma.

Ovarian cancer is a malignant gynecologic disease rarely diagnosed in the early stages. Among the various types of ovarian cancer, clear cell carcinoma has a poor prognosis due to its malignant potential. MicroRNAs (miRNAs/miRs) regulate gene expression in cells by suppressing the translation of target genes or by degrading the target mRNA. miRNAs are also secreted from the cells in the blood, binding to proteins or lipids and assisting in cell-cell communication. Therefore, serum miRNAs may be considered potential diagnostic biomarkers for ovarian cancer. The present study investigated and identified specific miRNAs associated with ovarian clear cell carcinoma and compared them to those in ovarian endometrioma samples and healthy controls. CA125, an ovarian tumor marker, did not differ between patients with ovarian clear cell carcinoma, endometriosis or healthy controls. Subsequently, four miRNAs (miR-146a-5p, miR-191-5p, miR-484 and miR-574-3p) were analyzed. The expression levels of miR-146a-5p and miR-191-5p were significantly increased in the serum samples from patients with ovarian clear cell carcinoma compared with those in the healthy controls, but there was no significant difference compared with in patients with endometriosis. Furthermore, the bioinformatics analysis showed that CCND2 and NOTCH2 were the candidate target genes of miR-146a-5p and miR-191-5p. In conclusion, the results of the present study demonstrated that miR-146a-5p and miR-191-5p may be useful as early and non-invasive diagnostic tools in ovarian clear cell carcinoma. These miRNAs can help in distinguishing between ovarian clear cell carcinoma and ovarian endometrioma. To the best of our knowledge, no previous studies have screened any candidates specifically for ovarian clear cell carcinoma.

Characterization of circulating miRNAs in the treatment of primary liver tumors.

BACKGROUND AND AIM: Circulating micro RNAs (miRNAs) indicate clinical pathologies such as inflammation and carcinogenesis. In this study, we aimed to investigate whether miRNA expression level patterns in could be used to diagnose hepatocellular carcinoma (HCC) and biliary tract cancer (BTC), and the relationship miRNA expression patterns and cancer etiology. METHODS: Patients with HCC and BTC with indications for surgery were selected for the study. Total RNA was extracted from the extracellular vesicle (EV)-rich fraction of the serum and analyzed using Toray miRNA microarray. Samples were divided into two cohorts in order of collection, the first 85 HCC were analyzed using a microarray based on miRBase ver.2.0 (hereafter v20 cohort), and the second 177 HCC and 43 BTC were analyzed using a microarray based on miRBase ver.21 (hereafter v21 cohort). RESULTS: Using miRNA expression patterns, we found that HCC and BTC could be identified with an area under curve (AUC) 0.754 (v21 cohort). Patients with anti-hepatitis C virus (HCV) treatment (SVR-HCC) and without antiviral treatment (HCV-HCC) could be distinguished by an AUC 0.811 (v20 cohort) and AUC 0.798 (v21 cohort), respectively. CONCLUSIONS: In this study, we could diagnose primary hepatic malignant tumor using miRNA expression patterns. Moreover, the difference of miRNA expression in SVR-HCC and HCV-HCC can be important information for enclosing cases that are prone to carcinogenesis after being cured with antiviral agents, but also for uncovering the mechanism for some carcinogenic potential remains even after persistent virus infection has disappeared.

Suppression of antitumor cytokine IL­24 by PRG4 and PAI­1 may promote myxoid liposarcoma cell survival.

