The Expression Of USP22at Human Materno-Fetal Interface During Early Pregnancy And Its Effects On Invasiveness Of Trophoblast

Trophoblast cells behavior like tumor invasiveness and cell proliferation at human materno-fetal interface during the early stages of normal pregnancy. The interaction of trophoblast cell and endometrial cell initiates series of sigaling cascader compose of adhesion, migration, dissolve extracellular matrix, angiogenesis and invasion of the uterine epithelium during embryo implantation.Then three physiological processes of adhasion, invasion and migration are completed. But the invasion of trophoblast which is different from the behavior of tumor cell is limited only to the endometrium and to the proximal third of the myometrium after placentation. The invasion of trophoblasts is precisely controlled but errors can happen and might have fatal consequences under some circumstances. For example, over aggressive invasion (deeper than the proximal third of the myometrium or beyond the extent of myometrium) is associated with gestational trophoblastic disease (eg. hydatidiform mole, choriocarcinoma and placental site tumors). In contrast, shallow invasion is associated with preeclampsia, spontaneous abortion and intrauterine growth retardation. Therefore, it is very imporiant to explore the mechanism of the invasion of trophoblasts and it will be helpful to prevent and cure diseases associated with trophoblasts abnormal invasion.Ubiquitin specific peptidase22(USP22) is a newly discovered potential cancer stem cell marker. Encoding products of USP22gene are highly expressed in a variety of tumors and paticipate in the regulation of gene transcription about cell transformation and cell cycle. The current research about UPS22participate in trophoblast invasion has not been yet reported. To explore the expression and mechanism of USP22in materno-fetal interface, our research investigated the localization and expression in villus and decidua of USP22in normal early pregnancy and recurrent miscarriage patients by immunohistochemistry, realtime quantitative polymerase chainreaction (PCR) and western blot. Then we investigate the invasion behavior of choriocarcinoma JEG-3cell after down-regulating expression of USP22by siRNA, MMP-2and MMP-9 expression of trophoblast cells by realtime PCR and western blot. The objective of this research was to explore the expression of USP22at human materno-fetal interface during early pregnancy and its effects on invasiveness of trophoblast at the level of cytology and molecular biology. A fully understanding of the role of USP22in the physiological state and pathological disease is critical for new therapeutic strategies.Part I The expression of USP22gene at the maternal-fetal interface during early pregnancyObjective:To investigate the localization and expression of USP22in normal early pregnancy and in patients with recurrent spontaneous abortion.Methods:The localization, mRNA and protein expression levels of USP22in villi and decidua from20cases of normal early pregnant women and15patients with unexplained recurrent spontaneous abortion were detected by immunohistochemistry, fluorescence real-time quantitative PCR and western blot.Results:(1) Immunohistochemical result showed that there were both expression in villi and decidua of normal pregnancy and patients with recurrent spontaneous abortion, and the expression in villi was higher than decidua and increased with the progress of pregnancy early pregnancy(P<0.05); Compared with normal pregnancy group, USP22expression levels of villi and decidua in patients with recurrent spontaneous abortion were significantly decreased (P<0.05).(2) Real-time quantitative PCR showed that the expression of USP22mRNA was in villi and decidua; compared with normal pregnancy, USP22mRNA expression level of villi in patients with recurrent spontaneous abortion were significantly decreased (P<0.05), and there were not statistically significant difference in decidua(P>0.05).(3) Western blot detection also indicated that the expression of USP22protein was in villi and decidua; compared with normal pregnancy, USP22protein expression level of villi in patients with recurrent spontaneous abortion were significantly decreased (P<0.05), and there were not statistically significant difference in decidua (P>0.05). Conclusion:USP22was expressed in the maternal-fetal interface, and it participated in regulation of trophoblast invasion in the normal early pregnancy and maintenance of normal pregnancy. The down-regulation of USP22expression in the maternal-fetal interface may be one of the causes of spontaneous abortion. Part II:Preparation and infection of lentivirus to interfere USP22Objective:To construct a lentivirus mediated siRNA expression vector for shRNA oligonucleotide fragments transcripted by USP22gene, and to establish a stable JEG-3cells which down-regulate USP22gene by infecting lentivirus.Methods:siRNA oligonucleotide sequence was prepared for USP22gene according to USP22gene sequence analysis software, and it was cloned into lentiviral vector PGCL-GFP to establish recombinant plasmid PGCL-USP22, liposomal transfected and packaged293T cells. After24h, the supernatant was collected, concentrated, and infected JEG-3cells. USP22silencing efficiency in JEG-3cells were detected by realtime quantitative PCR and Western blot.Results:The sequencing results of the recombinant plasmid PGCL-USP22confirmed that shRNA expression template was successfully cloned on PGCL vector, completely correct nucleotide insertion sequence without mutations and deletions. Realtime quantitative PCR and Western blot showed that:there were varying degrees of USP22downregulation in three PGCL-RNAi cell clones, USP22shRNA-3were downregulated more than70%which identified as effectively silence of USP22.Conclusion:Lentivirus-mediated SiRNA expression vector was constructed successfully, it packaged virus and infected target cells, and we establish a stable JEG-3cells which down-regulate USP22gene. Part III:Effect of down-regulation of USP22on choriocarcinoma JEG-3cell invasion behaviorObjective:To investigate the effect of down-regulate expression of USP22by siRNA on biological behavior and related molecules expression of choriocarcinoma trophoblast cells.Methods:The invasion of choriocarcinoma JEG-3cell after down-regulate expression of USP22by transwell invasion test, MMP-2and MMP-9expression of trophoblast cells by Western blot.Results:Transwell experiment showed that the number of invasion JEG-3cell in down-regulate USP22group (12.145±3.125) was significantly reduced compared to control group (20.143±3.128)(P<0.01). The expression of MMP-2and MMP-9mRNA levels in interference group were significantly lower than that of the control group (P<0.01); The expression of MMP-2and MMP-9protein levels in interference group were significantly lower than the control group (P<0.01).Conclusion:The ability of invasion of JEG-3cells was significantly reduced after inhibiting the expression of USP22. USP22promoting invasion of JEG-3cells could be associated by MMP-2, MMP-9transcriptional activation.