Upregulation of miR-23a∼27a∼24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells

miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. Here, we show that the over expression of miR-23a∼27a∼24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway as proved by the annexin assay, caspase activation, release of cytochrome-c and AIF (apoptosis inducing factor) from mitochondria. Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Bioinformatically, FADD is predicted to be the target of hsa-miR-27a and interestingly, FADD protein was found to be up regulated consistent with very less expression of hsa-miR-27a in HEK293T cells. This effect was direct, as hsa-miR-27a negatively regulated the expression of FADD 3′UTR based reporter construct. Moreover, we also showed that over expression of miR-23a∼27a∼24-2 sensitized HEK293T cells to TNF-α cytotoxicity. Taken together, our study demonstrates that enhanced TNF-α induced apoptosis in HEK293T cells by over expression of miR-23a∼27a∼24-2 cluster provides new insights in the development of novel therapeutics for cancer

Human 3D brain organoids: steering the demolecularization of brain and neurological diseases

Abstract Understanding of human brain development, dysfunction and neurological diseases has remained limited and challenging due to inability to recapitulate human brain-specific features in animal models. Though the anatomy and physiology of the human brain has been understood in a remarkable way using post-mortem, pathological samples of human and animal models, however, modeling of human brain development and neurological diseases remains a challenge owing to distinct complexity of human brain. In this perspective, three-dimensional (3D) brain organoids have shown a beam of light. Tremendous growth in stem cell technologies has permitted the differentiation of pluripotent stem cells under 3D culture conditions into brain organoids, which recapitulate the unique features of human brain in many ways and also offer the detailed investigation of brain development, dysfunction and neurological diseases. Their translational value has also emerged and will benefit the society once the protocols for the upscaling of brain organoids are in place. Here, we summarize new advancements in methods for generation of more complex brain organoids including vascularized and mixed lineage tissue from PSCs. How synthetic biomaterials and microfluidic technology is boosting brain organoid development, has also been highlighted. We discuss the applications of brain organoids in studying preterm birth associated brain dysfunction; viral infections mediated neuroinflammation, neurodevelopmental and neurodegenerative diseases. We also highlight the translational value of brain organoids and current challenges that the field is experiencing

Correction to: HSP60 critically regulates endogenous IL-1β production in activated microglia by stimulating NLRP3 inflammasome pathway

Upon publication of the original article [1], it was noticed that there is an error in Fig. 10, the dialog box in panel (b) was missing. The correct Fig. 10 is shown below

Sensitivity to apoptosis is increased after transfection of p(23a∼27a∼24-2).

<p>A. Annexin V-PE binding in HEK293T cells i) untransfected cells, ii) cells transfected with 4 µg p(23a∼27a∼24-2) for 24 h, and iii) cells treated with 20 ng TNF-α iv) cells transfected with 4 µg p(23a∼27a∼24-2) for 24 h and treated with 20 ng TNF-α. B. Mitochondrial membrane potential in HEK293T cells detected by DiOC6 staining and flow-cytometry analysis, i) untransfected cells, ii) 24 h after transfection with 4 µg p(23a∼27a∼24-2), iii) treated with 20 ng TNF-α, and iv) 24 h after transfection with 4 µg p(23a∼27a∼24-2) and treated with 20 ng TNF-α. C. Effect of overexpressed p(23a∼27a∼24-2) on the protein expression of FADD, pro-caspase 8, TRAF2, RIP. GAPDH/β-actin was used as an internal control. Data are representative of a typical experiment repeated three times with similar results.</p

Hypothetical model for p(23a∼27a∼24-2) induced apoptosis.

<p>The green arrows indicate down regulation and red arrows indicate up regulation.</p

Dose dependent expression of miR-27a and FADD and luciferase assay confirms that miR-27a targets the FADD 3′ UTR.

<p>A. Detection of mature miRNA after transfection of clone expressing p(23a∼27a∼24-2), the same blot was probed for U6 expression for normalization. B. Western blotting for FADD after transfection of clone expressing miR-27a, the same blot was probed for β-actin for normalization. The fold change in expression of FADD is also presented after densitometric analysis. C. Luciferase assay proves that 3′UTR of FADD is a target of miR-27a. *p(23a∼27a∼24-2) (2 µg) – Transfection of 2 µg of p(23a∼27a∼24-2). p(23a∼27a∼24-2) (4 µg) – Transfection of 4 µg of p(23a∼27a∼24-2).</p

Biological effect of over expression of p(23a∼27a∼24-2) in HEK293T cells.

<p>A. Annexin V-PE binding in HEK293T cells i) untransfected cells, ii) cells transfected with 4 µg p(23a∼27a∼24-2) for 24 h, and iii) cells transfected with p(23a∼27a∼24-2) for 24 h and treated with caspase inhibitor. B. Mitochondrial membrane potential in HEK293T cells, i) untransfected cells, ii) 24 h after transfection with 4 µg p(23a∼27a∼24-2), and iii) positive control, detected by DiOC6 staining and flow-cytometry analysis. C. ROS production in HEK293T cells i) untransfected cells, ii) 24 h after transfection with 4 µg p(23a∼27a∼24-2), and iii) positive control.</p

Overexpression of p(23a∼27a∼24-2) in HEK293T cells causes a change in precursor and mature form of mir-23a, miR-27a and miR-24-2.

<p>A. Northern Blot depicting change in miRNA expression after p(23a∼27a∼24-2) transfection (i) U6 loading control (ii) miR-23a (iii) miR-27a (iv) miR-24-2. B. Semiquantitative PCR confirms the increase in the miRNA transcript levels of miR-24-2 and miR-27a but not much change in miR-23a after p(23a∼27a∼24-2) transfection at 8-, 16- and 24- hours post-transfection, M refers to DNA ladder. The constitutive expression of β-actin is shown as the loading control. C. Real Time PCR for (i) pre-miR-24-2, (ii) pre-miR-27a and TaqMan assay for (iii) Mature miR-24-2, (iv) Mature miR-27a.</p