Molecular and Cellular Biochemistry最新文献

Methyltransferase like 3 (METTL3) has been reported to promote tumorigenesis of multiple myeloma (MM), however, the molecular mechanism still needs further research. The N6-methyladenosine (m6A) level in tissues or cells was measured by m6A kit and dot blot assay. The mRNA and protein expression were detected by quantitative real-time PCR (RT-qPCR) and Western blot, respectively. The cell counting kit-8 and colony formation assay were used to detect the cell proliferation. Coimmunoprecipitation (Co-IP) experiment verified the binding of two proteins. The luciferase reporter experiment demonstrated the targeted binding of miR-182-5p and CaMKII inhibitor 1 (CAMK2N1). More importantly, tumor growth was measured in xenograft mice. Our data showed that the expression of METTL3 was significantly increased in MM patients' samples and MM cells. METTL3 overexpression promoted MM cells proliferation, and METTL3 knockdown inhibited MM cells proliferation. Mechanically, METTL3-dependent m6A participated in DiGeorge syndrome critical region 8 (DGCR8)-mediated maturation of pri-miR-182. Upregulation of miR-182-5p further enhanced the promoting proliferation effect of METTL3 overexpression on MM cells. Moreover, the luciferase reporter gene experiment proved that miR-182-5p targetedly regulated CAMK2N1 expression. Xenograft tumor in nude mice further verified that METTL3 promoted MM tumor growth through miR-182/CAMK2N1 signal axis. In summary, the METTL3/miR-182-5p/CAMK2N1 axis plays an important role in MM tumorigenesis, which may provide a new target for MM therapy.

Bone and cartilage diseases are often associated with trauma and senescence, manifested as pain and limited mobility. The repair of bone and cartilage lesion by mesenchymal stem cells is regulated by various transcription factors. WW domain-containing protein 1 (WWP1) and WW domain-containing protein 2 (WWP2) are named for WW domain which recognizes PPXY (phono Ser Pro and Pro Arg) motifs of substrate. WWP1and WWP2 are prominent components of the homologous to the E6-AP carboxyl terminus (HECT) subfamily, a group of the ubiquitin ligase. Recently, some studies have found that WWP1 and WWP2 play an important role in the pathogenesis of bone and cartilage diseases and regulate the level and the transactivation of various transcription factors through ubiquitination. Therefore, this review summarizes the distribution and effects of WWP1 and WWP2 in the development of bone and cartilage, discusses the potential mechanism and therapeutic drugs in bone and cartilage diseases such as osteoarthritis, fracture, and osteoporosis.

Several commercially available triorganotin compounds were previously found to function as agonist ligands for nuclear retinoid X receptor (RXR) molecules. Triphenyltin isoselenocyanate (TPT-NCSe), a novel selenium atom containing a derivative of triorganotin origin, was found to represent a new cognate bioactive ligand for RXRs. TPT-NCSe displayed a concentration- and time-dependent decrease in the cell viability in both human breast carcinoma MCF-7 (estrogen receptor positive) and MDA‑MB‑231 (triple negative) cell lines. Reactive oxygen species levels generated in response to TPT-NCSe were significantly higher in both carcinoma cell lines treated with TPT-NCSe when compared to mock-treated samples. Treatment with 500 nM TPT-NCSe caused a decrease in SOD1 and increased SOD2 mRNA in MCF-7 cells. The levels of SOD2 mRNA were more increased following the treatment with TPT-NCSe along with 1 μM all-trans retinoic acid (AtRA) in MCF-7 cells. An increased superoxide dismutase SOD1 and SOD2 mRNA levels were also detected in combination treatment of 500 nM TPT-NCSe and 1 μM AtRA in TPT-NCSe-treated MDA-MB-231 cells. The data have also shown that TPT-NCSe induces apoptosis via a caspase cascade triggered by the mitochondrial apoptotic pathway. TPT-NCSe modulates the expression levels of apoptosis‑related proteins, Annexin A5, Bcl‑2 and BAX family proteins, and finally, it enhances the expression levels of its cognate nuclear receptor subtypes RXRalpha and RXRbeta.

