The Role Of Matrix Metalloproteinases And F10 Gene On The Pathological Of Abnormal Trophoblast Invasion-related Diseases

Invasion of trophoblast cells into uterine endometrial stroma and the inner third of the myometrium is a key process for the development of the definitive maternal-fetal circulation and for successful pregnancy in humans. It is reported that trophoblast invasion and malignant tumors use the same biochemical mediators to facilitate invasion. However, there are significant differences between these two model systems. Trophoblast cell invasion is initiated immediately after embryo implantation and is precisely regulated by a series of integrated adhesion and signaling events. It is controlled both temporally and spatially, occurs only during the first and early second trimesters of pregnancy,and does not generally go beyond the myometrium; tumor invasion, on the other hand, is uncontrolled and does not respect tissue boundaries. Dysregulation of the finely controlled process of trophoblast invasion can lead to a wide spectrum of pregnancy abnormalities. Excessively shallow invasion has been implicated in miscarriage, fetal growth restriction (FGR)and pre-eclampsia. In contrast, excessive invasion can result in abnormally deep utero-placental infiltration leading to invasive hydatidiform mole and even choriocarcinoma. These pregnancy abnormalities show varying degrees of local trophoblast invasion and can be classified as a category of diseases called "abnormal trophoblast invasion-related diseases".In recent years, more and more pathogenesis, prevention and cure research have been well studied. The precise molecular mechanisms that regulate trophoblast invasion during gestation and its relationship to feto-placental development have already been largely known, such as several proteinase, cytokines, adhesion molecule and growth factors appear to be involved. Among these, matrix metalloproteinases(MMPs) are metal-dependant endopeptidases capable of degrading extracellular matrix. MMPs. and their regulators, tissue inhibitors of metalloproteinase (TIMPs),appear to play a critical role in mediating trophoblast invasion. But the functional complexity of the MMPs and TIMPs on human trophoblast invasion has not yet fully elucidated.Hydatidiform mole is a benign gravid trophocyte diseases, which the main pathology of sertoli cell hyperplasia, for invasding the blood sinus wall of the uters easier. Hydatidiform mole could transform to malignancy as invasive molar and choriocarcinoma. A human novel gene, F10 (Acccession No. AB196290 in Genebank),which was a homo sapiens F10 mRNA for hypothetical protein,complete cds. We cloned F10 gene EST cDNA which up-regulated in hydatidiform mole by suppression subtractive hybridization (SSH). F10 gene, with no functional annotation,was found to be overexpressed in several types of trophoblastic tumor and adenocarcinoma suggesting its potential implication in tumorigenesis in previous researches (data not shown). However, no study has been made on the relationship among F10, trophoblast invasion and the development of placental. Therefore, it is important to do further research on biological functions of F10 gene in order to reveal the generation mechanism of trophoblast invasion and novel treatment and precaution for trophoblast invasion-related disease.In the present study, we first compared the protein levels of MMPs, TIMPs and F10 in groups from pregnancies complications, normal vaginal and caesarean deliveries. Therefore, this clinical-pathological correlation study aimed to investigate the relationship between these invasion-associated proteins and the pathogenesis of these two complicated pregnancies. In addition, we detected the expression of MMPs,TIMPs and F10 proteins in hydatidiform mole and normal villus to verify again the relationship of these genes between degrees of trophoblast invasion with abnormal trophoblast invasion-related complications. Last we established a HTR8/SVneo cell line with the F10 transgenic systemto provide additional evidence for the function and preliminary mechanism of F10 gene. These studies were of instructive significance in researching the pathogenesis of trophoblast invasion-related diseases and guidance clinical diagnosis and treatment.CHAPTER 1 THE RELATIONSHIP BETWEEN MATRIX METALLOPROTEINASES AND RE-ECLAMPSIA AND FETAL GROWTH RESTRICTIONOBJECTIVESeveral molecular studies show that, normal human pregnancy in which trophoblast cells proliferate and differentiate to form the placenta in a regulated manner, members of the MMP and TIMP family have been implicated in trophoblast-induced changes, as pre-eclampsia and FGR. Although the volume of literature on MMPs and TIMPs in pregnancy has recently increased, no study has simultaneously compared the protein levels of