Hydrogen Sulfide Involved In The Regulation Of PGDH In Human Chorion And Its Mechanism

Pre-term birth as the common diseases of obstetrics, is one of the main causes of perinatal death. Its morbidity is8.1%. One of the reasons is the exact mechanism of the incitation of labor is not sure. Prostaglandin (PG) produced within the intrauterine tissues play an important role in the initiation and progression of labor in human, such as chorion tissues, amniotic membrane, uterine muscle and so on. The quantity of PG is due to the balance between cyclooxygenase (COX-2) and15-hydroxyprosglandin dehydrogenase (PGDH).It has been known that both PGDH and COX-2can highly express in chorion. So we take human chorionic trophoblast cells as the object of study. And research was carried out from the two aspects of PGDH and COX-2.The purpose of the experiment is to express who affects the quantity of PG in chorion, and to explain the machine of this regulation.Over the last decade, more and more attention has been put on hydrogen sulfide, which is redefined as one of new gaseous signal molecules. In vivo, H2S is generated from L-cysteine by cystathionine β-synthase (CBS) and/or cystathionine y-lyase (CSE), distributing in different tissues. Study has found that the production rate of H2S in chorion, which were from preterm labor or un-preterm labor gravid, was difference.But the exact mechanism has not yet been elucidated. So we conducted the experiments to determine the effects of H2S on the expression and enzyme activity of PGDH and COX-2. Furthermore, to examine which one of CBS and CSE play an important role in the production of endogenous H2S, we constructed CBS siRNA and CSE siRNA.MicroRNA exists in almost all living organisms, which can inhibit the expression of target protein or silent genes by regulating the precession of protein translation. More and more research display that they can take vital important role in many pathological and physiological process. But how did they take part in the pregnancy and the on-set of labor is unknown. We design the experiment to determinate the direct effects of microRNAs on PGDH and COX-23’UTR in chorionic trophoblast cells by DLR detection system. And then to research the effects of H2S on PGDH and COX-2by microRNAs by real time RT-PCR and Western blot analysis. At last, we conducted experiments to observe mRNA expression level of microRNAs in chorion. Those chorion are from term labor, term un-labor and non-infectious spontaneous premature labour woman.Main results and conclusions are as follows: 1. Hydrogen sulfide involved in the regulation of PGDH in human chorionThe mRNA and protein expression of CBS and CSE were detected in chorionic trophblast cells, and real time PCR results revealed that the mRNA expression of CBS was more than CSE.Sodium hydrosulfide and L-cysteine were equally effective in promoting PGDH expression and enzyme activity. And those effects were disappearing in the presence of AOAA. D-cysteine has no effects on the PGDH expression and enzyme activity. Those results suggested that endogenous H2S was produced by CBS mainly, and regulating the expression and enzyme activity of PGDH.2. Mechanism of H2S on PGDH protein expression by microRNAsOver-expression miR-26b and miR-199a can inhibit the protein expression of PGDH. Suppression miR-26b and miR-199a could promote the protein expression of PGDH.miR-26a has no effects on the expression of PGDH.miR.-26b and miR-199a has direct effects on PGDH3’UTR, and miR-26a has no effects on PGDH3’UTR. Results suggested that PGDH was the target gene of miR-26b and miR-199a. Treated cells with L-cysteine and sodium hydrosulfide, miR-26b and miR-199a mRNA expression were reduced, and miR-26a expression was increased. This displayed that H2S can affect the microRNAs expression. Cells wre treated with the mimics of miR-26b and miR-199a for24hours, then treated with L-cysteine and sodium hydrosulfide. Results showing that the promoting effects of H2S on PGDH was disappeared.3.The expression of CBS,PGDH anddifferent microRNAs in the chorion tissue of term labor,term un-labor and spontaneous pre-term laborCompared with term un-labor, the mRNA and protein expression level of CBS and PGDH were reduced in the tissue of term labor, and further reduced in the tissue of spontaneous pre-term labor.The mRNA expression of miR-26b and miR-199a were significantly increased in the tissue of term labor, and further increased in the tissue of spontaneous pre-term labor. And the expression of miR-26a was opposite. The results suggested that miR-26a, miR-26b and miR-199take part the initiation of labor.In summary, the above results demonstrated: These date suggested that endogenous H2S is mainly produced by CBS, which can promote the expression and enzyme activity of PGDH in human chorionic trophoblast cells, and then reduced the concentration of PG in intrauterine tissues. Also H2S can down-regulate the expression of miR-26b and miR-199a,at the same time can up-regulate the expression of miR-26a.The mechanism of H2S on the PGDH expression is inhibiting the expression of miR-26b and miR-199a.