Subdural hematoma (SDH) drains into the extracranial lymphatic system through the meningeal lymphatic vessels (mLVs) but the formation of SDH impairs mLVs. Because vitamin D (Vit D) can protect the endothelial cells, we hypothesized that Vit D may enhance the SDH clearance. SDH was induced in Sprague-Dawley rats and treated with Vit D or vehicle. Hematoma volume in each group was measured by H&E staining and hemoglobin quantification. Evans blue (EB) quantification and red blood cells injection were used to evaluated the drainage of mLVs. Western blot analysis and immunofluorescence were conducted to assess the expression of lymphatic protein markers. We also examined the inflammatory factors levels in subdural space by ELISA. Vit D treatment significantly reduced SDH volume and improved the drainage of SDH to cervical lymph nodes. The structure of mLVs in SDH rats were protected by Vit D, and the expressions of LYVE1, PROX1, FOXC2, and VE-cadherin were increased after Vit D treatment. The TNF-α, IL-6, and IL-8 levels were reduced in Vit D group. In vitro, Vit D also increased the VE-cadherin expression levels under inflammation. Vit D protects the structure of mLVs and enhances the absorption of SDH, partly by the anti-inflammatory effect of Vit D.

The epidermal growth factor receptor 1 (EGFR) plays a crucial role in the progression of various malignant tumors and is considered a potential target for treating triple-negative breast cancer (TNBC). However, the effectiveness of representative tyrosine kinase inhibitors (TKIs) used in EGFR-targeted therapy is limited in TNBC patients. In our study, we observed that the TNBC cell lines MDA-MB-231 and MDA-MB-468 exhibited resistance to Gefitinib. Treatment with Gefitinib caused an upregulation of Fascin-1 (FSCN1) protein expression and a downregulation of miR-221-3p in these cell lines. However, sensitivity to Gefitinib was significantly improved in both cell lines with either inhibition of FSCN1 expression or overexpression of miR-221-3p. Our luciferase reporter assay confirmed that FSCN1 is a target of miR-221-3p. Moreover, Gefitinib treatment resulted in an upregulation of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in MDA-MB-231 cells. Using Stattic, a small-molecule inhibitor of STAT3, we observed a significant enhancement in the inhibitory effect of Gefitinib on the growth, migration, and invasion of MDA-MB-231 cells. Additionally, Stattic treatment upregulated miR-221-3p expression and downregulated FSCN1 mRNA and protein expression. A strong positive correlation was noted between the expression of STAT3 and FSCN1 in breast cancer tissues. Furthermore, patients with high expression levels of both STAT3 and FSCN1 had a worse prognosis. Our findings suggest that elevated FSCN1 expression is linked to primary resistance to EGFR TKIs in TNBC. Moreover, we propose that STAT3 regulates the expression of miR-221-3p/FSCN1 and therefore modulates resistance to EGFR TKI therapy in TNBC. Combining EGFR TKI therapy with inhibition of FSCN1 or STAT3 may offer a promising new therapeutic option for TNBC.

Polypyrimidine tract-binding protein 1 (PTBP1) is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which plays a key role in alternative splicing of precursor mRNA and RNA metabolism. PTBP1 is universally expressed in various tissues and binds to multiple downstream transcripts to interfere with physiological and pathological processes such as the tumor growth, body metabolism, cardiovascular homeostasis, and central nervous system damage, showing great prospects in many fields. The function of PTBP1 involves the regulation and interaction of various upstream molecules, including circular RNAs (circRNAs), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). These regulatory systems are inseparable from the development and treatment of diseases. Here, we review the latest knowledge regarding the structure and molecular functions of PTBP1 and summarize its functions and mechanisms of PTBP1 in various diseases, including controversial studies. Furthermore, we recommend future studies on PTBP1 and discuss the prospects of targeting PTBP1 in new clinical therapeutic approaches.

The neutrophil-to-platelet ratio (NPR) is considered to be an indicator of inflammatory status. The value of the NPR in predicting in-hospital adverse events (AEs) and long-term prognosis after percutaneous coronary intervention (PCI) in coronary artery disease (CAD) patients has not yet been reported. Meanwhile, the mechanisms behind its predictive value for long-term prognosis remain unreported as well. The study retrospectively enrolled 7284 consecutive patients with CAD undergoing PCI from January 2012 to December 2018. Multivariable logistic regression analysis, multivariable Cox regression analysis, Kaplan‒Meier (KM) curve analysis, restricted cubic spline (RCS) curve analysis, and sensitivity analysis were used in the study. All-cause death was the endpoint of the study. According to the median value of the NPR, the patients were divided into two groups: the high group (NPR ≥ 0.02, n = 3736) and the low group (NPR < 0.02, n = 3548). Multivariate logistic regression analysis demonstrated that a high NPR was a risk factor for in-hospital AEs [odds ratio (OR) = 1.602, 95% CI 1.347-1.909, p = 0.001]. During a mean follow-up period of 3.01 ± 1.49 years, the multivariate Cox regression analysis showed that a high NPR affected the long-term prognosis of patients (HR 1.22, 95% CI 1.03-1.45, p = 0.025) and cardiac death (HR 1.49, 95% CI 1.14-1.95, p = 0.003). The subgroup analysis showed that the NPR was affected by age and sex. The mediation analysis identified that the effect of the NPR on long-term outcomes is partially mediated by serum creatinine (Scr) and triglycerides. The NPR may be a convenient indicator of in-hospital AEs and poor long-term and cardiac outcomes in CAD patients. It might have impacted prognosis through effects on kidney function and lipid metabolism.

