The Study Of Angiogenin’s Regulation And The Related MiRNA-mechanism On The Invasion And Migration Functions Of Trophoblast Cells

Trophoblast cell(TB) is one of many important functional cells in mammalian placenta. In the normal course of early pregnancy, through the controllable and physiological invasion, trophoblast cells could reach the decidua tissues which lie in about one third of myometrium, facilitate the spiral arteries recast and expansion, and provide adequate nutrient supply and blood supply between maternal and fetal organization. Under normal physiological conditions, this "physiological attack" has clearly temporal and spatial characteristic. The invasion of trophoblast only occurs at early stages of pregnancy, reaches the peak at 12 th week of pregnancy, and gradually gets weaken during 14th-20 th week of pregnancy. Meanwhile, the trophoblastic invasion only reaches one third of the myometrium and has no effect on other tissues. However, under pathological conditions, dysfunction of trophoblastic invasion could directly lead to pregnancy-related diseases. For example, the excessive invasion of trophoblast cells would damage the surrounding tissues and ltimately lead to cancer, such as choriocarcinoma, and etc. In the process of preeclampsia(PE), due to inadequate trophoblastic implantation depth by a variety of factors, the implants and recast of spiral artery are restricted, which could result in miscarriage, placental ischemia, growth restriction, and etc. Thus, this physiological invasion of trophoblast is an important prerequisite for the successful implantation of the fertilized egg and embryo development, but the specific regulatory mechanism of its invasion function is poorly understood until now.In our previous studies, we found that, compared to the respectively control group, the invasion and migration functions of HTR-8/SVneo cells treated by 5% of endometrial stromal conditioned medium(DSCM) were significantly increased, while the level and activity of MMP-2 expression were significantly enhanced. Meanwhile, we also found that the invasion and migration functions were significantly reduced after treated by 20% DSCM, and the expression of MMP-2 was significantly reduced. In order to reveal the exact mechanism, then we used the protein microarrary to analyze the different expression of cytokines in treated trophoblasts. We found that the expression of angiogenin was significantly increased after 5% DSCM treatment and was significantly reduced after 20% DSCM treatment, which was closely related to the ability of trophoblastic invasion and migration. Therefore, we speculate that angiogenin might play an important regulatory role in trophoblastic invasion and migration functions. Objective and MethodBased on the previous studies, we intended to compare the expression of angiogenin in different cell lines and clinical samples by q PCR and Western blot methods. After the construction of angiogenin overexpression plasmid and si RNA, we would verify the regulation effects of angiogenin on trophoblastic invasion and migration capabilities by scratch and transwel experiments. Through the methods such as q PCR, Western blot, reporter assay methods and etc, we would elucidate the related mi RNA mechanisms involved in the regulation of angiogenin. Finally, we would test the expression of angiogenin, MMP-2 and related mi RNA in PE patients and normal pregnacy, analyze the correlationship among them, and further demonstrate the role of angiogenin in the pathological development of PE. ResultsThe results can be divided into four parts, as follows:1. Expression of angiogenin in cells after different DSCM treatment and clinical samples. First, we verified the Chip results by q PCR method, we found that, consistent with the microarray results, there was a significant correlation between the angiogenin level and the invasion functions of trophoblast cells after DSCM treatment. Meanwhile, the expression of angiogenin in JEG-3, the choriocarcinoma cell line, which has stronger invasion ability, was significantly higher than normal trophoblast cell line, HTR8/SVneo. Angiogenin levels in preeclampsia placenta were significantly lower than normal pregnant patients. In conclusion, these results suggested that Angiogenin might be involved in the regulation of trophoblastic invasion and migration.2. Angiogenin influences the invasion and migration functions of trophoblast cells. In order to demonstrate the effect of angiogenin on trophoblast cell function, we first designed and constructed angiogenin over-expression plasmid named p CMV-ANG. By gene sequencing and Western blot, we confirmed that the Angiogenin over-expression plasmid was successfully constructed plasmid. After overexpression of angiogenin by transfected in trophoblast cell line HTR8/SVneo, we found that the increased angiogenin significantly promoted the invasion and migration functions of trophoblast cells, and significantly upregulated the expression of MMP-2. Then downregulated the expression of angiogenin by transfection with angiogenin si RNA in HTR8/SVneo cells, we found that reduced angiogenin level significantly inhibited the trophoblastic invasion and migration functions and the expression of MMP-2. In order to confirm the correlation between angiogenin and MMP-2 in the regulation of trophoblast functions, we designed rescued functional experiments by the MMP-2 si RNA and found that the downregulated expression of MMP-2 significantly inhibited the promoting effects of angiogenin in trophoblastic invasion and migration abilities. Those results suggested that angiogenin promoted trophoblastic invasion and migration functions, which was dependent on MMP-2.3. Research on the mi RNA related mechanism of angiogenin regulation. In order to clarify the exactly regulatory mechanism of angiogenin on trophoblastic functions, we firstly analyzed the previous papers data about related mi RNA microarray. Through q PCR validation, we finally found that mi R-519d-3p was differentially expressed in trophoblast cells during pregnacy. After transfection of mi R-519d-3p mimics and inhibitor, transwel and scratch experiments showed that overexpression of mi R-519d-3p by mimics inhibited trophoblastic invasion and migration functions, downregulated expression by inhibitor promoted trophoblastic invasion and migration functions. Through the luciferase reporter gene assay, q PCR and Western blot, we found that there was a binding site of mi R-519d-3p in MMP-2 3’UTR, by which mi R-519d-3p inhibited the m RNA and protein expression of MMP-2. Through functional experiments and rescued experiments, we found that mi R-519d-3p regulated the trophoblastic invasion and migration by targeting MMP-2. Finally, we designed rescued experiments by transfection of angiogenin si RNA and mi R-519d-3p inhibitor, the results confirmed that the mechanism of mi R-519d-3p was participated in the regulation of angiogenin on trophoblastic invasion and migration.4. The expression of Angiogenin, mi R-519d-5p and MMP-2 in clinical sample. In order to confirm the regulation of Angiogenin/mi R-519d-3p/MMP-2 pathway in the pathogenic process of trophoblastic related diseases such as PE, we tested the expression and the correlation of angiogenin, mi R-519d-3p and MMP-2 in the placenta of s PE patients and normal pregnancy by q PCR and Pearson correlation analysis. q PCR results showed that, compared with normal pregnacy, the expression of angiogenin and MMP-2 in s PE patients’ placenta were significantly decreased, and the mi R-519d-3p expression were significantly increased. Meanwhile, Pearson correlation and linear regression analysis confirmed that, in the placenta of s PE patients, the expression of angiogenin, mi R-519d-3p and MMP-2 were significantly correlated, which suggested that the angiogenin/mi R-519d-3p/MMP-2 regulatory pathway might be involved in the pathogenic procedure of PE. ConclusionBy the results of the whole study, we could get the conclusions as below:1. Angiogenin is involved in the regulation of trophoblastic function. It can significantly promote the trophoblastic invasion and migration functions depending on regulating the expression of MMP-2.2. mi R-519d-3p can inhibit the trophoblastic invasion and migration functions by targeting MMP-2.3. Angiogenin play an important role in regulating the trophoblastic invasion and migration by mi R-519d-3p/MMP-2 pathway. Meanwhile, Angiogenin/mi R-519d/MMP-2 pathways was probably involved in the pathogenesis of PE.