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Objective:To investigate the expression and correlation of H19, matrix metalloproteinase (MMP)-2 and MMP-9 in patients with recurrent spontaneous abortion (RSA).Methods:Human extravillous trophoblast cell line HTR-8 was cultured in vitro. Lentivirus was used to infect the HTR-8 cell line to over-express or knockdown the expression of H19. The concentrations of MMP-2 and MMP-9 protein in cell culture supernatant were detected by enzyme linked immunosorbent assay (ELISA). The expression levels of H19, MMP-2 and MMP-9 mRNA in villi of patients with RSA were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Spearman correlation analysis was used to understand the correlation between H19 and the expression levels of MMP-2 and MMP-9. Results:After overexpression of H19, the expression levels of MMP-2 and MMP-9 mRNA and protein concentration in Lv-ph19 group were significantly higher than those in Lv-vector group ( P<0.05); After interfering with the expression of H19, the expression levels of MMP-2 and MMP-9 mRNA and protein concentration in Lv-shH19 group were significantly lower than those in Lv-shcon control group ( P<0.05). The number of spontaneous abortions in patients with recurrent spontaneous abortion was significantly higher than that in the control group ( P<0.05). qRT-PCR showed that the expression levels of H19, MMP-2 and MMP-9 mRNA in villi of patients with RSA were significantly lower than those in the control group ( P<0.05). There was a positive correlation between H19 and the expression levels of MMP-2 and MMP-9 ( P<0.05). Conclusions:H19 regulates the expression of MMP-2 and MMP-9 of trophoblast during early pregnancy, and the abnormal expression of H19, MMP-2, and MMP-9 in human first-trimester villous tissues was related with the incidence of early miscarriage.
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The imprinted gene H19, one of the maternally imprinted gene which had been found long time ago, is highly expressed in early embryo. It mainly concentrates in endoderm and mesoderm which plays an important role in regulating the development of embryos and in control of off spring behavior. More and more studies have shown that the alterations of methylation status in CpG islands of H19 gene imprinted control region will lead to the decline of male sperm quality and female oocyte quality, then affecting human fertility. Imprinting gene H19 is also related to the development of the embryo. This article reviews the progress of methylation of the imprinted gene H19 in assisted reproductive technology.
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OBJECTIVES@#To investigate the single nucleotide polymorphism （SNP） and haplotypes in differentially methylated region （DMR） upstream of H19 gene in Guangdong Han population.@*METHODS@#The PIA typing and restriction enzyme McrBC and HpaⅡ were used to digest the genomic DNA and obtain the individual uniparental DNA template strand. The data of uniparental SNP alleles, genotypes and haplotypes in DMR upstream of H19 gene were obtained by sequencing.@*RESULTS@#A total of 13 SNPs （rs10840167, rs2525883, rs12417375, rs4930101, rs2525882, rs2735970, rs2735971, rs11042170, rs2735972, rs10732516, rs2071094, rs2107425, and rs4930098） and one mutation locus （g7351c） were found. All loci followed the Hardy-Weinberg equilibrium （P>0.05） by statistical analysis. Except for rs12417375 （DP=0.279） locus, the DP of remaining 12 SNPs were 0.446-0.614, and the g7351c mutation locus （DP=0.013） was the particular loci of the Southern Chinese Han population. Eight haplotypes （designated as haplotype 1-8） were detected, in which 3 haplotypes had not yet been reported and the DP, PIC, PE and H were 0.891, 0.714, 0.524 and 0.758, respectively.@*CONCLUSIONS@#Obtained by PIA typing, the SNP in DMR upstream of H19 gene and its haplotypes genetic marker system have a high determination power and show a good practical value in forensic identification.
