The role of long non-coding RNAs and circular RNAs in cervical cancer: modulating miRNA function

Cervical cancer (CC) is a primary global health concern, ranking as the fourth leading cause of cancer-related death in women. Despite advancements in prognosis, long-term outcomes remained poor. Beyond HPV, cofactors like dietary deficiencies, immunosuppression, hormonal contraceptives, co-infections, and genetic variations are involved in CC progression. The pathogenesis of various diseases, including cancer, has brought to light the critical regulatory roles of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). The aberrant expression of these miRNAs, lncRNAs, and circRNAs plays a pivotal role in the initiation and progression of CC. This review provides a comprehensive summary of the recent literature regarding the involvement of lncRNAs and circRNAs in modulating miRNA functions in cervical neoplasia and metastasis. Studies have shown that lncRNAs and circRNAs hold great potential as therapeutic agents and innovative biomarkers in CC. However, more clinical research is needed to advance our understanding of the therapeutic benefits of circRNAs and lncRNAs in CC.


Introduction
Uterine cervix carcinoma ranks among the most frequently diagnosed gynecological malignancies and is recognized as the fourth most fatal cancer in women on a global scale (He et al., 2020).In 2020, the approximate incidence, prevalence, and fatality rate accounted for 604,127, 1.495.211, and 341,831, respectively (Bray et al., 2018;Singh et al., 2023).Cervical cancer (CC) predominantly manifests in two primary histological forms: squamous cell carcinomas (SCC) and adenocarcinoma (ADC).Adenosquamous carcinoma (ADSC) is the less common type of CC (Nicolás-Párraga et al., 2017;Small et al., 2017).Notably, there has been an increased upsurge in ADC incidence, which is more prevalent among young adult women (Fujiwara et al., 2014).The primary risk factor contributing to CC is the presence of oncogenic human papillomavirus (HPV) (Savira et al., 2022).Previous studies have revealed that HPV 16 and 18 types account for approximately 70% of HPV infections (Aalijahan and Ghorbian, 2019).HPV18 is particularly common in individuals with ADC, while HPV16 is predominant in SCC cases (He et al., 2020).Despite advancements in CC prognosis, long-term outcomes remain poor due to resistance and recurrence (He et al., 2020).Addressing this issue necessitates further research to identify new biomarkers for precise CC progression monitoring and therapeutic targets to enhance survival rates.
The progression from HPV infection to cancer necessitates the involvement of other cofactors, including specific dietary deficiencies, immunosuppression, prolonged use of hormonal contraceptives, coinfection with Chlamydia trachomatis (CT), co-infection with herpes simplex virus type 2 (HSV-2) and HIV, high parity, and tobacco smoking, among other potential cofactors (Aalijahan and Ghorbian, 2019).Furthermore, individual genetic variations and distinct epigenetic modifications are pivotal in carcinogenesis.DNA methylation, histone modifications, and non-coding RNAs (ncRNAs) influence gene expression without altering the DNA sequence.The ncRNAs are prevalent throughout the entire genome and can be classified into two subclasses: small ncRNAs with 20-200 nucleotides and long ncRNAs (lncRNAs) with over 200 nucleotides (Hosseini et al., 2017).