Suppression of the antitumor cytokine interleukin-24 (IL-24) is critical for the survival of myxoid liposarcoma (MLS) cells. It has been previously demonstrated by the authors that an MLS-specific chimeric oncoprotein, translocated in liposarcoma-CCAAT/enhancer-binding protein homologous protein (TLS-CHOP), supresses IL24 mRNA expression via induction of proteoglycan 4 (PRG4) to sustain MLS cell proliferation. However, IL-24 has also been revealed to be suppressed by the ubiquitin-proteasome system in human ovarian and lung cancer cells. Therefore, the aim of the present study was to elucidate the mechanism of IL-24 suppression in MLS cells. The results revealed that the proteasome inhibitor, MG-132, induced cell death in MLS cells in vitro; this effect was reduced following IL-24 knockdown. This indicated that proteasomal degradation of IL-24 may be an important process for MLS cell survival. In addition, it was also previously revealed by the authors that knockdown of plasminogen activator inhibitor-1 (PAI-1), a TLS-CHOP downstream molecule, suppressed the growth of MLS cells, thus instigating the investigation of the effect of PAI-1 on IL-24 expression in MLS cells. Double knockdown of PAI-1 and IL-24 negated the growth-suppressive effect of PAI-1 single knockdown in MLS cells. Interestingly, PAI-1 single knockdown did not increase the mRNA expression of IL24, but it did increase the protein abundance of IL-24, indicating that PAI-1 suppressed IL-24 expression by promoting its proteasomal degradation. Moreover, treatment of MLS cells with a PAI-1 inhibitor, TM5275, induced IL-24 protein expression and apoptosis. Collectively, the results of the present as well as previous studies indicated that IL-24 expression may be suppressed at the transcriptional level by PRG4 and at the protein level by PAI-1 in MLS cells. Accordingly, PAI-1 may represent an effective therapeutic target for MLS treatment.

Recurrence prediction in clear cell renal cell carcinoma using machine learning of quantitative nuclear features.

The recurrence of non-metastatic renal cell carcinoma (RCC) may occur early or late after surgery. This study aimed to develop a recurrence prediction machine learning model based on quantitative nuclear morphologic features of clear cell RCC (ccRCC). We investigated 131 ccRCC patients who underwent nephrectomy (T1-3N0M0). Forty had recurrence within 5 years and 22 between 5 and 10 years; thirty-seven were recurrence-free during 5-10 years and 32 were for more than 10 years. We extracted nuclear features from regions of interest (ROIs) using a digital pathology technique and used them to train 5- and 10-year Support Vector Machine models for recurrence prediction. The models predicted recurrence at 5/10 years after surgery with accuracies of 86.4%/74.1% for each ROI and 100%/100% for each case, respectively. By combining the two models, the accuracy of the recurrence prediction within 5 years was 100%. However, recurrence between 5 and 10 years was correctly predicted for only 5 of the 12 test cases. The machine learning models showed good accuracy for recurrence prediction within 5 years after surgery and may be useful for the design of follow-up protocols and patient selection for adjuvant therapy.

Differential toxicity and localization of arginine-rich C9ORF72 dipeptide repeat proteins depend on de-clustering of positive charges.

Arginine-rich dipeptide repeat proteins (R-DPRs), poly(PR) and poly(GR), translated from the hexanucleotide repeat expansion in the amyotrophic lateral sclerosis (ALS)-causative C9ORF72 gene, contribute significantly to pathogenesis of ALS. Although both R-DPRs share many similarities, there are critical differences in their subcellular localization, phase separation, and toxicity mechanisms. We analyzed localization, protein-protein interactions, and phase separation of R-DPR variants and found that sufficient segregation of arginine charges is necessary for nucleolar distribution. Proline not only efficiently separated the charges, but also allowed for weak, but highly multivalent binding. In contrast, because of its high flexibility, glycine cannot fully separate the charges, and poly(GR) behaves similarly to the contiguous arginines, being trapped in the cytoplasm. We conclude that the amino acid that spaces the arginine charges determines the strength and multivalency of the binding, leading to differences in localization and toxicity mechanisms.

Smooth muscle differentiation of coronary intima in autopsy tissues after sirolimus-eluting stent implantation.

