Tsai-Der Chuang

The Roles of Non-Coding RNAs in the Pathogenesis of Uterine Fibroids

Uterine fibroids are benign smooth muscle tumors that affect ~70% of women, with Black women being affected at a disproportionate rate. The growth of these tumors is driven by estrogen and progesterone. Driver mutations in genes such as MED12, HMGA2, and FH also play roles in the development and growth of fibroids. Despite their high prevalence, the pathogenesis of fibroids remains largely unknown, leading to a lack of effective therapeutic options. Non-coding RNAs (ncRNAs), including miRNAs (e.g., miR-21, miR-29, miR-200), lncRNAs (e.g., H19, MIAT, XIST), and circRNAs, are important regulatory RNAs that are becoming increasingly implicated in the aberrant expression of protein-coding genes functionally associated with ECM production, cell proliferation, apoptosis, and inflammation in fibroids. Race/ethnicity, MED12 mutations, and ovarian steroids influence the expression of ncRNA expression, further implicating their relevance to fibroid pathogenesis. Therapeutic targeting of these dysregulated ncRNAs in fibroids could enable more precise and individualized non-hormonal-based treatment for this common gynecologic tumor.

Comparative Analysis of Differentially Expressed Long Non-Coding RNA in Pre- and Postmenopausal Fibroids

Uterine fibroids (leiomyomas) are benign tumors whose growth is influenced by estrogen and progesterone. This study aimed to compare the profiles of differentially expressed long non-coding RNAs (lncRNAs) in fibroids from postmenopausal and premenopausal women to identify hormone-responsive lncRNAs. RNA sequencing was performed on six pairs of fibroid (Fib) and adjacent myometrium (Myo) tissues from postmenopausal women. Out of 7876 normalized lncRNAs, 3684 were differentially expressed (≥1.5-fold), with 1702 upregulated and 1982 downregulated in Fib. Comparative analysis with a previously published premenopausal dataset identified 741 lncRNAs that were altered based on their menopausal status, including 62 lncRNAs that were uniquely dysregulated in postmenopausal samples. Overall, 9 lncRNAs were selected for validation by PCR in an expanded cohort of 31 postmenopausal and 84 premenopausal paired samples. Several lncRNAs, including LINC02433, LINC01449, SNHG12, H19, and HOTTIP, were upregulated in premenopausal Fib but not in postmenopausal ones, while ZEB2-AS1 displayed the opposite pattern. CASC15 and MIAT were elevated in Fib from both groups, although the increase was less pronounced in the postmenopausal group. LINC01117 was significantly downregulated in postmenopausal Fib, with no change observed in premenopausal samples. Additionally, analysis based on MED12 mutation status revealed that lncRNAs such as LINC01449, CASC15, and MIAT showed limited or reduced differential expression (mutation-positive vs. mutation-negative) in postmenopausal patients compared to the premenopausal group. These findings indicate that lncRNA expression in fibroids is modulated by menopausal status, likely reflecting hormonal influence. Hormone-responsive lncRNAs may play key roles in fibroid pathogenesis and represent potential targets for therapeutic intervention.

The Functional Role of the Long Non-Coding RNA LINCMD1 in Leiomyoma Pathogenesis

Existing evidence indicates that LINCMD1 regulates muscle differentiation-related gene expression in skeletal muscle by acting as a miRNA sponge, though its role in leiomyoma development is still unknown. This study investigated LINCMD1′s involvement in leiomyoma by analyzing paired myometrium and leiomyoma tissue samples (n = 34) from patients who had not received hormonal treatments for at least three months prior to surgery. Myometrium smooth muscle cells (MSMCs) were isolated, and gene expression of LINCMD1 and miR-135b was assessed via qRT-PCR, while luciferase assays determined the interaction between LINCMD1 and miR-135b. To examine the effects of LINCMD1 knockdown, siRNA transfection was applied to a 3D MSMC spheroid culture, followed by qRT-PCR and Western blot analyses of miR-135b, APC, β-Catenin and COL1A1 expression. The results showed that leiomyoma tissues had significantly reduced LINCMD1 mRNA levels, regardless of patient race or MED12 mutation status, while miR-135b levels were elevated compared to matched myometrium samples. Luciferase assays confirmed LINCMD1′s role as a sponge for miR-135b. LINCMD1 knockdown in MSMC spheroids increased miR-135b levels, reduced APC expression, and led to β-Catenin accumulation and higher COL1A1 expression. These findings highlight LINCMD1 as a potential therapeutic target to modulate aberrant Wnt/β-Catenin signaling in leiomyoma.

Differential Expression of Small Non-Coding RNAs in Uterine Leiomyomas

We performed next-generation sequencing (NGS) on RNA from 19 paired leiomyoma (Lyo) and myometrium (Myo) specimens, stratified by race/ethnicity (White: n = 7; Black: n = 12) and mediator complex subunit 12 (MED12) mutation status (mutated: n = 10; non-mutated: n = 9). Analysis identified 2,189 small non-coding RNAs (sncRNAs) with altered expression in Lyo compared to paired Myo (≥1.5-fold change), including small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs). Among these, 17 sncRNAs showed differential expression in the MED12-mutated group versus Myo, while minimal changes were observed in the non-mutated group. Additionally, 31 sncRNAs displayed differential expression in Black women compared to White women. For validation, five novel miRNAs (miR-19a-3p, miR-99a-5p, miR-3196, miR-499a-5p, and miR-30d-3p) and five piRNAs (piR-009295, piR-020326, piR-020365, piR-006426, and piR-020485) were analyzed in 51 paired Lyo samples using qRT-PCR. Reduced expression of the selected sncRNAs was confirmed in Lyo versus Myo, with miR-19a-3p, miR-3196, miR-30d-3p, piR-006426, and piR-020485 linked to MED12 status, while miR-499a-5p and miR-30d-3p were associated with race/ethnicity. These findings suggest that sncRNA dysregulation contributes to altered gene expression in Lyo, influenced by MED12 mutation and racial background.

