Received 2024-08-07

Revised 2024-09-01

Accepted 2024-10-09

Introduction

The most common tumors found in the female genital tract are uterine leiomyomas, which were first described by a British pathologist from St. George's Hospital, Matthew Baillie [1, 2]. Several gynecological issues, as well as a heavy financial and health care cost, have been associated with them since then. The truth is that leiomyomas affect anything from half a million to eighty million persons [3, 4]. Their estimated yearly direct and indirect costs to the United States are $34.4 billion [5, 6]. Uterine fibroids (leiomyomas) are affected by growth hormones, signaling pathways, and other substances that decrease nucleotide excision repair (NER).

Regardless, steroid hormones influence a wide range of biological processes through binding to certain receptors, including development, reproduction, energy balance, metabolism, immunology, and behavior [7, 8]. Sex steroids (including androgens, oestrogens, and progesterones) and corticosteroids (including glucocorticoids and mineralocorticoids) are two ways to classify them. Nanomolar concentrations of steroid hormones in bodily fluids allow the endocrine system to maintain constant communication with the neurological and immunological systems. The body's natural and synthetic components, known as endocrine-disrupting chemicals (EDCs), are increasingly implicated in a host of harmful health effects, including reproductive dysfunction [9, 10], cognitive impairment [11, 12], cognitive deficits [13], metabolic diseases and disorders [14] and cancers [15, 16]. Due to their hormonal properties, which include oestrogen and androgen activity, EDCs have been associated with an elevated risk of tumors when present.

Although EDCs are still largely unknown, they play a significant role in regulating the tumor microenvironment. Therefore, RACK1 is an immunological EDC target that has been extensively studie [17] and this molecular actor mediates the progression of cancer.

Several investigations have found that uterine fibroids are associated with obesity, a positive family history, low vitamin D levels, and high TGF-β3 blood concentrations. Two other studies also found a correlation between vitamin D and uterine fibroids. The growth of smooth muscles and UFs in the uterus are controlled by a TGF-β receptor. Laboratory investigations have shown that the uterine smooth muscle directly touching the fibroid expresses a large amount of TGF-β [18, 19]. Expression of TGF- β is nearly double in UF tissue compared to normal smooth muscle, the control group [20, 21]. In contrast to healthy myometrium tissue, UF tissue contains roughly five times the amount of TGF- β 3 [22, 23]. The process of TGF- β signaling is highly involved in creating UFs. Myometrium and UF cancers express different types of TGF- β and the receptors that bind to them. By suppressing proliferation and inducing apoptosis, TGF- β effectively suppresses tumors in smooth muscle cells. In addition, UFs show an overexpression of TGF- β, which seems to be crucial for their development and advancemen [24-26].

Although uterine fibroids have a huge influence, the specific pathobiology of them is still a mystery. As a result, there is currently no viable medical therapy option. Leiomyoma formation is thought to begin with the proliferation of a smooth muscle cell [27, 28]. More changes to the signaling pathways are supposedly required [29].

Identifying the role of innate immunity in cancer development and comprehending endocrine-mediated microenvironment modulation may depend on how EDCs regulate RACK1 in both settings. Hence, our review covered the fact that RACK1 is involved in reacting both immune and cancer cells to EDCs. The alterations to these signaling pathways and their linkages in order to identify possible treatment targets.Our research focuses on uterine fibroids (leiomyomas) and the mechanisms by which growth hormones, signaling pathways, and other substances inhibit NER.

Materials and Methods

Search Strategy

Protocol for the Reporting of Systematic Reviews and Meta-Analyses, or PRISMA, was followed throughout the course of this review (Figure-1). We utilized search phrases such “fibroid, leiomyomata, leiomyoma, TGF-*, vitamin D, RACK 1, WNT, miR, Mutant gene” to extensively explore the Ovid, PubMed, and Cochrane Library databases. Phrases related to the outcome and the population were used in all database searches in the same way. From 2000-01-30 through 2023-12-30, all databases were searched. Only English dates and publications were supplied.