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Humans , Alleles , Asian People/genetics , China , DNA , Gene Frequency , Genetic Markers , Genetics, Population , Genotype , Haplotypes/genetics , Polymorphism, Single NucleotideABSTRACT
Objective To investigate SNP and distribution of haplotypes in differentially m ethylated region (DMR) upstream of H19 gene in Chinese Korean nationality in order to provide basic data for forensic application and population genetics research. Methods One hundred and one blood sam ples from unrelat-ed Chinese Korean individuals and 14 blood sam ples from 5 Chinese Korean intergenerational fam ilies which known genetic relationship were collected. The SNP in DMRupstream of H19 gene were investi-gated by PCR-cycle sequencing and McrB Cdigestion followed by PCR . The haplotypes detected by parentally im printed allele (PIA) m ethod and relevant genetic param eters were calculated. Results Thirteen SNPs (rs10840167, rs2525883, rs12417375, rs4930101, rs2525882, rs2735970, rs2735971, rs11042170, rs2735972, rs10732516, rs2071094, rs2107425, and rs4930098) and five haplotypes were detected in 1 174 bp target product in DMRupstream of H19 gene, with 9 SNPs having high discrim ination power as good genetic markers. The average gene diversity (GD) of haplotypes was 0.714. The maternal haplotype was confirm ed correctly by PIAm ethod from McrB C-digested products of genom ic DNA. Conclusion High polym orphism sexist in DMRupstream of H19 gene in Chinese Korean nationality. And determ ination of the maternal haplotype could furtherm ore enhance the forensic identification efficiency of im printed gene.
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Objective To obtain the expression pattern of imprint gene H19 in JEG-3 cell in order to explore the regulation mechanism of H19 on trophoblast cellular biological behavior .Methods After correct identification with sequencing for the recom-binant eukaryotic expression plasmid pRc/CMV which including the whole length of H19 cDNA ,the plasmid was transfected to the cell line JEG-3 .The expression of H19 mRNA was observed and the gene expression profile of three groups of JEG-3 cell were de-tected with Affymetri :U133 plus 2 .0 Array .Results After being transfected with target H 19 gene ,the expression of the mRNA level was significantly increased compared with control group .And the gene expression profile was changed significantly .19 genes were up-regulated ,77 genes were down-regulated .Expression levels of HES1 gene which being choosed as a different expression gene were detected by fluorescence quantitative PCR in severe preeclampsia placenta tissue and normal late pregnant placenta .The expression level of HES1 mRNA in severe preeclampsia placenta decreased significantly than normal late pregnant placenta tissues . Conclusion Many genes induced by H19 have been screened by high-throughput gene chip method .It provides the experimental ba-sis for advanced studying the regulation the cellular biological behavior with H 19 gene .
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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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Human uniparental gestations such as gynogenetic ovarian teratomas provide a model to evaluate the integrity of parent-specific gene expression - i.e. imprinting - in the absence of a complementary parental genetic contribution. The few imprinted genes characterized so far include the insulin-like growth factor-2 gene (IGF2) coding for a fetal growth factor and H19 gene whose normal function is unknown but it is likely to act as an mRNA. IGF2 is expressed by the paternal allele and H19 by the maternal allele. This reciprocal expression is quite interesting because both H19 and IGF2 genes are located close to each other on chromosome 11p15.5. In situ RNA hybridization analysis has shown variable expression of the H19 and IGF2 alleles according to the tissue origin in 11 teratomas. Especially, Skin, derivative of ectoderm, is expressed conspicuously. We examined imprinting of H19 and IGF2 in teratomas using PCR and RT-PCR of exonic polymorphism. H19 and IGF2 transcript could be expressed either biallelically or monoallelically in the teratomas. Biallelic expression (i.e., loss of imprinting) of IGF2 occured in 5 out of 6 mature teratomas and 1 out of 1 immature teratoma. Biallelic expression of H19 occured in 4 out of 10 mature teratomas and 1 out of 1 immature teratoma. Expression levels of H19 and IGF2 transcript using the semi-quantitative RT-PCR had no relation between monoallelic and biallelic expression. Moreover, IGF2 biallelic expression did not affect allele-specificity or levels of H19 expression. These results demonstrate that both genes, H19 and IGF2, can be imprinted, expressed and regulated independently and individually of each other in ovarian teratoma.