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Objective To study the effects of PHLDA2 overexpression on radiosensitivity and the underlying mechanisms in human osteosarcoma U2OS cell line.Methods To obtain the subclone,cells were exposed to G418 persistently after transfection of pEGFP-C3-PHLDA2 vector into U2OS cells.Three groups of blank control (U2OS),negative control (U2OS-neo) and transfected group (U2OS-PHLDA2) were used.The expression of PHLDA2 in the subclone cells was determined by Western blot.After exposure to X-ray irradiation,cellular growth activity and survival were detected by CKK-8 assay and colony formation assay,respectively.The cell apoptosis was measured by the Annexin V/PI staining,and the apoptotic protein was analyzed by Western blot.The in-vivo effects of PHLDA2 on irradiation were evaluated by xenografts.Results Compared with U2OS group and U2OS-neogroup,the sabclone cells were successfully obtained by G418 selection,in which the expression of PHLDA2 was upregulated(t =13.73,16.28,P ＜ 0.05).In vitro,PHLDA2 overexpression significantly enhanced the response to radiation in U2OS cells with a reduction of colony survival and proliferation with the increase of doses (t =5.00-8.23,P ＜0.05;t =-2.52--1.26,P ＜ 0.05).In vivo,PHLDA2-upregulated xenografts had more radiosensitivity than control groups with a significant inhibition of tumor growth (t =3.27,2.91,P ＜ 0.05).After 8 Gy irradiation,the apoptosis was significantly increased (t =10.11,9.61,P ＜ 0.05),accompanied with the activation of Caspased-3 in U2OS-PHLDA2 cells,which was presented by upregulation of cleaved Caspase-3 (t =11.26,10.72,P ＜ 0.05).Conclusions Exogenetic expression of PHLDA2 could significantly enhance the radiosensitivity of human osteosarcoma cells,which may be attributed to the activation of Caspase-3 that increases irradiation-induced apoptosis.
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Objective To study the expression of the imprinted gene H19 and IGF-Ⅱ in newborn placenta,and to discuss its influence on the birth body mass of the neonate. Methods The fresh placental tissues from full-term newborn (without trimester of pregnancy complica-tion and placenta and funic abnormality) with normal,high and low birth body mass (12,10 and 8 samples respectively)were collected. The expression of imprinted gene H19 and IGF-Ⅱ mRNA in the placenta were estimated by reakime fluorescence quantitative PCR Results The ex-pression of H19 mRNA in the placenta was negative correlation to the birth body mass (r =-0.403,P = 0.027).The expression of of IGF-H mRNA was positive correlated to the birth body mass (r = 0.444,P = 0.014). The H19 mRNA expression level in the high birth weight neonates (0.21 0.31) was significantly lower than that in the low birth body mass neonates (1.51 2.04)(P= 0.013). But the expression level of IGF-Ⅱ mRNA in the high birth body mass neonates (2.67±3.41) was significantly higher than that in the low birth body mass neonates (0.39±0.33)(P =0.013). Conclusion The expression of H19 and IGF-Ⅱ mRNA was significantly different in the placenta of normal,high and low birth body mass newboms. These two genes may be related to the birth body mass,and there may be some realation-ship between these two genes.
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Genomic imprinting plays a fundamental role in mammal fetal growth and behavior. Abnormal expression of imprinted genes is associated with some genetic diseases and cancers. Imprinted insulin-like growth factor 2 (IGF-2) controls fetal growth by regulating nutrient transportation in placenta. Paternal uniparental disomy, duplication of paternal allele and loss of imprinting are 3 molecular mechanisms of IGF-2 overexpression that can cause Beckwith-Weidemann’s syndrome (BWS). Some assisted reproductive techniques may cause some epigenetic changes that affect embryonic and postnatal development.
