Kaposi's sarcoma-associated herpesvirus viral protein kinase augments cell survival.

Oncogenic viruses have developed various strategies to antagonize cell death and maintain lifelong persistence in their host, a relationship that may contribute to cancer development. Understanding how viruses inhibit cell death is essential for understanding viral oncogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three different cancers in the human population, including Kaposi's sarcoma (KS), the most common cancer in HIV patients. Previous studies have indicated that the KSHV-encoded viral protein kinase (vPK) impacts many processes dysregulated in tumorigenesis. Here, we report that vPK protects cells from apoptosis mediated by Caspase-3. Human umbilical vein endothelial cells (HUVECs) expressing vPK (HUVEC-vPK) have a survival advantage over control HUVEC under conditions of extrinsic- and intrinsic-mediated apoptosis. Abolishing the catalytic activity of vPK attenuated this survival advantage. We found that KSHV vPK-expressing HUVECs exhibited increased activation of cellular AKT kinase, a cell survival kinase, compared to control cells without vPK. In addition, we report that vPK directly binds the pleckstrin homology (PH) domain of AKT1 but not AKT2 or AKT3. Treatment of HUVEC-vPK cells with a pan-AKT inhibitor Miransertib (ARQ 092) reduced the overall phosphorylation of AKT, resulting in the cleavage of Caspase-3 and the induction of apoptosis. Furthermore, vPK expression activated VEGF/VEGFR2 in HUVECs and promoted angiogenesis through the AKT pathway. vPK expression also inhibited the cytotoxicity of cisplatin in vitro and in vivo. Collectively, our findings demonstrate that vPK's ability to augment cell survival and promote angiogenesis is critically dependent on AKT signaling, which is relevant for future therapies for treating KSHV-associated cancers.

Reduced Vitellogenesis and Female Fertility in Gper Knockout Zebrafish.

The role G-protein coupled estrogen receptor (GPER) plays in vertebrate reproduction remains controversial. To investigate GPER's reproductive role, we generated a gper zebrafish mutant line (gper-/- ) using TALENs. Gper mutant females exhibited reduced fertility with a 40.85% decrease in embryo production which was associated with a significant decrease in the number of Stage V (730-750 µm) ovulated oocytes. Correspondingly, the number of early vitellogenic follicles (Stage III, 400-450 µm) in gper-/- ovaries was greater than that in wildtypes (wt), suggesting that subsequent follicle development was retarded in the gper-/- fish. Moreover, plasma vitellogenin levels were decreased in gper-/- females, and epidermal growth factor receptor (Egfr) expression was lower in Stage III vitellogenic oocytes than in wt counterparts. However, hepatic nuclear estrogen receptor levels were not altered, and estrogen levels were elevated in ovarian follicles. These results suggest that Gper is involved in the control of ovarian follicle development via regulation of vitellogenesis and Egfr expression in zebrafish.

Impaired oocyte maturation and ovulation in membrane progestin receptor (mPR) knockouts in zebrafish.

Accumulating evidence suggest that membrane progestin receptor α (mPRα) is the membrane receptor mediating nongenomic progestin signaling that induces oocyte maturation in teleost. However, the involvement of other members of mPR family in oocyte maturation is still unclear. In this study, we found impaired oocyte maturation in zebrafish lacking mPRα1, mPRα2, mPRß, or mPRγ2. In contrast, no difference was observed in oocyte maturation in the single knockout of mPRγ1, mPRδ, or mPRε. To study possible redundant functions of different mPRs in oocyte maturation, we generated a zebrafish line lacking all seven kinds of mPRs (mprs-/-). We found oocyte maturation was further impaired in mprs-/-. In addition, oocyte ovulation delay was observed in mprs-/- females, which was associated with low levels of nuclear progestin receptor (Pgr), a key regulator for ovulation. We also found reduced fertility in mprs-/- female zebrafish. Furthermore, eggs spawned by mprs-/- females were of poor quality.

