Negative Regulation Of Human Endometrial Stromal Cells On HOXA11 Gene In Co-operation With Progesterone

Background : Homeobox genes give identity of developing body segment and embryonic anteroposterior patterning in vertebrates, which is a family of regulatory genes controlling embryonic development and cell differentiation, contain a common 180-nucleotic sequence homeobox and code for specific nuclear proteins( homeoproteins) that act as transcription factors.HOXA11 gene ,one of the homeobox genes is, mainly expressed in uterus and cervix of mammal adults. Recent investigations indicate that HOXA11 gene plays an essential role in endometrial development and implantation of adult human.Many studies presume that HOX gene mediate the effects of sex steroids on endometrium. Many kinds of gynecological diseases are related to the abnormal expression of HOX genes. We have found that in vivo, the expression of HOXA11 gene in human endometrium had a cyclical variation with the mobile level of gonadal hormone. The expression of HOXA11 gene in the endometrial glandular epithelia was obviously decreased or disappeared in the mid-secretory phase, which suggested that progesterone down-regulated HOXA11 gene expreesion. We also found that stromal cells might product some kind(s) of progesterone dependent factor(s) which took part in down-regulated HOXA11 gene expreesion of endometrial epithelial cells by progesterone in vivo. But the progesterone dependent factors are poorly understood.Objective:In this study, we will approach that if the progesterone dependent factors are related to the downstream genes expression products regulated by HOXA11 gene of stromal cells. Methods: At first, we have blocked the human HOXA11 mRNA (Entrez Gene ID 3207, NM 005523, length 2295 bp) expression in stomal cells by RNAi. The target sequence is cgc agt ctc gtc caa ttt cta ( 5'-3': 432bp- 452bp ). Hs_HOXA11_4HP siRNA, as the special RNAi product, was transfected in primary cultured stromal cells. The best time of HOXA11 gene blocked was defined by semi-quantitative RT-PCR. The result showed that the optimum time was 12 hours, and the interference efficacy of HOXA11 gene was about 77.5%. Then the stromal cells culture medium (SCM) was collected. The endometrial epithelial cells in primary culture were incubated with 30% SCM and progesterone for 48 and 4 hours respectively. The HOXA11 mRNA and protein expression levels of the endometrial epithelial cells were detected by semi-quantitative RT-PCR and Western-blot.Results: The epithelial HOXA11 expression of adding 30% SCM was less than the controls .Among the all groups, HOXA11 gene (or protein) expression was minimum in endometrial epithelial cells incubated with SCM which had been treated with only progesterone or Hs_HOXA11_4HP siRNA and Progesterone. On the contrary, the HOXA11 gene (or protein) expression in the epithelial cells, which was incubated with SCM that had been treated with Hs_HOXA11_4HP siRNA or Hs_HOXA11_4HP siRNA and RU486 before adding progesterone, was not different from that of controlsConclusions:1. Hs_HOXA11_4HP siRNA, the special RNAi production of HOXA11 gene, shows its instantaneity on inhibiting the HOXA11 gene in stromal cells.2. The endometrial stromal cells may produce some kind(s) of progesterone dependent factor(s), which can participate in the negative effect of progesterone on the endometrial epithelial cells.3. The progesterone dependent factor(s) maybe have no conclusive relationship to the expression products of the downstream genes controlled by HOXA11 gene.