MiR-27a-3p promotes the osteogenic differentiation by activating CRY2/ERK1/2 axis.

BACKGROUND: Osteoporosis seriously disturbs the life of people. Meanwhile, inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. It was reported that miR-27a-3p reduced the symptoms of osteoporosis. However, the mechanism by which miR-27a-3p in osteogenic differentiation remains largely unknown. METHODS: To induce the osteogenic differentiation in MC3T3-E1 cells, cells were treated with osteogenic induction medium (OIM). RT-qPCR was used to evaluate the mRNA expression of miR-27a-3p and CRY2 in cells. The protein levels of CRY2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and the phosphorylation level of extracellular regulated protein kinases (ERK) 1/2 in MC3T3-E1 cells were evaluated by western blotting. Meanwhile, calcium nodules and ALP activity were tested by alizarin red staining and ALP kit, respectively. Luciferase reporter gene assay was used to analyze the correlation between CRY2 and miR-27a-3p. RESULTS: The expression of miR-27a-3p and the phosphorylation level of ERK1/2 were increased by OIM in MC3T3-E1 cells, while CRY2 expression was decreased. In addition, OIM-induced increase of calcified nodules, ALP content and osteogenesis-related protein expression was significantly reversed by downregulation of miR-27a-3p and overexpression of CRY2. In addition, miR-27a-3p directly targeted CRY2 and negatively regulated CRY2. Meanwhile, the inhibitory effect of miR-27a-3p inhibitor on osteogenic differentiation was reversed by knockdown of CRY2 or using honokiol (ERK1/2 signal activator). Furthermore, miR-27a-3p significantly inhibited the apoptosis of MC3T3-E1 cells treated by OIM. Taken together, miR-27a-3p/CRY2/ERK axis plays an important role in osteoblast differentiation. CONCLUSIONS: MiR-27a-3p promoted osteoblast differentiation via mediation of CRY2/ERK1/2 axis. Thereby, miR-27a-3p might serve as a new target for the treatment of osteoporosis.

Global mRNA and Long Non-Coding RNA Expression in the Placenta and White Adipose Tissue of Mice Fed a High-Fat Diet During Pregnancy.

BACKGROUND/AIMS: Gestational diabetes mellitus (GDM) is a common complication of pregnancy, but the mechanisms underlying the disorders remain unclear. The study aimed to identify mRNA and long non-coding RNA (lncRNA) profiles in placenta and gonadal fat of pregnant mice fed a high-fat diet and to investigate the transcripts and pathways involved in the development of gestational diabetes mellitus. METHODS: Deep and broad transcriptome profiling was performed to assess the expression of mRNAs and lncRNAs in placenta and gonadal fat from 3 mice fed an HFD and chow during pregnancy. Then, differentially expressed mRNAs and lncRNAs were validated by quantitative real-time PCR. The function of the differentially expressed mRNAs was determined by pathway and Gene Ontology (GO) analyses, and the physical or functional relationships between the lncRNAs and the corresponding mRNAs were determined. RESULTS: Our study revealed that 82 mRNAs and 52 lncRNAs were differentially expressed in the placenta of mice fed an HFD during pregnancy, and 202 mRNAs and 120 lncRNAs were differentially expressed in gonadal fat. GO and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed differentially expressed mRNAs of placenta were closely related to extracellular matrix interactions, digestion, adhesion, and metabolism, whereas the differentially expressed mRNAs in adipose tissue were related to metabolic and insulin signalling pathways. The gene network demonstrated that Actg2, Cnfn, Muc16, Serpina3k, NONMMUT068202, and NONMMUT068203, were the core of the network in placental tissue, and the genes Tkt, Acss2, and Elovl6 served as the core of the network in gonadal fat tissue. CONCLUSION: These newly identified key genes and pathways in mice might provide valuable information regarding the pathogenesis of GDM and might be used to improve early diagnosis, prevention, drug design, and clinical treatment.

First Report of a Case with Nondeletional Hb H Disease Caused by IVS-I-116 (A>G) of the α2-Globin Gene.

In this report, we describe a prenatal case with cardiomegaly and increased middle cerebral artery-peak systolic velocity (MCA-PSV) at 22 weeks' gestation. Fetal blood sampling revealed moderate anemia (Hb 7.4 g/dL) and increased Hb Bart's (γ4) level (28.2%), indicative of Hb H (ß4) disease. Molecular analysis of the family members revealed that the pregnant woman carried a heterozygous IVS-I-116 (A>G) (HBA2: c.96-2A>G) mutation of α2-globin gene, and the fetus was a compound heterozygote for IVS-I-116 and the Southeast Asian (- -SEA) deletion. This is the first reported case of nondeletional Hb H disease caused by the IVS-I-116 (A>G) mutation associated with fetal anemia identified by ultrasound.

Staphylococcus Aureus Induces Osteoclastogenesis via the NF-κB Signaling Pathway.

