Differential alternative splicing landscape identifies potentially functional RNA binding proteins in early embryonic development in mammals.

Alternative splicing (AS) as one of the important post-transcriptional regulatory mechanisms has been poorly studied during embryogenesis. In this study, we comprehensively collected and analyzed the transcriptome data of early embryos from human and mouse. We found that AS plays an important role in this process and predicted candidate RNA binding protein (RBP) regulators that are associated with reproductive development. The predicted RBPs such as EIF4A3, MAK16, SRSF2, and UTP23 were found to be associated with reproductive disorders. By Smart-seq2 sequencing analysis, we identified 5445 aberrant alternative splicing events in Eif4a3-knockdown embryos. These events were preferentially associated with RNA processing. In conclusion, our work on the landscape and potential function of alternative splicing events will boost further investigation of detailed mechanisms and key factors regulating mammalian early embryo development and promote the inspiration of pharmaceutical approaches for disorders in this crucial biology process.

Activation of HAND2-FGFR signaling pathway by lncRNA HAND2-AS1 in adenomyosis†.

Heart and neural crest derivatives expressed transcript 2 (HAND2) is a critical mediator of progesterone action in endometrial stromal cells. Silencing of Hand2 expression in mouse uterus leads to an unopposed FGFR-mediated action that causes female mice infertility. To investigate the involvement of HAND2-FGFR signaling in pathogenesis of adenomyosis, immunohistochemistry, in situ hybridization, and quantitative real-time PCR were employed to assess gene expression in the normal endometrium, the paired eutopic endometrium and ectopic lesions obtained from women with adenomyosis. DNA methylation in the regions of HAND2 promoter and the first exon was also monitored in these samples. Our results revealed that HAND2 expression were dramatically reduced, but FGF9 expression and FGFR-ERK1/2-mediated MAPK signaling pathway were enhanced in the eutopic endometrium and ectopic lesions of patients with adenomyosis compared to the normal controls. Interestingly, expression of HAND2-AS1, a long noncoding RNA that resides adjacent to HAND2 in genome, was also reduced in adenomyosis. DNA methylation analysis revealed that the bidirectional promoter between HAND2 and HAND2-AS1, and the first exon of HAND2 gene was heavily methylated in the eutopic endometrium and the ectopic lesions of adenomyosis. To investigate the regulation of gene expression by HAND2-AS1, HAND2-AS1 expression was silenced in human endometrial stromal cells. In contrast to the downregulation of HAND2 in response to HAND2-AS1 silencing, FGF9 expression was augmented significantly. Endometrial stromal cells lacking HAND2-AS1 exhibited enhanced proliferation and migration potentials. Collectively, our studies revealed a new molecular mechanism by which HAND2-AS1 is involved in the pathogenesis of adenomyosis via modulating HAND2-FGFR-mediated signaling.

Cerebral delivery of redox-responsive lenalidomide prodrug plus methotrexate for primary central nerve system lymphoma combination therapy.

Primary central nervous system lymphoma (PCNSL) is an extremely malignant CNS tumor with high incidence and mortality rates. Its chemotherapy in the clinic has been restricted owing to unsatisfactory drug distribution in the cerebral tissues. In this study, a redox-responsive prodrug of disulfide-lenalidomide-methoxy polyethylene glycol (LND-DSDA-mPEG) was successfully developed for the cerebral delivery of lenalidomide (LND), and methotrexate (MTX) via subcutaneous (s.c.) administration at the neck for combined anti-angiogenesis and chemotherapy on PCNSL. Both the subcutaneous xenograft tumor model and orthotopic intracranial tumor model demonstrated that the co-delivery of LND and MTX nanoparticles (MTX@LND NPs) may significantly inhibit the growth of lymphoma and effectively prevent liver metastasis by downregulating CD31 and VEGF expression. Moreover, an orthotopic intracranial tumor model further verified that through s.c. administration at the neck, redox-responsive MTX@LND NPs could bypass the blood-brain barrier (BBB), efficiently distribute into brain tissues, and effectively inhibit lymphoma growth in the brain, as detected by magnetic resonance imaging (MRI). Taken together, this biodegradable, biocompatible, and redox-responsive nano-prodrug with highly effective targeted delivery of LND and MTX in the brain through the lymphatic vasculature may provide a facile and feasible treatment strategy for PCNSL in the clinic.

