The Study Of Genetic Susceptibility Genes Of Complete Hydatidiform Mole And The Biological Function And Mechanism In Choriocarcinoma Cells

Complete hydatidiform mole(CHM)is a benign trophocyte disease with a potentially invasive ability that can spread to distant sites.The data of epidemiology shows,CHM has an unbalanced distribution among the different areas worldwide,which might conjecture the possible role of genetic factors in the genesis of CHM.As the results of the advanced cell genetics and molecular biology show,hydatidiform mole(HM)is the result of over expression of paternal genes,and the CHM is the result of androgenesis,whose genome is all from father;and family-recurrent CHM(FRCHM)whose genome is from parents is the outcome of maternal genome silencing by the variants of imprint gene NLRP7 and/or KHDC3L.However,we did not find any studies on the association between sporadic CHM and genetic loci up to now,possibly due to rarity of this disease.Nowadays,with the development of sequencing technique and based on the array of high throughput sequencing,the next-generation sequencing(NGS)was born.It made the gene variants associated to the diseases found easier.Whole exome sequencing(WES)is one technique of the NGS that targets on the coding sequence of proteins in the human genome.The sequence of exome is less than 1%of global human genome,but more than 85%variants associated to the disease is in the exome.As the research shows,WES could find the lower frequency variants of familial heredity disease and some heredity disease with complicated symptom.It means that the WES would be helpful in finding the susceptibility gene in the sporadic CHM.Choriocarcinoma is an extreme malignancy carcinoma derived from trophoblast cell,following the hydatidiform mole,abortion or term delivery.Compared with other solid tumor,it can metastasis to other places of the body earlier and easier through the blood,in contrast,other solid tumors metastasis come up at the advanced period.The literatures show that it hasn’t been investigated that the relation between the susceptibility gene MIIP of CHM and this kind of malignancy disease up to now.The study performed whole-exome sequencing analysis on CHM patients and normal healthy women with more than one health baby to find the possible susceptibility genes,and re-screened the candidate variants determined through bioinformatics analysis by mass spectrometry with enlarged samples;and finally validated the candidate genetic polymorphisms by direct sanger sequencing.After literature review,we choose the MIIP gene which could control the cell in migration and invasion to explore the biological functions in the malignancy disease of Choriocarcinoma cell.And investigate the possible mechanism to fully know the biological function and mechanism of susceptibility gene MIIP of CHM in choriocarcinoma cancer cells.Part ⅠWhole-exome sequencing reveals genetic variants of ERC1 and KCNG4 in Chinese Han women with complete hydatidiform molesObjectiveComplete hydatidiform mole(CHM)is an uncommon pregnancy-related disease with an invasive potential.The genetic background of the sporadic form of CHM has not been addressed previously except for the possible mechanisms of maternal genetic variants involved in those with biparental origin.This study is aiming to investigate the possible susceptibility genes of sporadic Complete hydatidiform mole.MethodsWe performed whole-exome sequencing analysis(WES)on the DNA from 51 CHM patients and 47 normal healthy women,re-screened the candidate variants by mass spectrometry in 199 CHM patients and 400 normal healthy women,and finally validated the candidate genetic polymorphisms by direct Sanger sequencing in 247 cases and 599 controls with another 205 new controls.Find the possible mechanism by referring to the literature.Results1.398,594 sequence nucleotide variants were found during the whole-exome sequencing,Functional analysis of the SNVs showed that there were 5,301 deleterious rare variants with 2,033 single-variant genes and 1,054 multiple-variant genes.2.During the second-scan of Mass-ARRAY,three gene variants may be associated with the sporadic complete hydatidiform mole.They were c.G48C(p.Q16H)in ERC1(p=0.01244),c.G1114A(p.G372S)in KCNG4(p=0.01156)and c.C739T(p.R247W)in MIIP(p=0.023152).3.After Sanger test by enlarged samples,only two variant still have the statistical significance.They were c.G48C(p.Q16H)in ERC1(p=0.0008361),c.G1114A(p.G372S)in KCNG4(p=0.017728).Conclusion:1、Two single nucleotide polymorphisms,c.G48C(p.Q16H)in ERC1 and c.G1114A(p.G372S)in KCNG4may be associated with an increased risk for CHM(p<0.05).2、ERC1 and KCNG4 may affect the stretch of centrosome during meiosis,then affect the procession of meiosis.Part IIThe study of biological function and mechanism of MIIP in choriocarcinoma cancer cellsObjectiveTo investigate the biological regulation of MIIP with the cell proliferation,apoptosis,invasion,migration,tumor-burdened experiment in nude-mice and other biological behaviors of choriocarcinoma cancer cells.The identification of the possible mechanism of MIIP in choriocarcinoma cancer cells.MethodsBy real-time RT PCR and western blot,the mRNA and protein levels of MIIP in choriocarcinoma cancer cell lines and normal trophoblast cell line were detected.Choriocarcinoma cell line JAR and JEG-3 were transfected with lentivirus of interfering RNA targeting MIIP or negative control to down-regulate MIIP expression.Normal trophoblast cell line SWAN were transfected with MIIP plasmid or the empty vector to up-regulate MIIP expression.Cell proliferation experiments were conducted by CCK-8 assay.Cell apoptosis analysis were carried out by flow cytometry technique.Cell migration and invasion were detected by transwell assays and Wound healing experiment.The tumor-burdened experiment was operated in SPF nude-mice with small animals living imager.We detected the possible molecule pathway by signal western blot.Results1.Interfering MIIP gene expression in Choriocarcinoma cell line JAR and JEG-3 enhacned cell proliferation,cell migration and invasion and had no influence on apoptosis.2.Up regulation of MIIP in normal trophoblast cell line SWAN inhibited the cell proliferation,cell migration and invasion and also had no influence on apoptosis.3.Down regulation of MIIP in Choriocarcinoma cell line JAR enhanced the ability of tumor-burden experiment.4.The variation of MIIP affected the expression of HDAC6 and acetylation-a-tubulinConclusion:1.The regulation of MIIP could affect part of the biological behavior with the role of cancer suppressor gene in choriocarcinoma.2.HDAC6 gene and acetylation-a-tubulin may participate in the biological behavior regulation of MIIP in choriocarcinoma.