Objective To investigate the expression level and clinical significance of circular RNAs(circRNAs)in serum extracellular vesicles(EVs)of patients with recurrent spontaneous abortion(RSA).Methods The serum samples from 3 RSA patients and 3 nor-mal pregnant women(controls)visited our hospital from May 2021 to March 2023 were collected and the gene expression profiling chips were used to screen for differentially expressed circRNAs in serum EVs.Ten RSA patients and 10 normal pregnant women were enrolled in a training set and 80 RSA patients and 64 normal pregnant women were enrolled in a validation set.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the levels of differentially expressed circRNAs between the two groups.The predictive value of differentially expressed circRNAs for RSA was evaluated by the receiver operating characteristic(ROC)curve.Results A total of 57 563 circRNAs in serum EVs were detected by the gene expression profiling chips.Compared with the control group,3 516 circRNAs were differentially expressed in RSA patients,including 2 377 up-regulated and 1 139 down-regulated.The de-tection results of qRT-PCR in the training set and validation set showed that the expression levels of hsa＿circ＿0054403 in serum EVs of RSA patients(2.07[1.00,6.68])was significantly higher than that in the control group(1.00[0.42,1.46],U=1 239,P＜0.01),while those of hsa＿circ＿0020897(0.33[0.11,1.40]vs 1.00[0.46,3.66],U=1 712,P＜0.01)and hsa＿circ＿0072745(0.49[0.22,1.60]vs 1.00[0.51,7.93],U=1 714,P＜0.01)were significantly lower than that in the control group.The ROC curve showed that the hsa＿circ＿0054403,hsa＿circ＿0020897,and hsa＿circ＿0072745 in serum EVs had high predictive value for RSA.Their AUCROC were 0.758,0.666,and 0.664,respectively,and the corresponding sensitivity and specificity were 47.5% and 100.0%,50.0% and 81.2%,and 62.5% and 70.3%,respectively.When the three circRNAs were combined,it's AUCROC,sensitivity,and specificity were 0.842,73.7%,and 79.7%,respectively.Conclusion The hsa＿circ＿0054403,hsa＿circ＿0020897,and hsa＿circ＿0072745 in serum EVs of RSA patients are abnormally expressed,which may serve as the potential markers for predicting RSA.

Objective To investigate the expression level and clinical significance of circular RNAs(circRNAs)in serum extracellular vesicles(EVs)of patients with recurrent spontaneous abortion(RSA).Methods The serum samples from 3 RSA patients and 3 nor-mal pregnant women(controls)visited our hospital from May 2021 to March 2023 were collected and the gene expression profiling chips were used to screen for differentially expressed circRNAs in serum EVs.Ten RSA patients and 10 normal pregnant women were enrolled in a training set and 80 RSA patients and 64 normal pregnant women were enrolled in a validation set.Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the levels of differentially expressed circRNAs between the two groups.The predictive value of differentially expressed circRNAs for RSA was evaluated by the receiver operating characteristic(ROC)curve.Results A total of 57 563 circRNAs in serum EVs were detected by the gene expression profiling chips.Compared with the control group,3 516 circRNAs were differentially expressed in RSA patients,including 2 377 up-regulated and 1 139 down-regulated.The de-tection results of qRT-PCR in the training set and validation set showed that the expression levels of hsa＿circ＿0054403 in serum EVs of RSA patients(2.07[1.00,6.68])was significantly higher than that in the control group(1.00[0.42,1.46],U=1 239,P＜0.01),while those of hsa＿circ＿0020897(0.33[0.11,1.40]vs 1.00[0.46,3.66],U=1 712,P＜0.01)and hsa＿circ＿0072745(0.49[0.22,1.60]vs 1.00[0.51,7.93],U=1 714,P＜0.01)were significantly lower than that in the control group.The ROC curve showed that the hsa＿circ＿0054403,hsa＿circ＿0020897,and hsa＿circ＿0072745 in serum EVs had high predictive value for RSA.Their AUCROC were 0.758,0.666,and 0.664,respectively,and the corresponding sensitivity and specificity were 47.5% and 100.0%,50.0% and 81.2%,and 62.5% and 70.3%,respectively.When the three circRNAs were combined,it's AUCROC,sensitivity,and specificity were 0.842,73.7%,and 79.7%,respectively.Conclusion The hsa＿circ＿0054403,hsa＿circ＿0020897,and hsa＿circ＿0072745 in serum EVs of RSA patients are abnormally expressed,which may serve as the potential markers for predicting RSA.