In Vivo Effects of Bay 11-7082 on Fibroid Growth and Gene Expression: A Preclinical Study

Current medical therapies for fibroids have major limitations due to their hypoestrogenic side effects. Based on our previous work showing the activation of NF-kB in fibroids, we hypothesized that inhibiting NF-kB in vivo would result in the shrinkage of tumors and reduced inflammation. Fibroid xenografts were implanted in SCID mice and treated daily with Bay 11-7082 (Bay) or vehicle for two months. Bay treatment led to a 50% reduction in tumor weight. RNAseq revealed decreased expression of genes related to cell proliferation, inflammation, extracellular matrix (ECM) composition, and growth factor expression. Validation through qRT-PCR, Western blotting, ELISA, and immunohistochemistry (IHC) confirmed these findings. Bay treatment reduced mRNA expression of cell cycle regulators (CCND1, E2F1, and CKS2), inflammatory markers (SPARC, TDO2, MYD88, TLR3, TLR6, IL6, TNFα, TNFRSF11A, and IL1β), ECM remodelers (COL3A1, FN1, LOX, and TGFβ3), growth factors (PRL, PDGFA, and VEGFC), progesterone receptor, and miR-29c and miR-200c. Collagen levels were reduced in Bay-treated xenografts. Western blotting and IHC showed decreased protein abundance in certain ECM components and inflammatory markers, but not cleaved caspase three. Ki67, CCND1, and E2F1 expression decreased with Bay treatment. This preclinical study suggests NF-kB inhibition as an effective fibroid treatment, suppressing genes involved in proliferation, inflammation, and ECM remodeling.

The immune landscape of uterine fibroids as determined by mass cytometry

To study the differences in immune cell profiles in uterine fibroids (Fibs) and matched myometrium (Myo). Observational study. Laboratory study. The study included tissue that was collected from 10 pairs of Fib and matched Myo from women, not on hormonal medications, undergoing hysterectomy and myomectomy. None. Differences in immune cell and cytokine composition between Fib and matched Myo. The mass cytometry analysis indicated that Fibs had a significantly higher number of natural killer (NK) cells, total macrophages, M2 macrophages, and conventional dendritic cells when compared with matched Myo from the same patient. In contrast, Fibs had significantly fewer CD3 and CD4 T cells when compared with Myo. The mass cytometry analysis results did not show any significant difference in the number of resting mast cells. Immunoflurorescent and immunohistochemical imaging confirmed the cytometry by time of flight results, showing a significantly higher number of NK cells, tryptase-positive mast cells indicative of mast cell activation, total macrophages, and M2 cells in Fibs and a significantly lower number of CD3 and CD4 T cells. The cytokine assay revealed significantly increased levels of human interferon α2, interleukin (IL)-1α, and platelet-derived growth factor AA and significantly lower levels of macrophage colony-stimulating factor and IL-1 receptor antagonist in Fib. Our results show significant differences in immune cell populations and cytokine levels between Fib and Myo. These differences could account for the increased inflammation in fib and a potential mechanism by which these tumors evade the immune system. These findings provide a foundation for further studies exploring the role of immune cells in Fib development.

The Effect of Race/Ethnicity and MED12 Mutation on the Expression of Long Non-Coding RNAs in Uterine Leiomyoma and Myometrium

The objective of this study was to elucidate the expression of long non-coding RNA (lncRNA) in leiomyomas (Lyo) and paired myometrium (Myo) and explore the impact of race and MED12 mutation. Fold change analysis (Lyo/paired Myo) indicated the expression of 63 lncRNAs was significantly altered in the mutated group but not in the non-mutated Lyo. Additionally, 65 lncRNAs exhibited an over 1.5-fold change in the Black but not the White group. Fifteen differentially expressed lncRNAs identified with next-generation sequencing underwent qRT-PCR confirmation. Compared with Myo, the expression of TPTEP1, PART1, RPS10P7, MSC-AS1, SNHG12, CA3-AS1, LINC00337, LINC00536, LINC01436, LINC01449, LINC02433, and LINC02624 was significantly higher, while the expression of ZEB2-AS1, LINC00957, and LINC01186 was significantly lower. Comparison of normal Myo with diseased Myo showed significant differences in the expression of several lncRNAs. Analysis based on race and Lyo MED12 mutation status indicated a significantly higher expression of RPS10P7, SNHG12, LINC01449, LINC02433, and LINC02624 in Lyo from Black patients. The expression of TPTEP1, PART1, RPS10P7, MSC-AS1, LINC00337, LINC00536, LINC01436, LINC01449, LINC02433, and LINC02624 was higher, while LINC01186 was significantly lower in the MED12-mutated group. These results indicate that Lyo are characterized by aberrant lncRNA expression, which is further impacted by race and Lyo MED12 mutation status.