Regulation of HAND2 Expression by LncRNA HAND2-AS1 in Ovarian Endometriosis Involving DNA Methylation.

HAND2 is a critical mediator of progesterone receptor signaling in endometrium. Silencing of HAND2 expression is associated with female infertility and endometrial cancers. We recently observed that lncRNA HAND2-AS1 and HAND2 are expressed coordinately in human endometrial stromal cells. To investigate involvement of HAND2-AS1 and HAND2 in pathogenesis of endometriosis, we employed immunohistochemistry, in situ hybridization, and quantitative real-time PCR to assess their expression in normal endometrium and the ectopic lesions obtained from patients with ovarian endometriosis. HAND2 promoter methylation was also monitored in these samples. Our results revealed that HAND2 and HAND2-AS1 expression levels were reduced but promoter methylation was enhanced significantly in ectopic endometrium when compared with the normal controls. Fluorescence in situ hybridization showed that HAND-AS1 is predominantly localized in the nuclei of endometrial stromal cells in contrast to the cytoplasmic distribution in epithelial cell compartment. To further investigate regulation of HAND2 expression by HAND2-AS1, HAND2-AS1 was silenced or overexpressed in human endometrial stromal cells. Our studies showed that expression levels of HAND2 and its direct target IL15 were attenuated markedly in HAND2-AS1 silenced cells but enhanced significantly in the overexpressed human endometrial stromal cells. Silencing of HAND2-AS1 also impaired endometrial stromal cell decidualization as indicated by downregulation of decidual biomarkers IGFBP1 and PRL. In addition, HAND2 promoter methylation was also enhanced upon HAND2-AS1 silencing. RNA immunoprecipitation studies further revealed that HAND2-AS1 is capable of binding to DNA methyltransferase DNMT1, indicating that HAND2-AS1 governs HAND2 expression epigenetically involving DNA methylation.

A 1-kb human CDCA8 promoter directs the spermatogonia-specific luciferase expression in adult testis.

Cell division cycle associated 8 (CDCA8) is a component of the chromosomal passenger complex and plays an essential role in mitosis, meiosis, cancer growth, and undifferentiated state of embryonic stem cells. However, its expression and role in adult tissues remain largely uncharacterized. Here, we studied the CDCA8 transcription in adult tissues by generating a transgenic mouse model, in which the luciferase was driven by a 1-kb human CDCA8 promoter. Our previous study showed that this 1-kb promoter was active enough to dictate reporter expression faithfully reflecting endogenous CDCA8 expression. Two founder mice carrying the transgene were identified. In vivo imaging and luciferase assays in tissue lysates revealed that CDCA8 promoter was highly activated and drove robust luciferase expression in testes. Subsequently, immunohistochemical and immunofluorescent staining showed that in adult transgenic testes, the expression of luciferase was restricted to a subset of spermatogonia that were located along the basement membrane and positive for the expression of GFRA1, a consensus marker for early undifferentiated spermatogonia. These findings for the first time indicate that the CDCA8 was transcriptionally activated in testis and thus may play a role in adult spermatogenesis. Moreover, the 1-kb CDCA8 promoter could be used for spermatogonia-specific gene expression in vivo and the transgenic lines constructed here could also be used for recovery of spermatogonia from adult testes.

Neddylation Inhibition Causes Impaired Mouse Embryo Quality and Blastocyst Hatching Failure Through Elevated Oxidative Stress and Reduced IL-1ß.