Downregulation of nuclear progestin receptor (Pgr) and subfertility in double knockouts of progestin receptor membrane component 1 (pgrmc1) and pgrmc2 in zebrafish.

The progestin receptor membrane components (Pgrmcs) contain two paralogs, Pgrmc1 and Pgrmc2. Our previous research into single knockout of Pgrmc1 or Pgrmc2 suggests that Pgrmc1 and Pgrmc2 regulate membrane progestin receptor or steroid synthesis and therefore female fertility in zebrafish. Additional roles of Pgrmcs may not be determined in using single Pgrmc knockouts due to compensatory roles between Pgrmc1 and Pgrmc2. To address this question, we crossed single knockout pgrmc1 (pgrmc1-/-) with pgrmc2 (pgrmc2-/-), and generated double knockouts for both pgrmc1 and pgrmc2 (pgrmc1/2-/-) in a vertebrate model, zebrafish. In addition to the delayed oocyte maturation and reduced female fertility, significant reduced ovulation was found in double knockout (pgrmc1/2-/-) in vivo, though not detected in either single knockout of Pgrmc (pgrmc1-/- or pgrmc2-/-). We also found significant down regulation of nuclear progestin receptor (Pgr) protein expression only in pgrmc1/2-/-, which was most likely the cause of reduced ovulation. Lower protein expression of Pgr also resulted in reduced expression of metalloproteinase in pgrmc1/2-/-. With this study, we have provided new evidence for the physiological functions of Pgrmcs in the regulation of female fertility by regulation of ovulation, likely via regulation of Pgr, which affects regulation of metalloproteinase expression and oocyte ovulation.

Subfertility and reduced progestin synthesis in Pgrmc2 knockout zebrafish.

Progestin receptor membrane component (Pgrmc1 & 2) is a heme-binding protein. Studies on Pgrmc1 have suggested possible roles in heme binding, activation of steroid-synthesizing P450s, along with binding and transferring of membrane proteins. However, the studies of Pgrmc1's paralog, Pgrmc2 are still lacking. In order to determine the physiologic function(s) of Pgrmc2, we generated a zebrafish mutant line (pgrmc2-/-). We found a reduction in both spawning frequency and the number of embryos produced in female pgrmc2-/-. This subfertility is caused by reduced oocyte maturation (germinal vesicle breakdown, GVBD) in pgrmc2-/- in vivo. Nonetheless, oocytes from pgrmc2-/- had similar sensitivity to 17α,20ß-dihydroxy-4-pregnen-3-one (DHP, a maturation induced progestin in zebrafish) compared with wildtype (wt) in vitro. Therefore, we hypothesized that oocyte maturation tardiness found in vivo, could be due to lack of progestin in pgrmc2-/-. Interestingly, we found significant reduced expression of hormones, receptors, and steroid synthesizing enzymes including lhcgr, egfra, ar, and esr2, cyp11a1 and hsd3b1. In addition, DHP levels in pgrmc2-/- ovaries showed a significant decrease compared to those in wt. In summary, we have provided a plausible molecular mechanism for the physiological functions of Pgrmc2 in the regulation of female fertility, likely via regulation of receptors and steroids in the ovary, which in turn regulates oocyte maturation in zebrafish.

Progestin and Nuclear Progestin Receptor Are Essential for Upregulation of Metalloproteinase in Zebrafish Preovulatory Follicles.

