Effect Of Differentially Expressed RPAIN On The Biological Behavior Of Placental Trophoblast By Complement Protein C1q And Its Mechanism

Preeclampsia is a pregnancy-specific syndrome that affects 3-5%of pregnancies and is traditionally diagnosed by the combined presentation of high blood pressure and proteinuria.Preeclampsia is one of the main causes of maternal,foetal and neonatal mortality,especially in low-income and middle-income countries.,however the exact aetiology and pathophysiology remain uncertain.Long non-coding RNA(LncRNA)are a class of transcripts whose lengths exceed 200 nt and do not encode proteins.These lncRNAs have a series of important functions and participate in the development of many diseases,including cancers,cardiovascular diseases and neurovascular-related disorders through multiple levels of transcription,post transcriptional and epigenetic.RPAIN is the IncRNA we found by IncRNA chip technique for preeclampsia-related differences.In order to find out the possible mechanism of RPAIN,we analyzed its position and nucleic acid sequence by UCSC and BLAST,and found that Clqbp is adjacent to it.It is speculated that Clqbp may be the regulatory target of RPAIN.The purpose of this study was to elucidate the function of RPAIN in preeclampsia At the same time,the correlation between RPAIN and C1qbp was further studied,clarify its target relationship and explore its possible mechanism in preeclampsia.Objective1.To detect the expression of RPAIN in placenta of normal pregnancy and preeclampsia.2.The overexpression strategy was used to demonstrate the function of RPAIN in trophoblast cells.3.The molecular mechanism of RPADIN leading to the alteration of trophoblast cells lines via C1qbp and then participate in the pathogenesis of preeclampsia was analyzed by the rescue strategy.Methods1.The expression of RPAIN in placenta of normal pregnancy and pre eclampsia was detected by real-time fluorescent quantitative PCR(qPCR).2.Construction of lentiviral vector,infection of human HTR-8/SVneo trophoblast cells,to build a stable expression of RPAIN cell lines,using qPCR method to detect overexpression efficiency.3.CCK8(Cell Counting Kit-8).transwell assay and flow cytometry were used to research the effect on the function of HTR-8/SVneo trophoblast cells(proliferation,invasion and apoptosis)after transfected with lentivirus RPAIN,as well as the changes in cell proliferation,cell invasion and apoptosis-related factors.4.Construction of C1qbp,RPAIN + Clqbp overexpression of lentiviral vector,co-infection of human HTR-8/SVneo trophoblast cells,qPCR was used to detect the expression efficiency.5.The proliferation,cell invasion and cell apoptosis of HTR-8/SVneo trophoblast cells were detected by CCK8,transwell assay and flow cytometry.To elucidate the molecular mechanism of RPAIN through Clqbp leading to the function of trophoblast cells and to participate in the preeclampsia.Result1.qPCR results showed that the expression of RPAIN in placenta of preeclampsia patients was higher than that in normal pregnancy group(P<0.01).2.qPCR results showed that the expression of RPAIN was obviously higher than that control group after transfected with lentivirus RPAIN in HTR-8/SVneo trophoblast cells(P<0.01).3.Lentivirus RPAIN was transfected into human HTR-8/SVneo trophoblast cells to observe the effect on the biological function of trophoblasts.The results show that RPAIN overexpression significantly suppressed HTR-8/SVneo cell proliferation and cell invasion(P<0.01)and promote cell apoptosis(P<0.01).4.The HTR-8/SVneo trophoblast cells were constructed to construct Clqbp,RPAIN+C1qbp overexpressing lentiviral vector,the results of qPCR showed that the expression of overexpression of Clqbp was higher than that of the control group(p<0.001).The expression of C1qbp was decreased after co-transfection with RPAIN +C1qbp(p<0.05).5.CCK-8 assay and transwell assay showed that cell proliferation and cell invasion was promoted when C1qbp was overexpressed of HTR-8/SVneo trophoblast cells(p<0.001),whereas co-overexpression of RPAIN and C1qbp restored cell invasion(p<0.001)and cell proliferation was not significantly changed(p>0.05)of HTR-8/SVneo trophoblast cells.Flow cytometry analysis revealed that Clqbp overexpression could decrease cell apoptosis of HTR-8/SVneo trophoblast cells(p<0.05),whereas co-overexpression of RPAIN and C1qbp also inhibited apoptosis of HTR-8/SVneo trophoblast cells(p<0.01).Conclusion1.The expression of RPAIN in placental tissue of patients with preeclampsia was significantly higher than that in normal pregnant women,while the expression of C1qbp in the preeclampsia was lower than that in normal pregnancy group.C1qbp as the target of RPAIN,RPAIN had a significant negative regulation on C1qbp of HTR-8/SVneo trophoblast cells.2.The overexpression of lentivirus RPAIN in HTR-8/SVneo trophoblast cells can restrain the cell proliferation and invasion ability and promote cell apoptosis ability.C1qbp overexpression can increase cell proliferation and cell invasion and decrease cell apoptosis of HTR-8/SVneo trophoblast cells.Co-overexpression of RPAIN and Clqbp restored cell invasion and inhibited apoptosis of HTR-8/SVneo trophoblast cells.Therefore,the high expression of RPAIN in placenta of preeclampsia may affect the biological function of placental trophoblast cells through C1qbp,which is participate in the development of preeclampsia.