Clinical, in vitro, and in vivo investigations were all part of this study. From 2000 to 2023, the proceedings of the following societies were hand-searched: Pacific Coast Ripeness Society, American Society for Regenerative Pharmaceutical, American Association of Gynecologic Laparoscopists, and Society for Gynecologic Examination. After perusing the titles, we moved on to the abstracts to find any relevant information. All potentially relevant citations were reviewed in full reports.

Selection Criteria

This review included studies about steroid signaling, signaling pathways, growth factors, miRNAs, vitamins, and mutant genes. A paper that lacked molecular investigation on UFs, a study focusing on another disease or a specific population of patients, a randomized controlled trial, a case study, a review, an editorial, or a letter was not included. 

Main Outcome Variables

Suppression of nucleotide excision repair (NER) in uterine fibroids (leiomyomas) was the primary outcome variable. Research on the suppression of nucleotide excision repair (NER) in uterine fibroids (leiomyomas) was included in all studies that assessed growth hormones, signaling pathways, or other agents. Following the guidelines laid out by the PICO (Population, Intervention/Exposure, Comparison, and Result) framework for effective surveys, we drafted the following audit address to guide the investigation: People with leiomyomas, or uterine fibroids, were the intended recipients. It failed to identify a particular intervention or comparison due to the extremely varied exposures for leiomyomas. The following factors were considered: Signaling based on steroids, TGF-ß, 25-hydroxyvitamin D, RACK1, Wnt/β-catenin, vitamin effects, mutant genes, and so on.

Data Extraction

All citations of included studies were exported to Mendeley. After deleting duplicates, all references were manually screened and categorized. To choose studies that could be included in the review, one of the writers looked at the titles and abstracts to see if they were eligible. Then, they read the entire texts to see if they were. Final decisions regarding manuscript screening and data extraction were made by one person for consistency. Finally, the required data of the full-text screening were exported into extraction table. In this study, general information (title, author, journal, year), cell source type, type of investigation, method of testing and effect on UF was collected from each of the studies (Table-2). Also, in Table-3,: patients characteristics, method of measurement of variables, animals (If the study is carried out on animals), cell line, steroid signaling, signaling pathway, grows factors, miRNA, vitamin, mutant gene and finally results were extracted from the included studies in this systematic review.

Risk of Bias Assessment

In order to evaluate the possibility of bias, we employed the revised Cochrane risk-of-inclination device (RoB-2), which consists of five distinct parts: randomization handle, deviations from planned treatments, lost result information, result estimation, and detailed result determination. The findings of the analysis for potential bias are presented in Table-2.

Results

About 24 papers out of 990 were evaluated that were initially reviewed (Figure-1). Table-1 shows the distribution of the research according to the authors, origin, and year. They were divided into sources of cells, methods, steroid signaling, signaling pathways, growth factors, miRNAs, vitamins, and mutant genes.

18 of these studies investigated human source cells, one studied both human and mouse, one studied both human and cell line, 3 studies examined cell line, and one study examined mouse cell line. (Table-1). Three studies investigated the influence of TGF-ß, four studied vitamins, two studied both vitamin and TGF-ß, three studied RACK1, three studied estrogen receptor, one studied miRNA, and two studies examined mutant genes.

Discussion

Our discussion indicates a convergent pathway for several signal transduction pathways. , TGF-β, vitamin D, RACK 1, WNT, miR/Wnt/β-catenin pathways are activated by growth factors, progesterone and vitamin D receptors on membranes. In cases where different pathways are treated additively, this phenomenon of convergence may have important therapeutic implications. In the following, the factors assessed in the studies included in this review are briefly mentioned.