BACKGROUND Osteomyelitis is one of the refractory diseases encountered in orthopedics, while Staphylococcus aureus (S. aureus) is the most common causative organism in osteomyelitis. However, the precise mechanisms underlying the bone loss caused by S. aureus infection have not been well defined. Here, we investigated the effect of S. aureus on osteoclast differentiation and the probable molecular mechanism. MATERIAL AND METHODS RAW 264.7 cells were treated for 5 days with live S. aureus, inactivated S. aureus, and S. aureus filtrate. Then, the formation of osteoclast-like cells and resorption pits was observed, and the expression of osteoclast-specific genes (TRAP, MMP-9, cathepsin K, CTR and Atp6v0d2) was detected by real-time PCR. Moreover, key proteins in the signaling pathway associated with osteoclast differentiation were detected with Western blot. RESULTS The data showed that live S. aureus, inactivated S. aureus, and S. aureus filtrate induced osteoclast formation, promoted bone resorption, and increased the expression of osteoclast-specific genes in a dose-dependent manner in the absence RANKL. In addition, we found that the S. aureus-induced osteoclastogenesis was related to the degradation of IκB-a, phosphorylation of NF-κB p65, and increased expression of NFATc1. Thus, we used JSH-23 to inhibit NF-κB transcriptional activity. The effect of the S. aureus-induced osteoclastogenesis and the expression of osteoclast-specific genes and NFATc1 were inhibited, which indicated that the NF-κB signaling pathway plays a role in S. aureus-induced osteoclastogenesis. CONCLUSIONS This study demonstrated that S. aureus induces osteoclastogenesis through its cell wall compound and secretion of small soluble molecules, and the NF-κB signaling pathway plays a role in this process.

Staphylococcus aureus Protein A induces osteoclastogenesis via the NF­κB signaling pathway.

Staphylococcus aureus (S. aureus) is the most common organism causing osteomyelitis, and Staphylococcus aureus protein A (SpA) is an important virulence factor anchored in its cell wall. However, the precise mechanisms underlying the bone loss caused by SpA have not been well understood. The present study aimed to investigate the effect of SpA on osteoclast differentiation, and the probable mechanism was investigated. Raw264.7 cells were treated with SpA in the absence or presence of receptor­activated (NF)­κB ligand for 5 days, and morphological and biochemical assays were used to assess osteoclastogenesis and explore the underlying mechanisms. Data demonstrated that SpA induced osteoclast differentiation and promoted bone resorption in a dose­dependent manner in the absence or presence of RANKL. In addition, the expression of osteoclast­specific genes, such as the tartrate resistant acid phosphatase, matrix metalloproteinase­9, cathepsin K, calcitonin receptors and d2 isoform of the vacuolar ATPase Vo domain, were enhanced by SpA. Furthermore, the SpA­induced osteoclast differentiation was associated with the degradation of inhibitor of κB­α, phosphorylation of NF­κB p65 and increased expression of nuclear factor of activated T­cells. However, by treatment with JSH­23, an NF­κB inhibitor, the formation of osteoclast­like cells and resorption pits was significantly reduced, and the expression of osteoclast­specific genes was also inhibited. Collectively, in the present study SpA induced osteoclast differentiation, promoted bone resorption, and the NF­κB signaling pathway was involved in this process.

The role of GPR1 signaling in mice corpus luteum.

Chemerin, a chemokine, plays important roles in immune responses, inflammation, adipogenesis, and carbohydrate metabolism. Our recent research has shown that chemerin has an inhibitory effect on hormone secretion from the testis and ovary. However, whether G protein-coupled receptor 1 (GPR1), the active receptor for chemerin, regulates steroidogenesis and luteolysis in the corpus luteum is still unknown. In this study, we established a pregnant mare serum gonadotropin-human chorionic gonadotropin (PMSG-hCG) superovulation model, a prostaglandin F2α (PGF2α) luteolysis model, and follicle and corpus luteum culture models to analyze the role of chemerin signaling through GPR1 in the synthesis and secretion of gonadal hormones during follicular/luteal development and luteolysis. Our results, for the first time, show that chemerin and GPR1 are both differentially expressed in the ovary over the course of the estrous cycle, with highest levels in estrus and metestrus. GPR1 has been localized to granulosa cells, cumulus cells, and the corpus luteum by immunohistochemistry (IHC). In vitro, we found that chemerin suppresses hCG-induced progesterone production in cultured follicle and corpus luteum and that this effect is attenuated significantly by anti-GPR1 MAB treatment. Furthermore, when the phosphoinositide 3-kinase (PI3K) pathway was blocked, the attenuating effect of GPR1 MAB was abrogated. Interestingly, PGF2α induces luteolysis through activation of caspase-3, leading to a reduction in progesterone secretion. Treatment with GPR1 MAB blocked the PGF2α effect on caspase-3 expression and progesterone secretion. This study indicates that chemerin/GPR1 signaling directly or indirectly regulates progesterone synthesis and secretion during the processes of follicular development, corpus luteum formation, and PGF2α-induced luteolysis.