Objective To investigate the expression level and clinical significance of hsa_circ_0005075 in serum extracellular vesicles(EVs)of patients with recurrent spontaneous abortion(RSA).Methods Fourteen RSA patients and 14 normal pregnant women from the Department of Obstetrics and Gynecology,Qilu Hospital of Shandong University were enrolled in a training set,and 64 RSA pa-tients and 48 normal pregnant women were enrolled in a validation set.The expression levels of hsa_circ_0005075 in serum EVs were detected by the quantitative real-time PCR(qRT-PCR),and their correlation with clinical pathological parameters of RSA patients were analyzed.Serum anti-thyroid globulin antibody(A-TG)and anti-thyroid peroxidase antibody(A-TPO)were detected by the elec-trochemiluminescence assay.Serum anticardiolipin(ACA)IgA,IgG,and IgM antibodies and anti-β2 glycoprotein 1(β2GP1)IgA,IgG,and IgM antibodies were determined by the chemiluminescence immunoassay.The correlation of these autoantibodies with the lev-els of hsa_circ_0005075 in serum EVs was analyzed by the Pearson correlation.The clinical application value of hsa_circ_0005075 in the diagnosis of RSA was evaluated by the receiver operating characteristic(ROC)curve.Results The detection results of the training set showed that the expression levels of hsa_circ_0005075 in serum EVs of RSA patients(7.69[4.74,42.15])were significantly high-er than that in normal pregnant women(1.02[0.51,4.23],U=28,P＜0.01].Similarly,in the validation set,the expression levels of hsa_circ_0005075 in RSA patients(4.96[1.73,8.89])were also significantly higher than that in normal pregnant women(1.00[0.24,2.96],U=693,P＜0.01).The ROC curve showed that hsa_circ_0005075 in serum EVs had good diagnostic value for RSA(AUCROC=0.774),with 70.3%of sensitivity and75.0%of specificity.In addition,the expression level of hsa_circ_0005075 in serum EVs was significantly correlated with A-TPO(r=0.298,P＜0.05).Conclusion The hsa_circ_0005075 in serum EVs is highly ex-pressed in RSA patients,which may have a potential differential diagnostic value for the diagnosis of RSA.

Objective To explore the correlation of serum microRNA-499a-5p (miR-499a-5p), fibroblast growth factor 9 (FGF9) and inflammatory factors with the severity and prognosis of acute lung injury (ALI) caused by sepsis. Methods A total of 151 septic patients were selected for the study. They were divided into sepsis with acute lung injury (ALI) group (68 cases) and general sepsis group (83 cases) based on the occurrence of ALI. According to the oxygenation index, septic patients with ALI were further categorized into mild (23 cases), moderate (26 cases), and severe (19 cases) groups. The survival status of septic patients with ALI was assessed within 28 days, and they were divided into good prognosis group (43 cases) and poor prognosis group (25 cases). Additionally, 151 healthy individuals undergoing physical examinations during the same period were selected as the control group. The differences and correlations among miR-499a-5p, FGF9 and inflammatory factors in each group were analyzed. The predictive efficacy of serum miR-499a-5p and FGF9 in patients with sepsis complicated with ALI was analyzed by receiver operating characteristic (ROC) curve. Results The levels of miR-499a-5p and FGF9 in the mild, moderate, and severe groups showed significant decrease in a sequential manner, while the inflammatory factors interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and C-reactive protein (CRP) levels showed significant increase in a sequential manner (P < 0.05). The miR-499a-5p and FGF9 levels in the poor prognosis group were significantly lower than those in the good prognosis group, while the IL-1, IL-6, TNF-α, and CRP levels were significantly higher in the poor prognosis group (P < 0.05). In patients with sepsis and ALI, miR-499a-5p and FGF9 were negatively correlated with IL-1, IL-6, TNF-α, and CRP levels (P < 0.05); miR-499a-5p could positively regulate FGF9 expression (P < 0.05). The diagnostic efficacy of predicting the prognosis of septic patients with ALI using a combination of miR-93 and miR-135a was significantly higher than that of individual prediction (P < 0.05). Conclusion The levels of miR-499a-5p, FGF9 and inflammatory factors are associated with the severity and prognosis of ALI in patients with sepsis. The miR-499a-5p and FGF9 may serve as potential biomarkers for the evaluation of prognosis in septic patients with ALI.

