MicroRNA-148b secreted by bovine oviductal extracellular vesicles enhance embryo quality through BPM/TGF-beta pathway

Background Extracellular vesicles (EVs) and their cargoes, including MicroRNAs (miRNAs) play a crucial role in cell-to-cell communication. We previously demonstrated the upregulation of bta-mir-148b in EVs from oviductal fluid of cyclic cows. This miRNA is linked to the TGF-β pathway in the cell proliferation. Our aim was to verify whether miR-148b is taken up by embryos through gymnosis, validate its target genes, and investigate the effect of miR-148b supplementation on early embryo development and quality. Methods Zygotes were cultured in SOF + 0.3% BSA (Control) or supplemented with: 1 μM miR-148b mimics during: D1-D7 (miR148b) or D1-D4 (miR148b-OV: representing miRNA effect in the oviduct) or D4-D7 (miR148b-UT: representing miRNA effect in the uterus) or 1 μM control mimics was used during: D1-D7 (CMimic). Embryos at ≥ 16-cells and D7 blastocysts (BD7) were collected to examine the mRNA abundance of transcripts linked to the TGF-β pathway (TGFBR2, SMAD1, SMAD2, SMAD3, SMAD5, BMPR2, RPS6KB1, POU5F1, NANOG), total cell number (TC), trophectoderm (TE), and inner cell mass (ICM) were also evaluated. One-way ANOVA was used for all analyses. Results We demonstrated that miR-148b can be taken up in both 16-cell embryos and BD7 by gymnosis, and we observed a decrease in SMAD5 mRNA, suggesting it's a potential target of miR-148b. Cleavage and blastocysts rates were not affected in any groups; however, supplementation of miR-148b mimics had a positive effect on TC, TE and ICM, with values of 136.4 ± 1.6, 92.5 ± 0.9, 43.9 ± 1.3 for miR148b and 135.3 ± 1.5, 92.6 ± 1.2, 42.7 ± 0.8, for miR148b-OV group. Furthermore, mRNA transcripts of SMAD1 and SMAD5 were decreased (P ≤ 0.001) in 16-cell embryos and BD7 from miR148b and miR148b-OV groups, while POU5F1 and NANOG were upregulated (P ≤ 0.001) in BD7 and TGFBR2 was only downregulated in 16-cell embryos. pSMAD1/5 levels were higher in the miR148b and miR148b-OV groups. Conclusions Our findings suggest that supplementation of bta-miR-148b mimics during the entire culture period (D1 - D7) or from D1 - D4 improves embryo quality and influences the TGF-β signaling pathway by altering the transcription of genes associated with cellular differentiation and proliferation. This highlights the importance of miR-148b on embryo quality and development.

Circ-USP9X accelerates deep vein thrombosis after fracture by acting as a miR-148b-3p sponge and upregulates SRC kinase signaling inhibitor 1

Abstract Objectives: This study aims to elucidate the role of circUSP9X (Circular RNA Ubiquitin Specific Peptidase 9 X-Linked) in the development of venous thrombosis in the lower extremities. Methods: An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl2) were developed. The expression levels of cir-cUSP9X, microRNA-148b-3p (miR-148b-3p), and SRC Kinase Signaling Inhibitor 1 (SRCIN1) were quantified using quantitative reverse transcription Polymerase Chain Reaction and Western blot analysis. Cell cytotoxicity, viability, apoptosis, and inflammation in HUVECs were assessed via Lactate Dehydrogenase (LDH) assay, MTT assay, flow cytometry, Enzyme-Linked Immunosorbent Assay, and Western blot, respectively. Hematoxylin and Eosin staining were employed for histopathological examination of the venous tissues in the animal model. The interaction between circUSP9X, miR-148b-3p, and SRCIN1 was further explored through dual-luciferase reporter assays and RNA Immunoprecipitation experiments. Results: The present findings reveal a significant upregulation of circUSP9X and SRCIN1 and a concurrent downregulation of miR-148b-3p in DVT cases. Knockdown of circUSP9X or overexpression of miR-148b-3p ameliorated CoCl2-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl2's cytotoxic effects. The effects of manipulating circUSP9X expression were counteracted by the corresponding modulation of miR-148b-3p and SRCIN1 levels. Additionally, circUSP9X knockdown effectively inhibited the formation of DVT in the mouse model. A competitive binding mechanism of circUSP9X for miR-148b-3p, modulating SRCIN1 expression, was identified. Conclusion: circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.

Mechanism of miR⁃148b⁃3p regulating proliferation of keloid derived fibroblasts / 安徽医科大学学报

Objective @#To analyze the effect of miR⁃148b⁃3p on the proliferation of keloid derived fibroblasts. @*Methods @#The expression levels of miR⁃148b⁃3p and SPARC in human keloid derived fibroblasts (HKF) and normal human fibroblasts (NFS) were analyzed by real time PCR. The expression level of SPARC protein was detected by Western blot. The effects of miR⁃148b⁃3p and SPARC on HKF proliferation were analyzed by CCK⁃8 method the luciferase reporter plasmid was constructed. The targeted binding site of miR⁃148b⁃3p and the target gene was analyzed by luciferase reporter gene method.@*Results @#miR⁃148b⁃3p was low expressed in HKF and SPARC was high expressed in HKF. Transfection of miR⁃148b⁃3p in HKF cells could down regulate the expression of SPARC and inhibit cell proliferation. Online analysis software predicted that miR⁃148b⁃3p could target the 3 ′⁃ UTR binding SPARC ; The results of dual luciferase reporter gene further confirmed that miR⁃148b⁃3p could target the 3 ′⁃ UTR of SPARC. Transfection of SPARC eukaryotic expression plasmid into HKF transfected with miR⁃148b⁃3p could counteract the effect of miR⁃148b⁃3p and restore cell proliferation. @*Conclusion @# miR⁃148b⁃3p can inhibit the proliferation of HKF by targeting the 3 ′⁃ UTR of SPARC and inhibiting its expression.