Effects of sialidase NEU1 siRNA on proliferation, apoptosis, and invasion in human ovarian cancer.

Ovarian cancer is one of the most common malignancies encountered in the world. In ovarian cancer tissues of patients, NEU1 was expressed in a higher level than that in adjacent normal tissues. In this research, we aimed to elucidate the role of NEU1 siRNA on proliferation, apoptosis, and invasion of OVCAR3 and SKOV3 cells which expressed NEU1 notably. By cell viability assay and flow cytometry method, we found that NEU1 siRNA effectively inhibited the cancer proliferation, arrested cells cycle at G0/G1 phase, and induced apoptosis when compared to the Mock group. Result of transwell assay showed that invasion of cells in OVCAR3 and SKOV3 treated with NEU1 siRNA were suppressed significantly. Gene set enrichment analysis showed that lysosome and oxidative phosphorylation related signal pathway were associated with the NEU1 expression. In addition, Western blot revealed that expressions of Cln3 and Cln5 were depressed, and ATP5B and ATP5J expressions were also reduced. In conclusion, NEU1 siRNA can effectively inhibit proliferation, apoptosis, and invasion of human ovarian cancer cells by targeting lysosome and oxidative phosphorylation signaling, which can serve as a new target ovarian cancer treatment.

[Reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel].

OBJECTIVE: To investigate the reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel. METHODS: shRNA plasmid vector specifically targeting MDR1 and MDR3 genes was transfected into A2780/taxol cells. The early stage cell apoptosis and the effect of intracellular rhodamine 123 (Rh123) accumulation were detected by flow cytometry (FCM). The late stage cell apoptosis rate was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/taxol cells was determined by methyl thiazolyl tetrazolium (MTT) assay. MDR1 and MDR3 mRNA were assessed by RT-PCR, and caspase-3 protein was detected by western blot. RESULTS: After treatment with MDR1 and MDR3 shRNA plasmid vector, early apoptosis rate of A2780/taxol cells was (20.21 +/- 0.56)% and (10.87 +/- 1.24)%, respectively. MDR1 and MDR3 shRNA could increase cellular Rh123 accumulation (116.6 +/- 8.1 and 98.4 +/- 3.8, respectively). The late stage apoptosis rates detected by TUNEL displayed the same tendency as FCM results did. The IC(50) for paclitaxel of A2780/taxol cells was decreased significantly. The mRNA levels of MDR1 and MDR3 in A2780/taxol cells were decreased by (73.3 +/- 0.8)% and (51.6 +/- 0.4)% of control, and the reduction of MDR1 and MDR3 mRNA was in a time-dependent manner. The expression of caspase-3 protein of MDR1 and MDR3 shRNA vector transfected group in A2780/taxol cells was significantly increased [(80.8 +/- 2.6)% and (72.0 +/- 4.7)%, respectively]. CONCLUSION: MDR1 and MDR3 gene silencing could recover sensitivity of A2780/taxol cells to paclitaxel and induce cell apoptosis, thus reversing cell resistance to paclitaxel.

[Effects of epidural ropivacaine labor analgesia on duration of labor and mode of delivery].

OBJECTIVE: To study the effects of ropivacaine on the duration of labor and mode of delivery in the primigravidas using patient-controlled epidural analgesia (PCEA). METHODS: Retrospective analysis was performed. The 190 healthy, full-term, and single-fetus parturient primigravidas who received PCEA with 0.1% ropivacaine + fentanyl (1 microg/ml) were in the epidural analgesia group. Another 222 primigravidas who did not receive PCEA were in the control group. The duration of labor and modes of delivery, and the neonatal Apgar scores in both two groups were recorded and evaluated. RESULTS: Those in the epidural analgesia group experienced a significantly longer first stage [(426 +/- 161) minutes], longer second stage [(54 +/- 27) minutes] and longer full duration of delivery [(489 +/- 166) minutes] than those in the control one [(409 +/- 170) minutes, (364 +/- 167) minutes and (37 +/- 22) minutes]. The rate of using pitocin in the epidural analgesia group (30.2%) was significantly higher than that in the control group (4.1%). The cesarean section rate in epidural analgesia group (20.0%) was lower than that in the control one (28.4%); while the rate of instrumental delivery in the epidural analgesia group (20.0%) was significantly higher than that in the control one (6.3%). In summary, there were significant differences between two groups in the duration of labor, the rate of using pitocin, the rate of instrumental delivery and the rate of cesarean section. But there were no differences found for those newborn who had Apgar scores less than 7 at the point of both one and five minutes (7.9% and 4.5%, 2.6% and 0.5% respectively). CONCLUSION: Epidural ropivacaine labor analgesia lengthens the duration of labor and increases the rate of instrumental delivery, but it has no significant negative effects on the neonates.