| BackgroundEctopic pregnancy(EP)refers to the pregnancy that occurs outside the uterine cavity,the most prone to occur in the fallopian tube.At present,EP diagnosis mainly depends on vaginal ultrasonography.However,in early and early tubal pregnancy,because the ectopic mass is too small and B-ultrasound can not be recognized,biomarkers are needed to assist in the diagnosis of EP.The commonly used biomarkers in the clinic are the growth of HCG and progesterone,especially HCG.However,during the series of HCG examinations waiting for diagnosis,the ectopic pregnancy masses continually erode to the fallopian tubes,which may cause rupture of the fallopian tubes and cause massive hemorrhage in the abdominal cavity and even death.At present,there are more than 20 kinds of studies on EP diagnostic biomarkers,but most of them have high false positives or false negatives.The diagnostic results are not ideal,and they are not.well used in clinical practice,and these markers are not very good.Identify EP and spontaneous abortion(SA).Therefore,this study designed a cohort study to collect serum from early pregnancy patients with vaginal bleeding or abdominal pain,extract serum exosomes for sRNA sequencing,follow-up pregnancy outcomes,screen for EP,SA,and viable intrauterine pregnancy(VIP)Differentially expressed miRNAs were analyzed by qRT-PCR,and progesterone and HCG levels were correlated to explore biomarkers for the diagnosis of EP.Huayu Xiaozheng Granule is the in-house preparation of the First Affiliated Hospital of Guangzhou University of Traditional Chinese M edicine.It is the result of the team that led by Professor Deng Gaojun devoted to the clinical and basic research of tubal pregnancy for more than 20 years.MiR-184 is a differentially expressed miRNA in EP patients sequenced and validated in serum exosomes,and is expressed in many tumors.The invasion and migration behavior of early tubal gestational trophoblasts is similar to that of tumor cells.Therefore,based on previous studies,this study used human chorionic trophoblast cell HTR-8/SVneo as a carrier to study the mechanism of miR-184-mediated granules.Clinical researchObjectiveScreening-verified serum exosome miRNAs,combined with serum HCG and progesterone levels,were used to investigate the combination of individual biomarkers and multiple biomarker combinations for early EP.MethodsBy second-generation high-throughput sequencing of exosomal sRNA,with|Fold Change |>1.5,p<0.01 as the threshold,the miRNAs with significant differential expression of EP,SA and VIP were screened for qTR-PCR verification.Correlation analysis was performed between progesterone and HCG levels,and the area under the ROC curve(AUC)of the diagnostic EP biomarkers was calculated.The specificity of fixation was 90%and 95%,respectively.Results1.As a single biomarker for the diagnosis of EP,progesterone has the largest AUC 0.777,the highest fixation sensitivity is 90%,the highest specificity is 60%for microRNA-378d,the highest fixation sensitivity is 95%,and the highest specificity is 36%for microRNA-146a-5p.As a combination of multiple biomarkers for diagnosing EP,AUC was improved to 0.801-0.889,fixed sensitivity was 90%,specificity of HCG+P+microRNA-146a-5p and HCG+P+microRNA-215-5p were 76%,fixation sensitivity was 95%,and HCG+P+microRNA-146a-5p sensitivity was 40%.2.As a biomarker for diff-erential diagnosis of EP and SA,progesterone obtained the highest AUC 0.746 in a single biomarker,with a fixed sensitivity of 90%,and the highest specificity of 44.4%in microRNAs-378d and microRNAs-215-5p.Among the multiple biomarker combinations,HCG+P+miR-146a-5p also achieved an ideal AUC 0.783 with a fixed sensitivity of 90%,and HCG+P+miR-146a-5p had the highest specificity of 66.60%.Basic experimentObjectiveTo study the differential expression of miR-184 in the villus pregnancy and normal intrauterine pregnancy villus tissue;to explore the function of miR-184;to find