Steroid-based Signaling

Trilogy of studies examined steroid-based signaling on human source cells. These studies examined ß and α sterogen receptors. Myometrium and leiomyomas were both examined for their DNA methylation level in one study. In these patients, the leiomyomas usually had larger levels of ER- α mRNA than their myometriums. The increased levels of ER-α mRNA expression seen in uterine leiomyomas could be attributed to an aberrant DNA methylation status at the promoter region of the ER- α gene (Figure-2) [41]. Another study compared fibroids with normal myometrial tissue in terms of estrogen receptor α and ß expression and DNA-binding status in order to better understand the molecular basis for the altered estrogen responsiveness in leiomyomas. The ratio of estrogen receptor α-to-estrogen receptor α expression effects the potential growth of myomas for the percentage of estrogen receptor α DNA-binding homodimers, not the individual expression levels [42]. Patients with endometriosis or fibroids from Asian or Indian origins were examined for ER α T/C polymorphism in the third study. Further research is needed to establish the ER α C allele in Asian Indian women as a potential indicator for endometriosis and fibroids. Endometriosis and fibroids risk factors include dietary variables, racial background, and ethnicity. We need to conduct more research in this area [43].

TGF-ß

Six investigations were conducted on TGF-ß. One study used pairs of myometrium and leiomyoma tissues donated by 28 women who had hysterectomy. Each tissue's total RNA was collected and examined utilizing Northern blot technique in order to detect TGF-ß 1 messenger RNA. The total and active levels of transforming growth factor-ß 1 proteins were examined using an enzyme-linked immunosorbent assay. TGF-ß 1 (0.01-1 ng/mL), anti-transforming growth factor-ß antibody (0.01-10 ng/mL), or platelet-derived growth factor (10 ng/mL) were employed to measure the proliferation of pure human myometrial and leiomyoma cells using [3H]thymidine incorporation Also, transforming growth factor-ß 1 only promotes cell proliferation in leiomyoma cells at low doses, just like its bimodal and dose-dependent activities in smooth muscle cells elsewhere (Figure-3) [18]. In their study, Arici et al. assessed the amounts of TGF-β3 mRNA and protein in the myometrium and leiomyoma. In the subsequent steps, they injected TGF-β3 into the developed myometrial and leiomyoma cells. Leiomyoma samples contained 3.5 times more TGF-*3 mRNA than myometrial samples. The amount of TGF-β3 mRNA was five times higher in leiomyoma samples collected during the midsecretory phase compared to samples taken during the proliferative phase [30-33]. During the menstrual cycle, Eric Levens et al. found that FMOD expression was approved in leiomyoma and myometrium (N=20). Seven women were given gonadotropin-releasing hormone analogue (GnRHa) as treatment in the trial, whereas twenty women were given transforming growth factor (TGF)- β in addition to GnRHa. Immunohistochemistry and western blotting confirmed the presence of immunoreactive FMOD in LSMC, MSMC, vascular walls, and connective tissue fibroblasts. The treatment of MSMC with TGF- β 1 (2.5 ng/ml) led to an increase in the expression of FMOD, which was time- and cell-dependent. Nevertheless, upon treatment with GnRHa (0.1 m), FMOD expression was shown to be downregulated (P 0.05) in LSMC. The pretreatment of LSMC and MSMC with Smad3 SiRNA and U0126, an inhibitor of MEK1/2, reduced the impact of TGF- β and GnRHa on FMOD production [34]. The levels of TGF- β 3 in the myometrium and leiomyoma were assessed in a study carried out by Arici et al. The cells were subsequently treated with TGF-β 3. The level of TGF- β 3 mRNA was 3.5 times higher in leiomyoma samples compared to myometrial samples. Compared to leiomyoma samples taken during the proliferative phase, those taken during the midsecretory phase had TGF- β 3 mRNA levels that were five times greater [33]. The study conducted by Micha*Ciebiera et al. [50] found that ulipristal acetate (UPA) can decrease blood and tissue TGF- β 3 concentrations, which could significantly impact UF biology.

