| As an important member of assisted reproductive technologies(ARTs),preimplantation genetic diagnosis(PGD)prevents many of genetic diseases to transmit to offspring through gametes or embryos genetic analysis.PGD has been widely used for more than 20 years.Compared with other ARTS,the protocol required for PGD includes not only embryo culture and manipulations in vitro,but also the more invasive biopsy procedure.It has been reported that manipulations of embryo at early stage may be associated with negative health outcomes of offspring.However,there is no systematic assessment of PGD technology yet.We previously generated mouse model with cooperation to state key laboratory of reproductive biology to evaluate the effect of blastomere biopsy on health of offspring.Mice of biopsied group were born after blastomere biopsy and embryo transfer following in vitro culture for hours,while mice of control group were given birth through embryo in vitro culture and embryo transfer without blastomere biopsy.We found there was a potential high risk of lung adenocarcinoma among male mice of biopsied group when they were old.Thus,we aimed to study the molecular mechanism of the high risk.Alteration in DNA methylation patterns is one of the most frequent events that occur in human tumors.Recently,many studies has found that changes in DNA methylation was common at early stage of lung carcinoma and associated with the cancer progression.Additionally,blastomere was biopsied during the procedure of genome-wide DNA demethylation.So it probably induces DNA methylation pattern aberration in offspring.Therefore,we investigated the contribution of DNA methylation alterations to high risk of lung cancer in old-age biopsied male mice.We measured the DNA methylation pattern in lung tissue of adult mice from biopsied and control groups by MedIP-CHIP array.There were 6464 regions detected with different DNA methylation status between the two groups.And 5863 methylation difference regions showed hypomethylation in biopsied group,857 of them with more than 1.5 fold differences compared with control group.The other 601 methylation difference regions showed hypermethylation in biopsied group,but none of them with more than 1.5 fold differences compared with control group.There were about nighty percent of methylation difference regions showed hypomethylation in biopsied group.It indicated that whole-genome DNA hypomethylation occurred in lung tissue of adult mice born after blastomere biopsy.Hypomethylation may activate some oncogene and then promote tumorigenesis.Therefore,genes hypomethylated in biopsied group were supposed to include some important genes that associated with cancer.There were 47 hypomethylation regions in biopsied group with more than 1.8 fold differences compared to control group,and 41 of them were within promoter.So we focused the 41 genes with promoter hypomethylated in biopsied group.With the help of software SignalMap,we found 27 of the 41 regions with more than two probes showed significant methylation difference between two groups.So we thoughtmethylation difference to exist actually in the 27 regions.Bioinformatics analysis showed that,8 of 27 genes with promoter hypomethylated in biopsied group involved in cell proliferation,cell differentiation,cell cycle and neoplasm and so on.It suggested that blastomere biopsy may influence cell proliferation,differentiation and regulation of cell cycle.We further read these genes' functional reports,and two genes have been reported to be associated with lung carcinoma.Gene Gal encodes a classical neuropeptide and has been detected to be expressed in multiple carcinoma,such as pheochromocytoma,pituitary adenoma,gastrointestinal cancer,breast cancer and small cell lung cancer.After binding to receptor,protein Gal involves in activating ERK1/2 signal pathway and controlling cell proliferation,survival,migration and other biologic procedures.Gene Ladl encodes a protein which can activate phosphorylation,and involves in MEK5-ERK5 signaling activation through interacting with MEKK2.MEK5-ERK5 signaling has multiple functions,such as regulation of cell proliferation,differentiation.Activated ERK5 also involves in regulating gene expression,gene c-myc and c-fos are two of the targeted genes.And gene c-myc is an oncogene that has been reported to high express in lung carcinoma.We supposed that gene Gal and Ladl were two key genes,and further analyzed their contributions in lung carcinoma of old-age biopsied male mice.Bisulfite sequencing demonstrated that promoter of gene Gal and Ladl were both hypomethylated in biopsied group.It confirmed the result of MedIP-CHIP array.But promoter of gene Gal was just slight hypomethylated in biopsied group.It may be because only a part of difference methylation region showed in MedIP-CHIP was examined in this study.RT-qPCR showed that biopsied group with significant higher expression of gene Gal than that of control group.So we presumed that promoter of gene Gal was probably significant hypomethylated and induced high expression in biopsied group.However,we did not detect the methylation difference in this study.Promoter of gene Ladl was detected hypomethylated significant in biopsied group,and the methylation rate was lower about 10%than that of control group.And we found expression of Ladl in biopsied group nearly double to that of control group through RT-qPCR.RT-qPCR also demonstrated that gene MEK5,ERK5 and their downstream gene c-myc,c-fos significant higher expressed in biopsied group.All of gene MEK5,ERK5,c-myc and c-fos have been reported to be high expression in tumor.It suggested that hypomethylation within promoter of gene Ladl caused its increased expression in biopsied group.Consequently,the expression of the downstream gene MEK5,ERK5,c-myc and c-fos were increased.They may enhanced the risk of lung carcinoma occurred among old-age biopsied male mice.In conclusion,blastomere biopsy induces whole-genome DNA hypomethylation in lung tissue of adult mice.DNA hypomethylation causes genes high expression which involve in tumorigenesis.They are supposed to increase the risk of lung carcinoma occurred among old male mice which born after blastomere biopsy. |
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