Mammalian blastocyst hatching is an essential prerequisite for successful embryo implantation. As the rate-limiting step of current assisted reproductive technology, understanding the key factors regulating blastocyst hatching would be significantly helpful to improve the performance of the assisted reproductive practice. In early embryo development, the fine-tuned elimination of maternal materials and the balanced protein turnover are inevitable for the competent to hatch and implant into endometrium. Neddylation, a ubiquitination-like protein modification, has been shown to be involved in oocyte maturation and early embryo development. In this study, aiming to discover an unknown role of neddylation in the blastocyst hatching process, we provided functional evidence of neddylation in mammalian embryo quality and blastocyst hatching. Treatment with MLN4924, a specific neddylation inhibitor, lowered the embryo quality and dramatically reduced the hatching rate in mouse blastocysts. The transcriptional profile showed the upregulation of oxidative stress-related genes and aberrant expression of immune-related genes. The elevated oxidative stress was validated by qPCR and markers of apoptosis, DNA damage, reactive oxygen species, and cytoskeleton. Moreover, we found the secreted IL-1ß level was reduced in an NF-κB-independent manner, leading to the final poor embryo quality and blastocyst hatching failure. This is the first report of neddylation being of great importance in the mammalian blastocyst hatching process. Further investigations uncovering more detailed molecular mechanisms of neddylation regulation in blastocyst hatching would greatly promote not only the understanding of this crucial biological process but also the clinical application in reproductive centers.

The Influence of Heavy Metals on Gastric Tumorigenesis.

Objectives: This study aimed to observe the relationship among heavy metals concentration, microsatellite instability (MSI), and human epidermal growth factor receptor type 2 (HER2) gene amplification in gastric cancer (GC) patients. Methods: The concentrations of 18 heavy metals in the plasma of GC patients and healthy controls were measured by inductive coupled plasma emission spectrometry (ICP-MS). MSI detection was conducted by detecting 5 microsatellite repeat markers by PCR analysis. HER2 gene amplification was detected by fluorescence in situ hybridization (FISH). The relationship among heavy metal elements, tumor biomarkers, HER2 amplification, and MSI status was analyzed by Pearson correlation analysis. Results: A total of 105 GC patients and 62 healthy controls were recruited in this study. The concentration of arsenic (As), chromium (Cr), cuprum (Cu), mercury (Hg), manganese (Mn), lead (Pb), stibium (Sb), selenium (Se), stannum (Sn), strontium (Sr), thallium (Tl), vanadium (V), and zinc (Zn) were significantly different between GC patients and controls. Among 105 GC patients, including 87 microsatellite-stable (MSS) samples and 18 MSI samples, the concentration of Ga is significantly higher in the MSI group than that in the MSS group. Meanwhile, in 97 GC patients having detected HER2 gene amplification, 69 of 97 had negative HER2 gene amplification and the rest 28 GC patients had positive HER2 gene amplification. The concentration of Hg, Sn, and Tl is noticeably higher in the HER2 positive group than in the HER2 negative group. Only Sb was positively correlated with MSI, but none of these heavy metals was correlated with HER2 gene amplification. Conclusions: The results indicated that Sb has significant positive correlation with the MSI status, which suggests that Sb may cause MSI in GC. However, further research studies are required to elucidate the mechanisms in the near feature.

Target-Sequencing of Female Infertility Pathogenic Gene Panel and a Novel TUBB8 Loss-of-Function Mutation.

Genetic screening is an important approach for etiology determination and helps to optimize administration protocols in reproductive centers. After the first pathogenic gene of female infertility was reported in 2016, more and more new pathogenic genes were discovered, and we sought to develop an efficient and cost-effective method for genetic screening in patients. In this study, we designed a target-sequencing panel with 22 female infertility-related genes, namely, TUBB8, PATL2, WEE2, and PANX1 and sequenced 68 primary infertility (PI) and recurrent pregnancy loss (RPL) patients. We sequenced 68 samples reaching an average depth of 1559× and detected 3,134 variants. Among them, 62.2% were synonymous single-nucleotide variants (SNVs) and 36.3% were non-synonymous SNVs. The remaining 1.5% are indels (insertions and deletions) and stop-gains. DNAH11 and TUBB8 are the two genes that mutated most frequently. We also found a novel TUBB8 variant (c.898_900del; p.300_300del), proved its loss-of-function mechanism, and profiled the interactome of the wild-type (WT) and mutant TUBB8 proteins. Overall, this target-sequencing method provides an efficient and cost-effective approach for screening in IVF clinics and will support researchers for the discovery of new pathogenic variants.