25-hydroxyvitamin D

Chebiera, Micha investigated the impact of variables such as body mass index (BMI), serum levels of 25-hydroxyvitamin D and altering growth factor β 3 (TGF- β 3), and personal and family medical history on uterine fibroids formation. Factors that could increase the likelihood of uterine fibroids include a high body mass index (BMI), a positive genetic background, low vitamin D levels, and high concentrations of TGF- β 3 in the blood. None of the groups showed any difference in the expression of FBN-1. The expression of LTBP-1 and FBN-1 mRNA and protein was enhanced in cultured leiomyoma smooth muscle cells (LSMC) by 17 β -eestradiol (E2) (P<0.05). E2 did not influence FBN-1 or LTBP-1 levels when given alongside progesterone. A response to estrogen triggers the secretion of LTBP-1 and FBN-1 by leiomyomata. It is possible that the extracellular matrix metabolism varies in medium-sized leiomyomas [35]. Halder We hope to learn more about the effects of vitamin D3 on TGF-β3-induced protein fibrosis in immortalized human uterine leiomyoma (HuLM) cells in this study. Vitamin D3's effects on fibrosis-related protein production in immortalized human uterine leiomyoma (HuLM) cells were studied in relation to TGF- β 3. By lowering the nuclear translocation and phosphorylation of Smad2 and Smad3 in HuLM cells, vitamin D3 considerably diminished the effects of TGF- β 3 on fibrosis in human leiomyoma cells [37].

RACK1

The function of RACK1 has been the subject of research. Xi-Xi Cao set out to discover the molecular mechanisms by which RACK1 aids in the migration and metastasis of breast cancer. To perform migration tests, cell lines derived from breast carcinomas were utilized. Additionally, the Rho kinase inhibitor and siRNA targeting RACK1 were employed. RACK1/RhoA interactions were studied using immunoprecipitation and immunofluorescence. The RhoA/Rho kinase pathway is activated by RACK1 through interaction with RhoA in vitro. The RACK1 protein is highly correlated with RhoA and accepted tumor spread indicators in 160 samples studied by immunohistochemistry [38] (Figure-4). To learn how RACK1 contributes to breast cancer progression, invasion, and metastasis, Xi-Xi Cao et al. performed their own investigation. In vivo and in vitro investigations have shown that RACK1 promotes breast cancer cell growth and metastasis [39, 40].

Wnt/β-catenin

One study looked at the Wnt/β-catenin signaling pathway. Gene expression and DNMT activity were examined in Marla et al. E. using qRT-PCR and ELISA. There was an increase in DNMT1 gene expression in uterine fibroid tissue compared to myometrium. A potential therapeutic target was identified in HCC cells—a new positive feedback loop involving DNMT1/miR-378a-3p/TRAF1/NF- κ [44]. Twenty-one endometrial polyps and fifteen lipomas were subjected to DNA sequencing analysis using FFPE-samples obtained by Dominique et al. The combination of estrogen and mutant MED12 promotes the Wnt pathway, which in turn activates γ -catenin, leading to the development of lesions similar to leiomyomas in mice [51].

Effects of Vitamins

A vitamin's impact was the subject of one research. The results of every study corroborated its effectiveness. Uterine fibroids and surrounding normal myometrium were compared in terms of vitamin D receptor (VDR) protein expression by Sunil K. Hald et al. By lowering VDR levels, 1,25(OH)2D3 treatment for human uterine fibroids diminishes aberrant expression of key ECM-related to proteins in HuLM cells. One potential noninvasive, effective, and safe method for treating uterine fibroids in humans is the use of 1,25(OH)2D3 [32]. Christian et al. analyzed trace element and micronutrient levels in serum (vitamins A, C, D, and E). Uterine fibroids were associated with decreased serum calcium, vitamin D, and vitamin C levels in women [46]. Vinita Singh et al. discovered that compared to healthy controls, women who had uterine fibroids had far lower quantities of vitamin D3 in their blood. All women had their blood FSH and vitamin D levels checked after uterine ultrasounds [47]. This was done on the third day of menstruation. One study looked into beta-tocopherol (into). UFs are linked to elevated levels of alpha-tocopherol (AT), body mass index (BMI), a favorable family history, and low parity, as reported by Michal Ciebiera et al. [49], The impacts of miR-21, miR-29b, miR-23b, and miR-197 were examined in a study. The top five dysregulated microRNAs (miRNAs) in ULMs were determined by Wang et al. to be mutations in miR-21, miR-29b, miR-23b, and miR-32 [52].

