ABSTRACT
Bladder cancer is one of the leading causes of cancer-related death in men, however, there was only limited effective treatment for invasive bladder cancer. DAPK1 has been shown to play important role in apoptosis and autophagy to suppress cancer progression. Previous results have shown that DAPK1 promoter was hypermethylated in the majority of bladder cancer specimens, however, the prognostic significance of DAPK1 in bladder cancer has yet to be demonstrated. In the present study, we found that DAPK1 expression was negatively associated with tumor stage and a low level expression of DAPK1 in bladder cancer specimens were associated with shorter survival in bladder cancer patients in 3 independent bladder cancer datasets (n = 462). Further investigation showed that FGFR3 knockdown resulted in downregulation of DAPK1 in bladder cancer cell line, suggesting that FGFR3 may be an upstream factor of DAPK1. Further analysis of the 3 independent bladder cancer datasets have identified ACOX1, UPK2, TRAK1, PLEKHG6 and MT1X genes had their expression significantly correlated with that of DAPK1. Knockdown of DAPK1 in bladder cancer T24 cells resulted in downregulation of ACOX1, UPK2 and TRAK1. Interestingly, TRAK1, by itself, was a favorable prognostic marker in the 3 independent bladder cancer datasets. Importantly, by using connectivity mapping with DAPK1-associated gene signature, we found that vemurafenib and trametinib could possibly reverse DAPK1-associated gene signature, suggesting that inhibition of Raf/MEK pathway may be a potential therapeutic approach for bladder cancer. Indeed, treatment of vemurafenib in T24 bladder cancer cells resulted in upregulation of DAPK1 confirming our connectivity mapping, while knockdown of DAPK1 resulted in reduced sensitivity towards inhibition of Braf signaling by vemurafenib. Together, our results suggest that DAPK1 is an important prognostic marker and therapeutic target for bladder cancer and have identified possible therapeutic agents for future testing in bladder cancer models with low DAPK1 expression.
Subject(s)
Biomarkers, Tumor/metabolism , Death-Associated Protein Kinases/metabolism , Urinary Bladder Neoplasms/pathology , Biomarkers, Tumor/genetics , Cell Line, Tumor , Death-Associated Protein Kinases/genetics , Gene Expression Regulation, Neoplastic , Humans , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptor, Fibroblast Growth Factor, Type 3/genetics , Receptor, Fibroblast Growth Factor, Type 3/metabolism , Urinary Bladder Neoplasms/enzymologyABSTRACT
This study was to isolate microsatellite markers from Microtus fortis genome by magnetic beads enrichments. Through hybridization of biotin-labeled microsatellite oligonucleotide probes, which were captured by streptavidin-coated magnetic with the adaptor-ligated enzyme-digested genome fragments, single-stranded DNA fragments containing microsatellites were obtained. After PCR amplification, these fragments were then cloned into T vectors and were transformed into competent cells subsequently. Ninety-two microsatellite sequences were randomly isolated from 70 positive clones. Twenty-one out of 27 pairs of designed microsatellite primers were screened out from the microsatellite sequences, and 10 out of the 21 microsatellite loci were used to investigate the genetic diversity of three populations of M. fortis, Hunan (wild), Hunan (domesticated), and Ningxia (domesticated). All the 10 microsatellite loci used to analyze the genetic diversity exhibited a good level of polymorphism. The values of observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content (PIC) were all the highest in the Hunan (wild) population, lower in the Hunan (domesticated) population, and the lowest in the Ningxia (domesticated) population.
Subject(s)
Arvicolinae/classification , Arvicolinae/genetics , Genetic Variation/genetics , Microsatellite Repeats/genetics , AnimalsABSTRACT
The study aimed to reveal alterations in expression and methylation levels of the growth-related imprinted genes H19 and Igf2 in fetuses of diabetic mice. Diabetes was induced in female mice by intraperitoneal injection of streptozotocin. DNA and total RNA were extracted from fetuses obtained from diabetic and control dams on embryonic day (E) 14. Real-time RT-PCR analysis revealed that the mRNA expression of Igf2 in fetuses from diabetic mice was 0.65-fold of the control counterparts. Bisulfite genomic sequencing demonstrated that the methylation level of the H19-Igf2 imprint control region was 19.1% higher in diabetic fetuses than in those of control dams. In addition, the body weight of pups born to diabetic dams was 26.5% lower than that of the control group. The results indicate that maternal diabetes can affect fetal development by means of altered expression of imprinted genes. The modified genomic DNA methylation status of imprinting genes may account for the change in gene expression.
Subject(s)
DNA Methylation , Diabetes Mellitus, Experimental , Fetus/physiology , Genomic Imprinting , Insulin-Like Growth Factor II/genetics , Pregnancy in Diabetics , RNA, Untranslated/genetics , Adult , Animals , CpG Islands , Diabetes, Gestational/genetics , Endonucleases/metabolism , Female , Fetus/anatomy & histology , Humans , Male , Mice , Pregnancy , RNA, Long NoncodingABSTRACT
Insulin promotes early embryonic development, but whether this action affects postimplantation fetal development and alters the expression of imprinted genes remain to be determined. This study analyzed the expression and methylation levels of the growth-related imprinted genes H19 and insulin-like growth factor 2 (Igf2) in fetuses exposed to insulin before implantation. We cultured 2-cell embryos in either 0 or 0.25 microg/ml insulin until the blastocyst stage and then transferred them into pseudopregnant recipient mice. The number of embryos developing to blastocysts after insulin exposure was 16.4% higher than that of the control, and the birth body weight of the insulin-exposed group was 17.8% higher than that of the control group. Real-time reverse transcription-polymerase chain reaction analysis revealed that exposure of preimplantation embryos to insulin increased the mRNA expression of both Igf2 and H19 in embryonic day (E) 14 fetuses. Bisulfite genomic sequencing demonstrated that the methylation level of the H19-Igf2 imprint control region was 19.3% lower in insulin-exposed E14 fetuses than in controls. The present study indicates that insulin exposure during the preimplantation stage alters the expression of imprinted genes and affects fetal development.
Subject(s)
Blastocyst/drug effects , Gene Expression Regulation, Developmental/drug effects , Genomic Imprinting/drug effects , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Animals , Base Sequence , CpG Islands , DNA Methylation/drug effects , Embryo Implantation , Embryo Transfer , Embryonic Development , Female , Insulin-Like Growth Factor II/chemistry , Insulin-Like Growth Factor II/genetics , Male , Mice , Mice, Inbred ICR , Molecular Sequence Data , RNA, Long Noncoding , RNA, Messenger/metabolism , RNA, Untranslated/chemistry , RNA, Untranslated/genetics , Sequence Analysis, DNAABSTRACT
OBJECTIVE: To study the effect of irradiation on the susceptibility of radiation caries. METHODS: The structures of 56 teeth enamel and dentin of 63 roots were observed using SEM and the collagen fibre and the resistance to the acid were also investigated after irradiation of 30 Gy, 50 Gy and 70 Gy. RESULTS: The enamel structure changes were found after irradiation with different doses. The significant difference was found in the enamel changes between high or middle dose group and low dose group or control. The dentin morphology changed, some collagen fibre vanished and resistance to acid was reduced after irradiation with 50 Gy and 70 Gy. CONCLUSIONS: The radiation reduced the resistance of teeth to the acid and increased the caries susceptibility.
