â…  The Study Of Syncytin-1Methylation Patterns In Human Placentas With Preeclampsiaâ…¡ Preliminary Study Of Syncytin-1in Human Nonsmall Cell Lung Cancer

BackgroundPreeclampsia is a common multi-system disorder complicating2%-8%pregnancies. The disease seriously damage the heath of pregnant women and perinatal babies. As a major obstetric complications, it is responsible for maternal and fetal deaths. Clinical characteristers are hypertension (BP≥140/90mmHg) and proteinuria (≥0.3g/24h or random urine protein:+) after20weeks of gestation. Despite substantial research effort in the past several decades, the etiology and pathogenesis of PE is still unclear. In addition to the termination of pregnancy, lack of effective clinical treatment is always one of the main causes of maternal mortality. Therefore, the disease has been the hot research of obstetric field. Clinical symptoms of preeclampsia gradually disappear after delivery of the placenta. The studies of placenta become the focus of preeclampsia etiology. The placenta defects caused by abnormal differentiation, migration, invasion of trophoblasts are closely related with the incidence of preeclampsia. One of the pathological features of the preeclampsia is dysplasia of syncytiotrophoblast cells. In2000. the human endogenous retroviral family W, env(HERVWE) was first reported by Sha. Syncytin is its protein product, which is highly expressed in placenta tissue and has functions of mediating cell fusion, proliferation, antiapoptosis and immune suppression. Syncytin-1was the first functional fusogenic protein involved in mediation of cell-cell fusion and induction of syncytiotrophoblast described. The multi-nucleated syncytiotropho-blast layer is essential for normal placental function and is essential for nutrient exchange and secretion of honnones including human chorionic gonadotrophin (HCG), human chorionic somatomammotropin (HCS). A reduced expression of syncytin may contribute to decreased formation of syncytiotrophoblast, abnormal fusion and differentiation of cytotrophoblast, vascular dysfunction and villous hypoxia.These may be one of etiology of preeclampsia.In addition to traditional research, more and more people pay attention to the role of epigenetics in the pathogenesis of PE. DNA methylation is an important epigenetic modification. CpG dinucleotide cytosine was methylated to5-methylated cytosine by DNA methyltransferase. DNA methylation has a variety of biological functions including regulation of gene expression, gene imprinting, preservation of chromosomal integrity.There are few reports worldwide about syncytin-1methylation patterns in human placentas with preeclampsia and no reports about correlations between DNMT3L and syncytin-1methylation levels. In this study, we first examined the levels of syncytin-1mRNA expression in3rd trimester normal and preeclampsia placentas by quantitative real-time polymerase chain reaction (PCR). We then assessed DNA methylation by MSP, COBRA and sequencing. Our aim was to analyze the expression of syncytin-1and syncytin-1methylation patterns in human placentas with preeclampsia and explore its associations with pathogenesis of preeclampsia and provide the basis data for the diagnosis and treatment of preeclampsia.Part one Syncytin-1methylation patterns in human placentas with preeclampsiaObjective:To analyze the expression of syncytin-1and syncytin-1methylation patterns in human placentas with preeclampsia and explore its associations with pathogenesis of preeclampsia.Methods:1. Reverse transcription real-time PCR was used to detect the transcription activities of syncytin-1, ASCT2, HCS, GCMa, DNMT1, DNMT3a, DNMT3b and DNMT3L in the placentas of normal third trimester and preeclampsia;2. Immunohistochemistry was used to show the protein location of syncytin-1, HCS, DNMT1, DNMT3b, DNMT3L and5mC.The level of syncytin-1protein was measured by Western blot in3N and3P placentas. 3. MSP, COBRA and bisulfite sequencing were carried out to analyze the methylation status of syncytin-15’LTRs in placentas of preeclamptic and normal pregnancies.Results:1. GAPDH was used as internal reference gene, compared with normal third trimester, DNMT3a, DNMT3b, syncytin-1and HCS mRNA levels were decreased in placentas of preeclamptic pregnancies(P<0.05), whereas, DNMT1and DNMT3L were upregulated in PE compared with the normal control group (P<0.05). There were no significant difference in GCMa and ASCT2between the two groups(P>0.05). PCNA was used as internal reference gene, DNMT1, DNMT3b and DNMT3L were increased in3P compared with3N(P<0.05). DNMT3a was decreased in3P compared with3N(P<0.05).2. The