Clinical Effect of Nimodipine Combined with Magnesium Sulfate on Pregnancy-Induced Hypertension Syndrome.

The incidence of pregnancy-induced hypertension in China is 9.4%, which is at a relatively high level. Its serious impact on maternal and infant health is the main reason for maternal and perinatal morbidity and mortality. There are many factors affecting pregnancy-induced hypertension. The incidence of pregnancy-induced hypertension is different due to different levels of cultural knowledge, health awareness, economic income, nutrition, and medical support. Since its etiology has not been elucidated thus far, there is no known treatment of the disease, and the main principles are spasmolysis, hypotension, expansion, and timely termination of pregnancy. Observe the effect of nimodipine combined with magnesium sulfate on serum heat shock protein 70 (HSP70) and pentamer 3 (PTX3) levels in patients with pregnancy-induced hypertension. Ninety-six patients with pregnancy-induced hypertension syndrome admitted to our hospital from May 2016 to February 2019 are selected and randomly divided into two groups according to the 1 : 1 principle, with 48 cases in each group. The single drug group is treated with magnesium sulfate, and the combined group is treated with nimodipine combined with magnesium sulfate. Changes in blood pressure, HSP70, PTX3, placental growth factor (PLGF), and vascular endothelial cell injury markers are recorded in the two groups, and adverse reactions and pregnancy outcomes are observed. After treatment, the blood pressure and levels of HSP70, PTX3, endothelin-1 (ET-1), and nitric oxide (NO) in the two groups decreased, and the level of PLGF increased. The diastolic blood pressure, systolic blood pressure, and levels of HSP70, PTX3, ET-1, and NO in the combined group are lower than those in the single drug group, and the level of PLGF is higher than that in the single drug group (P < 0.05). During the treatment period, the adverse reaction rate of the combined group is 6.25% compared with 8.33% of the single agent group, and the difference is not statistically significant (P > 0.05). Follow-up visits found that the cesarean section rate and abnormal fetal heart rate in the combined group are 16.67% and 4.17%, respectively, which are lower than 35.42% and 16.67% in the single drug group, and the difference is statistically significant (P < 0.05). Compared with 14.58%, 12.50%, and 2.08% in the single drug group, the neonatal asphyxia rate, premature birth rate, and stillbirth rate in the combined group are 6.25%, 4.17%, and 0.00%, respectively, and the difference is not statistically significant (P > 0.05). Nimodipine combined with magnesium sulfate can effectively control blood pressure in patients with pregnancy-induced hypertension, reduce vascular endothelial damage, regulate the expression of HSP70, PTX3, and PLGF, and improve pregnancy outcomes without increasing adverse reactions.

Redox-responsive self-assembled polymeric nanoprodrug for delivery of gemcitabine in B-cell lymphoma therapy.

Gemcitabine, as a standard and classic strategy for B-cell lymphoma in the clinic, is limited by its poor pharmacodynamics. Although stimuli-responsive polymeric nanodelivery systems have been widely investigated in the past decade, issues such as complicated procedures, low loading capacity, and uncontrollable release kinetics still hinder their clinical translation. In view of the above considerations, we attempt to construct hyperbranched polyprodrug micelles with considerable drug loading via simple procedures and make use of the particularity of the tumor microenvironment to ensure that the micelles are "inactivated" in normal tissues and "activated" in the tumor microenvironment. Hence, in this work, a redox-responsive polymeric gemcitabine-prodrug (GEM-S-S-PEG) was one-pot synthesized via facile esterification and acylation. The self-assembled subsize (< 100 nm) GEM-S-S-PEG (GSP NPs) with considerable loading capacity (≈ 24.6%) exhibited on-demand and accurate control of gemcitabine release under a simulated tumor microenvironment and thus significantly induced the apoptosis of B-cell lymphoma in vitro. Moreover, in the A20 tumor xenograft murine model, GSP NPs efficiently decreased the expansion of tumor tissues with minimal systemic toxicity. In summary, these redox-responsive and self-assembling GSP NPs with a facile one-pot synthesis procedure may hold great potency in clinical translation for enhanced chemotherapy of B-cell lymphoma. STATEMENT OF SIGNIFICANCE: A redox-responsive polymeric gemcitabine-prodrug (GEM-S-S-PEG) was one-pot synthesized via facile esterification and acylation. The self-assembled subsize (< 100 nm) GEM-S-S-PEG (GSP NPs) exhibited significant tumor therapeutic effects in vitro and in vivo. The polyprodrug GEM-S-S-PEG prepared in this study shows the great potential of redox-responsive nanodrugs for antitumor activity, which provides a reference value for the optimization of the design of functional polyprodrugs.