Biogenesis of lncRNAs, circRNAs, and microRNAs
The biogenesis of lncRNAs can be divided into various categories, including exonic, intronic, intergenic, and overlapping (Hosseini et al., 2017).The lncRNAs, through both cis-and transregulation, can alter the phenotypes of cancer cells by targeting their final genes (Guttman and Rinn, 2012).Given their capacity to induce dysregulation in various human diseases and foster cancer development, lncRNAs hold significant potential as active biomarkers and, specifically, as therapeutic targets in cancer (He et al., 2020).
Small non-coding RNAs, particularly microRNAs (miRNAs), have been identified as key players in various cellular processes such as cell cycle regulation, inflammation, apoptosis, migration, and differentiation.Additionally, they influence messenger RNA (mRNA) translation and stability (Di Leva et al., 2014).Studies have substantiated that miRNAs can function as oncogenes or tumor suppressors in various cancer types (Romano et al., 2017;Rupaimoole and Slack, 2017).Understanding the regulatory mechanisms governing miRNA expression is closely intertwined with cancer diagnosis, prognosis, treatment, and underlying pathogenesis (Shen et al., 2020).
Circular RNAs (circRNAs) are a large family of non-coding RNAs in the transcript of eukaryotes.They appear as covalently closed loops formed by successive exons in the nucleus.CircRNAs have polar 5′ and 3′ ends and polyadenylated tails.There are many types of circRNAs in different cell lines and species, involving diverse ranges of biological processes between cells (Chen and Yang, 2015).After transferring to the cytoplasm, circRNAs perform various biological actions by binding to microRNAs and proteins (Ren et al., 2019).They play a vital role in transcriptional regulation and are potential biomarkers (Liang et al., 2021).Additionally, circRNAs are secreted into body fluids both in free form and encapsulation in multi-vesicular endosomes and exosomes along with lipids, proteins, and nucleic acids, controlling various biological functions in different areas of the body

GRAPHICAL ABSTRACT
The role of lncRNAs and circRNAs in cervical cancer.(Wang et al., 2019a), including fetal growth and brain synaptic plasticity (Liang et al., 2021).Moreover, abnormal expression of circRNAs is detected in the occurrence of diseases such as human malignancies, Alzheimer's, and Parkinson's disease (Nisar et al., 2021).

Biological functions of long noncoding RNAs
Long non-coding RNAs exhibit a broad spectrum of biological and regulatory functions, including proliferation, differentiation, development, and DNA damage repair (Wang and Chang, 2011).They interfere with transcription, translation, and post-transcriptional and post-translation processes, enabling a direct interaction with DNA, RNA, and proteins (He et al., 2020).Cis-acting lncRNAs are closely associated with specific transcription sites, affecting neighboring genes' chromatin structure or expression state on the same allele.In contrast, trans-acting lncRNAs act from a distance, triggering the expression of genes, proteins, or RNA molecules within the cell (Guttman and Rinn, 2012;Kopp and Mendell, 2018).
LncRNAs are involved in epigenetic gene regulation through three main mechanisms.First, they influence the transcription of target genes by regulating histone modifications and the chromatin status.Second, lncRNAs actively recruit repressors or cell transcription factors to the promoters of their target genes.Third, they tightly bind to proteins involved in transcription, preventing them from attaching to their specific DNA targets (Long et al., 2017).
In addition, lncRNAs act as competing endogenous RNAs (ceRNAs) by binding to miRNAs, thereby preventing miRNAmediated gene suppression (Gutschner and Diederichs, 2012).Some lncRNAs serve as templates or precursors for various small RNAs (Cheetham et al., 2013) and influence alternative mRNA splicing (Romero-Barrios et al., 2018).At the post-translational level, lncRNAs can bind to target proteins, affecting their localization and transportation.Previous studies have reported that lncRNAs function as scaffolds, facilitating protein-protein interactions, and the formation of protein complexes (Yoon et al., 2013) (Figure 1).

Relevant lncRNAs in cervical cancer
Long non-coding RNAs interact with proteins, mRNAs, and miRNAs and play a critical role in cancer progression.Several lncRNAs, including HOTAIR, CCAT2, H19, MALT1, SPRY4- Functions of long non-coding RNAs (lncRNAs) in cervical cancer.

