Bone-targeting engineered small extracellular vesicles carrying anti-miR-6359-CGGGAGC prevent valproic acid-induced bone loss.

The clinical role and underlying mechanisms of valproic acid (VPA) on bone homeostasis remain controversial. Herein, we confirmed that VPA treatment was associated with decreased bone mass and bone mineral density (BMD) in both patients and mice. This effect was attributed to VPA-induced elevation in osteoclast formation and activity. Through RNA-sequencing, we observed a significant rise in precursor miR-6359 expression in VPA-treated osteoclast precursors in vitro, and further, a marked upregulation of mature miR-6359 (miR-6359) in vivo was demonstrated using quantitative real-time PCR (qRT-PCR) and miR-6359 fluorescent in situ hybridization (miR-6359-FISH). Specifically, the miR-6359 was predominantly increased in osteoclast precursors and macrophages but not in neutrophils, T lymphocytes, monocytes and bone marrow-derived mesenchymal stem cells (BMSCs) following VPA stimulation, which influenced osteoclast differentiation and bone-resorptive activity. Additionally, VPA-induced miR-6359 enrichment in osteoclast precursors enhanced reactive oxygen species (ROS) production by silencing the SIRT3 protein expression, followed by activation of the MAPK signaling pathway, which enhanced osteoclast formation and activity, thereby accelerating bone loss. Currently, there are no medications that can effectively treat VPA-induced bone loss. Therefore, we constructed engineered small extracellular vesicles (E-sEVs) targeting osteoclast precursors in bone and naturally carrying anti-miR-6359 by introducing of EXOmotif (CGGGAGC) in the 3'-end of the anti-miR-6359 sequence. We confirmed that the E-sEVs exhibited decent bone/osteoclast precursor targeting and exerted protective therapeutic effects on VPA-induced bone loss, but not on ovariectomy (OVX) and glucocorticoid-induced osteoporotic models, deepening our understanding of the underlying mechanism and treatment strategies for VPA-induced bone loss.

Reciprocal negative feedback loop between EZH2 and miR-101-1 contributes to miR-101 deregulation in hepatocellular carcinoma.

Although the tumor suppressive role of miR-101 is well documented in hepatocellular carcinoma (HCC), how the expression of miR-101 itself is regulated remains elusive. In the present study, we demonstrated that the miR-101 precursor pre-miR-101-1 could be regulated by an important epigenetic regulator, the enhancer of zeste homolog 2 (EZH2). Reporter gene assays revealed that ectopic expression of EZH2 inhibited the transcriptional activities of miR-101-1 promoter. Subsequent analyses revealed that miR-101-1 directly represses the expression of EZH2, and miR-101-1 and EZH2 form a reciprocal negative feedback loop as indicated by the fact that ectopic mature miR-101 could induce endogenous pre-miR-101-1 expression. This mature miR-101-induced pre-miR-101 expression was specific to pre-miR-101-1 and depended on EZH2 activities. Moreover, our results also demonstrated that similar antitumor effects can be achieved either by ectopic miR-101 or EZH2 silencing in HCC cells. These findings show that elevated EZH2 contributes to miR-101 deregulation in HCC and highlight the coordinated role of miR-101 and EZH2 in hepatocarcinogenesis.

The TKTL1 gene influences total transketolase activity and cell proliferation in human colon cancer LoVo cells.

A plasmid carrying DNA to be transcribed into a small interfering RNA against transketolase-like-1 mRNA was constructed and transfected into a human colon cancer cell line. The mRNA expression of transketolase gene family in the human colon cell line was determined by real-time polymerase chain reaction. The effect of anti-transketolase-like-1 small interfering RNA on cell proliferation and cell cycle in the human colon cancer cell line cells was detected by flow cytometry and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide. The transketolase-like-1 gene was significantly downregulated in human colon cancer cell line cells transfected with small interfering RNA transketolase-like-1 constructs compared with the cells transfected with control vector and the cells without transfection. In addition, the anti-transketolase-like-1 small interfering RNA construct significantly decreased the level of transketolase in the transfected human colon cancer cell line cells, arrested them in G0/G1 phase and substantially inhibited cell proliferation. No significant difference was found in the other two genes (transketolase and transketolase-like-2 genes) between the transfected human colon cancer cell line cells and the controls (P>0.05). Our data demonstrated that the transketolase-like-1 gene plays an important role in total transketolase activity and in the cell proliferation of human colon cancer. Transketolase-like-1 may serve as a target for novel anticancer therapies.

[Effect of glucose-6-phosphate dehydrogenase on intracellular gsh level in Raji cells during oxidative stress].

AIM: To explore a role of G6PD in replenishment of intracellular GSH during oxidative stress. METHODS: In vitro Raji cell was cultured, intracellular GSH levels and G6PD, GR, GPX activities were determined at different time points after PMS treatment when G6PD activity was inhibited or not by DHEA. RESULTS: Intracellular GR, GPX, G6PD activities elevated significantly combined with GSH level decreased dramatically before 30 minutes, replenished gradually after 30 minutes and restore normal levels about 6 h after PMS treatment when G6PD was not inhibited. No change in GR and significant increase in GPX activity were shown following depleted GSH after PMS treatment when G6PD was inhibited by DHEA. CONCLUSION: G6PD contributes to replenish intracellular GSH and is a critical factor regulating GSH levels during oxidative stress.

Detection of three common G6PD gene mutations in Chinese individuals by probe melting curves.

OBJECTIVE: In order to develop and validate an assay for rapid detection of three common G6PD gene mutations in Chinese individuals. METHODS: In this report we design two sets of primers and fluorescently labeled hybridization probes recognizing adjacent sequences in the amplicon; after annealing, the fluorophores were in resonance energy transfer, providing real-time monitoring of the amplication process. At the completion of PCR, fluorescence was monitored as the temperature increased through the Tm of the probe/product duplex, and a characteristic melting profile for each mutation was obtained. By using the fluorescence method and PCR/RE, a total of 57 samples obtained from two groups of G6PD-deficient individuals were studied. RESULT: A rapid method for detection of three common G6PD gene mutations in Chinese individuals by probe melting curves was developed. This method shows 100% accordance with the traditional method. CONCLUSION: This fluorescent melting curve analysis is a simple, rapid, and effective method for clinical diagnosis and screening of G6PD deficiency.