Long Non-Cod­ing RNA Signature in Cervical Cancer

Overview

Background: Cervical cancer as a common urogenital cancer among women has caused significant health problems. Efforts have been made to identify its pathogenic process in order to find targeted ther­apies. Long non-cod­ing ribonucleic acids (lncRNAs) have been shown to regulate several cancer-related pathways and genes that contribute to pathogenesis of human malignancies, includ­ing cervical cancer. In the present review, we searched PubMed, Google scholar, Web of Science and Scopus databases for key words “cervical cancer” or “cervical neoplasm” and “long non-cod­ing RNA” or “lncRNA” (up to December 2017).

Aim: To elaborate the role of lncRNAs in cervical cancer.

Conclusions: LncRNAs affect cervical cancer pathogenesis through numerous mechanisms, such as mak­ing scaffolds for assembly of protein complexes, serv­ing as directors to recruit proteins, function­ing as transcriptional enhancers through chromatin remodeling, serv­ing as decoys to free up proteins from chromatin, or revers­ing the effects of other regulatory non-cod­ing RNAs, such as microRNAs. Pathway-based analysis showed that several lncRNAs modulate PI3K/ Akt/ mTOR, Wnt-β catenin and Notch pathways in the process of cervical cancer pathogenesis. In addition, expression of a handful of lncRNAs has been associated with human papilloma virus infection. Identification of lncRNAs that alter cancer-related signal­ing pathways and subsequent expression analysis of these lncRNAs in patients’ samples would help to design effective targeted ther­apies.

Key words:

lncRNA –  cervical cancer – oncogene – tumor suppressor gene

Introduction

In recent years, progress in genome ana­lyses has led to recognition of an evolv­ing class of non-cod­ing ribonucleic acids (ncRNAs) that participate in the modulation of gene expression and epigenetic reprogramming [1]. A signi­ficant number of these ncRNAs are longer than 200 nucleotides and instead of be­ing transcriptional “noise”, they use various routes to control gene expression [1]. These so-called long non-cod­ing RNA (lncRNAs) have tissue specific expression pattern [2] but are less conserved than protein cod­ing RNAs [3]. They are involved in almost every aspect of physiological processes, such as preservation of DNA integrity [4], telomere bio­logy [5], immune cell homeostasis [6], regulation of hormone receptors [7] as well as differentiation and homeostasis of metabolic tissues [8]. The differential expression of lncRNAs in malignant tissues compared with normal tissues of the same origin has been demonstrated in several studies [3,9– 14] what implies their role in pathogenesis of different cancers. Such speculation has been further supported by the presence of distinct single nucleotide polymorphisms within lncRNA cod­ing genes which alter the risk of cancer development [15,16].

Cervical cancer as a common uro­genital cancer among women is mostly associated with human papilloma virus (HPV) infection. However, as its incidence is much lower than the prevalence of HPV infection, other factors are thought to have synergic effects with HPV infection to induce cervical cancer [17]. Dysregulation of Wnt/ β-catenin signal­ing as well as PI3K/ Akt/ mTOR signal­ing pathway have also been implicated in the pathogenesis of cervical cancer [18]. Consider­ing the role of lncRNAs in the regulation of these pathways, we searched the literature to identify lncRNAs that modulate cervical cancer risk especially through alteration of these pathways or through modulation of HPV infection process.    

Search strategy 

We searched PubMed, Google scholar, Web of Science and Scopus databases with the key words “cervical cancer” or “cervical neoplasm” AND “long non-cod­ing RNA” or “lncRNA”. Original articles were chosen if they were written in English and had enough number of samples for expression analysis (at least 20 patients’ samples from exclusive clinical studies) and described the mechanism of lncRNA involvement in cervical cancer (for in vitro studies). Other types of papers were excluded from the study. Papers, which focused on analysis of lncRNAs at genomic level, were also excluded.

LncRNA involvement in cervical cancer

A recent study aimed at identification of expression profiles of lncRNAs, circular RNAs, microRNA (miRNA), and messenger RNA (mRNA) in HPV16 mediated ce­rvical squamous cell carci­noma have found 19 lncRNAs that are frequently differentially expressed in cervical cancer samples compared to normal samples. Such differentially expressed lncRNAs have been shown to participate in cervical cancer pathogenesis as revealed by the co-expression network and function prediction [19]. In addition to this high throughput studies, several studies have assessed the significance of lncRNAs in cervical cancer pathogenesis. Based on the importance of HPV infection and dysregulation of signal­ing pathways in the pathogenesis of cervical cancer, we subsequently analyzed lncRNAs based on their involvement in one of these mechanisms.