HOTAIR lncRNA
HOTAIR, "2.2 kb HOX transcript antisense intergenic RNA," is one of the extensively studied oncogenic lncRNAs.It is encoded by the antisense strand of the HOXC gene and is located on chromosome 12 at position q13.13 (Rüegg, 1985).Like other lncRNAs, HOTAIR recruits chromatin-modifying proteins and influences the epigenome of cancer (Ren et al., 2019).In CC, the levels of HOTAIR are tightly controlled by the HPV E7 protein (Sharma et al., 2015).Furthermore, HOTAIR functions as a sponge for specific miRNAs, resulting in the dysregulation of their respective target genes.For instance, HOTAIR has been reported to alter the miR-143-3p/BCL2 axis, promoting CC cell growth (Liu et al., 2018a).It also modulates the miR-23b/MAPK1 axis, which is involved in cell proliferation and metastasis (Li et al., 2018).Additionally, HOTAIR overexpression has regulatory effects on various metabolic functions, such as the activation of the Notch-Wnt signaling pathway in SiHa cells (Lee et al., 2016) and the mTOR pathway in different CC cell lines, including C33A, HeLa, and CaSki (Zhang et al., 2015).The expression of HOTAIR leads to the upregulation of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), and genes associated with the epithelial-mesenchymal transition (EMT), thereby promoting tumor aggressiveness in CC (Kim et al., 2015).It is worth noting that the HOTAIR enhancer contains a single nucleotide polymorphism, rs920778T, which is associated with HOTAIR overexpression and increased susceptibility to cancer (Xiao et al., 2011).In cases of HPV-positive CC, the rs920778 C HOTAIR allele is more prevalent, but its expression level may be reduced due to the binding of miR22 to the rs920778C sequence, leading to the suppression of the HOTAIR expression (Sh et al., 2016).

CCAT2 lncRNA
Substantial upregulation of the long non-coding RNA, known as colon cancer-related transcript 2 (CCAT2), has been observed in CC cells, including HeLa, SiHa, and CaSki, as well as in CC tissues (Lee et al., 2016).

CCAT1 lncRNA
The lncRNA CCAT1 is located within the c-Myc gene region, belonging to the Myc proto-oncogene family, which is known for its critical role in tumorigenesis.CCAT1, acting as a sponge for miR-181a-5p, promotes the proliferation and invasion of CC cells.This function is accomplished through the upregulation of heparin-binding EGF-like growth factor (HB-EGF) and matrix metalloproteinase-14 (MMP14) (Shen et al., 2019).Furthermore, upon binding to c-Myc, CCAT1 activates the Wnt/β-catenin signaling pathway, leading to increased cell progression (Zhang and Gao, 2017).

Metastasis-associated lung adenocarcinoma transcript 1 lncRNA
The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) consists of 8,000 nucleotides and is located in chromosome 11q13.1.It was first recognized in non-small cell lung cancer (Sun and Ma, 2019).Overexpression of MALAT1 has been observed in CC cell lines and cancer tissues infected with highrisk HPV (Jiang et al., 2014).MALAT1 is a sponge for several miRNAs, including miR-124, miR-145, and miR-20 (Lu et al., 2016).Consequently, downregulation of MALAT1 in both CC tissues and CC cell lines leads to a significant reduction in invasion and metastasis, achieved through the modulation of the MALAT1-miR-124-RBG2 axis and the inhibition of the epithelial-mesenchymal transition (Lee et al., 2016).Additionally, MALAT1 plays a role in the mechanisms of radio resistance by acting as a sponge for miR-145 during CC radiotherapy (Lu et al., 2016).

SPRY4-IT1 lncRNA
Intron two of the SPRY4 gene generates SPRY4 intronic transcript 1 (SPRY4-IT1), a molecule that assumes the role of either a tumor suppressor or an oncogenic factor in various cancer types (Xie et al., 2015).However, in CC, SPRY4-IT1 expression levels surpass those of normal tissues and are closely associated with advanced clinical stages, ultimately diminishing the survival rates of CC patients (Cao et al., 2016).Recent research has unveiled the pivotal role of SPRY4-IT1 silencing in CC cell lines, particularly in inhibiting migration and invasion via the SPYR4-IT1/ miR-101-3p/ZEB1 axis.This function is directly linked to the active suppression of epithelial-mesenchymal transition (EMT) alterations, leading to increased E-cadherin levels and reduced expression levels of both N-cadherin and vimentin (Fan et al., 2019).

