ABSTRACT
Objective:To observe the changes of the expression level of long non-coding RNA (lncRNA) KCNQ1OT1 and microRNA (miR)-146a-3p in placenta tissues of preeclampsia (PE) patients, as well as their effect and mechanism on the biological functions of trophoblast cells.Methods:A total of 45 cases of hospitalized PE patients in Hainan General Hospital from July 2017 to July 2018 were selected as the PE group, 55 normal pregnant women during the same period were chosed as the control group. The expression level of KCNQ1OT1 mRNA and miR-146a-3p in the placenta tissues between two groups were detected by using quantitative real time (qRT)-PCR. Pearson′s test was furtherly analyzed the correlation between them. Human trophoblast cell line (HTR8/SVneo) were randomly divided into control and lipopolysaccharide (LPS) groups, and then LPS group were divide into four sub-groups,included LPS group, short hairpin RNA (sh)-KCNQ1OT1 (after silencing the expression of KCNQ1OT1), miR-146a-3p inhibitor and sh-KCNQ1OT1+miR-146a-3p inhibitor. The targeting relationship between KCNQ1OT1 and miR-146a-3p were predicted by bioinformatics software and confirmed by luciferase assay. The cell proliferation and invasion capacities were respectively detected by cell counting kit-8 (CCK-8) and transwell assay. The expression level of KCNQ1OT1 mRNA and miR-146a-3p were detected by qRT-PCR and the protein expression level of CXC chemokine ligand 12 （CXCL12） and CXC chemokine receptor type 4 (CXCR4) were tested by western blot.Results:(1) The mRNA expression level of KCNQ1OT1 in the placenta of PE group was lower than that of control group (0.23±0.03 vs 0.51±0.04, P<0.05), and the miR-146a-3p expression level was higher than that of the control group (0.49±0.03 vs 0.31±0.03, P<0.05), there were statistical significant differences between the two groups. (2) Luciferase assay showed that there was a targeting relationship between KCNQ1OT1 and mir-146a-3p. Compared with the control group, the mRNA expression level of KCNQ1OT1 in the LPS group were significantly decreased (0.91±0.03 vs 0.35±0.03, P<0.05), and the expression level of miR-146a-3p were significantly increased (0.22±0.03 vs 0.63±0.04, P<0.05). The cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 significantly reduced in the LPS group compared with control group (all P<0.05). The mRNA expression level of KCNQ1OT1 (0.23±0.03) in the sh-KCNQ1OT1 group were further decreased, the expression of miR-146a-3p (0.85±0.03) were further increased, and the cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 were all further reduced compared with control group，there were significant difference between two groups (all P<0.05). Comparing the miR-146a-3p inhibitor group, and sh-KCNQ1OT1+miR-146a-3p inhibitor group with the sh-KCNQ1OT1 group, respectively, the expression level of KCNQ1OT1 mRNA (0.78±0.04 vs 0.50±0.03) increased, and the expression level of miR-146a-3p (0.42±0.03 vs 0.46±0.03) decreased, the cell proliferation, invasion and migration capacities and the protein expression of CXCL12 and CXCR4 were all increased ,there were statistically significant differences (all P<0.05). Conclusion:KCNQ1OT1 could target the regulation of miR-146a-3p through CXCL12/CXCR4 pathway in the proliferation, invasion an migration of HTR8/SVneo cells, which may be involved in the pathogenesis of PE.
ABSTRACT
Objective:To study the expression and significance of ADMA/NOS/NO/cGMP pathway in the maternal-fetal interface of patients with preeclampsia. Methods: 60 patients were selected and divided into severe preeclampsia group, mild preeclampsia group and gestational hypertensive group according to the disease condition, meanwhile 20 healthy pregnant women of singleton receiving cesarean section were chosen as control group. Levels of NO and cGMP in placenta of the four groups were detected and compared,and activities of total NOS in placenta were measured and compared. The expression of eNOS and iNOS in placental tissue was detected by immunohistochemistry SP method. High-performance liquid chromatography was used to detect the level of ADMA in HUVECs. Results:The levels of NO in placenta of severe and mild preclampsia groups were (7. 6±3. 6) and (11. 4±4. 3) μmol/g, which were statistically significantly lower than that in control group(P<0. 05). The levels of cGMP in placenta of severe and mild pre-clampsia groups were ( 3. 26 ± 0. 31 ) and ( 4. 53 ± 0. 42 ) pmol/g, which were aslo significantly lower than that in control group ( P<0. 05). In the four groups,the level of cGMP in placenta was positively correlated with the level of NO in placenta(r=0. 672). The activities of total NOS in placenta of severe and mild preeclampsia groups were (10. 4±3. 0)and (14. 8±1. 6)U/mg protein,which were statistically significantly lower than that in control group ( P<0. 05 ) . In placenta of the four groups, the activity of total NOS was positively correlated with the level of NO(r=0. 785). The expression of eNOS in placenta of severe and mild preeclampsia groups was statistically significantly lower than that in control group ( P<0. 05 ) , the expression of iNOS in severe preeclampsia group was significantly higher than that in the control group(P<0. 05). The levels of ADMA in HUVECs of severe preeclampsia group,mild pre-eclampsia group and gestational hypertensive group were statistically significantly higher than that in the control group(P<0. 05). The level of ADMA in HUVECs was negatively correlated with the level of NO in placenta of the four groups ( r=-0. 582 ) . Conclusion:ADMA/NOS/NO/cGMP pathway may play an important role in the pathogenesis of preeclampsia.