LncRNAs and HPV infection

HPVs as double-stranded circular DNA vi­ruses encode several proteins, which participate in their DNA repli­cation, gene transcription and cellular trans­formation. E6 and E7 proteins coded by high-risk HPV viruses participate in the pathogenesis of HPV-associated carcinomas [20]. Degradation of p53 and retinoblastoma protein (Rb) as two important tumor suppressor proteins is induced by the HPV onco­genic proteins E6 and E7, respectively. E6 also participates in carcinogenesis through induction of telomerase activation, while E7 alters the expression of synthesis phase genes by directly disturb­ing pRb/ E2F complex and en­hances cell survival by induc­ing ex­pres­-sion of interleukin-648 and anti­apo­-ptotic Mcl-126 and trigger­ing the Akt/ PKB pathway [20]. The cooperation of HPV oncogenes and lncRNAs in cervical cancer context has been first revealed for metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). This lncRNA has been over-expressed in cervical cancer cell lines compared with normal cervical squamous cell samples. However, its expression has been decreased follow­ing the E6/ E7 knockdown in CaSki cells. Further asses­sment of clinical samples has confirmed exclusive expression of MALAT1 in HPV--positive cervical squamous cells, but not in HPV-negative normal cervical squamous cells [21]. The positive association between MALAT1 expression levels and HPV infection has also been documented in cervical epithelial tissues by microarray analysis [22].

HOX transcript antisense RNA (HOTAIR) participates in epigenetic regulation of gene expression through recruitment of chromatin remodel­ing polycomb repressive complex 2 (PRC2). This lncRNA has been recognized as a target of E7 in HPV16 related cervical cancers. HOTAIR expression has been shown to be progressively decreased in a linear manner from HPV negative controls to HPV16 positive non-malignant and cervical cancer samples. Such down-regulation was concomitant with up-regulation of HOTAIR target, HOXD10, and enhancement of cancer related pathways in most cervical cancer cases. Conversely, a minority of them had considerably higher HOTAIR expression, associated with high E7 expression and enhancement of metastatic pathways. The interaction between HOTAIR and E7 has further been supported by observation of a positive correlation between E7 expression and expressions of both HOTAIR and PRC2 complex members (EZH2 and SUZ12) in cervical cancer cases. In addition, both in silico analysis and RNA immunoprecipitation endorsed the functional inactivation of HOTAIR by direct interaction with E7. Consequently, HOTAIR has been identified as a downstream target of HPV16 E7 in the process of cervical cancer pathogenesis [23].

LncRNAs and PI3K/ Akt/ mTOR pathway

Maternally expressed 3 (MEG3) as a tumor suppressor lncRNA has been impli­cated in cervical cancer. Its over-expres­sion in cervical cancer cells resulted in down-regulation of PI3K, Akt, MMP-2, MMP-9 and Bcl-2 expression while up-regulat­ing Bax and P21 expression. Consequently, lncRNA MEG3 inhibits cervical cancer by modification of PI3K/ Akt/ Bcl-2/ Bax/ P21 and PI3K/ Akt/ MMP-2/ 9 signal­ing pathway [24]. GAS5 as another tumor suppressor has been shown to modulate cellular growth and drug resistance through the PTEN/ PI3K Akt/ mTOR pathway. The low level of GAS5 leads to PTEN down-regulation by interact­ing with miR-21 because PTEN is one of the genes in the PI3K/ Akt/ mTOR pathway whose expression is decreased by GAS5. Eventually, the low expression of PTEN triggers the PI3K/ Akt pathway, therefore produc­ing a circulation. Moreover, GAS5 and miR-21 modulate cisplatin resistance in cervical cancer cells via the PI3K/ Akt pathway [25].

Mitogen activated protein kinases (MAPK) pathway

TCONS_00026907 as a newly identified lncRNA enhances expression of cyclin D1 and Bcl-2 in vivo and in vitro. Its knock-down inhibits growth of cervical tumors and modulates the expression of ELK1, p-ELK1, c-fos, cyclin D1 and Bcl-2 in vivo. Consider­ing the role of ELK1 as a nuclear target for the Ras-Raf-MAPK signal­ing cascade [26], the oncogenic effect of this lncRNA in cervical cancer might be due to its effect on MAPK pathway.

LncRNAs and Wnt/ β-catenin pathway

The Wnt/ β-catenin signal­ing pathway is a quintessential survival pathway which modulates several cellular processes includ­ing proliferation, growth, survival and metabolism. XIST silenc­ing in cervical cancer cells decreased the protein level of β-catenin and inhibited the protein expression of two Wnt/ β-catenin down­stream genes – cyclin D1 and c-Myc [27]. Besides, CCAT-1 role in enhancement of proliferation and suppression of apo­ptosis of cervical cancer cells is also through induction of the Wnt/ β-catenin pathway [28].

LncRNAs and Notch pathway

Evaluation of the essential signal­ing cascades regulated by Notch in HOTAIR--overexpress­ing cells has shown that HOTAIR overexpression in SiHa cells has led to increased NOTCH1, HES1 and p300 expression [29].

