Preliminary Study Of Long Non-Coding RNA GAS5 Associating With Preeclampsia And Regulating Biological Behaviors Of Trophoblast

Objective: Preeclampsia is a lethal pregnancy specific hypertensive disorder involving multisystem,especially the early-onset type,carries a significantly higher risk of maternal and fetal complications which often deteriorates rapidly.Despite advances in technology,extensive studies have been conducted,the pathogenesis still remains largely unknown.Termination of pregnancy is still the only effective treatment which induces iatrogenic preterm birth and adverse maternal and fetal outcomes.The two-stage model hypothesis is the most widely accepted theory.During normal pregnancy,cytotrophoblasts emigrate from the chorionic villi and invade the uterus deeply,replace the maternal spiral arteries’ endothelial lining and insert themselves amongst the smooth muscle cells.However,the invasion is inadequate and the implantation is shallow in patients with preeclampsia.As a result,ischemic placenta releases damaging factors effecting on systemic vascular endothelial cell and induces the clinical symptoms subsequently.Numbers of studies have examined different expression of protein-coding genes in preeclampsia placentas.However,about 75% of the genome is transcribed into non-coding RNA transcripts during biological activities.Long non-coding RNAs(lnc RNAs)are a diverse class of non-translated RNAs which play an important role in the regulation of gene expression.They act as antisense decoys,signals and scaffolds,likewise,engaging in interferences of transcription.Lnc RNA GAS5 is a transcript of the growth arrest-specific 5(gas5)gene,it is dysregulated in multiple human cancers and tumor cells confirming a tumor suppressor role.GAS5 can act as the molecular spongy binding numerous micro RNAs but the details are still largely unclear.Negative regulation between GAS5 and micro RNA-21(mi R-21)has been certificated in various tumors and other diseases,and the probable mechanism is the disruption of mi R-21 to form RNA-induced silencing complex(RISC),which makes the mi R-21 inhibited cancer suppressor genes like PTEN(Phosphatase and tensin homolog deleted on chromosome 10)reactivated,involving the PI3K/AKT(phosphatidylinositol 3-kinase,PI3K/serine-threonine kinase,AKT)signaling pathway and the downstream proteins.The implantation of trophoblast is similar with the biological behaviors of tumor cells.Until now,role of GAS5 in preeclampsia has not been reported.Previous work of our research group showed that in GEO Profiles,high throughput sequencing affirmed the high expression level of GAS5 in human placental tissue and the different expression between preeclampsia and normal pregnancy.Therefore,this research firstly attempted to verify the GAS5 involvement of preeclampsia,the different clinical features of preeclampsia and the correlation between GAS5 and clinical parameters.Subsequently,we want to determine the influence of GAS5 on the biological behavior of trophoblast cells.Finally,GAS5 was proved to target mi R-21 and affect PI3K/AKT signaling pathway as well as the downstream proteins.Thus the relevant mechanism of GAS5 involved the preeclampsia pathogenesis was preliminarily explored.Methods: In this study,40 patients(20 cases with early-onset preeclampsia and 20 cases with late-onset)and 32 gestational age-matched normotension pregnant women(12 cases with placenta previa associated with placenta accreta spectrum disorders(PASD)were set as early-onset control group,requiring early termination of pregnancy,and 20 cases with normal pregnancy in the third trimester)were recruited.Using quantitative real-time polymerase chain reaction(q RT-PCR),we detected the expression of GAS5 in placentas and figured out its correlation with clinical parameters by Pearson correlation analysis.Using student’s t test,the differences of GAS5 expression between the groups with or without maternal,fetal adverse outcome were determined.Then we constructed the GAS5 lentivirus expression vectors and transfected into human trophoblast cell lines JEG-3 and HTR-8/SVneo.Using in vitro cell culture studies,we found the effect of GAS5 on trophoblast biological behaviors including proliferation,apoptosis,migration and invasion by MTT assay,flow cytometry,scratch test assay and Transwell assay.Then we did further mechanistic analysis to reveal the relation between GAS5 and micro RNA-21(mi R-21)by q RT-PCR and rescue experiment.In addition,the involvement of PI3K/AKT signaling pathway and its downstream proteins including MMP-9 and TP53 in regulation of GAS5 was studied by both q RT-PCR and western blot analysis.Results: We found higher level of GAS5 in placentas of preeclampsia affected women especially in early-onset group(P<0.05).The clinical parameters of preeclampsia groups demonstrated obvious differences compared with controls indicating the involvement of multiple organ.The early-onset and the late-onset showed their unique characters that the pathogenesis may not be identical.The expression of GAS5 existed strongly in correlation with Thrombin Time,fibrinogen indicating coagulation function and serum protein level (0.5<r<0.8).However,GAS5 was not significantly associated with adverse maternal and fetal outcomes(P>0.05).After successful construction of GAS5 lentivirus overexpression and knockdown vectors,we found that knockdown of GAS5 enhanced the proliferation of the two trophoblast cell lines and the overexpressed GAS5 inhibited the proliferation ability(P<0.05).Overexpression of GAS5 inhibited the migration and invasion of the trophoblasts(P<0.05),but knockdown of GAS5 did not affect these abilities(P>0.05).However,no effect on apoptosis was detected.To further explore the relevant mechanism,we firstly detected the increased expression level of mi R-21 after GAS5 knocking down,on the contrary,the level of mi R-21 decreased when GAS5 overexpressed(P<0.05).After the successful construction of mir-21 lentiviral vectors,both the overexpression vectors of GAS5 and mir-21 were co-transfected into the two types of trophoblasts,the expression of GAS5 was down-regulated by the overexpressed mir-21.In contrast,after co-transfection of knocking down vectors,the expression of GAS5 was up-regulated by knocking down of mir-21(P<0.05).Further,knockdown of GAS5 activated PI3K/AKT signal pathway in both RNA and protein levels(P<0.05),this alteration in Western Blot assay was more significant.The downstream MMP-9 and TP53 were also activated,but due to the low expression quantity,this phenomenon was only verified in q RT-PCR.In trophoblast cells,GAS5 did not affect the RNA and protein expression of PTEN(P>0.05).Conclusion: This is the first report of the relation between GAS5 and preeclampsia.1.The expression of GAS5 increased in placental tissues of preeclampsia,especially in early-onset preeclampsia,and the clinical characteristics of early-onset and late-onset were not identical.GAS5 highly correlated with thrombin time,fibrinogen indicating coagulation function and plasma protein level.Nevertheless,GAS5 was not relevant to the occurrence of adverse maternal and fetal outcomes.2.Overexpression of GAS5 inhibited the proliferation,migration and invasion of JEG-3 and HTR-8/SVneo trophoblasts,but knocking down of GAS5 only promoted the proliferation of trophoblast without effect on migration and invasion.GAS5 showed no effect on apoptosis in trophoblasts.3.GAS5 negatively regulated mi R-21.Knocking down of GAS5 activated the PI3K/AKT signaling pathway and the downstream proteins MMP-9 and TP53,and the overexpression of GAS5 inhibited this pathway and the downstream proteins,noticing the expression amount of MMP-9 and TP53 are rare.GAS5 couldn’t alter the expression of PTEN gene in trophoblast cells.