Expression of MiR-148b-3p in Lung Adenocarcinoma and Its Correlation with Prognosis / 中国肺癌杂志

BACKGROUND@#MiR-148b-3p is an important microRNA that has been reported to be significantly related to various types of cancer, but its role in lung adenocarcinoma remains elusive. The purpose of this study is to detect the expression level of miR-148b-3p in lung adenocarcinoma specimens, and to analyze its correlation with the clinicopathological features as well as the prognosis of patients with lung adenocarcinoma.@*METHODS@#A total of 123 tumor specimens from lung adenocarcinoma patients who underwent surgical resection in our department from January 2011 to December 2012 were collected. The expression of miR-148b-3p was detected by quantitative real-time PCR (qRT-PCR), and its correlation with clinicopathological features of patients with lung adenocarcinoma was analyzed. Multivariate Cox proportional hazard models were used to analyze independent predictors of overall survival in patients with lung adenocarcinoma. The overall survival (OS) of patients in miR-148b-3p high expression group and miR-148b-3p low expression group were estimated by means of the Kaplan-Meier method and were compared using the Log-rank test method.@*RESULTS@#Of the 123 patients with lung adenocarcinoma, 71 were in miR-148b-3p high expression group and 52 in low expression group. MiR-148b-3p was significantly associated with tumor grade (P=0.001) and tumor size (P=0.007), but not with age, gender, smoking history, history of alcohol, tumor thrombus, pleural invasion, node status or metastasis status. Multivariate Cox proportional hazard model analysis showed that tumor size (P=0.032), node status (P=0.005) and miR-148b-3p expression level (P=0.047) were significant independent predictors of overall survival of patients with lung adenocarcinoma. Kaplan-Meier survival analysis showed that the overall survival of patients with high expression of miR-148b-3p was significantly better than that of patients with low expression (P=0.010).@*CONCLUSIONS@#MiR-148b-3p was significantly associated with tumor grade and tumor size in lung adenocarcinoma, and served as an independent predictor of overall survival of patients with lung adenocarcinoma. The overall survival of patients with high expression level of miR-148b-3p was significantly better than that of patients with low expression.

Chemotherapeutic drugs affect methylation of ER-α in breast cancer cells by down-regulating miR-148b / 中华内分泌外科杂志

Objective To investigate the effects of chemotherapeutic drugs on ER-α expression and methylation in breast cancer cells.Methods Human breast cancer cells MCF-7(ER+,Luminal A) were induced by paclitaxel(PTX) and epirubicin(EPI) for more than 6 months,with an incremental dose,respectively.The expression and methylation status of ER-α in MCF-7 cells were detected before and after drug treatment.miRNAs with consistent expression changes in MCF-7 cells after two drugs' treatment were screened by microarray,and verified by quantitative PCR (qPCR).Targets of the most significantly down-regulated miRNA were analyzed by bioinformatics.miRNA inhibitor was transfected into MCF-7 cells,miRNA mimic was transfected into MCF-7/PTX and MCF-7/EPI cells,then ER-α and DNA methyltransferase 1 (DNMT1) expression were detected by Western blot,and ER-α methylation was detected by quantitative methylation-specific PCR (qMSP).Results PTX resistant MCF-7/PTX cell line and EPI resistant MCF-7/EPI cell line were established.Both drug treatments caused a decrease in ER-α protein expression and an increase in methylation levels,with up-regulation of DNMT1 and his tone deacetylase 1 (HDAC 1) expression.miRNAs with consistent expression changes in MCF-7 cells after drug treatments were screened and verified by qPCR,the most significant down-regulation among which was miR-148b.Bioinformatics analysis,and further confirmed by luciferase reporter gene assay (Luciferas) that DNMT1 was a direct target of miR-148b.miR-148b inhibitor induced decreased expression of ER-α and increased methylation level in MCF-7 cells,accompanied by increased expression of DNMT1;whereas miR-148b mimic caused an increased expression of ER-α and decreased methylation level in MCF-7/PTX and MCF-7/EPI cells,with a decreased expression of DNMT1.Conclusion Chemotherapeutic drugs (represented by PTX and EPI) induce aberrant miRNA expression in breast cancer MCF-7 cells,and down-regulate miR-148b further to attenuate the inhibition of DNMT1 expression,which promote,hypermethylation and down-regulation of ER-α.

p53 inhibits the proliferation of lung cancer cell PC-9 by regulating miR-148b / 实用医学杂志

Objective To explore the function of p53 on regulating the expression of miR-148b in lung cancer cell line PC-9 and its corresponding molecular mechanism and the impact on cell proliferation. Methods Transient transfection of p53 eukaryotic expressing plasmids into lung cancer cell line PC-9 was performed to establish a cell model over-expressing p53. RT-PCR was used to explicit the impact of p53 on the expression of miR-148b. A reporter vector containing miR-148b promoter was used to investigate the function of p53 on regulating the transcription of miR-148b. Low-expressing miR-148b by transfecting its specific inhibitors , a CCK-8 assay was performed to explore the influence of miR-148b on the lung cancer cell proliferation inhibited by p53. Results Over-expression of p53 promoted miR-148b expression in lung cancer cell line PC-9. P53 could increase the luciferase activity driven by miR-148b promoters. Knockdown of miR-148b attenuated the impact of p53 on inhibiting the proliferation of PC-9 cells. Conclusion P53 inhibits the proliferation of lung cancer cell line PC-9 partially depending on miR-148b.