Regulation Of Non-coding RNA On The Function Of Trophoblast Cells And Mechanism Exploration In Preeclampsia

Preeclampsia(PE), a kind of hypertensive disorders complicating pregnancy, is a pregnancy-specific disease with a new onset of hypertension and proteinuria after 20 weeks of pregnancy as its main clinical manifestation, and even can cause damage to multiple organs. It is a leading contributor to the increased maternal and perinatal morbidity and mortality, with adverse impacts to maternal and child health. The incidence of the disease is high, while the pathogenesis is not yet completely understood. There have been many mechanisms associated with the pathogenesis, such as: the insufficient remodeling of uterine spiral arteries(commonly known as "shallow placental implantation"), endothelial dysfunction(including inflammatory mediators and oxidative stress, etc.), genetic factors and nutritional deficiencies, and insulin resistance. Among these, endothelial dysfunction and insufficient remodeling of uterine spiral arteries are closely associated with the behavioral change of extravillous trophoblast, to some extent. Then further exploration of the various factors affecting extravillous trophoblast’s biological functions and the relevant regulatory mechanisms, will provide great help to further interpretation of the pathogenesis of PE, and its prevention as well. In recent years, a large number of studies have reported the role of non-coding RNA(nc RNA) on multiple cells through a variety of regulatory mechanisms, thereby affecting the occurrence and development of human disease. Even prediction was made by some scientists thatnc RNA might has no less importance than proteins in the process of biological development. Thus in this study, the regulatory role of two members in nc RNAs family including micro RNA and lnc RNA on trophoblast cells in the pathogenesis of PE were explored: 1. The aberrant expression of mi R-101 in PE placentas could modulated trophoblast cells HTR-8/SVneo apoptosis and then modified endoplasmic reticulum stress(ERS) induced apoptotic proteins by targeting ERp44 in vitro, thereby contributing to the pathogenesis of PE; 2. The dysregulated of long noncoding RNA MVIH in PE placentas might participate in the remodeling of spiral arteries by regulating trophoblast cells proliferation, migration, invasion and tube formation ability in both and trophoblast cells endothelial cells as well.Part I:Mi R-101 regulates apoptosis of trophoblast cells HTR-8/SVneo by targeting Endoplasmic reticulum(ER) protein 44 during preeclampsiaObjective:1. To analyze the difference of mi R-101 expression level between human preeclampsia(PE) and normal placenta tissues. To investigate the co-relationship and possible targeted binding between mi R-101 and endoplasmic reticulum protein 44(ERp44) according to the differentiated expression level of mi R-101 in preeclampsia and combined with our previous work on ERp44.2. To explore whether the endoplasmic reticulum stress and induced trophoblast apoptosis could be regulated importantly by the combining of ERp44 and mi R-101.Methods:1. A total number of 60 placenta villus tissues(including 30 PE and 30 normal) were obtained from primipara women who underwent cesarean deliveries from Desember, 2011 to March, 2012 in our hospital. To detect the expression level of mi R-101 in 30 PE and 30 normal placentas by using the real-time quantitative polymerase chain reaction(q PCR). Combined with our previous work on the expression detection of ERp44 proteins in these tissue samples, the Person co-relation analysis was used to analyze the co-relationship between mi R-101 and ERp44 proteins. Also the luciferase reporter assay was applied to verify the targeted binding between these two factors.2. For further analysis of the effect of mi R-101 on the biological behaviors(apoptosis)of the functional cells of placenta(extravillous trophoblast cells), the human extravillous trophoblast HTR-8/SVneo cells were overexpressed and knock-down with mi R-101 expression respectively. Then the q PCR assay was used to clarify the over-expression and knockout efficiency, the Western blotting assay to detect the expression of ERp44 proteins as well.3. The flow cytometry and Hoechst staining were utilized for trophoblast apoptosis analysis after the HTR-8/SVneo cells were overexpressed and down-expressed with mi R-101. And Western blotting assay was adopted to detect the expression of apoptotic proteins Caspase-3 and Bcl- 2.4. The Western blotting assay was used to evaluate the activation of endoplasmic reticulum stress-related signaling pathway(the proteins expression level of Caspase-12, ATF-6, PERK, CHOP, IRE1 p and ATF-4) after that the HTR-8 / SVneo cells were overexpressed and down-expressed with mi R-101.5. To further verify the activated degree of endoplasmic reticulum stress by the knockout of mi R-101, Western blotting analysis was to determine the exression of above-mentioned proteins under the treatment of endoplasmic reticulum stress inhibitor phenyl butyric acid(4-Phenylbutyric acid, 4-PBA).Resuts:1.