PVT1 lncRNA
Plasmacytoma variant translocation 1 (PVT1) is an exceptionally conserved long non-coding RNA (lncRNA) located downstream of the MYC gene.It is frequently co-amplified with MYC in various types of cancer (Li et al., 2019).PVT1 downregulates the expression levels of miR-195 and miR-200b in CC through direct binding or the enhancement of histone H3K27me3 in the promoter regions of miR-195 and miR-200b (Zhang et al., 2016;Shen et al., 2017).MiR-195 is associated with chemoresistance and the epithelial-mesenchymal transition, while miR-200b plays a role in cellular proliferation, invasion, and migration of CC cells.A positive correlation between serum PVT1 levels and the expression of PVT1 in cancer tissues has been reported, suggesting PVT1 as a potential diagnostic biomarker for CC (Yang et al., 2016).

GAS5 lncRNA
Growth arrest-specific transcript 5 (GAS5) exhibits tumor suppressor activity in various cancer types (Schneider et al., 1988;Pickard and Williams, 2016).Therefore, in CC, lower GAS5 expression levels are linked to tumor progression and poorer clinical outcomes for individuals with CC (Cao et al., 2014).The inhibition of GAS5 in CC cells has been observed to enhance proliferation, migration, and invasion, further underscoring its potential as a tumor suppressor in the development of CC (Cao et al., 2014).Notably, GAS5 functions as a sponge for miR-106b, resulting in the increased expression of IER3 (immediate early response 3) and enhanced radio sensitivity of CC cells (Gao et al., 2019).

Maternally expressed gene 3 lncRNA
Maternally expressed gene 3 (MEG3), a long non-coding RNA derived from the DLK1-MEG3 locus at the chromosomal location 14q32.3, is well-established as a cancer-suppressive factor (Zink et al., 2018), which was initially recognized as the ortholog of gene trap locus 2 (Gtl2) in mice.MEG3 overexpression demonstrates preventive effects on cell proliferation and enhances apoptosis in cancer cells, both in a p53-dependent and independent manner (Paci et al., 2014).A notable reduction in the MEG3 expression level was observed in CC tissues and cell lines.Enhancing MEG3 in CC cell lines prevented proliferation and induced cell cycle arrest and apoptosis.It was achieved by direct binding to P-STAT3, leading to subsequent ubiquitination and degradation of P-STAT3 (Zhang and Gao, 2019).Additionally, MEG3 exerts its cancer-suppressive effect by downregulating the levels of miR-21-5p in CC cell lines (Avila et al., 2023).Knockdown of MEG3 in HeLa and CaSki cells resulted in a significant upregulation of miR21-5p expression (Yadav et al., 2023).

Biological functions of circular RNAs
Circular RNAs (circRNAs) represent a recently discovered class of widespread endogenous non-coding RNA molecules characterized by their covalent circular structures, which serve as transcription factors, miRNA sponges, and RNA-binding proteins (RBPs) (Hsiao et al., 2017).CircRNAs have been observed to exhibit abnormal expression LncRNA effects in metabolic pathways in cervical cancer.
patterns in cancer cells and play diverse roles in the development and progression of cancer.Due to their unique stable loop-like structure, circRNAs have shown promise as potential biomarkers and therapeutic targets for various types of cancer (Zhu et al., 2021).CircRNAs have various biological functions such as regulation of gene expression (Hansen et al., 2013).They act as microRNA sponges, sequestering and inhibiting the activity of microRNAs, thus modulating the expression of target genes (Hansen et al., 2013).CircRNAs can interact with proteins (Du et al., 2016) and regulate alternative splicing of mRNA by interacting with splicing factors, thereby regulating the diversity of the transcriptome (Ashwal-Fluss et al., 2014).They also play roles in cellular proliferation, apoptosis, and cellular senescence (Abdelmohs et al., 2015;Li et al., 2015).CircRNAs are also associated with various aspects of cancer, including tumorigenesis, metastasis, and resistance to therapy (Guarnerio et al., 2016).Additionally, they have been implicated in neurological disorders, synaptic function, neuronal development, and neurodegenerative diseases (Rybak-Wolf et al., 2015) (Figure 3).