BACKGROUND: In coronary atherosclerotic disease, the proliferation of intimal smooth muscle cells (SMCs) is regarded as beneficial with respect to stable and unstable plaques, but is thought detrimental in discussions on coronary stent restenosis. To resolve this discrepancy, we focused on the quality, not quantity, of intimal SMCs in coronary atherosclerotic disease. METHODS: Autopsied coronary artery specimens from seven patients implanted with bare metal stents (BMS), three with paclitaxel-eluting stents (PES), and 10 with sirolimus (rapamycin)-eluting stents (SES) were immunostained for SMC markers. Cultured human coronary artery SMCs were also treated with sirolimus and paclitaxel. RESULTS: Intimal SMC differentiation, estimated by the ratio of h-caldesmon+ cells to α-smooth muscle actin+ (α-SMA+) cells, was significantly increased whereas dedifferentiation, estimated from the ratio of fibroblast activation protein alpha (FAPα)+ cells to α-SMA+ cells, was significantly decreased, in tissues of SES compared with BMS cases. No difference in the degree of differentiation was found between PES and BMS cases or between the three groups in nonstented arteries used as controls. Correlation analyses for each field of view revealed a significant positive correlation between h-caldesmon and calponin staining but significant negative correlations with FAPα staining in α-SMA+ cells. Cultured SMCs were shorter (dedifferentiated) and showed an increased FAPα/α-SMA protein when treated with paclitaxel, whereas they became elongated (differentiated) and showed increased calponin/α-SMA proteins with sirolimus. CONCLUSIONS: The SMCs of the coronary intima may differentiate after SES implantation. SMC differentiation may explain both the plaque stabilization and reduced risk of reintervention associated with SES.

Development of multiple AI pipelines that predict neoadjuvant chemotherapy response of breast cancer using H&E-stained tissues.

In recent years, the treatment of breast cancer has advanced dramatically and neoadjuvant chemotherapy (NAC) has become a common treatment method, especially for locally advanced breast cancer. However, other than the subtype of breast cancer, no clear factor indicating sensitivity to NAC has been identified. In this study, we attempted to use artificial intelligence (AI) to predict the effect of preoperative chemotherapy from hematoxylin and eosin images of pathological tissue obtained from needle biopsies prior to chemotherapy. Application of AI to pathological images typically uses a single machine-learning model such as support vector machines (SVMs) or deep convolutional neural networks (CNNs). However, cancer tissues are extremely diverse and learning with a realistic number of cases limits the prediction accuracy of a single model. In this study, we propose a novel pipeline system that uses three independent models each focusing on different characteristics of cancer atypia. Our system uses a CNN model to learn structural atypia from image patches and SVM and random forest models to learn nuclear atypia from fine-grained nuclear features extracted by image analysis methods. It was able to predict the NAC response with 95.15% accuracy on a test set of 103 unseen cases. We believe that this AI pipeline system will contribute to the adoption of personalized medicine in NAC therapy for breast cancer.

MiR-34a induces myofibroblast differentiation from renal fibroblasts.

BACKGROUND: Renal fibrosis is the common outcome of progressive kidney diseases. To avoid dialysis, the molecular mechanism of renal fibrosis must be explored further. MicroRNAs play key roles in renal fibrosis. MiR-34a is a transcriptional target of p53, which regulates the cell cycle and apoptosis. Previous studies demonstrated that miR-34a promotes renal fibrosis. However, the distinct roles of miR-34a in renal fibrosis have not been fully elucidated. Here, we identified the roles of miR-34a in renal fibrosis. METHOD: We first analyzed p53 and miR-34a expression in kidney tissues in s UUO (unilateral ureteral obstruction) mouse model. Then, to confirm the effects of miR-34a in vitro, we transfected a miR-34a mimic into a kidney fibroblast cell line (NRK-49F) and analyzed. RESULTS: We found that the expression of p53 and miR-34a was upregulated after UUO. Furthermore, after transfection of the miR-34a mimic into kidney fibroblasts, the expression of α-SMA was upregulated dramatically. In addition, α-SMA upregulation was greater upon transfection of the miR-34a mimic than upon treatment with TGF-ß1. Moreover, high expression of Acta2 was maintained despite sufficient removal of the miR-34a mimic by changing the medium 4 times during the 9-day culture. After transfection of the miR-34a mimic into kidney fibroblasts, we did not detect phospho-SMAD2/3 by immunoblotting analysis. CONCLUSION: Our study revealed that miR-34a induces myofibroblast differentiation from renal fibroblasts. Moreover, the miR-34a-induced upregulation of α-SMA was independent of the TGF-ß/SMAD signaling pathway. In conclusion, our study indicated that the p53/miR-34a axis promotes the development of renal fibrosis.