Effects of the Zishen Yutai Pill Compared With Placebo on Live Births Among Women in a Fresh Embryo Transfer Cycle: A Randomized Controlled Trial.

OBJECTIVE: To assess the efficacy of the Zishen Yutai Pill compared with placebo on live birth rates among women after fresh embryo transfer cycles. METHODS: We conducted a double-blind, multicenter, placebo-controlled, randomized trial to investigate whether administration of the Zishen Yutai Pill would improve pregnancy outcomes among women undergoing fresh embryo transfer after in vitro fertilization or intracytoplasmic sperm injection. The primary outcome was live birth rate. Secondary outcomes were rates of implantation, biochemical pregnancy, clinical pregnancy, pregnancy loss, cycle cancellation, and maternal, fetal, and neonatal complications. A total sample size of 2,265 women (1:1 in two groups) was used to detect a live birth rate difference between the Zishen Yutai Pill and placebo. Participants were enrolled and randomized to receive 5 g of the Zishen Yutai Pill or placebo orally, three times per day during the study. RESULTS: Recruitment was completed between April 2014 and June 2017, with 2,580 patients screened. Two thousand two hundred sixty-five patients were randomized: 1,131 to the Zishen Yutai Pill and 1,134 to placebo. Characteristics were similar between groups. In intention-to-treat analysis, the rates of live birth in the Zishen Yutai Pill (ZYP) group and placebo group were 26.8% and 23.0% (rate ratio [RR], 1.16; 95% CI 1.01-1.34; P=.038), respectively. The implantation rates were 36.8% and 32.6% in the ZYP and placebo groups, respectively (RR 1.13; 95% CI 1.01-1.25; P=.027). The biochemical pregnancy rate for the ZYP group was 35.5% compared with 31.1% in the placebo group (RR 1.14; 95% CI 1.02-1.28; P=.026). The rates of clinical pregnancy in the ZYP and placebo groups were 31.2% compared with 27.3%, respectively (RR 1.14; 95% CI 1.00-1.30; P=.043). There were no significant between-group differences in the rates of pregnancy loss, maternal, or neonatal complications (all P>.05). CONCLUSION: The Zishen Yutai Pill increased the rate of live birth after fresh embryo transfer compared with placebo. CLINICAL TRIAL REGISTRATION: Chictr.org.cn, Chictr-TRC-14004494.

A ratiometric fluorescent nanoprobe for signal amplification monitoring of intracellular telomerase activity.

Telomerase is considered a valuable diagnostic and prognostic cancer biomarker. Accurate and reliable detection of telomerase activity is of great value in clinical diagnosis, screening of inhibitors, and therapeutics. Here, we developed a novel amplified fluorescence resonance energy transfer (FRET) nanoprobe for highly sensitive and reliable monitoring of intracellular telomerase activity. The nanoprobe (QDSA@DNA) was composed of a streptavidin-modified quantum dot (QDSA) which was functionalized with a telomerase primer sequence (TP) and Cy5-tagged signal switching sequence (SS) through biotin-streptavidin interaction. When the nanoprobe was assembled, the Cy5 was in close proximity to the QDSA, resulting in high FRET efficiency from the QDSA to Cy5. In the presence of telomerase, the TP could be extended to produce telomeric repeat units, which was complementary to the loop of SS. Thus, the SS could hybridize with elongated sequences to form a rigid double-stranded structure, which forced the Cy5 away from the surface of the QDSA, causing low FRET efficiency. Furthermore, due to the production of multiple repeat units by telomerase, multiple hairpin structures could be opened, yielding significant fluorescence ratio (FQDsa/FCy5) enhancement for sensing of telomerase activity. In this way, the combination of a FRET and target-assisted strategy in a nanoprobe improved the detection accuracy and amplified the detection signal, respectively. The QDSA@DNA nanoprobe also showed high selectivity, excellent nuclease stability, and good biocompatibility. More importantly, this nanoprobe was found to be an excellent platform for efficient monitoring of intracellular telomerase activity, providing a potential platform in tumor diagnosis and screening of telomerase-related inhibitors.