Regulation of circRNA biogenesis
CircRNA biogenesis is intricately controlled by repetitive intronic elements, particularly sequences derived from transposons (Wilusz, 2015).The production of circRNA involves alternative splicing of one or several exons of a gene in the presence of an RNA-mediated silencing complex (Liang et al., 2021).Splice sites bring exons closer together and facilitate the formation of closed loops (Wilusz, 2015).Backsplicing, mediated by inversion repeats flanking cyclic exons, further aids circRNA production.When producing circRNAs, different versions of the closed loop are created depending on repeated base pairs.Therefore, the specific arrangement of intronic repeats regulates the potential function of protein-coding genes (Wilusz, 2015).

Protein factors in circRNA biogenesis
Numerous protein factors play pivotal roles in circRNA biogenesis.NF90/NF110 is a crucial regulator that assembles exons to form circRNAs in the nucleus and interacts with mature circRNAs in the cytoplasm (Li et al., 2017).NOVA2, an enriched RNA-binding protein, plays a crucial role in the circRNA expression by binding to specific sites inside the introns flanking circRNA exons (Knupp et al., 2021).Heterogeneous nuclear ribonucleoprotein M (HNRNPM) is another factor involved in controlling circRNA biogenesis by preventing misplaced exon splicing and backsplicing after binding to homeostatic gene transcripts (Ho et al., 2021).The RNA-binding protein SFPQ regulates circRNA biogenesis by overseeing long-intron splicing, ensuring precise splicing, and preserving the intron integrity (Stagsted et al., 2021).

Circular RNA and modulation microRNAs in cervical cancer
circRNAs have emerged as a novel therapeutic target in CC (Chaichian et al., 2020).A recent study highlighted the role of circRNA-VPRBP in blocking galectin-1-mediated CC metastasis by Functions of circular RNAs (circRNAs) in cervical cancer.
Frontiers in Cell and Developmental Biology frontiersin.orgbinding to RACK1 O-GlcNAcylation (Zhang et al., 2023).Aberrant expression of circRNAs has been linked to the initiation of pathological processes in cervical tissue, influencing cell proliferation, migration, and apoptosis.CircRNAs are considered a potential clinical biomarker for early detection of CC, offering a prognosis for early treatment (Chen et al., 2021).
Circular RNA (hsa_circ_0001772) is implicated in cancer tumorigenesis, and its knockdown reduces tumor growth, migration, invasion, and glycolysis and promotes CC cell apoptosis (Ding et al., 2021).Hsa_circ_0001772 functions as a sponge for miR-758-3p, modulating PUM2 expression, which reverses the suppressive influence of upregulated miR-758-3p on CC cell malignant behaviors.Hsa_circ_0001772 fosters CC advancement by absorbing miR-758-3p and enhancing PUM2 expression.

