Pivotal Role of Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) in Uterine Leiomyoma

Abstract

Uterine leiomyomas are smooth-muscle tumors originating in the myometrium and are the most common pelvic tumors in women of reproductive age. Symptomatic tumors may result in abnormal uterine bleeding, bladder dysfunction, pelvic discomfort, and reproductive issues, such as infertility and miscarriage. There are currently few non-invasive treatments for leiomyoma, but there are no practical early intervention or preventive methods. In this study, human uterine leiomyoma and myometrial tissues were used to detect the protein and mRNA expression levels of UCHL1. To explore the effects of UCHL1 knockdown and inhibition in leiomyoma and myometrial cells, we determined the mRNA expressions of COL1A1 and COL3A1. Collagen gel contraction and wound-healing assays were performed on myometrial and leiomyoma cells. We found that UCHL1 expression was considerably higher in uterine leiomyomas than in the myometrium. COL1A1 and COL3A1 expression levels were downregulated after inhibition of UCHL1 in human leiomyoma cells. Furthermore, the elimination of UCHL1 significantly decreased the migration and contractility of leiomyoma cells. In conclusion, these results indicate that UCHL1 is involved in the growth of leiomyoma in humans. For the treatment of uterine leiomyoma, targeting UCHL1 activity may be a unique and possible therapeutic strategy.

Keywords: LDN57444; UCHL1; collagen production; leiomyoma; ubiquitin carboxyl-terminal hydrolase L1.

Figure 1

UCHL1 was highly expressed in human leiomyoma. (A–C) Strong expression of UCHL1 was observed by (A) immunohistochemistry, (B) real-time PCR, and (C) Western blotting in leiomyoma. The scale bar shows 50 µm. The bar shows the standard error (* p < 0.05, ** p < 0.01).

Figure 2

Inhibition of UCHL1 resulted in decreased collagen production in leiomyoma. mRNA expressions of COL1A1 and COL3A1 were analyzed by quantitative real-time PCR. (A) Downregulation of COL1A1 was observed by LDN57444 in myometrial cells. (B) No downregulation of COL3A1 was observed in myometrial cells. (C,D) The expression of COL1A1 and COL3A1 was downregulated by LDN57444 in leiomyoma cells. (E,H) UCHL1 knockdown efficiency was established by quantitative real-time PCR analysis of UCHL1. (F,G) The expression of COL1A1 and COL3A1 did not decrease in myometrial cells in UCHL1 shRNA transduction. (I,J) The expression of COL1A1 and COL3A1 significantly decreased in leiomyoma cells in UCHL1 shRNA transduction (* p < 0.05, *** p < 0.001, **** p < 0.0001).

Figure 2

Inhibition of UCHL1 resulted in decreased collagen production in leiomyoma. mRNA expressions of COL1A1 and COL3A1 were analyzed by quantitative real-time PCR. (A) Downregulation of COL1A1 was observed by LDN57444 in myometrial cells. (B) No downregulation of COL3A1 was observed in myometrial cells. (C,D) The expression of COL1A1 and COL3A1 was downregulated by LDN57444 in leiomyoma cells. (E,H) UCHL1 knockdown efficiency was established by quantitative real-time PCR analysis of UCHL1. (F,G) The expression of COL1A1 and COL3A1 did not decrease in myometrial cells in UCHL1 shRNA transduction. (I,J) The expression of COL1A1 and COL3A1 significantly decreased in leiomyoma cells in UCHL1 shRNA transduction (* p < 0.05, *** p < 0.001, **** p < 0.0001).

Figure 3

The ability of collagen gel contraction by leiomyoma cells was controlled by UCHL1. Collagen gel contraction decreased in UCHL1 shRNA of leiomyoma cells when compared to that of the control group. This finding was not observed in myometrial cells (** p < 0.01).

Figure 4

The wound-healing assay with or without UCHL1 knockdown. The wound closure of the UCHL1 shRNA group was significantly decreased in leiomyoma and myometrial cells. The scale bar shows 1000 µm (* p < 0.05).