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Objective To explore the pathogenesis-related imprinted genes in osteosarcoma and investigate the role of imprinted genes expression in osteosarcoma pathogenesis.Methods The model of malignant transformed immortalized human osteoblastic cells was established and the malignant transformed cells were collected on day 40,55,70,90.The untreated cells passaged from the normal cells on day 90 served as control.After the total RNA extraction,the finally synthesized biotinylated cDNAs were hybridized to CapitalBio Genechip CAP-F0017.2 of the differentially expressed genes.Two genes were chosen at random to further confirm the array results using the SYBR Green real-time PCR in samples of control,day 40 and day 90. Results By an entrance limit of ≥2.0 or ≤0.5,ten imprinted genes which biological functions are mainly related with cell growth/maintenance and signaling transduction were detectable for notablely differential expression,including down-regulated genes of CD81,GRB10,NDN,MEST and up-regulated genes of H19,MKRN3,SLC22A1L,TSSC3,CDKN1C in the malignant transformation of immortalized human osteoblastic cells.The array results were further confirmed by the real-time RT-PCR.Conclusion Ten imprinted genes were detectable for notablely differential expression.Our results will promote our understanding of the molecular mechanisms of imprinted genes in the course of osteosarcoma pathogenesis.
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Objective To establish a simple and high-performance analytical technique for detecting DNA methylation markers and SNPs simultaneously,and obtain the population genetics data of some SNPs in the hypermethylated region upstream of the human H19 gene.Methods The haplotypes of H19FR1 and H19FR2 which located in the promoter region upstream of the human H19 gene were investigated from 232 unrelated Chinese individuals living in Wuhan by means of PCR and subsequent denaturing gradient gel electrophoresis(DGGE).Based on the methylation status of the genomic DNA,selective detection of the parental alleles for H19FRs was examined by using the methylation-sensitive restriction enzyme(msRE) Hpa II or Hha I.Results Five haplotypes and nine phenotypes were observed for H19FR1 in Chinese Han population in Wuhan,and the power of discrimination(DP),polymorphism information content(PIC) and probability of paternity exclusion(PE) were 0.803,0.58 and 0.322 respectively.For the H19FR2,two haplotypes and three phenotyes were detected,and the DP,PIC and PE were 0.626,0.37 and 0.162 respectively.Sequencing results showed that there were 3 SNPs,a7342g,a7357g and g7547a,and one g7351c point mutation in H19FR1.In the H19FR2,there was only one SNP,a8097g.The msRE,HpaⅡ or HhaⅠ,could digest the maternal allele,and only a single band derived from the paternal allele was detected by post-digestion PCR-DGGE(PDP-DGGE) technique.Conclusion PDP-DGGE is a simple,sensitive and effective technique for analyzing DNA methylation status and SNPs simultaneously,and can be used for discriminating the parental origin of alleles.
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Objective To perfect gene profile expressed in pre-implantation embryos. Methods Using nested RT-PCR to investigate the expression of seven imprinted genes: P57~ KIP2, LIT1, TSSC3, GRB10, PEG3, ARHI, and ZAC1 in human oocytes and pre-implantation embryos. Results Transcripts of P57~ KIP2 and ZAC1 were detected in human oocytes and at all stages of pre-implantation; LIT1 was expressed only in stages of 8-cell and blastocyst; transcripts of TSSC3 could not be detected; GRB10 mRNA could be detected in oocytes and pre-implantation embryos except for 2-cell embryo; ARHI was expressed in oocytes and 2,8-cell embryos and blastocyst; Peg3 mRNA existed in 4,8-cell embryos and blastocyst. Conclusion Except for TSSC3, transcripts of the other six imprinted genes are detected in human pre-implantation development, which are helpful for pre-implantation diagnosis of imprinted diseases, and provide the theoretical basis for understanding the correlation among assisted reproductive technology, genetic imprinted diseases and tumor.
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Objective To study the expression of ARHI mRNA,an imprinted suppressor gene,in ovarian serous carcinoma and the methylation status of its three CpG islands,and to determine the possible role of aberrant methylation of ARHI gene in pathogenesis of ovarian serous carcinoma.Methods Twenty normal ovarian specimens and 20 ovarian serous carcinoma specimens were analyzed.Total RNA was extracted and semi-quantitative RT-PCR was employed to detect the mRNA expression of ARHI gene.Methylation status of three CpG islands were examined by DNA bisulfite treatment,PCR amplification and methylation specific restriction enzymes,TagI for CpG I and III,BstUI for CpG II.Results The average of ARHI/G3PDH was 0.73?0.09 in normal ovarian specimens,and it was 0.43?0.37 in ovarian carcinoma specimens(P