MMPs and TIMPs in groups from pregnancies complications, normal vaginal and caesarean deliveries. Therefore, this study was due to elucidate the relationship between the expression of matrix metalloproteinases and pregnancy complications, as pre-eclampsia and fetal growth restecition.METHODSSamples of human placental tissues were obtained from pregnancies hospitalized in Nanfang Hospital (Guangzhou, China) between January 2012 and June 2013,including preeclampsia (N=24), fetal growth restriction (N=10), normal social factorscesarean section (N=15)and normal normal vaginal deliveries (N=15).We used Western blot and immunohistochemical method to detecting the expression of MMP-2, MMP-8, MMP-9, MMP-11, MMP-19, MT2-MMP, MT3-MMP,MT5-MMP, TIMP-1 and TIMP-3 in the four group placental tissues. One-Way ANOVA is adopted to analyze the datas in the multiple groups, Bonferroni test is applied to analyzed the comparison between two groups.RESULTS1. Demographic data of the study populationMaternal age, BMI, fetal weight and placental weight were no significant difference between pre-eclampsia / FGR group and normal delivery pregnancies.However, placental weight were significantly higher in normal vaginal deliveries(P<0.05) than in pre-eclampsia group and fetal weight and placental weight were significantly higher in normal vaginal deliveries (P<0.05) than in FGR group. No significant difference was found in maternal age and BMI between normal caesarean pregnancies and vaginal pregnancies (P>0.05).2. The expression of MMPs and TIMPs in four groups2.1 In the four groups, we detected protein levels of eight MMPs and two TIMPs.Compared with those in normal pregnancies, the expression of MMP-2, -8, -9 and -11 was downregulated in villous tissues of pre-eclampsia cases (P<0.05). TIMP-1 and-3 were increased in pre-eclampsia (P<0.05). There were no significant difference between normal vaginal delivery and preeclampsia group on the expression of MMP-19, MT2-MMP, MT3-MMP, MT5-MMP (P>0.05).2.2 Compared with those in normal pregnancies, the expression of MMP-2, -8,-9 and -11 was downregulated in villous tissues of FGR cases (P<0.05). TIMP-1 and-3 were increased in FGR (P<0.05). There were no significant difference between normal vaginal delivery and FGR group on the expression of MMP-19, MT2-MMP,MT3-MMP, MT5-MMP (P>0.05).2.3 No significant difference was found between normal vaginal deliveries and caesarean deliveries (P>0.05).CONCLUSIONThis study reported the alterations in MMPs and TIMPs related to both pre-eclampsia and fetal growth restriction. We speculate that the change in invasion-associated proteinase expression will affect placental development and may thus contribute to the development of complicated pregnancies.CHARPTER 2 THE EXPRESSION OF A NOVEL GENE F10 IN PRE-ECLAMPSIA AND FETAL GRWOTH RESTRICTIONOBJECTIVEA novel related gene F10 expressed highly in hydatidiform mole was detected and decreased cDNA library between normal villus early pregnant choriocarcinoma and hydatidiform mole tissues achieved by preliminary study with suppression subtractive hydridization (SHH) technique. Previous studies showed that the expression of F10 gene was positive and its intensity increased gradually in hydatidiform mole, invasive mole and choriocarcinoma. In addition, the expression of F10 gene was positive in several tumor tissues, as ovarian cancer and endometrial cancer. But there was no publishing on the regulation of F10 in the human trophobalst cells. In our previous study, we found that the expression of MMPs and TIMPs were abnormal. We supposed that these were relation with the trophoblast shadow invasion.In light of the similar of the invasion behavior between trophoblast and tumor cells,we hypothesized the F10 gene involving the process of trophoblast shadow invasion.METHODSSamples of human placental tissues were obtained from pregnancies hospitalized in Nanfang Hospital (Guangzhou, China) between January 2012 and June 2013,including preeclampsia (N=24), fetal growth restriction (N=10), normal social factorscesarean section (N=15) and normal normal vaginal deliveries (N=15).We used western blot and immunohistochemical method to detecting the expression of F10 in the four group placental tissues. One-Way ANOVA is adopted to analyze the datas in the multiple groups, Bonferroni test is applied to analyzed the comparison between two groups.RESULTS1. F10 is down-regulated in pre-elcamptic placental tissues Levels of F10 were determined and compared in the tissues homogenates of the third trimester placenta, normal pregnancies with vaginal delivery and cesarean V delivery, preeclampsia, and FGR by Western blot analysis. A decrease in F10 levels was observed in pre-eclampsia placenta compared with those in the normal pregnancy placenta. Semi-quantitative analysis indicated