Mutant Genes

Mutant genes were the subject of two investigations. Markowski looked at MED12, which is a quality code for a go-between subcomplex 12, and HMGA2, which is a quality code for a high-mobility bunch protein 2, in one of these. Based on these results, it seems that the pathophysiology of fibroids can vary. Separate clonal outgrowths may occur if an unidentified "mutator" mutated MED12 in the majority of these populations. There is a modest but recognized risk of MED12-mutated fibroids turning into cancers following a leiomyoma, hence the latter mutation cannot be utilized as a signal for benign development.-Leomyosarcoma sequence -STUMP (small tumors of smooth muscle with unclear cancer risk) [53, 55, 56]. According to the results of an RT-PCR and FISH analysis, PCOLCE is a potential chromosomal 7 locus whose product, PCPE, cleaves sort I procollagen C-propeptide. The following reasons make PCOLCE an intriguing gene for the study of uterine fibroids: its high levels of expression in the uterus, the fact that rodent fibroblasts undergo anchorage-independent development when subjected to retroviral-mediated insertional mutagenesis, and, lastly, its position on the human chromosomee (7q21.3*q22) [54, 55].

Conclusion

Uterine leiomyomas are characterized by cell proliferation and an excess of disorganized extracellular matrix (ECM). This pair of activities is carefully controlled by a network of signaling pathways. For instance, the ECM contains dormant transforming growth factors. Once tissue proteases have activated cell surface receptors, they are ready to be used. Two key features of leiomyoma signaling pathways are interconnectedness and convergence. They need to communicate with one other and work together in a number of contexts. Leiomyoma cells' intricate connection to the ECM is an intriguing illustration of this. Cellular secretion of transforming growth factors increases collagen synthesis and, by extension, ECM stiffness. A number of research compounds, such as liarozole, use this relationship as an advantage. Our discussion indicates a convergent pathway for several signal transduction pathways. , TGF-β, vitamin D, RACK 1, WNT, miR/Wnt/β-catenin pathways are activated by growth factors, progesterone and vitamin D receptors on membranes. Important therapeutic implications may arise from this phenomenon of convergence in situations where various pathways are addressed additively. Targeting numerous signaling pathways at once is the foundation of dual targeting and multitargeting. As a result, additive or synergistic effects may occur. Some cancers, like prostate cancer, are currently being treated using this multifocal signal modulation approach.

Future research on multifocal targeting in leiomyoma could benefit from a better understanding of signaling pathways in the tumor. It is suggested that future studies on the issue related to the factors of the present study be done with a larger sample size than the existing studies and in the form of cohort designs. In addition, the use of modern techniques in a homogeneous manner in future studies to perform meta-analysis on the obtained results can help significantly in providing treatment protocols. Furthermore, it should be emphasized that these results should be taken with caution, even though the risk of predisposition assessment is minimal for most of the components discussed in this research.

Conflict of Interest

None declared.

GMJ Copyright© 2024, Galen Medical Journal. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/) Email:info@gmj.ir

 Correspondence to: Fatemeh Asadi, Department of Genetics, Izeh Branch, Islamic Azad University, Izeh, Khuzestan, Iran Telephone Number: +987143311172 Email Address: fatemehasadi@miau.ac.ir

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Table 1. Distribution of the Studies according to the Authers, orgine and Year

Reference	Main Auther	orgine	Year
[18]	Arici	New Haven, Conn	2003
[32]	Halder	Nashville, Tennessee	2013
[33]	Arici	USA	2000
[34]	Levens	USA	2005
[35]	MichałCiebiera	Poland	2016
[36]	Yangyu Zhao	USA	2007
[37]	Halder	Tennessee	2011
[38]	Xi-Xi Cao	China	2011
[39]	Xi-Xi Cao	China	2010
[40]	Buoso	Italy	2019
[41]	Asada	Japan	2008
[42]	Panagiotis	Greece	2008
[43]	Govindan, Sujatha	Russia	2009
[44]	Zhu	China	2021
[45]	Gallagher	USA	2018
[46]	Makwe	NGA	2021
[47]	Vinita	India	2019
[48]	Li	China	2020
[49]	Ciebiera	Poland	2018
[50]	Ciebiera	Poland	2018
[51]	Markowski	Germany	2012
[52]	Wang	Newark, NJ	2007
[53]	Markowski	Bremen	2014
[54]	Jokinen	Finland	2023