[Impact of the expression of DNA methyltransferase 1 on the differentiation of spermatogonial stem cells in mice].

OBJECTIVE: To investigate the influence of the expression of DNA methyltransferase 1 (DNMT1) on the differentiation of spermatogonial stem cells (SSC) in mice. METHODS: SSCs were isolated from the testis tissue of 1-week-old BALB/c male mice by two-step enzyme digestion. DNMT1-siRNA and negative control siRNA (NC-siRNA) were transfected into the third-generation SSCs after isolation and purification, and the untransfected cells were used as the control. At 24 hours after transfection, the mRNA and protein expressions of DNMT1 were detected by real-time quantitative PCR (RT-qPCR) and Western blot, respectively, and the methylation level of DNMT1 was determined. The SSCs were induced to differentiate into spermatocytes using the stem cell growth factor, and the expressions of the germ cell proliferation-related protein (Nanos2), promyelocytic leukemia zinc finger protein (PLZF) and retinoic acid-stimulated protein 8 (Stra8) were measured by RT-qPCR and Western blot after 48 hours of differentiation. RESULTS: At 24 hours after transfection, the relative mRNA and protein expressions of DNMT1 and the DNA methylation level were significantly decreased in the DNMT1-siRNA group compared with those in the control and DNMT1-NC groups (P < 0.05), but showed no statistically significant difference between the latter two (P > 0.05). The relative mRNA and protein expressions of Nanos2 and PLZF were also decreased while those of Stra8 increased in the DNMT1-siRNA group in comparison with those in the control and DNMT1-NC groups after 48 hours of differentiation (P < 0.05), but none exhibited any statistically significant difference between the control and DNMT1-NC groups (P > 0.05). CONCLUSION: Knockdown of DNMT1 promotes the differentiation of SSCs into spermatocytes in mice, which may be related to the reduction of the genome methylation level, inhibition of the expressions of Nanos2 and PLZF, and promotion of the expression of Stra8.

Clinical Significance of Monitoring Circulating Free DNA and Plasma Heat Shock Protein 90alpha in Patients with Esophageal Squamous Cell Carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the predominant histological type of esophageal cancer in China and has an extremely poor prognosis. Circulating free DNA (cfDNA) and plasma heat shock protein 90alpha (Hsp90a) are two novel noninvasive biomarkers for diagnosis and prognostic prediction of several types of cancer. However, to the best of our knowledge, the roles of the two biomarkers in ESCC are still unknown. METHODS: Here, we recruited 93 primary ESCC patients and detected plasma concentrations of the two markers at different time points, including 1-3 days pre-chemotherapy, 1-7 days pre-surgery and 7-14 days post-surgery. Baseline concentrations of the two markers were associated with main characteristics of ESCC patients which were collected at first diagnosis. Correlation between the two markers and traditional serum biomarkers at baseline was also examined. Furthermore, dynamic changes of the cfDNA and Hsp90α concentrations among different time points and the potential clinical significance were assessed. RESULTS: Consequently, there was no significant association between baseline concentrations of the two markers and clinical features. Especially, cfDNA demonstrated stronger correlation with other circulating biomarkers than Hsp90α at baseline level. Importantly, both cfDNA and Hsp90α concentrations were significantly increased after surgery. Kaplan-Meier survival analysis showed that a change in concentration of cfDNA (ΔcfDNA) but not Hsp90α (ΔHSP90ɑ) between pre-surgery and post-surgery had significant effect on the overall survival of surgical patients with ESCC. CONCLUSION: Thus, ΔcfDNA evaluation could be a promising prognostic marker for surgical ESCC patients. Our findings may improve the understanding of the function of cfDNA and Hsp90α in ESCC.