Ovulation requires proteinases to promote the rupture of ovarian follicles. However, the identity of these proteinases remains unclear. In our previous studies using RNA-seq analysis of differential expressed genes, we found significant down-regulation of five metalloproteinases: adam8b (a disintegrin and metalloproteinase domain 8b), adamts8a (a disintegrin and metalloproteinase with thrombospondin motif 8a), adamts9, mmp2 (matrix metalloproteinase 2), and mmp9 in the nuclear progestin receptor knockout (pgr -/-) zebrafish that have failed to ovulate. We hypothesize that these metalloproteinases are responsible for ovulation and are regulated by progestin and Pgr. In this study, we first determined the expression of these five metalloproteinases and adamts1 in preovulatory follicles at different times within the spawning cycle in pgr -/- and wildtype (wt) zebrafish and under varying hormonal treatments. We found that transcripts of adam8b, adamts1, adamts9, and mmp9 increased drastically in the preovulatory follicular cells of wt female zebrafish, while changes of adamts8a and mmp2 were not significant. This increase of adam8b, adamts9, and mmp9 was significantly reduced in pgr -/-, whereas expression of adamts1 was not affected in pgr -/- zebrafish. Among upregulated metalloproteinases, adamts9 mRNA was found to be expressed specifically in follicular cells. Strong immunostaining of Adamts9 protein was observed in the follicular cells of wt fish, and this expression was reduced drastically in pgr -/-. Interestingly, about an hour prior to the increase of metalloproteinases in wt fish, both Pgr transcript and protein increased transiently in preovulatory follicular cells. The results from in vitro experiments showed that adamts9 expression markedly increased in a dose, time and Pgr-dependent manner when preovulatory follicles were exposed to a progestin, 17α,20ß-dihydroxy-4-pregnen-3-one (DHP). Taken together, our results provide the first evidence that upregulation of adamts9 occurs specifically in preovulatory follicular cells of zebrafish prior to ovulation. Progestin and its receptor (Pgr) are essential for the upregulation of metalloproteinases.

Pgrmc1 Knockout Impairs Oocyte Maturation in Zebrafish.

Recent investigations suggest progestin receptor membrane component 1 (PGRMC1) associates with and transports a wide range of molecules such as heme, cytochromes P450, steroids with 21 carbons, membrane progestin receptor alpha (mPRα/Paqr7), epidermal growth factor receptor (EGFR), and insulin receptor. It is difficult to discriminate the true functions of PGRMC1 from the functions of its associated molecules using biochemical and pharmacological approaches. To determine the physiological function(s) of PGRMC1, we generated global knockouts for pgrmc1 (pgrmc1 -/-) in zebrafish. We found a reduction in both spawning frequency and the number of embryos produced by female mutants. We also observed reduced sensitivity of fully-grown immature oocytes to a progestin hormone and a reduced number of oocytes undergone meiotic maturation both in vivo and in vitro in pgrmc1 -/-. This reduced sensitivity to progestin corresponds well with significant reduced expression of mPRα, the receptor mainly responsible for mediating oocyte maturation and meiosis resumption in fish. The results provide in vivo and in vitro evidence for the physiological functions of Pgrmc1 in oocyte maturation and fertility, as well as a plausible molecular mechanism via regulation of mPRα, which in turn directly regulates oocyte maturation and affects fertility in zebrafish.

Basic Transcription Factor 3 Is Required for Proliferation and Epithelial-Mesenchymal Transition via Regulation of FOXM1 and JAK2/STAT3 Signaling in Gastric Cancer.

Gastric cancer (GC) is the most common epithelial malignancy worldwide. Basic transcription factor 3 (BTF3) plays a crucial role in the regulation of various biological processes. We designed experiments to investigate the molecular mechanism underlying the role of BTF3 in GC cell proliferation and metastasis. We confirmed that BTF3 expression was decreased in GC tissues and several GC cell lines. Lentivirus-mediated downregulation of BTF3 reduced cell proliferation, induced S and G2/M cell cycle arrest, and increased apoptosis. Knockdown of BTF3 significantly reduced the expression of Forkhead box M1 (FOXM1). Upregulation of FOXM1 significantly inhibited the decrease in cell proliferation due to BTF3 silencing, S and G2/M cell cycle arrest, and increase in apoptosis. Knockdown of BTF3 decreased Ki-67 and PCNA expression, whereas it increased p27 expression, which was inhibited by upregulation of FOXM1. Knockdown of BTF3 significantly decreased the ability to invade and migrate. Moreover, knockdown of BTF3 increased E-cadherin expression, whereas it decreased N-cadherin and ZEB2 expression, indicating a decrease in epithelial-mesenchymal transition (EMT). Phosphorylation of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) was significantly inhibited by knockdown of BTF3. IL-6-stimulated phosphorylation of STAT3 and JAK2 markedly suppressed inhibition of EMT due to BTF3 silencing. Silencing of BTF3 decreased tumor volume and weight and reduced peritoneal nodules in implanted tumors. Our findings provide a novel understanding of the mechanism of GC and highlight the important role of BTF3/FOXM1 in tumor growth and BTF3/JAK2/STAT3 in EMT and metastasis.

