microRNA Let‐7b: A Novel treatment for endometriosis

Abstract Endometriosis is an oestrogen‐dependent, chronic inflammatory disease that affects 10% of reproductive‐aged women. Current treatment options depend on female sex steroid hormone modulation; however, all have side effects and are not useful in women who want to conceive. microRNAs treatments have provided promising results for some chronic diseases and cancers. We have previously shown the microRNA Let‐7b is repressed in endometriosis and that loss of Let‐7 contributes to the pathophysiology of the disease. Here, we propose using microRNA Let‐7b for the treatment of endometriosis in a murine model. Endometriosis was treated using microRNA Let‐7b or a scrambled control microRNA. Let‐7b treatment resulted in reduced endometriosis lesion size. Decreased gene expression was noted in several genes known to promote endometriosis growth including ER‐α, ER‐ß, Cyp19a, KRAS 4A, KRAS 4B and IL‐6. These results indicate that microRNA Let‐7b has a pleiotropic role in endometriosis pathophysiology affecting oestrogen signalling, inflammation and growth factor receptors. Local treatment of endometriosis with Let‐7b is a promising therapy for endometriosis that simultaneously affects multiple pathways driving endometriosis without systemic hormonal side effects.


| INTRODUCTION
Endometriosis has many symptoms that negatively affect both the reproductive capability and professional/social life of affected women. 1 This disease is characterized by the proliferation and growth of endometrial tissue outside of the uterine cavity that causes pelvic pain and infertility. 2,3 The incidence of the endometriosis is approximately 10% in reproductive-aged women and 20%-50% of women with infertility or pelvic pain. 4 The pathophysiology of endometriosis is not well understood; therefore, current treatment options for endometriosis are limited to manipulation of female sex steroid hormones. 5 in a sequence-specific fashion, blocking translation or leading to mRNA degradation. 7 miRNAs have a major role in regulation of development and in cellular homeostasis. 8,9 Additionally, aberrant microRNA expression is linked to many diseases such as cancer, 10 cardiovascular disorders 11 and inflammatory diseases. 12 microRNAs are the potent regulators of gene expression in endometriosis. 13 Distinct miRNA expression profiles have been identified in microarray studies of eutopic and ectopic endometrial tissue samples. 13 We previously reported that circulating microRNAs, including Let-7b-5p, were significantly decreased in the serum of disease. 14,15 Further, we have reported decreased Let-7 family members in endometriosis tissue where they are involved in the regulation of genes such as KRAS and aromatase. 16,17 These studies prompted us to hypothesize that miRNA Let-7b-5p might have a major role in the pathogenesis and treatment of endometriosis.
Here, we report the therapeutic use of miRNA Let-7b-5p in the treatment of endometriosis in a murine model.

| Animals
Six-to eight-week-old C57BL/6J wild-type female mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were maintained in the animal facility of Yale School of Medicine.
They housed five animals per cage in a 12-hour light, 12-hour dark cycle (7 AM-7 PM) with ad libitum access to food and water.
All animals were treated under an approved protocol by Yale University Institutional Animal Care and Use Committee. Mice were acclimated at least 1 week, and vaginal cytology analysis was performed to determine oestrous cycle stage of individual animals prior to surgery. Ten animals that were in diestrous stage were used as recipient, and three mice that were in oestrus stage were used as donors.