The prevention and control of Novel Coronavirus Pneumonia caused by Novel Coronavirus is at a critical period. Nucleic acid detection, as the definite diagnosis tool, plays an important role in rapid diagnosis, therapeutic efficacy, epidemic prevention and control. However, the disease is outbreak, and the time of nucleic acid detection in clinical application is short. So the insufficient method verification and clinical evaluation has been made. "False negative" is observed in clinical practice, and the result of nucleic acid detection is not matched with the clinical diagnosis. Therefore, it is urgent to optimize the current methods and improve detection sensitivity. Based on latest studies of Novel Coronavirus, this article reviews the current status and application prospects of nucleic acid detection. Also, this article provides references for clinicians and researchers.

Objective To evaluate the diagnostic value of CEA, CA19-9, CA72-4, AFP, and CA125 in gastric stromal tumors. Methods 41 patients with gastric stromal tumors and 11 patients with gastric leiomyoma were recruited in this study from Qilu Hospital of Shandong University during May 2014 to December 2017. The tissue was collected by surgery, and HE staining was done. Immunohistochemistry was employed to detect the expression of CD34, CD117, and DOG-1. Serum of all cases and 41 healthy volunteers in the same hospital were collected. The levels of CEA, CA19-9, CA72-4, AFP and CA125 were examined by electrochemiluminescence assay, and the differences in each group were compared by M-W test or K-W test. Then the relationship between those biomarkers and the clinical parameters of gastric stromal tumors was analyzed. Moreover, AUC (Area Under the curve), cut-off value, sensitivity, specificity, positive predictive value, negative predictive value with drawing ROC curve (receiver operating characteristic curve) were also calculated. Results Spindle cells or epithelioid cells were observed in the tissue of gastric stromal tumors. The expression of CD34, CD117, DOG-1 were positive. The level of the serum CEA 1.53 (1.15, 2.22) ng/ml in patients with gastric stromal tumor patients was higher than that in healthy controls 1.06 (0.62, 1.48) ng/ml and that in patients with gastric leiomyoma 0.79 (0.39, 1.39) ng/ml (the U value was 446.5, and 113.0 respectively, P<0.05). The level of CA19-9 in gastric stromal tumors 9.30 (4.95, 12.70) U/ml was higher than that in healthy controls 6.62 (4.56, 8.82) U/ml (the U value was 615.5, P<0.05). There were no significant differences in AFP, CA125, and CA72-4 of three groups (the H value was 4.348, 1.073, and 3.897, P>0.05). Furthermore, the level of CEA was closely related to TNM stage (the U value was 129.0, P<0.05). The diagnostic value of CEA and CA19-9 was statistically not significant (P>0.05). However, the combination of two markers might increase diagnostic efficiency. The sensitivity, specificity, positive predictive value, negative predictive value and AUC was 92.7％, 48.8％, 64.4％, 87.0％ and 0.752 respectively. Conclusion The combination of CEA and CA19-9 has better sensitivity and negative predictive value in auxiliary diagnosis of gastric stromal tumors.

Objective To explore the effect of hypoxia on the proliferation of human umbilical vein endothelia cell (HUVEC) and its underlying mechanism.Methods HUVEC were incubated at different oxygen concentrations (1％,5％,21％) and at 5％ CO2 at a temperature of 37 ℃ for 24 hours.Proliferation rate was detected by WST-1.The target gene of miR-99a was predicted by bioinformatics and verified by double luciferase reporter assay.MiR-99a mimics,miR-99a siRNA,mTOR siRNA,or mTOR cDNA were transfected in HUVEC by lipofectamine 2000,and then incubated in hypoxia.Mir-99a and mTOR were respectively detected by qRT-PCR and RT-PCR or Western blot.Results The proliferation of HUVEC was obviously decreased under hypoxia (1％ O2) (33.3％ ± 6.5％),as compared with normoxia(21％ 02) (P =0.004).Hypoxia could up-regulate the expression level of miR-99a (1.94 ± 0.24,P =0.001),which further inhibited the proliferation of HUVEC.Bioinformatics and double luciferase reporter assay indicated that mTOR was the target gene of miR-99a.Under hypoxia,the gene and protein level of mTOR were down-regulated by miR-99a.Further research demonstrated that the proliferation of HUVEC was inhibited under hypoxia by miR-99a-mTOR pathway.Conclusions Hypobaric treatment could significantly inhibit the proliferation of HUVEC,which might be associated with miR-99a-mTOR pathway.

