Downregulation of miR-183 inhibits apoptosis and enhances the invasive potential of endometrial stromal cells in endometriosis

Abstract

Endometriosis is a common gynecological disease, yet its pathogenesis remains poorly understood. Recent studies have demonstrated that the aberrant expression of certain microRNAs (miRNAs) may correlate with the development and progression of endometriosis. In this study, we profiled several differentially expressed miRNAs in the normal, eutopic and ectopic endometrium by miRNA microarray screening analysis, among which, miR-183 was found to be downregulated in the ectopic and eutopic tissues, and the result was further confirmed by real-time PCR (qPCR). Functional analysis indicated that miR-183 plays a promotional role in endometrial stromal cell (ESC) apoptosis and has a negative regulatory impact on the invasive ability of cells, although it has no effect on ESC proliferation. Ovarian steroids (17β-estradiol and progesterone) and inflammatory factors (tumor necrosis factor-α and interleukin-6) decreased the expression of miR-183 in the ESCs. This regulatory function may further manifest the growth and invasive potential of ESCs by altering the expression of miR-183. These findings suggest that the downregulation of miR-183 expression is involved in the development and progression of endometriosis.

Figure 1

Hierarchical clustering of the 3 groups (ectopic endometrium, eutopic endometrium and normal endometrium). (A) Ectopic vs. eutopic endometrium, and (B) eutopic vs. normal endometrium. Distinguishable miRNA expression profiling is observed. Red indicates high relative expression, green indicates low relative expression and black represents zero.

Figure 2

Validation of miR-183 expression by qPCR. (A) Ectopic and eutopic endometrium from women with endometriosis (n=20) and normal control endometrium from women without endometriosis (n=20). (B) Endometrial stromal cells (ESCs) from women with endometriosis and endometriosis-free women. miR-183 expression data are presented as the fold change compared with the normal group (^*p<0.05). EM, endometrium.

Figure 3

Stable lentiviral transduction of anti-miR-183-5p and miR-183 plasmid into endometrial stromal cells (ESCs). (A) Transduction efficiency was determined by fluorescence microscopy following transduction with recombinant lentivirus. (B) The expression levels of endogenous miR-183 were determined by qPCR. ^**P<0.01, significantly different from both the untransduced and empty lentivirus control groups.

Figure 4

Effect of overexpression and downregulation of miR-183 on the invasive potential of endometrial stromal cells (ESCs). Matrigel invasion data when ESCs infected with miR-183-lentivirus, In-miR-183-lentivirus (miR-183 inhibitor) and green fluorescent protein (GFP)-lentivirus (control) in upper well were incubated in 2% FCS medium and lower well was filled with 10% FCS medium. After 24 h, the number of cells that invaded through Matrigel was counted in at least 10 fields/well. (A) Representative images reveal the ESCs that invaded through Matrigel. The ESCs in which miR-183 expression was downregulated, had a greater invasive potential. (B) Counting accuracy was guaranteed by optical density quantification. CON, control; NC, negative control.

Figure 5

Effect of overexpression and downregulation of miR-183 on growth of endometrial stromal cells (ESCs). (A) Cell apoptosis was measured by Annexin V-FIFC. (B) Growth curves of ESCs transfected with miR-183-lentivirus, In-miR-183-lentivirus (miR-183 inhibitor) and green fluorescent protein (GFP)-lentivirus (control). Cell proliferation was measured by MTT assay. Data are presented as the means ± SD, ^*p<0.05 and ^**p<0.001. CON, control; NC, negative control.

Figure 6

Effects of (A) ovarian steroids and (B) inflammatory factors on miR-183 expression in endometrial stromal cells (ESCs) assessed by qPCR. miR-183 expression data are presented as the foldachange relative to the normal group, ^*p<0.05. 17β-E2, 17β-estradiol; P, progesterone; TNF, tumor necrosis factor; IL, interleukin.