Downregulations of miR-449a and miR-145-5p Act as Prognostic Biomarkers for Endometrial Cancer.

This investigation aimed to explore the underlying prognosis-associated microRNA (miRNA) biomarkers in endometrial cancer. Homo sapiens miRNA data set GSE35794 and miRNA data in TGGA database were downloaded and applied to screen the differentially expressed miRNAs (DE-miRNAs) using unpaired t-test in limma package in R. Basing on Venn analysis, the overlapped DE-miRNAs were screened and their potential targets were predicted according to miRWalk followed by target functional enrichment analyses and protein-protein interaction network visualized using Cytoscape. Finally, according to the information provided by the The Cancer Genome Atlas (TCGA) database, correlations between miRNAs or targets and patient prognosis were analyzed by survival package in R. A total of 24 overlapped DE-miRNAs were identified between endometrioid endometrial cancer samples and normal samples. Then, the miRNA-target regulatory network was constructed, including 11 upregulated miRNAs (e.g., miR-200a, miR-200b, and miR-200c) and five downregulated miRNAs (e.g., miR-449a, miR-145-5p, and miR-145-3p). Lymphocyte enhancer factor-1 (LEF1) was predicted to be a target of miR-449a and SOX11 was a target of miR-145-5p. Functional enrichment analyses of these targets were significantly related to the biological process of "negative regulation of transcription from RNA polymerase II promoter" and "positive regulation of transcription from RNA polymerase II promoter" (e.g., NOTCH1, LEF1, and SOX11). In addition, survival analysis showed that miR-449a, miR-145-5p, and LEF1 were approximately correlated with the overall survival prognosis of endometrial cancer patients. Downregulations of miR-449a and miR-145-5p might be involved in the pathogenesis of endometrial cancer and could act as prognostic biomarkers for endometrial cancer patients.

The antiresoptive effects of recombinant Lingzhi-8 protein against retinoic acid-induced osteopenia.

The antiresorptive agents still are the mainstay of osteoporosis treatment. This study aimed to investigate the efficacy of recombinant Lingzhi-8 (rLZ-8) on osteoclast in vitro and bone resorption in vivo. The rLZ-8 protein was derived from Ganoderma lucidum transformation and produced by a genetic system. Receptor activator of nuclear factor kappa-Β ligand induced RAW 264.7 cells to differentiate into osteoclastic cells in vitro. Cells were exposed to different doses of rLZ-8 for 7 days to measure differences of osteoclastic differentiation, apoptosis rate and gene expression. rLZ-8 was labeled with Alexa Fluor 568 to observe its intracellular distribution under super-resolution light microscopy. In addition, retinoic acid was administered to female rats for 14 days to develop osteopenia changes. Different doses of rLZ-8 were simultaneously administered to rats treated with retinoic acid to observe changes of bone mineral density, biochemical parameters and organ weight ratio. Results indicated that rLZ-8 regulated receptor activator of nuclear factor kappa-Β (RANK) - tumor necrosis factor receptor-associated factor 6 (TRAF6) - c-Jun N-terminal kinase (JNK) signaling pathway, by which rLZ-8 inhibited osteoclastic differentiation and promoted osteoclastic apoptosis. Through 3D-structured illumination microscopy, it was observed that rLZ-8 entered RAW264.7 cells and accumulated gradually into the cytoplasm but little into nucleus. Administration with rLZ-8 reversed loss of bone mass and improved ALP activity in osteoporotic rats. Low-to high-dose rLZ-8 treatments displayed little toxic effects on rat organs and did not seem to impact their overall health. All data suggested that rLZ-8 has possible action against osteoporosis.

Serum miR-1181 and miR-4314 associated with ovarian cancer: MiRNA microarray data analysis for a pilot study.

OBJECTIVE: This study aims to identify serum microRNAs (miRNAs) related to ovarian cancer. STUDY DESIGN: MiRNA profiling data (GSE79943) were generated from the Gene Expression Omnibus, including 3 serum samples from healthy individuals and 4/3/16/6 serum samples from patients with ovarian cancer stage I/II/III/IV. Differentially expressed miRNAs (DEmiRNAs) were identified between controls and ovarian cancer stage I/II/III/IV by using limma package (p-value <0.05 and |log2 fold change| ≥0.5). miRWALK2.0 database was used to find experiment-validated targets of DEmiRNAs, and CTD database was utilized to screen known genes related to ovarian cancer. clusterProfiler package was used to perform pathway enrichment analysis of DEmiRNAs. Targets of DEmiRNAs were validated by using GSE40595, involving 8 normal ovarian stroma, 31 ovarian cancer stroma, 6 human ovarian surface epthelium, and 32 ovarian tumor epthelial component. RESULTS: Between stage I/II/III/IV and control, 39/143/29/39 DEmiRNAs were identified, which were regarded as key miRNAs. Between 4 DEmiRNA sets, 15 common DEmiRNAs were identified (e.g. up-regulated hsa-miR-1181 and hsa-miR-4314). Hsa-miR-1181 participated in "Jak-STAT signaling pathway" and "miRNAs in cancer"; hsa-miR-4314 took part in cancer-related pathways. STAT3 and KRAS, known marker genes of ovarian cancer, were targeted by hsa-miR-1181 and hsa-miR-4314, respectively. Besides, FOXP1 was targeted by hsa-miR-1181; FOXP1-AS1 and FOXP1-IT1 were down-regulated in ovarian cancer. GRWD1, IP6K1, and NEGR1 were targeted by hsa-miR-4314; GRWD1, IP6K1, and NEGR1 were down-regulated in ovarian tumor. CONCLUSION: MiR-1181 and miR-4314 might promote ovarian tumorigenesis via down-regulating FOXP1 and GRWD1/IP6K1/NEGR1, respectively. In addition, the 15 common DEmiRNAs might provide directions for ovarian cancer diagnosis.