the miR-184 target gene and miR-184-based phlegm-killing granules to kill embryos And molecular mechanisms.Methods1.Clinical organization verificationThe expression of miR-184 in the ovarian pregnancy villus tissue and normal intrauterine pregnancy villus tissue was verified by qRT-PCR.2.MiR-184 function verificationExogenously transfected miR-184 mimics,inhibitor and respective NC of HTR-8/SVneo cells overexpressed or inhibited miR-184,and qRT-PCR detected miR-184 expression in HTR-8/SVneo cells.Cell proliferation was detected by CCK8 assay,apoptosis was detected by flow cytometry with AnnexinV/PI double staining,cell migration was detected by scratch assay,cell invasion was detected by transwell assay,and HCG was detected by ELLISA.3.MiR-184 target gene prediction and verificationThe target genes of miR-184 were predicted by TargetScan,miRDB,miRanda,and CLIP biosignal analysis,and target gene screening was performed according to mirSVR values in combination with literature and research purposes.Overexpression or inhibition of miR-184 EPB41L5 mRNA and protein levels was detected by qRT-PCR and Western blot.It was verified by dual luciferase reporter gene experiments whether miR-184 directly targets the 3' UTR end of EPB41L5 mRNA.SiRNA interference analysis can silence the change of HTR-8/SVneo cell invasion and migration induced by miR-184 overexpression or inhibition after silencing EPB41L5.4.Effect of Huayu Xiaozhen Granule on the proliferation,invasion,migration and expression of HCG in trophoblastic cells HTR-8/SVneoHTR-8/SVneo cells were treated with different concentrations of Huayu Xiaozheng Granules,cell proliferation was detected by CCK8 assay,cell mi gration was detected by scratch test,cell invasion was detected by transwell assay,and HCG changes were detected by ELLISA.5.Effect of Huayu Xiaozhen Granule on the expression of miR-184 and target gene EPB41L5 in HTR-8/SVneo cellsHTR-8/SVneo cells were treated with high,medium and low doses of Huayu Xiaozheng Granule.The expressions of miR-184 and EPB41L5 mRNA in HTR-8/SVneo cells were detected by qRT-PCR.The expression of EPB41L5 protein was detected by Western blot.Results1.miR-184 was lowly expressed in the villus pregnancy,which was statistically different from normal intrauterine pregnancy(P<0.05).2.Transfected miR-184 mimics,inhiitor and NC 48h,the level of miR-184 in HTR-8/SVneo cell overexpression group was up-regulated,and the level of miR-184 in the interference group was down-regulated,which was statistically different from the respective NC group(P<0.05).There was no significant difference in HTR-8/SVneo cell proliferation,apoptosis and HCG expression between the groups overexpressing or interfering with miR-184(P>0.05).At 72 h after transfection,the migration distance of miR-184 overexpression group decreased,and the cell migration distance of the interference group increased.The number of transmembrane cells in the miR-184 overexpression group increased,and the number of transmembrane cells in the miR-184 interference group decreased,which was statistically different from the NC group(P<0.05).3.? The target gene of miR-184 was predicted to be EPB41L5 by biosignal analysis.?Western blot showed that the expression of EPB41L5 protein in HTR-8/SVneo cells was down-regulated in miR-184 overexpressing group and transfected with miR-184 mimicking agent,interfering agent and each negative control for 72h,and the expression of EPB41L5 protein in the interference group was up-regulated.There was a statistical difference(P<0.05).qRT-PCR confirmed that the same trend of EPB41L5 mRNA changes after 48 hours of the same method(P<0.05).?double luciferase reporter gene experiments confirmed that miR-184 directly acts on the 3' UTR end of EPB41L5,thereby inhibiting the expression of EPB41L5.After 72 hours of silencing of EPB41L5 by 4SiRNA,HTR-8/SVneo cell migration was inhibited;HTR-8/SVneo cell invasion was inhibited,and