Effects of bovine pregnancy-associated glycoproteins on gene transcription in bovine endometrial explants

Pregnancy-associated glycoproteins (PAGs) are a complex gene family, whose members are expressed by trophoblasts of ruminants and related species. In cattle, the PAGs accumulate at the trophoblast-uterine interface and many can enter the maternal circulation. However, very little is known about the role they play in pregnancy although preliminary results suggest that PAGs at the placenta-uterine interface play roles involving matrix turnover and immune modulation. This study was designed to provide further insight into the biological roles of bovine PAGs by measuring changes in endometrial transcript abundance for some matrix metalloproteinases (MMPs) and chemokines/cytokines. PAGs for these experiments were purified from mid-gestation bovine placental extracts by affinity chromatography. Heifers were synchronized and bred by artificial insemination with high fertility semen (n = 14) or dead semen (n = 5). Heifers were slaughtered at day 18 post-insemination and the reproductive tracts were obtained and flushed to determine if a conceptus was present. Endometrial explants were collected and split between 4 groups: pregnant with and without 15 [mu]g/ml PAG (n = 10) and nonpregnant with and without 15 [mu]g/ml PAG (n = 9). Endometrial explants were cultured with or without added PAGs for up to 96 hours at 37 degreesC and 5 percent CO2 and samples were harvested at 24 hour intervals for extraction of RNA and fixation. This study focuses on the 48 and 72 hour collection points. Transcript abundance for target genes was analyzed in the endometrial tissue by quantitative PCR. The normalization control transcript was peptidylprolyl isomerase A (PPIA). After 48 and 72 hours, significant increases in CXCL1, CXCL2, and CXCL5 as well as MMP1, MMP3 and MMP13 were measured in the PAG-treated endometrium from both pregnant and non-pregnant animals (P<0.05). CCL11 was upregulated at both time points in the pregnant endometrium but only after 72 hours in the nonpregnant endometrium. There were also significant decreases in message for CCL2, CCL8d CCL16 in the PAG-treated groups from both pregnant and non-pregnant animals at each time point (P<0.05). Significant decreases in CXCL10, CXCL12, and Regakine message were seen only in PAG-treated endometrium from pregnant animals (P<0.05). Structural differences in the luminal and glandular epithelium were seen in the PAG-treated biopsies from both non-pregnant and pregnant heifers. These results suggest that PAGs are capable of inducing structural changes as well as changes in transcript abundance in bovine endometrial explants, which suggests that this model system might be useful to assess PAG function at the placenta-uterine interface.

Screening of Reference Genes for RT-qPCR in Chicken Adipose Tissue and Adipocytes

Reverse transcription quantitative real-time PCR is the most commonly used method to detect gene expression levels. In experiments, it is often necessary to correct and standardize the expression level of target genes with reference genes. Therefore, it is very important to select stable reference genes to obtain accurate quantitative results. Although application examples of reference genes in mammals have been reported, no studies have investigated the use of reference genes in studying the growth and development of adipose tissue and the proliferation and differentiation of preadipocytes in chickens. In this study, GeNorm, a reference gene stability statistical algorithm, was used to analyze the expression stability of 14 candidate reference genes in the abdominal adipose tissue of broilers at 1, 4, and 7 weeks of age, the proliferation and differentiation of primary preadipocytes, as well as directly isolated preadipocytes and mature adipocytes. The results showed that the expression of the TATA box binding protein (TBP) and hydroxymethylbilane synthase (HMBS) genes was most stable during the growth and development of abdominal adipose tissue of broilers, the expression of the peptidylprolyl isomerase A (PPIA) and HMBS genes was most stable during the proliferation of primary preadipocytes, the expression of the TBP and RPL13 genes was most stable during the differentiation of primary preadipocytes, and the expression of the TBP and HMBS genes was most stable in directly isolated preadipocytes and mature adipocytes. These results provide reference bases for accurately detecting the mRNA expression of functional genes in adipose tissue and adipocytes of chickens.

A Critical Role of Peptidylprolyl Isomerase A Pseudogene 22/microRNA-197-3p/Peptidylprolyl Isomerase A Axis in Hepatocellular Carcinoma

The burden of hepatocellular carcinoma (HCC) worldwide is increasing over time, while the underlying molecular mechanism of HCC development is still under exploration. Pseudogenes are classified as a special type of long non-coding RNAs (lncRNAs), and they played a vital role in regulating tumor-associated gene expression. Here, we report that a pseudogene peptidylprolyl isomerase A pseudogene 22 (PPIAP22) and its parental gene peptidylprolyl isomerase A (PPIA) were upregulated in HCC and were associated with the clinical outcomes of HCC. Further investigation revealed that PPIAP22 might upregulate the expression of PPIA through sponging microRNA (miR)-197-3p, behaving as competing endogenous RNA (ceRNA). PPIA could participate in the development of HCC by regulating mRNA metabolic process and tumor immunity based on the functional enrichment analysis. We also found a strong correlation between the expression levels of PPIA and the immune cell infiltration or the expression of chemokines, especially macrophage, C-C motif chemokine ligand 15 (CCL15), and C-X-C motif chemokine ligand 12 (CXCL12). Our findings demonstrate that the PPIAP22/miR-197-3p/PPIA axis plays a vital role in the progression of HCC by increasing the malignancy of tumor cells and regulating the immune cell infiltration, especially macrophage, through CCL15-CCR1 or CXCL12-CXCR4/CXCR7 pathways.