protein staining of syncytin-1and HCS was weaker in3P placentas than that of in3N; DNMT3b and DNMT3L protein expression were more stronger in3P than those in3N placentas. The staining results of DNMT1and5mC did not show significant difference.Western blot also show higher expression of syncytin-1in3N placentas than that of3P.3. MSP,COBRA and sequencing displayed that syncytin-15’-LTRs were found to be more highly methylated in PE placenta tissues than in the normal control group(P<0.05).4. Spearman analyse, there was a significant positive correlation between syncytin-1and HCS mRNA levels (r=0.713.P<0.05)and negative correlation between syncytin-1and DNMT3b mRNA levels(r=-0.544.P<0.05). There was a significant negative correlation between syncytin-1mRNA levels and its methylation levels(r=-0.505,P<0.05). There were significant positive correlations between DNMT3b mRNA levels and syncytin-1methylation levels(r=0.45,P<0.05), DNMT3L and syncytin-1methylation levels (r=0.536. P<0.05), DNMT3b and DNMT3L mRNA levels(r=0.554, P<0.05); Methylation levels of syncytin-15’LTRs in3N and3P were analyzed by three methods:MSP, COBRA and sequencing. There were positive correlations between COBRA and MSP (rs=0.443,P<0.05), COBRA and sequencing (rs=0.862,P<0.01).Conclusions:1. Decreased expression of syncytin-1was related to preeclampsia, but the receptor ASCT2was not associated with PE.2. Aberrant methylation of HERVWE15’LTRs was involved in the occurrence of PE. The mechanism may be hypermethylation of syncytin-1repressed its transcription.3. DNMTs regulate methylation of syncytin-1. PCNA as internal reference genes, the expression of DNMT3L and DNMT3b increased in PE, both were positive correlations with methylation of syncytin-1. Cooperation of DNMT3L and DNMT3b upregulated promoter CpG methylation levels of syncytin-1and repressed syncytin-1mRNA.Part two Preliminary study of syncytin-1in human nonsmall cell lung cancer BackgroundThere are many similarities between tumorigenesis and placenta formation, growth and development process. Such as high proliferation rate, lack of contact inhibition of cell growth, strong migratory and invasive, rich vascular network and blood supply, immune escape. Lung cancer is one of the leading causes of cancer in most countries in the world. In China, lung cancer has the highest incidence and mortality of all cancers, the morbidity and mortality of this malignant tumor was serious harm to human health and life. Surgery, chemotherapy and radiotherapy are common methods to treat cancers. As it’s diagnostic and theraprutic technology is limited, the rate of5-year-survival is still very low. How to improve the five-year survival rate of patients with lung cancer? What factors are closely related to the survival and prognosis of patients with lung cancer? These problems have become the focus of the research of many scientists. The study about the relationship between syncytin-1and prognosis with cancers have few reports. Syncytin-1and the prognosis with NSCLC has not been reported. In order to study the impact of the tumor microenvironment immune molecules on the survival and prognosis of patients with lung cancer. Firstly, we collected the cases who underwent surgical resection for lung cancer5years ago, and conformed whether they are survival or died by telephone. Then, tissue specimen resected from the patients were immunohistochemically analyzed for syncytin-1, TIM-3,CD4+and CD8+T cells expression. Furthermore, we analyzed the relationship between them and the5-year-survival rate of NSCLC. These are experimental basis to search an effective method and reasonable therapy way to promote the5years survival rate for lung cancer.Objective:To detect the expression of syncytin-1, TIM-3, CD4+and CD8+T cells in nonsmall lung cancers(NSCLC) tissues and to explore the prognosic value of immune molecules in the tumor microenvironment of patients with NSCLC.Methods:Imunohistochemistry was used to detect the expression of syncytin-1, TIM-3.CD4+and CD8+T cells in NSCLC tissues. The difference between5-years-survival group and5-years-non-survival group was analyzed.Results:The expression of syncytin-1and TIM-3cells in5-years-survival group were lower and CD8+T cells were higher than that of in5-year-non-survival (P<0.05). There was no significant difference observed in CD4+T cells between two groups (P>0.05).Conclusions:The expression of syncytin-1. TIM-3and CD8+T cells can be one of standards to judge the prognosis of NSCLC. High numbers of CD8+T cells is a strong indicator for a favorable clinical outcome, but high expression of syncytin-1and TIM-3show a poor prognosis.