compared with Si-EPB41L5 NC group,Si-EPB41.5 group had a decrease in transmembrane cells and statistically significant(P<0.05).4.?Huayu Xiaozheng Granule can inhibit the proliferation of HTR-8/SVneo cells in a concentration-and time-dependent manner.Compared with the blank group,the cell viability of Huayu Xiaozheng Granule 1,2 g/L group was significantly different after 12 hours of treatment(P<0.01).The cell viability of Huayu Xiaozheng Granule 0.125,0.25,0.5,1,2 g/L group was significantly different after 24 hours and 48 hours of treatment(P<0.05).The cell viability of HTR-8/SVneo cells treated with Huayu Xiaozheng Granules at the same concentration for 12,24 and 48 hours decreased significantly(P<0.05)in 0.25,0.5,1 and 2 g/L groups.? Huayu Xiaozheng Granule 0.5g/L could significantly inhibit HTR-8/SVneo cell migration.? After 24 hours of drug intervention on HTR-8/SVneo cells,the number of cell membrane penetration decreased in Huayu Xiaozheng granule 0.4 and 0.5g/L groups,which was significantly different from that in NC group(P<0.01).Compared with MTX group,the number of invasive cells in Huayu Xiaozheng Ke 0.3 g/L and 0.4 g/L groups was higher(P<0.05),and the number of invasive cells in 0.5 g/L group was lower(P<0.01).? Compared with the blank group,the expression of beta-HCG in Huayu Xiaozheng Granule 0.4 and 0.5g/L groups was down-regulated,and the difference was statistically significant(P<0.01).MTX could inhibit the secretion of beta-HCG in HTR-8/SVneo cells.Compared with the blank group,the difference was significant(P<0.01),and the difference was significant(P<0.01)compared with the Huayu Xiaozheng Granule 0.3g/L group.5.? Huayu Xiaozheng Granules(0.5g/L,0.4g/L and 0.3g/L)could up-regulate the expression of microRNA-184 in HTR-8/SVneo cells for 8 hours,which was significantly different from that in the.NC group(P<0.05).?0.5g/L,0.4g/L and 0.3g/L Huayu Xiaozheng Granules could reduce the expression of EPB41L5 in HTR-8/SVneo cells for 24 hours,which was significantly different from that in NC group(P<0.05).Conclusion1.Clinical research(1)In HCG,progesterone and 6 candidate miRNA single markers,progesterone diagnosis EP has the best discrimination,miR-378d specificity is the highest,but because clinical diagnosis of progesterone EP is not ideal,so this experiment The single marker in the diagnosis of EP is not effective.In the multi-marker combination for the diagnosis of EP,the discrimination of different combinations was similar,and HCG+P+miR-146a-5p and HCG+P+miR-215-5P were the most specific,both being 76%(fixed sensitivity 90%).HCG+P+miR-146a-5p was the most sensitive when the fixed sensitivity was 95%,which was 40%.(2)As a marker for differential diagnosis of EP and SA,progesterone achieved the highest AUC of 0.746,miR-378d and miR-215-5P with the highest specificity of 44.4%(fixation sensitivity 90%)in a single biomarker.,wherein miR-378d AUC is higher,at 0.684.Among the multiple biomarker combinations,HCG+P+miR-146a-5p achieved an ideal AUC of 0.783,with the highest specificity of 66.6%(fixation sensitivity of 90%).2.Basic experiment(1)miR-184 is down-regulated in the villus tissue of the fallopian tube.miR-184 has the function of inhibiting invasion and migration of HTR-8/SVneo cells.(2)Biosignal analysis predicted and confirmed that EPB41L5 is a target gene of miR-184 by qRT-PCR,western and dual luciferase reporter genes.The 3' UTR end of EPB41L5 mRNA is a direct target of miR-184.(3)Huayu Xiaozheng Granule can inhibit the proliferation,invasion,migration and HCG secretion of HTR-8/SVneo cells.(4)Huayu Xiaozheng Granule can up-regulate the expression of miR-184 in HTR-8/SVneo cells and down-regulate the expression of its target gene EPB41L5 protein and mRNA.It is suggested that Huayu Xiaozheng Granule may exert the killing effect by regulating miR-184 to inhibit the invasion and migration of trophoblast cells. |
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