Long non-coding RNA RP11-284F21.9 functions as a ceRNA regulating PPWD1 by competitively binding to miR-769-3p in cervical carcinoma

Cervical carcinoma is the most common gynecological cancer in women worldwide. Emerging evidence has shown that long non-coding RNAs (lncRNAs) participate in multiple biological processes of cervical carcinoma tumorigenesis. We aimed to investigate the function of a novel lncRNA RP11-284F21.9 in cervical carcinoma. We found that RP11-284F21.9 was down-regulated in cervical carcinoma tissues and cell lines. Overexpression of RP11-284F21.9 inhibits proliferation, invasion and migration of cervical carcinoma cells in vitro. Further, we identified that RP11-284F21.9 directly interacted with miR-769-3p and functioned as the miR-769-3p sponge. Mechanistically, we showed that miR-769-3p regulated peptidylprolyl isomerase domain and WD repeat-containing protein1 (PPWD1) expression by targeting PPWD1 3′-UTR. Furthermore, xenograft tumor model revealed that overexpression of RP11-284F21.9 inhibited tumor growth of cervical carcinoma in vivo. Taken together, our results demonstrate that RP11-284F21.9 functions as tumor suppressor and regulates PPWD1 expression through competitively binding to miR-769-3p in cervical carcinoma, suggesting that RP11-284F21.9/miR-769-3p/PPWD1 axis could serve as a promising prognostic biomarker and therapeutic target for cervical carcinoma.

Liraglutide Treatment Ameliorates Neurotoxicity Induced by Stable Silencing of Pin1

Post-translational modulation of peptidylprolyl isomerase Pin1 might link impaired glucose metabolism and neurodegeneration, being Pin1 effectors target for the glucagon-Like-Peptide1 analog liraglutide. We tested the hypotheses in Pin1 silenced cells (SH-SY5Y) treated with 2-deoxy-d-glucose (2DG) and methylglyoxal (MG), stressors causing altered glucose trafficking, glucotoxicity and protein glycation. Rescue by liraglutide was investigated. Pin1 silencing caused increased levels of reactive oxygen species, upregulated energy metabolism as suggested by raised levels of total ATP content and mRNA of SIRT1, PGC1α, NRF1; enhanced mitochondrial fission events as supported by raised protein expression of FIS1 and DRP1. 2DG and MG reduced significantly cell viability in all the cell lines. In Pin1 KD clones, 2DG exacerbated altered mitochondrial dynamics causing higher rate of fission events. Liraglutide influenced insulin signaling pathway (GSK3b/Akt); improved cell viability also in cells treated with 2DG; but it did not revert mitochondrial dysfunction in Pin1 KD model. In cells treated with MG, liraglutide enhanced cell viability, reduced ROS levels and cell death (AnnexinV/PI); and trended to reduce anti-apoptotic signals (BAX, BCL2, CASP3). Pin1 silencing mimics neuronal metabolic impairment of patients with impaired glucose metabolism and neurodegeneration. Liraglutide rescues to some extent cellular dysfunctions induced by Pin1 silencing.

Biological Actions of the Hsp90-binding Immunophilins FKBP51 and FKBP52

Immunophilins are a family of proteins whose signature domain is the peptidylprolyl-isomerase domain. High molecular weight immunophilins are characterized by the additional presence of tetratricopeptide-repeats (TPR) through which they bind to the 90-kDa heat-shock protein (Hsp90), and via this chaperone, immunophilins contribute to the regulation of the biological functions of several client-proteins. Among these Hsp90-binding immunophilins, there are two highly homologous members named FKBP51 and FKBP52 (FK506-binding protein of 51-kDa and 52-kDa, respectively) that were first characterized as components of the Hsp90-based heterocomplex associated to steroid receptors. Afterwards, they emerged as likely contributors to a variety of other hormone-dependent diseases, stress-related pathologies, psychiatric disorders, cancer, and other syndromes characterized by misfolded proteins. The differential biological actions of these immunophilins have been assigned to the structurally similar, but functionally divergent enzymatic domain. Nonetheless, they also require the complementary input of the TPR domain, most likely due to their dependence with the association to Hsp90 as a functional unit. FKBP51 and FKBP52 regulate a variety of biological processes such as steroid receptor action, transcriptional activity, protein conformation, protein trafficking, cell differentiation, apoptosis, cancer progression, telomerase activity, cytoskeleton architecture, etc. In this article we discuss the biology of these events and some mechanistic aspects.

Cyklofiliny – białka o wielu funkcjach

Aktywne formy białek powstają w wyniku translacji oraz zmian zachodzących w trakcie, bądź po tym procesie (fałdowanie, potranslacyjne modyfikacje, kierowanie do odpowiedniego przedziału komórkowego). Struktura przestrzenna białka uzależniona jest od jego sekwencji aminokwasowej co udowodnił Christian Anfinsen, który badając fałdowanie rybonukleazy A, skupił się na odzyskiwaniu aktywności zdenaturowanego enzymu w wyniku prowadzonego procesu renaturacji (nagroda Nobla w dziedzinie chemii w 1972 r. razem z Stanfordem Moore’em i Williamem H. Steinem). Fałdowanie polipeptydów stabilizowane jest i wspomagane przez dwie grupy białek, są to czaperony (nazywane również białkami opiekuńczymi) oraz izomerazy: disulfidoizomerazy (PDI, ang. protein disulfide isomerase, EC 5.3.4.1) i izomerazy peptydyloprolilowe (PPI, ang. peptidylprolyl isomerase EC 5.2.1.8). Czaperony asystują podczas fałdowania białek utrzymując ich stany przejściowe i zapobiegają tworzeniu nieprawidłowych struktur, natomiast izomerazy prowadzą izomeryzację wiązań dwusiarczkowych (PDI) i peptydyloprolilowych (PPI). Praca ta w całościpoświęcona jest charakterystyce cyklofilin, należących do rodziny PPI ze szczególnym uwzględnieniem ich funkcji w procesach związanych z regulacją i patogenezą.