Conclusion and future directions
Due to various limitations and shortcomings, such as challenges in early detection, a lack of targeted therapies, limited HPV vaccination coverage, treatment side effects, and resistance to therapy, CC stands as one of the leading causes of death among women worldwide.LncRNAs and circRNAs serve pivotal roles in modulating miRNA activity, thereby influencing the initiation and progression of cervical carcinogenesis.These non-coding RNAs engage in intricate regulatory networks, directly interacting with miRNAs to impact downstream target mRNA expression.Examples of such interactions include lncRNA HOTAIR, which acts as a sponge for miR-23b-3p, relieving the suppression of its target gene ZEB1.This leads to enhanced EMT and increased metastatic potential in CC cells.Similarly, circRNA CDR1as sequesters miR-7, leading to increased expression of its target gene EGFR, promoting cell proliferation and migration in CC.The dysregulation of these non-coding RNA-mediated interactions contributes significantly to key aspects of cervical carcinogenesis, including uncontrolled proliferation, evasion of apoptosis, and increased metastatic potential.Furthermore, the intricate interplay between lncRNAs, circRNAs, and miRNAs contributes to the establishment of a conducive tumorigenic microenvironment.Numerous studies have explored the diagnostic and therapeutic potential of miRNAs in various cancers.This review focuses on summarizing the role of lncRNAs and circRNAs in the context of CC.Understanding these regulatory mechanisms at the molecular level provides valuable insights into the complexity of CC pathogenesis.Targeting the dysregulated interactions between lncRNAs, circRNAs, and miRNAs holds promise for developing innovative diagnostic and therapeutic strategies against cervical carcinogenesis.Thus, elucidating these molecular networks enhances our comprehension of CC biology and informs potential avenues for precision medicine in its management.Additionally, the review emphasizes the potential of lncRNAs as biomarkers and therapeutic targets in CC.It provides a detailed examination of specific lncRNAs implicated in CC development, such as HOTAIR, CCAT2, H19, MALAT1, SPRY4-IT1, PVT1, GAS5, MEG3, and C5orf66-AS1.We present a comprehensive list of circRNAs and their functions in CC, detailing how they act as miRNA sponges, influencing mRNA expression, and contributing to the pathogenesis of CC.
This article is a valuable resource for researchers, highlighting the potential of lncRNAs and circRNAs as promising targets for diagnostic and therapeutic interventions in CC.Efforts to identify early detection biomarkers in CC focus on specific lncRNA-miRNA-target mRNA networks associated with initial stages of the disease.The dysregulation of a lncRNA, which modulates miRNA activity and influences the expression of oncogenic mRNAs, holds potential as an early diagnostic biomarker, enabling timely intervention and improving prognosis.Investigating diverse interactions within lncRNA-miRNA-target mRNA networks in CC subtypes can lead to the development of molecular signatures for precise subtyping.These specific networks may indicate distinct subtypes, aiding in the formulation of tailored treatment strategies and enhancing therapeutic outcomes.Additionally, exploring liquid biopsy approaches to detect lncRNAs, miRNAs, and target mRNAs in bodily fluids offers non-invasive diagnostic possibilities.Circulating nucleic acids associated with regulatory networks can serve as liquid biopsy markers, providing a convenient method for monitoring disease progression and treatment response.Integrating diverse lncRNA-miRNA-target mRNA networks with established molecular markers contributes to comprehensive molecular profiling, enhancing CC diagnosis accuracy by considering multiple layers of molecular information.This understanding guides clinicians in developing personalized treatment plans.Furthermore, identifying specific lncRNA-miRNA-target mRNA networks linked to treatment response or resistance serves as therapeutic monitoring markers.Monitoring dynamic changes in these networks provides real-time insights, allowing for timely adjustments in treatment strategies.In conclusion, recognizing the potential of lncRNAs and circRNAs as diagnostic markers in CC prompts specific exploration of molecular networks for practical clinical applications.Spanning from early detection to subtypespecific diagnostics and non-invasive monitoring, these applications offer actionable directions for translating molecular discoveries into tangible benefits for CC patients.However, the utilization of these emerging lncRNAs and circRNAs as biomarkers for both the prognosis and diagnosis of CC requires further exploration through extensive clinical research in this field.

TABLE 1
Related lncRNAs and functions in cervical cancer.

TABLE 2
Related circRNAs and functions in cervical cancer.