Downregulation of miR-200a Protects Mouse Leydig Cells Against Triptolide by Triggering Autophagy.

BACKGROUND: MicroRNAs play important roles in testicular development and spermatogenesis. Previous research has indicated that the level of miR-200a was significantly upregulated in patients with different spermatogenic impairments. However, the mechanism by which miR-200a regulated spermatogenic impairments remains unclear. METHODS: Leydig cells were treated with triptolide (TP) to mimic spermatogenic impairments. CCK-8 and flow cytometry were used to detect the proliferation and apoptosis in Leydig cells, respectively. In addition, Western blot assay was used to examine ATG7, ATG5, p62 protein levels in MLTC-1 cells. RESULTS: TP dose-dependently upregulated the expression of miR-200a in MLTC-1 cells. In addition, TP inhibited the proliferation of MLTC-1 cells via inducing apoptosis and oxidative stress; however, these phenomena were notably reversed by miR-200a antagomir. Furthermore, luciferase reporter assay identified that ATG7 was the direct binding target of miR-200a. TP treatment markedly inhibited the activation of autophagy in MLTC-1 cells via inhibition of ATG7. Conversely, downregulation of miR-200a significantly induced autophagy in TP-treated MLTC-1 cells by activation of ATG7. Meanwhile, the cell protective effects of miR-200a against TP were reversed by autophagy inhibitor 3MA, indicating that autophagy plays an important role. CONCLUSION: These results indicated that downregulation of miR-200a could protect MLTC-1 cells against TP by inducing autophagy. Therefore, miR-200a might serve as a new therapeutic target for the treatment of male hypogonadism.

Overcoming therapeutic failure in osteosarcoma via Apatinib-encapsulated hydrophobic poly(ester amide) nanoparticles.

Anti-angiogenic tyrosine kinase inhibitors (TKIs) have been proved to be effective in prolonging progression-free survival in advanced osteosarcoma. However, osteosarcoma stem-like cells persist for a long time and ultimately cause disease recurrence and therapy resistance. Here, we reveal that inefficient accumulation of Apatinib, an anti-angiogenic TKI, induces the expression of ribosome-associated genes in osteosarcoma, and confers apoptosis resistance. An engineered nanoscale delivery system based on hydrophobic poly(ester amide) has been established to effectively deliver Apatinib to improve the treatment. Notably, the considerable uptake by osteosarcoma cells enables this nanodrug to distribute increasingly inside the tumor. Furthermore, the delivered nano-Apatinib can suppress osteosarcoma stemness and enhance osteosarcoma stem-like cell apoptosis, and overcomes the crucial bottleneck of the unfavorable stem-like cell residue for TKI therapy. Importantly, nano-Apatinib significantly inhibits the osteosarcoma stem-like cell-derived tumor growth in contrast with free Apatinib, with minimal side effects. These results suggest that this Apatinib-loaded nano delivery system may serve as a promising strategy to solve the issue of TKI therapeutic resistance existing in advanced osteosarcoma.

Hepatic HuR modulates lipid homeostasis in response to high-fat diet.

Lipid transport and ATP synthesis are critical for the progression of non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms are largely unknown. Here, we report that the RNA-binding protein HuR (ELAVL1) forms complexes with NAFLD-relevant transcripts. It associates with intron 24 of Apob pre-mRNA, with the 3'UTR of Uqcrb, and with the 5'UTR of Ndufb6 mRNA, thereby regulating the splicing of Apob mRNA and the translation of UQCRB and NDUFB6. Hepatocyte-specific HuR knockout reduces the expression of APOB, UQCRB, and NDUFB6 in mice, reducing liver lipid transport and ATP synthesis, and aggravating high-fat diet (HFD)-induced NAFLD. Adenovirus-mediated re-expression of HuR in hepatocytes rescues the effect of HuR knockout in HFD-induced NAFLD. Our findings highlight a critical role of HuR in regulating lipid transport and ATP synthesis.

MicroRNA­181 exerts an inhibitory role during renal fibrosis by targeting early growth response factor­1 and attenuating the expression of profibrotic markers.