this reduced expression of F10 in pre-eclamptic placentae was significant (P<0.001). Importantly, in the pre-eclampsia groups, only few cases showed a weak expression in syncytiotrophoblast cells(P=0.001).2. F10 is down-regulated in FGR placental tissuesWestern blot studies were conducted to examine F10 protein expression in the FGR placenta and control group. The bands representing the F10 protein expression were weaker in FGR placentas as compared with the controls. Semi-quantitative analysis showed that the mean value of F10 expression was decreased in FGR(P=0.001) , as compared with the controls. The results from immunohistochemical analysis were the same as the western blot (P=0.002).3. There was no difference between normal vaginal deliveries and normal cesarean deliveries pregnancyNo significant difference was observed in normal vaginal deliveries and normal cesarean deliveries pregnancy (P>0.03). We also assessed F10 by immunohistochemistry and confirmed it was localized to the syncytiotrophoblast in both normal and complicated pregnancy placenta.CONCLUSIONDown-regulation of F10 was detected in both pre-eclampsia and FGR placenta tissues,which is consistent with the previous result about MMPs and TIMPs abnormal. These results further support our hypothesis that F10 and MMPs may underlie the pathogenesis of trophoblast shadow invasion. In addition,down-regulation of F10 may play a role in the regulation of MMPs and TIMPs in the pathogenesis of pre-eclampsia and FGR.CHAPTER 3 THE EXPRESSION OF MATRIX METALLOPROTEINASES AND F10 GENE IN THE HYDATIDIFORM MOLEOBJECTIVEHydatidiform mole is a group of benign pregnant trophoblastic cell diseases which related to trophoblast invasion. Being the potential malignancy, it may develop in to invasive hydatidiform mole and choriocarcinoma. But up to now, the pathologival mechanism of the development and neoplasia of hydatidiform mole has been not clear. In our previous studies, F10 and MMPs may underlie the pathogenesis of trophoblast shadow invasion. Similarly,we hypothesis that the hydatidiform mole related gene F10, trophoblast invasion regulators MMP and TIMPs might involved the pathogenesis of hydatidiform mole. In this study, we investigated the effect of F10 and MMPs/TIMPs on the hydatidiform mole disease.METHODSVillus samples were obtained from pregnancies hospitalized and outpatients in Nanfang Hospital (Guangzhou, China) between January 2012 and December 2013,including hydatidiform mole (N=6) as experimental group and induced abortion (N=6)as the control group. We used western blot and immunohistochemical method to detecting the expression of F10, MMPs and TIMPs in the two group villus tissues.Comparison between two groups was done with independent sample T-test analysis.RESULTS1. Demographic data of the study populationNo significant difference was found in maternal age and the gestational age between control group and hydatidiform mole (P>0.05).2. The expression of MMPs and TIMPs in two groupsIn the two groups, we detected protein levels of eight MMPs and two TIMPs.Compared with those in normal villus, the expression of MMP-2 (P<0.001)、-8(P=0.002)、-9 (P<0.001)、-11 (P<0.001) were up-regulated in villous tissues of hydatidiform mole cases by Western blot analyzing. TIMP-1 (P=0.003) and -3(P<0.001 ) were decreased in hydatidiform mole (P<0.001). No significant difference was found on the expression of MMP-19, MT2-MMP, MT3-MMP and MT5-MMP between two groups (P>0.05).Results from immunohistochemical analyzing,we found the expression of MMP-2 (P=0.004) 、-8 (P=0.004) 、-9 (P=0.001 )、-11 (P=0.003) were up-regulated in villous tissues of hydatidiform mole cases and the expression of TIMP-1 (,P=0.004)、-3 (P<0.001) were down-regulated.3. The expression of F10 proteins were up-regulated in hydatidiform moleCompared with those in normal villus, the expression of F10 was up-regulated in villous tissues of hydatidiform mole cases (P<0.001).CONCLUSIONCompared with normal villus, the expression of F10 and TIMP-1, TIMP-3 up-regulated in hydatidiform mole, meanwhile, the expression of MMP-2, MMP-8,MMP-9, MMP-11 were down-regulated which were contrary to the results from pre-eclampsia and FGR. It suggested that F10 gene might be play a role in the trophobalst invasion with the company of MMPs and TIMPs.CHAPTER 4 IDENTIFICATION OF F10 AS A NOVEL CELL CTCLE REGULATOR IN HUMAN TROPHOBLAST PROLIFERATIONOBJECTIVEOur previous studies found that the abnormal expression of F10 and MMPs/TIMPs in trophoblast invasion-related diseases. F10 were suggested as a key regulator in the processing of trophoblast invasion by adjusting the expression of MMPs and TIMPs. In the present study, we employed both gain and loss function approaches to investigate the role of F10 in trophoblast cells to study the character and function of F10 gene