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Table 2. Summary of Studies

Cell source type	Type of investigation	Method of testing	Effect on UF	Risk of bias	Reference
Human	TGF-β1	Northern blot analysis and ELIZA	At low doses, factor-β1 primarily induces cell proliferation in leiomyoma cells.	Risk of bias attributable to the randomization process: low Concerns with the risk of bias stemming from departures from the targeted interventions. Insufficient outcome data: little Risk of bias in outcome measurement: low Risk of bias in the selection of the reported outcome: low	[18]
Human	1,25-Dihydroxyvitamin D3	Analyzed by Western blotting using antibodies against VDR and β-actin	VDR expression is lower in human uterine fibroids in comparison to the surrounding normal myometrium.	The randomization approach poses a low risk of bias. Potential for bias as a result of interventions that deviate from the intended course: some worries Insufficient data on outcomes: low Potential for bias in outcome measurement: little Very little chance of prejudice in choosing this result to report	[32]
Human	TGF-β3	Northern analysis was used to assess the levels of TGF-β3 and fibronectin messenger RNA.	In comparison to the myometrial samples, the leiomyoma samples had a 3.5-fold higher quantity of TGF-β3 mRNA.	The randomization approach poses a low risk of bias. Potential for bias as a result of interventions that deviate from the intended course: some worries Insufficient data on outcomes: low Potential for bias in outcome measurement: little Very little chance of prejudice in choosing this result to report	[33]
Human	TGF-β3	A vaginal ultrasound scan, blood and tissue samples, and the measurement of TGF-β3 concentrations in serum and tissues	One significant aspect of ulipristal acetate’s (UPA) impact on UF biology may be the decrease in serum and tissue TGF-β3 concentrations in UFs.	Potential for bias as a result of randomization: little Concerns about the possibility of bias arising from treatments that deviate from their intended goals Results data missing: minimal There is little chance of bias in the results. Selective bias in reporting results: little	[34]

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Table 3. Studies included in the Systematic Review

Auther	Patients	Method	Animals	Cell line	Steroid signaling	Signaling pathway	Grows factors	miRNA	Vitamin	Mutant gene	Results
Arici [18]2003	Pairs of myometrium and leiomyoma tissues were collected from 28 hysterectomy patients.	Tissue pairs of myometrium and leiomyoma were obtained from 28 patients who had hysterectomy. Northern blot analysis was employed to identify TGF-β1 messenger RNA following the extraction of total RNA from each tissue sample. The quantity of active and total transforming growth factor-β1 protein was quantified using an enzyme-linked immunosorbent assay. The proliferation of cultured human myometrial and leiomyoma cells was assessed via the [3H]thymidine incorporation method following anti-transforming growth factor-β antibody (0.01-10 ng/mL),  treatment with TGF-β1 (0.01-1 ng/mL), or platelet-derived growth factor (10 ng/mL).					The assessment was conducted using TGF-β1 (0.01-1 ng/mL), anti-transforming growth factor-β antibody (0.01-10 ng/mL), or platelet-derived growth factor (10 ng/mL).				Transforming growth factor-β1 has bimodal and dose-dependent effects on smooth muscle cells in other tissues, which is consistent with the observation that, at low doses, it primarily increases cell proliferation in leiomyoma cells.

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Growth Factors, Signaling Pathways and other Biochemical Markers effect on NER Suppression in Leiomyomas

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Growth Factors, Signaling Pathways and other Biochemical Markers effect on NER Suppression in Leiomyomas

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