Evaluation of the expression level of microRNA-21, microRNA-15a, microRNA-372 in human follicular fluid stem cells-derived oocyte-like cells (OLCs).

OBJECTIVE: Today, researchers have succeeded in achieving oocyte-like cells through the in vitro differentiation of stem cells. MicroRNAs are key regulators of oocyte development. In this study we decided to evaluate the expression pattern of microRNA-21, microRNA-15a, and microRNA-372 in oocyte-like cells, to determine the maturation stage of oocyte-like cells. METHODS: Human follicular fluid samples were collected and centrifuged, and their cells were divided into 3 groups; day 7 as control group, days 14 and 21. During this period, the cells were evaluated for their morphological appearance and viability by inverted microscopy. RNA isolation was performed and cDNA was reversely transcribed by specific stem-loop RT primers. Real-time RT-PCR was used to detect microRNA expression. RESULTS: The relative expression of microRNA-21 and microRNA-15a on day 21 was significantly down-regulated compared to the control group (day 7), but microRNA-372 did not show a significant difference. Also, on day 14 compared to the control group (day 7), microRNA-21 did not show a significant difference; but microRNA-15a and microRNA-372 were significantly down-regulated. MicroRNA-21 and microRNA-15a on day 21 compared to day 14 revealed down-regulated levels, but microRNA-372 revealed up-regulated levels. CONCLUSIONS: Our results showed significant decreases in the expression of microRNA-21 and microRNA-15a in oocyte-like cells, as well as in oocytes, which may lead to cytoplasmic maturation, germinal vesicle break down and the completion of meiosis І. In addition, down-regulation expression of microRNA-372 maybe a confirmation that mesenchymal stem cells have differentiated into germ cells, and these cells were differentiated into oocyte-like cells.

Genosensing Applications of Glassy Carbon Electrodes Modified with Multi-Walled Carbon Nanotubes Non-Covalently Functionalized with Polyarginine.

We report the advantages of glassy carbon electrodes (GCE) modified with multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with polyarginine (PolyArg) for the adsorption and electrooxidation of different DNAs and the analytical applications of the resulting platform. The presence of the carbon nanostructures, and mainly the charge of the PolyArg that supports them, facilitates the adsorption of calf-thymus and salmon sperm double-stranded DNAs and produces an important decrease in the overvoltages for the oxidation of guanine and adenine residues and a significant enhancement in the associated currents. As a proof-of-concept of possible GCE/MWCNTs-PolyArg biosensing applications, we develop an impedimetric genosensor for the quantification of microRNA-21 at femtomolar levels, using GCE/MWCNTs-PolyArg as a platform for immobilizing the DNA probe, with a detection limit of 3fM, a sensitivity of 1.544 × 103 Ω M-1, and a successful application in enriched biological fluids.

Label-free graphene oxide-based SPR genosensor for the quantification of microRNA21.

This work is focused on the development of a genosensor for microRNA-21 quantification using surface plasmon resonance (SPR) to transduce the hybridization event. The biosensing platform was built by self-assembling two bilayers of poly(diallyldimethylammonium chloride) (PDDA) and graphene oxide (GO) at a gold surface modified with 3-mercaptopropane sulfonate (MPS), followed by the covalent attachment of the DNA probe. GO was used in two directions, to allow the anchoring of the probe DNA and to increase the sensitivity of the biosensing event due to its field enhancer effect. The new bioanalytical platform represents an interesting alternative for the label-free biosensing of microRNA-21, with a linear range between 1.0 fM and 10 nM, a sensitivity of 5.1 ± 0.1 moM-1 and a detection limit of 0.3fM. The proposed sensing strategy was successfully used for the quantification of microRNA-21 in enriched urine samples. Graphical abstract.

Effect of microRNA-21 on the proliferation of human degenerated nucleus pulposus by targeting programmed cell death 4

This study aims to explore the effect of microRNA-21 (miR-21) on the proliferation of human degenerated nucleus pulposus (NP) by targeting programmed cell death 4 (PDCD4) tumor suppressor. NP tissues were collected from 20 intervertebral disc degeneration (IDD) patients, and from 5 patients with traumatic spine fracture. MiR-21 expressions were tested. NP cells from IDD patients were collected and divided into blank control group, negative control group (transfected with miR-21 negative sequences), miR-21 inhibitor group (transfected with miR-21 inhibitors), miR-21 mimics group (transfected with miR-21 mimics) and PDCD4 siRNA group (transfected with PDCD4 siRNAs). Cell growth was estimated by Cell Counting Kit-8; PDCD4, MMP-2,MMP-9 mRNA expressions were evaluated by qRT-PCR; PDCD4, c-Jun and p-c-Jun expressions were tested using western blot. In IDD patients, the expressions of miR-21 and PDCD4 mRNA were respectively elevated and decreased (both P<0.05). The miR-21 expressions were positively correlated with Pfirrmann grades, but negatively correlated with PDCD4 mRNA (both P<0.001). In miR-21 inhibitor group, cell growth, MMP-2 and MMP-9 mRNA expressions, and p-c-Jun protein expressions were significantly lower, while PDCD4 mRNA and protein expressions were higher than the other groups (all P<0.05). These expressions in the PDCD4 siRNA and miR-21 mimics groups was inverted compared to that in the miR-21 inhibitor group (all P<0.05). MiR-21 could promote the proliferation of human degenerated NP cells by targeting PDCD4, increasing phosphorylation of c-Jun protein, and activating AP-1-dependent transcription of MMPs, indicating that miR-21 may be a crucial biomarker in the pathogenesis of IDD.