in human trophoblast cells and to approach which signal transduction were affected in F10 interfere cell proliferation.METHODSStable F10 over-expression and silenced-expression HTR8/SVneo cell lines were structured. The effects of F10 depletion or over-expression on cell proliferation were examined by MTT The expression of cell proliferation-related factors PCNA and cyclinE were examined by immunofluorescence. And apoptosis-related factors caspase-3 were examined by Western blot. Flow cytometry assay was used for detecting the cell apoptosis gated. The expression of signal proteins MEK1/2,ERK1/2, PI3K and STAT3 were examined by western blot analysis. One-Way ANOVA is adopted to analyze the datas in the multiple groups, Bonferroni test is applied to analyzed the comparison between two groups.RESULTS1. The establishment of silent and overexpression vector and virus, setting up the stable cloneBy RT-PCR (P<0.001) and Western blot (P<0.001) analysis, the establishment of F10 stable over-expression and silenced-expression HTR8/SVneo cell clone was successfully constructed.2. Modelation of F10 expression in cell proliferation2.1 The number of F10 over-expression cells was induced from second day after plating. respectively, compared with the control cells by MTT analysis(P<0.001).2.2 The F10 over-expression cells group reveal the protein expression levels of PCNA (P<0.001) and CyclinE (P<0.001) was significantly high as compared to the control. But no significant difference was found between silenced-expression group and control group(P>0.05). From these results, it was clear that F10 could effectively promote the cell proliferation.3. Effects of F10 expression on cell apoptosis3.1 Flow cytometry and Western blot analysis were used in order to obtain more accurate and comprehensive information about the impact of F10 on cell apoptosis.While HTR-8/SVneo cells were transfected over-expression vector with F10, a significant decrease of apoptosis was observed as compared to those control cells using both flow cytometry (P<0.001). In contrast,the cells infected with F10 silenced-expression didn’t show an apparent increase of apoptosis in contrast to those control cells (P>0.05). There was no difference in the number of apoptotic cells between control group and F10 gene silenced-expression group.3.2 Western blot analysis indicated that the expression level of caspase-3 protein increased in the cells treated with F10 over-expression (P<0.001). Meanwhile there was no difference between control group and F10 gene silenced-expression group(P>0.05).4. The expression of signal proteins in F10 suppresses cell proliferationTo examine which signal pathway F10 promotes EVT cell invasion, we used Western blot analysis. Results with an anti-active p-ERKl/2 (P<0.001) , p-MEK1/2(P<0.001) antibody showed strong signals for over-expression transfectants. We further found that F10 expression did not change the expression level of p-PI3K and p-STAT3. Also,we found no significant differences between binding of above anti-active total signal protein antibody to F10 over-expression trophoblast cells compared with HTR8/SVneo.CONCLUSIONCollectively, our observations indicate that over-expression of F10 may promote trophoblast cell proliferation. Furthermore, F10 gene might induced cell proliferation through stimulate of the MEK/ERK signal proteins. Taken together, these data suggest that F10 is involved in regulating proliferation and invasion of trophoblast cells.SUMMARY1. Compare with normal subjects, significant down-regulation of MMP-2,MMP-8, MMP-9, MMP-11 and up-regulation of TIMP-1, TMP-3 were detected in pre-eclampstic and FGR placentas. IThe expression of MMPs and TIMPs were abnormal, which may underlies the pathogenesis of pre-eclampsia and FGR.2. The novel gene F10 was found down-regulation in pre-eclampsia and FGR placentas with marked reduing MMPs protein concentration. There might be a relationship between MMPs and F10 gene. F10 gene might be the upstream stimulating factor by regulating the expression of MMPs involving the trophoblast invasion process.3. In contrast, the expression of MMP-2, MMP-8, MMP-9, MMP-11 were up-regulation with marked elevated F10 protein concentration and TIMP-1, TIMP-3 were down-regulation in hydatidiform mole villus. We once again proved that F10 gene might be the upstream stimulating factor by regulating the expression of MMPs involving the trophoblast invasion process.4. A clone of stable F10 over-expression and silence human HTR8/SVneo cells was successfully constructed. It was a basis step for further research the character and function of F10 gene. The cell clone of overexpression F10 gene induced increased of cell growth and suppressed cell apoptosis. This processing might be through activating of the MEK/ERK signal pathways by F10 gene.