Progressive renal fibrosis is a common complication of chronic kidney disease that results in end­stage renal disorder. It is well established that several microRNAs (miRs) function as critical regulators implicated in fibrotic diseases. However, the role of miR­181 in the development and progression of renal fibrosis remains unclear, and the precise mechanism has not yet been fully defined. The present study identified the functional implications of miR­181 expression during renal fibrosis. miR­181 exhibited significantly reduced expression in the serum of renal fibrosis patients and in the kidneys of mice with unilateral ureteral obstruction (UUO). In addition, miR­181 downregulated the expression of human α­smooth muscle actin (α­SMA) in response to angiotensin II stimulation. Transfection with miR­181 mimics significantly suppressed the expression levels of α­SMA, connective tissue growth factor, collagen type I α1 (COL1A1) and collagen type III α1 (COL3A1) in NRK49F cells. Notably, early growth response factor­1 (Egr1) was identified as a direct target gene of miR­181. Furthermore, in vivo experiments revealed that treatment with miR­181 agonist strongly rescued kidney impairment induced by UUO, as supported by Masson's trichrome staining of kidney tissues and reverse transcription­quantitative polymerase chain reaction analysis of COL1A1 and COL3A1 mRNA levels. Therefore, miR­181 may be regarded as an important mediator in the control of profibrotic markers during renal fibrosis via binding to Egr1, and may be a promising new target in the diagnosis and therapy of renal fibrosis.

Overexpression of RASAL1 Indicates Poor Prognosis and Promotes Invasion of Ovarian Cancer.

RAS protein activator like-1 (RASAL1) exists in numerous human tissues and has been commonly demonstrated to act as a tumor suppressor in several cancers. This study aimed to identify the functional characteristics of RASAL1 in ovarian adenocarcinoma and a potential mechanism of action. We analyzed RASAL1 gene expression in ovarian adenocarcinoma samples and normal samples gained from the GEO and Oncomine databases respectively. Then the relationship between RASAL1 expression and overall survival (OS) was assessed using the Kaplan-Meier method. Furthermore, the biological effect of RASAL1 in ovarian adenocarcinoma cell lines was assessed by Quantitative real time-PCR (qRT-PCR), Cell Counting Kit-8 (CCK-8), western blot, wound healing and transwell assay. The statistical analysis showed patients with higher RASAL1 expression correlated with worse OS. The in vitro assays suggested knockdown of RASAL1 could inhibit cell proliferation, cell invasion and migration of ovarian adenocarcinoma. Moreover, the key proteins in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathway were also decreased in ovarian adenocarcinoma cells with RASAL1 silencing. These findings provide promising evidence that RASAL1 may be not only a powerful biomarker but also an effective therapeutic target of ovarian adenocarcinoma.

Ovarian IL-1α and IL-1ß levels are associated with primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is characterized by premature ovarian failure and lack of menstrual periods in women under the age of 40. Ample evidence suggests that interleukin 1 (IL-1) family proteins are involved in the processes of ovarian follicular development and depletion. The association of these cytokines with POI disorder and the underlying molecular mechanism in regulation of the ovarian functions remain largely undetermined. In this study, follicular fluids and serum samples were harvested from POI patients and healthy women who underwent follicular aspiration during in vitro fertilization. Enzyme-linked immunosorbent assay (ELISA) and the quantitative real-time PCR (qPCR) were employed to assess the levels of protein secretion and mRNA expression of IL-1α and IL-1ß, as well as the molecules that are critical for apoptotic pathways. Our results showed that IL-1α level in serum samples and follicular fluid of POI patients was significantly elevated in comparison to that in healthy women. Interestingly, the follicular levels of both IL-1α and IL-1ß and TNF-α were significantly higher than their serum levels. Our qPCR analysis further revealed that there was a significant upregulation of apoptotic Bax mRNA expression, but expression of anti-apoptotic factor Bcl-2 mRNA expression was downregulated in POI patients. In conclusion, our studies revealed that the elevated level of IL-1 in follicle fluids of POI patients may be the major causal factor for follicular apoptosis that consequently impairs follicle reserve and ovarian functions by follicle depletion.