| Induction of endometriosis in mice
Endometriosis was induced in ten mice using a modified version of the syngeneic endometriosis protocol that has been used previously in our laboratory. 18 In accordance with this model, identical sizes of uterine tissue fragments were sutured onto the peritoneal surface. Three mice in oestrus stage were killed using a CO 2 chamber, both uterine horns from each mouse were removed and opened longitudinally and divided into equal fragments measuring 4 mm 2 . These fragments were preserved on ice in DMEM/F12 Ham 1:1 media (Gibco; Grand Island, NY, USA) until transplantation. For implantation, ten mice were anaesthetized by inhalation of isoflurane (Isothesia; Henry Schein, OH, USA) and laparotomy was performed by midline incision. Two uterine fragments were sutured on each right and left peritoneal surface area using 5-0 polyglactin sutures (Vicryl; Ethicon, Somerville, NJ, USA). Subsequently, the peritoneum and skin were closed with same sutures.   Table 1. The specificity of the amplified transcript and absence of primer dimers were confirmed by a melting curve analysis. Gene expression was normalized to that of β-actin as an internal control. Relative mRNA expression was calculated using the comparative cycle threshold (Ct) method (2 −ΔCT ). 19,20 All experiments were carried out three times and each in duplicate.   Student t test was used for evaluating of normally distributed variables. P < 0.05 was considered as statistically significant. Let-7b-treated and control mice. We also specifically measured the area of endometrial tissue and determined that it was also significantly reduced in Let-7b-treated mice compared to control mice, as shown in Figure 1E.

| Differential expression of genes that are involved in endometriosis
The effect of Let-7b treatment on expression of genes that are involved in endometriosis was determined by qRT-PCR in the lesions and compared with expression in the control group. We observed decreased expression of several genes known to mediate endometriosis growth or endometriosis-associated inflammation.

| DISCUSSION
We identify a novel, nonhormonal therapy for endometriosis that is Aromatase P450 has an essential role in oestrogen synthesis and has been demonstrated to regulate local oestrogen production in endometriosis. 42 Shibahara et al 43 showed that high Let-7f expression was significantly correlated with low aromatase protein levels in primary breast cancer stromal cells. They also identified a Let-7f binding site in Cyp19a1, indicating that the aromatase gene is a direct target of Let-7f. 43 We demonstrated similar results in endometrial stromal cells from endometriosis patients and in Ishikawa cells. 17 In our study, significantly increased Let-7b and Let-7f expression levels were determined after aromatase inhibitor treatment. Further, decreased aromatase expression and reduced endometrial cell migration were shown after Let-7f mimic transfection. 17 High levels of Let-7b after aromatase inhibitor treatment indicated that Let-7b has a role in aromatase regulation and decreased aromatase levels after Let-7b treatment supported that Let-7b treatment reduces local oestrogen production and action.
Endometriosis is also a chronic inflammatory disease, and macrophages have a principal role in this inflammatory process. An increase in M1 type macrophages is seen in endometriosis. 44 Let-7b may regulate inflammation through its known target gene TLR-4, which regulates M1 macrophage response. 45 We observed a trend towards decreased levels of TLR-4 in the Let-7b treatment group compared to controls. Another inflammatory marker, IL-6, levels were significantly suppressed in the Let-7b treatment group. These results suggest that Let-7b treatment does reduce inflammation associated with endometriosis.
In this study, a local treatment route was used for miRNA Let-7b treatment. The effective delivery of the oligonucleotides to target cells after systemic administration is not very effective as they are eliminated from the bloodstream by hepatic degradation. Therefore, many of the systemic oligonucleotide treatment studies have focused on liver disease. 46 Various types of oligonucleotide carriers have been used to resolve these problems; however, none of them has fully accomplished targeted oligonucleotide delivery. 47 The most effective oligonucleotide-based drugs approved by the U.S. Food where local delivery is very effective. 48 We suggest that this drug would work best as an intraperitoneal therapy delivered at the time of surgery or by injection. Further, dose response and safety studies will be required prior to human application.
In conclusion, microRNA Let-7b treatment of endometriosis resulted in decreased oestrogen signalling (ER and Cyp19A1), decreased KRAS and decreased inflammatory signalling (IL-6). The pleiotropic effects of Let-7b treatment suggest that multiple complementary mechanisms were responsible for the actions of Let-7b in endometriosis. These numerous effects suggest the potential for a more comprehensive endometriosis therapy without the systemic effects, a common feature of current drugs.

ACKNOWLEDG EMENTS
This work was supported by NIH U54 HD052668 and R01 HD076422.

CONFLI CT OF INTEREST
The author(s) declared no potential conflict of interests with respect to the research, authorship and/or publication of this article.