miR-483-3p suppresses the proliferation and progression of human triple negative breast cancer cells by targeting the HDAC8>oncogene

Triple negative breast cancer (TNBC) is a heterogeneous subclass of breast cancer (BC) distinguished by lack of hormone receptor expression. It is highly aggressive and difficult to treat with traditional chemotherapeutic regimens. Targeted-therapy using microRNAs (miR) has recently been proposed to improve the treatment of TNBC in the early stages. Here, we explore the roles of miR-483-3p/HDAC8 HDAC8 premiR-vector on tumorigenicity in TNBC patients. Clinical TNBC specimens and three BC cell lines were prepared. miR-483-3p and expression levels were measured using quantitative real-time polymerase chain reaction. Cell cycle progression was assessed by a flow-cytometry method. We also investigated cell proliferation by 3-2, 5-diphenyl tetrazolium bromide assay and colony formation assay. We used a to overexpress miR-483-3p, and a HDAC8-KO-vector for knocking out the endogenous production of HDAC8. Our data showed significant downregulation of miR-483-3p expression in TNBC clinical and cell line samples. The HDAC8 was also upregulated in both tissue specimens and BC cell lines. We found that increased levels of endogenous miR-483-3p affects tumorigenecity of MDA-MB-231. Downregulation of HDAC8 using the KO-vector showed the same pattern. Our results revealed that the miR-483-3p suppresses cellular proliferation and progression in TNBC cell lines via targeting HDAC8. Overall, our outcomes demonstrated the role of miR-483-3p as a tumor suppressor in TNBC and showed the possible mechanism via HDAC8. In addition, targeted treatment of TNBC with miR-483-3p might be considered in the future.     

Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence

High glucose (HG)‐induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co‐transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG‐induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence‐associated beta‐galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT‐PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2‐NBD‐glucose. HG increased ECs senescence markers and oxidative stress, down‐regulated eNOS expression and NO formation, and induced the expression of VCAM‐1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX‐4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H₂O₂ in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG‐induced ECs senescence and SGLT1 and 2 expression. Thus, HG‐induced ECs ageing is driven by the local angiotensin system via the redox‐sensitive up‐regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.     

Significance of neutrophil microparticles in ischaemia‐reperfusion: Pro‐inflammatory effectors of endothelial senescence and vascular dysfunction

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia‐reperfusion, we examined leucocyte‐derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol‐myristate‐acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence‐associated β‐galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12‐hour incubation, the endothelial‐dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro‐thrombotic by up‐regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro‐coagulant endothelial microparticles. They initiated an early pro‐inflammatory response by inducing phosphorylation of NF‐κB, MAP kinases and Akt after 1 hour, and up‐regulated VCAM‐1 and ICAM‐1 at 24 hours. Accordingly, VCAM‐1 and COX‐2 were also up‐regulated in the coronary artery endothelium and eNOS down‐regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil‐ and monocyte‐derived microparticles. A 80% immuno‐depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro‐inflammatory, pro‐coagulant and pro‐senescent responses in endothelial cells through redox‐sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia‐reperfusion–driven inflammation and delay its dysfunction.     

Does survivin overexpression enhance the efficiency of fibroblast cell-based wound therapy?

Molecular Biology Reports - Cell-based wound therapy is faced with some limiting factors that decrease the therapeutic efficacy of transplanted cells. In this study, we aimed to genetically modify...     

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6

Molecular Biology Reports - Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have...     

A robust CRISPR–Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.     

Differential Roles of Intra-accumbal Orexin Receptors in Acquisition and Expression of Methamphetamine-Induced Conditioned Place Preference in the Rats

Neurochemical Research - A large amount of document has revealed that the orexin system in the reward circuity, including the nucleus accumbens (NAc), contributes to the modification of drug...     

Assessment of sugar and sugar accumulation-related gene expression profiles reveal new insight into the formation of low sugar accumulation trait in a sweet orange (Citrus sinensis) bud mutant

Molecular Biology Reports - The accumulation of soluble sugars in fleshy fruits largely determines their sweetness or taste. A spontaneous sweet orange mutant ‘Hong Anliu’ (HAL, Citrus...     

Multinuclear Yeast Magnusiomyces (Dipodascus,Endomyces) magnusii is a Promising Isobutanol Producer

Higher alcohol isobutanol is a promising liquid fuel. During alcoholic fermentation, Saccharomyces cerevisiae produces only trace amounts of isobutanol. Screening the collection of nonconventional yeasts show that Magnusiomyces magnusii accumulates 440 mg of isobutanol per L in rich YPD medium. Here, the transformation protocol for M. magnusii is adapted based on the use of the dominant markers conferring resistance to nourseothricin or zeocin; the strong constitutive promoter TEF1 is cloned and a reporter system based on LAC4 gene from Kluyveromyces lactis coding for β‐galactosidase is constructed. In order to increase isobutanol production in M. magnusii, the heterologous gene ILV2 from S. cerevisiae is expressed in M. magnusii under control of the TEF1 promoter. The best stabilized transformants produce 620 mg of isobutanol per L in YPD medium and 760 mg L^?1 in the medium with 2‐oxoisovalerate. This suggests that M. magnusii is a promising organism for further development of a robust isobutanol producer.