The alteration of inflammatory phenotype by macrophage polarization plays an important role in diabetic wound repair. Apigenin has been reported to be anti-inflammatory and promote tissue repair; however, whether it regulates macrophage polarization to participate in diabetic wound repair remains to be investigated. We found that apigenin promoted miR-21 expression in LPS-stimulated RAW264.7 cells, inhibited cellular M1-type factor TNF-α and IL-1β secretion and increased M2-type factor IL-10 and TGF-β secretion, and accelerated macrophage conversion from M1 type to M2 type, whereas this protective effect of apigenin was counteracted by a miR-21 inhibitor. Moreover, we established a macrophage-HUVECs cell in vitro co-culture system and found that apigenin accelerated the migration, proliferation, and VEGF secretion of HUVECs by promoting macrophage miR-21 expression. Further, mechanistic studies revealed that this was mediated by the TLR4/Myd88/NF-κB axis. In in vivo study, diabetic mice had significantly delayed wound healing compared to non-diabetic mice, accelerated wound healing in apigenin-treated diabetic mice, and decreased M1-type macrophages and increased M2-type macrophages in wound tissues.

Phenotypic change of vascular smooth muscle cells (VSMCs) is the main contributor of vascular pathological remodeling in atherosclerosis. The endoplasmic reticulum (ER) is critical for maintaining VSMC function through elimination of misfolded proteins that impair VSMC cellular function. ER-associated degradation (ERAD) is an ER-mediated process that controls protein quality by clearing misfolded proteins. One of the critical regulators of ERAD is HRD1, which also plays a vital role in lipid metabolism. However, the function of HRD1 in VSMCs of atherosclerotic vessels remains poorly understood. The level of HRD1 expression was analyzed in aortic tissues of mice fed with a high-fat diet (HFD). The H&E and EVG (VERHOEFF'S VAN GIESON) staining were used to demonstrate pathological vascular changes. IF (immunofluorescence) and WB (western blot) were used to explore the signaling pathways in vivo and in vitro. The wound closure and transwell assays were also used to test the migration rate of VSMCs. CRISPR gene editing and transcriptomic analysis were applied in vitro to explore the cellular mechanism. Our data showed significant reduction of HRD1 in aortic tissues of mice under HFD feeding. VSMC phenotypic change and HRD1 downregulation were detected by cholesterol supplement. Transcriptomic and further analysis of HRD1-KO VSMCs showed that HRD1 deficiency induced the expression of genes related to ER stress response, proliferation and migration, but reduced the contractile-related genes in VSMCs. HRD1 deficiency also exacerbated the proliferation, migration and ROS production of VSMCs induced by cholesterol, which promoted the VSMC dedifferentiation. Our results showed that HRD1 played an essential role in the contractile homeostasis of VSMCs by negatively regulating ER stress response. Thus, HRD1 in VSMCs could serve as a potential therapeutic target in metabolic disorder-induced vascular remodeling.

Electrical stimulation (ES) plays an important role in regulating cell osteoblast differentiation. As a noninvasive rehabilitation therapy method, Es has a unique role in postoperative recovery. Bone morphogenetic protein-2 (BMP-2) is the most commonly used bioactive molecule in in situ tissue engineering scaffolds, and it plays an important regulatory role in the whole process of bone injury repair. In this study, the osteogenic regulation of MC-3T3-E1 cells was studied by combining pulsed electrical stimulation (PES) and different concentrations of BMP-2. The results showed that PES and BMP-2 could synergically promote the proliferation of MC-3T3-E1 cells. The qPCR results of osteoblast-related genes showed that PES was synergistic with BMP-2 to promote osteoblast differentiation mainly through the regulation of the Smad/BMP and insulin like growth factor 1 (IGF1) signaling pathways. The expression level of alkaline phosphatase (ALP) and alizarin red staining further demonstrated the synergistic effect of PES and BMP-2 on promoting osteogenic differentiation and mineralization of cells. PES and BMP-2 could also synergically promote cell proliferation, expression of collagen I (COL-I) and ALP, and cell mineralization on the 3D-printed polylactic acid scaffold. These results suggest that the use of PES can enhance the osteogenic effect of in situ bone repair scaffolds containing BMP-2, reduce the dose of BMP-2 alone, and reduce the possible side effects of high-dose BMP-2 in vivo.