MiR-200a promotes epithelial-mesenchymal transition of endometrial cancer cells by negatively regulating FOXA2 expression.

Endometrial cancer is the most common gynecological cancer. Epithelial-mesenchymal transition (EMT) plays a critical role in tumor invasion and metastasis, which limits the success of treatment. Here, we investigated the roles of forkhead box A2 (FOXA2) and microRNA-200a (miR-200a) in regulating the EMT of endometrial cancer cells RL95-2. Empty vector or FOXA2 was stably transfected into RL95-2 cells. MTT assay measured cell proliferation, apoptosis assay measured apoptosis, Transwell invasion assay measured cell invasion, and Western blot measured the protein expression of FOXA2, E-cadherin, and vimentin. ChIP assay determined the binding of FOXA2 to E-cadherin promoter. For miR-200a analysis, the cells with stable FOXA2 expression were transfected with miR-negative control or miR-200a. Forced expression of FOXA2 decreased the proliferation and invasion, and increased the apoptosis of RL95-2 cells. FOXA2 also affected the EMT-associated proteins: FOXA2 increased the protein expression of E-cadherin and decreased the expression of vimentin. Moreover, FOXA2 positively regulated the promoter of E-cadherin in RL95-2 cells. Luciferase reporter assay identified FOXA2 as a target of miR-200a, which negatively regulated FOXA2. Western blot results showed that overexpression of miR-200a decreased the expression of E-cadherin but increased the expression of vimentin in the endometrial cancer cells by downregulating FOXA2 expression. FOXA2 may act as a tumor suppressor and inhibit EMT of endometrial cancer cells. FOXA2 expression is controlled by miR-200a, which promotes EMT of the endometrial cancer cells.

Effect of siRNA PERK on fluoride-induced osteoblastic differentiation in OS732 cells.

The purpose of this work is to study the action of fluoride on osteoblastic function through knocking down double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) mRNA in OS732 cells (human osteoblast-like cell line). The previous researches had demonstrated that fluoride induced endoplasmic reticulum (ER) stresses in other cells or tissues. PERK as one branch of UPR to combat ER stress played a role in mediating the proliferation and differentiation of osteoblast. The mechanism of skeletal fluorosis by which fluoride regulated osteoblast was not fully defined. We used the real-time PCR and small interfering RNA techniques to determine the expression PERK signaling and osteoblastic and osteoclastic differentiation-related factors and investigated the role of PERK signaling in fluoride-stimulated osteoblastic function. Cells transfected with 50 nM small interfering RNA (siRNA)-PERK showed effectively decreased protein and gene expression of PERK and reduced protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2). Meantime, cells transfected with siRNA significantly decreased the protein level of alkaline phosphatase (ALP) and nuclear factor kappa B ligand (RANKL) in cells under fluoride exposure. It suggested that knockdown of PERK expression hardly stimulated osteoblastic and osteoclastic early differentiation induced by fluoride. Conversely, there were littler effect of siRNA PERK on expression of Runt-related transcription factor 2 (RUNX2) and osteoprotegerin (OPG) in cells, but fluoride exposure markedly stimulated their expression. This study proved that the mechanism underlying fluoride induced osteoblastic and osteoclastic differentiation possible was due to activation of ALP and RANKL mediated by PERK in OS732 cells.

Oxidative stress and activities of caspase-8, -9, and -3 are involved in cryopreservation-induced apoptosis in granulosa cells.

OBJECTIVE: The aim of this study is to determine whether the oxidative stress and the activities of caspase-8, -9, and -3 are involved in cryopreservation-induced apoptosis in granulosa cells. STUDY DESIGN: Granulosa cells from rats were cryopreserved with or without genistein. The level of superoxide dismutase (SOD) was measured. Moreover, the expressions of caspase-8, -9, and -3 in both mRNA and protein were measured. RESULTS: The SOD level in cryopreserved granulosa cells was significantly lower than that in a fresh control group, and the levels of caspase-8, -9, and -3 expression in both mRNA and protein in cryopreserved granulosa cells were significantly higher than those of fresh control granulosa cells. Furthermore, the levels of caspase-8 and -3 expression in both mRNA and protein of granulosa cells cryopreserved in presence of genistein were significant lower than those of granulosa cells cryopreserved in absence of genistein. CONCLUSION: Oxidative stress induced by cryopreservation in granulosa cells is involved in the activation of caspase-8 and -3. Cryopreservation-induced apoptosis in granulosa cells is mediated, at least in part, by activation of caspase-8, -9, and -3 dependent apoptotic pathways.

Optimizing cryoprotectant perfusion conditions for intact ovary: a bovine model.

PURPOSE: The aim of this study was to detect the effects of different perfusion pressure and different length of perfusion period on whole ovarian cryopreservation METHODS: Bovine whole ovaries were vitrified-warmed. The ovaries were divided into the experimental groups according to different perfusion pressure and different length of perfusion period. Follicular viability was assessed using the trypan blue test; the percentage of morphologically normal primordial follicles and the 17-ß estradiol level in the culture supernatants were measured. RESULTS: When perfusion pressure was 100 mmHg, and the length of perfusion period was 40 min, the viability of ovarian tissues in bovine whole ovarian cryopreservation were higher than other protocols. CONCLUSION: Protocol IIb (the perfusion pressure was 100 mmHg, and the length of perfusion period was 40 min) was appropriate for bovine whole ovarian cryopreservation.