(1) The comparison of clinical data about the 30 PE and 30 normal pregnant women included in this study proved that the maternal age, smoking history and other indicators showed no significant difference with p> 0.05 while the systolic blood pressure, diastolic blood pressure, proteinuria and birth weight were differentiated statistically with p﹤0.05 or p﹤0.01.(2) We found a 60% lower expression of mi R-101 in preeclamptic placentas than normal, with p<0.05. And there existed an inverse correlation between mi R-101 and ERp44 protein in PE placentas, with a inverse correlation coefficient-0.826. The luciferase reporter gene assay confirmed a targeted binding of mi R-101 to the 3’UTR region of ERp44, with a fluorescence inhibition rate of 30% when mi R-101 was co-transfected with wild ERp44 sequence.2. The expression level of mi R-101 in HTR-8/SVneo cells was 28-fold higher than control while a decreased expression of 45% was found in ant-mi R-101 group as compared to its control, which proved the success of transfection experiments. The expression of ERp44 protein was inhibited by 50% after mi R-101 was overexpressed, while a 2.39-fold increase was detected after mi R-101 was blocked, with significantly statistical difference.3. Upregulation of mi R-101 expression could inhibit apoptosis of HTR-8/SVneo cells, with a corresponding regulation to apoptosis-related proteins Caspase-3 and Bcl-2. However, a reverse was disclosed when mi R-101 was blocked.4. The endoplasmic reticulum stress signaling pathway was blocked when mi R-101 was over-expressed in HTR-8/SVneo cells, with improvement of Caspase-12、ATF-6、PERK、CHOP、IRE1p、ATF-4 proteins. Then an activation of endoplasmic reticulum stress signaling pathway was found when the expression of mi R-101 was inhibited, combined with decrease of Caspase-12、ATF-6、PERK、CHOP、IRE1p、ATF-4 proteins.5. The decrease of mi R-101 could partially reverse the inhibition to endoplasmic reticulum stress signaling pathway caused by 4-PBA.Conclusion:MiR-101 can contribute to the ER stress induced trophoblast cells apoptosis by targeting ERp44 in PE.Part II:Long Noncoding RNA MVIH regulates proliferation,migration & invasion and tube formation of trophoblast cells by inducing Jun B protein during the remodeling of spiral arteries in preeclampsiaObjective:1. To analyze the different expression level of MVIH in human preeclampsia(PE) placenta tissues compared with normal.2. To explore the regulated role of MVIH on biological functions including proliferation,migration & invasion and tube formation of trophoblast cells and endothelial cells during the remodeling of spiral arteries in PE.3. To explore the possible pathogenesis associated with MVIH and PE by selecting the proteins which is modulated by MVIH.Methods:1. A total number of 60 placenta villus tissues(including 30 PE and 30 normal) were obtained from primipara women who underwent cesarean deliveries from Desember, 2011 to March, 2012 in our hospital. To detect the expression level of MVIH in 30 PE and 30 normal placentas by using the real-time quantitative polymerase chain reaction(q PCR).2. For further analysis of the effect of MVIH on the biological behaviors of the functional cells of placenta(trophoblast cells), the human extravillous trophoblast HTR-8/SVneo and JEG-3 cells were overexpressed and knock-down with MVIH expression respectively. Then the q PCR assay was used to clarify the over-expressionand knockout efficiency.3. In order to explore the regulated role of MVIH in trophoblast cells’ proliferation, the MTT assay and flow cytometry were utilized to detect the change of trophoblast cells’ viability and the percent of each cell cycle after the HTR-8/SVneo and JEG-3 cells were overexpressed and down-expressed with MVIH.4. After both HTR-8/SVneo and