Genetic variability of genes in NER pathway influences the treatment outcome of gastric cancer.

We performed a study to investigate the role of ERCC1, ERCC2, ERCC5, XPA and XPC polymorphisms from perspective of the whole NER pathway in the prognosis of gastric cancer. A total of 410 gastric cancer patients were recruited between January 2010 and December 2011. Restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) was used to analyze genotypes of ERCC1 rs11615 and rs3212986, ERCC2 rs13181 and s1799793, ERCC5 rs17655, XPA rs1800975 and XPC rs2228001. Our study found that carriers of ERCC1 rs3212986 TT genotype showed significantly favorable survival than wide-type GG genotype in multivariate analysis (OR=6.38, 95% CI=2.54-19.03), and patients with variant CC genotype of ERCC2 rs13181 exhibited better response to chemotherapy than those with AA genotype (OR=2.21, 95% CI=1.17-4.25). By Cox proportional hazards model, patients with variant TT genotype of ERCC1 rs3212986 exhibited longer PFS and OS than those who had GG genotype (for PFS, HR=0.37, 95% CI=0.17-0.75; for OS, HR=0.36, 95% CI=0.13-0.87). For ERCC2 rs13181 polymorphism, carriers with CC genotype demonstrated significantly increased hazards of progression of disease and death in multivariate model (for PFS, HR=0.48, 95% CI=0.26-0.88; for OS, HR=0.44, 95% CI=0.20-0.91). In conclusion, our finding suggests that ERCC1 rs3212986 and ERCC2 rs13181 gene polymorphism could influence the response to chemotherapy and clinical outcome of gastric cancer.

miR-27a as an oncogenic microRNA of hepatitis B virus- related hepatocellular carcinoma.

microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through post-transcriptional interactions with mRNA. miRNAs have recently emerged as key regulators of various cancers. Although miR- 27a has been implicated in several other cancers, its role in hepatitis B virus-related hepatocellular carcinoma (HCC) is unknown. In this study, we showed miR-27a to be frequently up-regulated in HCC tissues and HCC cell lines (HepG2 and Huh7). Overexpression of miR-27a enhanced cell proliferation, promoted migration and invasion, and activated cell cycling in HepG2 and Huh7 cells. In summary, our results suggest that up-regulation of miR-27a may play an oncogenic role in the development of HCC and might thus be a new therapeutic target in HCC patients.

[Study on adsorption of nonpolar R-side amino acids on silver nanoparticles by FT-SERS].

The adsorption orientation and adsorption characteristics of aliphatic-nonpolar amino acids represented by L-methionine on silver nanoparticles were studied by FT-surface-enhanced Raman spectroscopy (FT-SERS) method with different pH and concentrations. According to the experiments, in order to get the best FT-SERS effects, the optimal concentration range of the L-methionine on silver sol is between 10(-3) mol x L(-1)-10(-4)(mol x L(-1). The FT-SERS effects and adsorption model change with pH; The SERS are poor when pH is near the isoelectric point or over 11 or 3. The interaction of L-methionine with Ag nanoparticles is the physical and chemical adsorption by NH3, COO-, S, which could also be effected by pH. From the results and previous reports on this aspect, the general rules of the adsorption model and the orientation of the aliphatic-nonpolar amino acids on silver nanoparticles were speculated, which would be useful for the study of amino acids, protein, peptide, enzyme etc.