Objective To explore the effect of hypoxia on the proliferation of human umbilical vein endothelia cell (HUVEC) and its underlying mechanism.Methods HUVEC were incubated at different oxygen concentrations (1％,5％,21％) and at 5％ CO2 at a temperature of 37 ℃ for 24 hours.Proliferation rate was detected by WST-1.The target gene of miR-99a was predicted by bioinformatics and verified by double luciferase reporter assay.MiR-99a mimics,miR-99a siRNA,mTOR siRNA,or mTOR cDNA were transfected in HUVEC by lipofectamine 2000,and then incubated in hypoxia.Mir-99a and mTOR were respectively detected by qRT-PCR and RT-PCR or Western blot.Results The proliferation of HUVEC was obviously decreased under hypoxia (1％ O2) (33.3％ ± 6.5％),as compared with normoxia(21％ 02) (P =0.004).Hypoxia could up-regulate the expression level of miR-99a (1.94 ± 0.24,P =0.001),which further inhibited the proliferation of HUVEC.Bioinformatics and double luciferase reporter assay indicated that mTOR was the target gene of miR-99a.Under hypoxia,the gene and protein level of mTOR were down-regulated by miR-99a.Further research demonstrated that the proliferation of HUVEC was inhibited under hypoxia by miR-99a-mTOR pathway.Conclusions Hypobaric treatment could significantly inhibit the proliferation of HUVEC,which might be associated with miR-99a-mTOR pathway.

Objecfive To investigate the effects of treatment for cerebrovascular disorder patients with heparin and low molecular weight heparin(LMWH) on serum PAPP-A concentrations and provide the basis for evaluating the clinical significance of PAPP-A in the following study.Methods Forty cases with cerebrovascular disease from Qilu Hospital from November 2009 to May 2010 were collected in this study.Blood samples were taken before and after drug administration.All cases were divided into four groups according to situation of medication.Group A consisted of 10 patients who received subcutaneous LMWH anticoagulation therapy, and blood samples were collected before LMWH injection, three hours after subcutaneous LMWH anticoagulation therapy in the first day, the second day and the seventh day and 24 hours after the last injection. Group B consisted of 10 patients who did not receive LMWH therapy, and blood samples were collected immediately after admission, the first day, the second day and the seventh day after admission. Group C consisted of 10 patients with percutaneous carotid intervention who received intravenous heparin at the beginning of stenting, and blood samples were collected from the arterial sheath just before angiography and heparin administration, and at 3, 5, 15, 40 and 100 min after heparin administration. Group D consisted of 10 patients who received carotid angiography but LMWH-free therapy,and blood samples were collected from the arterial sheath just before and after angiography. Serum PAPP-A concentrations were analyzed by ELISA to evaluate the differences of intra-groups and differences at different time points of inter-groups. Results In group A, PAPP-A concentrations were time dependent and elevated gradually from 12. 36 (9. 90-14. 32) mIU/L before LMWH injection to 21.80 (23.50-19.73) mIU/L at the seventh day after injection (M=38. 72, P < 0.01 ). In group C, there was a rapid increase of PAPP-A concentration from 12. 86 ( 9. 67-14. 05 ) mIU/L to 51.56 ( 44. 20-66. 00 ) mIU/L within 5 min after intravenous heparin injection (M=46. 06, P <0. 01 ). The PAPP-A concentration of one week after LMWH administration in group A was 21.80 (23.50-19.73) mIU/L, significantly higher than that in group B [11.81 (9. 21-12. 89) mIU/L] (U<0. O01, P<0.01). The PAPP-A concentration at 15 min after heparin administration in group C was 43.70 (37.70-54. 30) mIU/L, significantly higher than that after angiography in group D [14. 18 (11.25-15. 86) mIU/L] ( U<0. 001, P <0. 01 ). The peak level of blood PAPP-A after subcutaneous LMWH injection was significantly lower than that after intravenous heparin injection. The concentrations in group A and C were 21.80 ( 23.50-19. 73 ) and 51.56 (44. 20-66. 00) mIU/L respectively, and had a significant difference ( U=0. 999, P < 0. 01 ) . Conclusions Both intravenous heparin and subcutaneous LMWH administration induce an increase in serum PAPP-A concentration. The effect of drug should be considered when PAPP-A is selected as an evaluation indicator.

Endoplasmic reticulum stress,an critical component of cell stress,is a protective response for eukaryotic cells. The change of partial pressure of oxygen (for instance hypoxia) is a primary condition for cell stress,significantly impacting the biological phenotype and behavior of cells (including survival,migration and invasion et al). Through endoplasmic reticulum stress,intracellular concentration of unfolded protein is reduced,and agglutination inhibited. Recently,endoplasmic reticulum stress has been demonstrated to play an important role in pathogenesis of some diseases such as cancer,diabetes and inflammation,which are characterized by the micro-environment hypoxia in local tissue. Therefore,investigations on the mechanism of endoplasmic reticulum stress caused by hypoxia may promote the novel therapeutic strategy for tumors,cardiovascular diseases and diabetes.