JEG-3 cells were transfected with overexpression and down-regulated of MVIH respectively, the transwell assay(without the matrigel) was used to evaluate the role of MVIH in HTR-8/SVneo cells migration ability while the transwell assay(with the matrigel) was to detect the invasion potential of JEG-3 cells.5. In order to verify the role of MVIH in tube formation of trophoblast cells and endothelial cells. An Endothelial Tube Formation Assay Kit was used to analyze the effect of MVIH in tube formation potential of HTR-8/SVneo and human umbilical vein endothelial cells(HUVEC) which were overexpressed and blocked with MVIH. Then q PCR analysis was adopted after the two cells were treated as above to test the expression of angiogenesis-related factors such as vascular endothelial growth factor(VEGF), angiopoietin I and II(Ang I, Ang II).6. To explore the possible mechanism in which MVIH regulating the functions of trophoblast cells and endothelial cells. The protein profiling was used to select and analyze the dys-regulated proteins in HTR-8/SVneo cell after it was treated with MVIH overexpression and si-RNAs targeting MVIH.7. To detect the regulated role of the target protein on the above mentioned cell functions(proliferation and migration in HTR-8/SVneo cells and tube formation in HUVEC), we used an overexpression plasmid to transfect trophoblast cells and HUVEC. Then the target protein was co-transfected with si-MVIH to deeply verify the regulared role in cells’ functions which is directly modified by MVIH.Resuts:1.(1) The comparison of clinical data about the 30 PE and 30 normal pregnant women included in this study proved that the maternal age, smoking history and other indicators showed no significant difference with p> 0.05 while the systolic blood pressure, diastolic blood pressure, proteinuria and birth weight were differentiated statistically with p﹤0.05 or p﹤0.01.(2) We found a 70% lower expression of MVIH in preeclamptic placentas than normal, with p<0.01.2. The expression level of MVIH in HTR-8/SVneo cells was 39.31-fold higher than control while a decreased expression of 72% was found in si-MVIH group as compared to its control, while a 21.05-fold higher of MVIH expression was found in after over-expressed with MVIH and a 63% lower when MVIH was blocked in JEG-3 cells. These proved the success of transfection experiments.3. Upregulation of MVIH in both HTR-8/SVneo and JEG-3 cells could improve the cells viability, and accelerate the mitotic process by promoting the cells to go to S or G2 cycle from G1 cycle. However, when MVIH was blocked in both cells, the viability of cells was decreased and the mitotic process was inhibited in varying degrees with a lower cells percent of S or G2 cycle and an increase of G1 cycle.4. Migration ability of HTR-8/SVneo cells were accelerated by MVIH overexpression, while was decreased by the block of MVIH, which were prove by transwell assay(without the matrigel). Then in the JEG-3 cells, upregulation of MVIH could promote the invasion potential while an adverse results with down-regulation of MIVH.5. Endothelial Tube Formation Assay proved that both the branch point and total length of the tubes were increased in MVIH overexpression groups in both HTR-8/SVneo and HUVEC cells, while both decrease in si-MVIH groups. Also, the q PCR analysis presented an ascent of VEGF, Ang I and Ang II while a drop of s Flt-1.And a series of reverse results were found in both cells after block of MVIH expression.6. After tested by the protein profiling analysis, we selected that Jun B protein was induced in HTR-8/SVneo cell after treated with MVIH overexpression plasmid while was decreased in si-MVIH group compared with each corresponding control. Thus the Jun B protein might be regulated directly by MVIH.7.(1) Compared with control, HTR-8/SVneo cells which were over-expressed with Jun B presented an increase in cells proliferation and migration, while when co-transfected with si-MVIH, the promotion was partly inhibited.(2) These results proved that MVIH might participate in the modification of trophoblast and HUVEC cells’ functions which are invlloved in the remodeling of spiral arteries in PE through inducing Jun B proteins.Conclusion:MVIH participate in remodeling of spiral arteries in PE by modulating biological functions of trophoblast cells and endothelial cells including proliferation,migration & invasion and tube formation through inducing Jun B proteins.