LncRNA involvement in epithelial-mesenchymal transition (EMT) of cervical cancer cells

HOTAIR as an oncogenic lncRNA in cervical cancer has been shown to alter the expression of several genes participated in cell migration, invasion and EMT, such as vascular endothelial growth factor, MMP-9, E-cadherin, β-catenin, vimentin, Snail and Twist [30]. EMT-related levels have also been ele­vated in xenografts originated from HOTAIR-overexpress­ing SiHa cells com­pared with the control tumors [29]. In addition, HOTAIR enhances migration and invasion of HeLa cervical cancer cells, at least partially, through the modulation of vimentin expression [31]. HOTAIR role in EMT might also due to its effect on COX-2 stabilization, which leads to induction of matrix metalloproteinases and vascular endothelial growth factor (Schema 1) [32].

MALAT1 exerts its role in cervical cancer cell invasion and metastasis by enhancement of the EMT process through increas­ing the expression of Snail [33]. MALAT1 silenc­ing suppressed the invasion and metastasis of cervical cancer cells, increased expression of the epithelial markers E-cadherin and ZO-1, and simultaneously decreased expression of mesenchymal markers β-catenin and vimentin as well as the Snail transcription factor (Schema 2) [22].

The lncRNA taurine‐upregulated gene 1(TUG1) was also shown to increase migration and invasion of cervical can­cer cells by modulat­ing EMT‐related markers such as fibronectin, vimentin and cytokeratin [34].

HOXA11-AS has also been shown to participate in EMT. HOXA11-AS silenc­ing has led to increase in E-cadherin expression while decreas­ing levels of β-catenin, vimentin and the EMT-mediat­ing transcription factor Snail [35].

In addition, EZH2-bind­ing lncRNA in cervical cancer (lncRNA-EBIC) has a role in migration and invasion of cervical cancer cells through modulation of E-cadherin [36].

LncRNAs role in cervical cancer immune evasion

The human leukocyte antigen-G (HLA-G) as a member of the non-classical ma­jor histocompatibility complex family is recruited by cancer cells to beat attentive immuno-surveillance of the host. HOTAIR has been shown to en­hance HLA-G associated immune escape by competitively bind­ing to miR-148a (Schema 1) [37].

Discussion

Consistent with diverse mechanisms of lncRNAs participation in regulation of gene expression, lncRNAs can affect cervical cancer pathogenesis through various mechanisms. Over­all, the regulatory function of lncRNAs can be exerted through mak­ing scaffolds for assembly of protein complexes, serv­ing as guides to recruit proteins, function­ing as transcriptional enhancers through chromatin remodeling, serv­ing as decoys to free up proteins from chromatin, or revers­ing the effects of other regulatory ncRNAs, such as miRNAs [38]. Besides, the expression of HPV oncogenes as the most important causal factor in cervical cancer has been shown to be linked to expression levels of some lncRNAs. However, functional studies to reveal the exact mechanism of this association has been performed for only two lncRNAs, namely MALAT1 and HOTAIR. Moreover, expression of several lncRNAs has been shown to be dysregulated in tumor tissues as well as plasma samples from cervical cancer patients. Higher expression of certain lncRNAs in the plasma of cervical cancer patients compared to healthy subjects provides an applicable tool for screen­ing and follow-up of patients.

More importantly, methylation pat­tern of the MEG3 lncRNA has been demonstrated to be a dia­gnostic and prognostic marker of cervical cancer with the capability to predict high--risk HPV infection and lymph node metastasis [39]. Consider­ing the early onset of methylation alterations dur­ing carcinogenesis, identification of such marks is valuable in early detection of cancer.

Notable, several lncRNAs have been shown to affect cancer-related path­ways. Alterations in these pathways modulate response to conventional che­motherapeutic approaches as revealed for cisplatin resistance in cervical can­cer [25]. On the other hand, dysregulation of numerous signal­ing pathways, such as Notch and mTOR pathways, has been demonstrated in cervical cancers through transcriptome analysis. Consider­ing the therapeutic potential of these signal­ing pathways in at least some types of cervical cancer, targeted inhibition of Notch and mTOR pathways has been suggested as therapeutic options for cervical cancer patients [40]. Consequently, identification of lncRNAs that alter these signal­ing pathways and subsequent expression analysis of these lncRNAs in patients’ samples would help to better select patients for recruitment in these trials.

The authors declare they have no potential conflicts of interest concerning drugs, pro­ducts, or services used in the study.

The Editorial Board declares that the manu­script met the ICMJE recommendation for biomedical papers.

Submitted/Obdrženo: 25. 4. 2018

Accepted/Přijato: 1. 11. 2018

Soudeh Ghafouri-Fard, MD, PhD

Department of Medical Genetics

Shahid Beheshti University of Medical Sciences

Bldg No. 2 SBUMS

Arabi Ave, Daneshjoo Blvd, Velenjak

Tehran, Iran

e-mail: s.ghafourifard@sbmu.ac.ir