1. Introduction

The analysis of non-coding RNA (ncRNAs) expression in endometrial cancers (EC) might be

of importance in understanding the neoplastic transformation of the endometrium. These RNA

transcripts are possible biomarkers of early neoplastic transformation, but their significance might be

even greater.

Approximately only 2% of the genome in humans has protein encoding functions, so most of

the human genome is not translated into proteins. It has been called genomic dark matter, because no

specific function has been described for it [1]. However, many information-carrying genes have been

demonstrated to have significant regulatory functions, even if they remain untranslated. The ncRNAs

include transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), antisense RNAs (asRNA), micro RNAs

(miRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), competing endogenous

RNAs (ceRNAs) and piwi-interacting RNAs (piRNAs).

Recently, an additional group of ncRNAs has been discovered: they are called long-ncRNAs

(lncRNAs), as they are over 200 nucleotides in length, in contrast to other ncRNAs, which are

usually shorter [2,3]. lncRNAs are less comparable between species; protein-coding genes tend

to be homologous between mammals [4]. Four main mechanisms are involved in their expression

and they are sometimes defined as archetypes: signaling, decoying, scaffolding and guidance [5,6].

lncRNAs seem to have an importance in physiological regulation, and their dysfunction has been

implicated in cardiovascular and neurodegenerative diseases as well as in cancers [7,8]. lncRNAs

seem to be important modulators in biological mechanisms and significant cofactors of tumorogenesis.

lncRNA gene polymorphisms have been linked to cancer risk [9]. Systematic reviews on the association

between lncRNA single-nucleotide polymorphisms (SNPs) and the overall risk of developing a cancer

are starting to appear in the literature. A total of 17 SNPs in four common lncRNA genes have

already been described by Zhi et al. in a meta-analysis [9]. However, the involvement of lncRNAs

in endometrioid endometrial adenocarcinoma (EEC) remains to be fully addressed. Indeed, RNAs

are expressed in different ways in the normal, hyperplastic and dysplastic endometrium [6]; hence,

interference in RNA expression could actually lead to the clinical control of neoplastic transformation

and disease progression. We offer an overview of the current knowledge of the relationship between

ncRNAs and endometrial adenocarcinoma (EC) that shows promise in the diagnosis and treatment of

this condition.

Long Non-Coding RNAs: General Introduction

lncRNAs cover a large part of the non-coding information of the human DNA, which represents

more than 90% of the whole genome. They constitute a wide and complex group of molecules with

more than 200 nucleotides, usually lack an open reading frame, and in various ways are involved in

the pathophysiology of cancer [2,3,5]. Their roles in the regulation of gene expression, imprinting,

transcription, and post-translational processing have been documented in several types of cancer.

Thousands of long intergenic ncRNAs (lincRNAs) have been identified in various mammals in genome

sequencing studies. Some of these RNAs have been conserved during the evolution of various species

and might have a fundamental importance in the regulation of cell mechanisms [2–5].

3. Competing Endogenous RNAs, miRNA, lncRNA Profiling Regulation and Cancer

The miRNAs are a class of small ncRNAs that have been associated with several diseases,

including cancer [21]. They bind to target mRNAs via sequence complementarity, leading to translation

inhibition and mRNA destabilization. Several lncRNAs are capable of binding to miRNAs interfering

with this mechanism, thus blocking their action on target mRNAs. These lncRNAs are called

ceRNAs, and several examples having a role in tumorigenesis and evolution have been published.

Many lncRNA–miRNA interactions have been reported or suggested recently [22]. For example,

the lncRNA named MEG3 was recently implicated as an influencer of the signal transducer and

activator of transcription 3 (STAT3) expression, by altering miR-21 expression in ovarian cancer; it

has been shown to function as a tumor suppressor in many cancer types [23]. Maternally expressed

gene 3 (MEG3) is a maternally imprinted lncRNA with a tumor suppressor role in various tumors.

Sun et al. [24] found significantly lower MEG3 expression in endometrial carcinoma tissues than in

normal endometrial tissues.

A cross downregulation of miRNA and lncRNAs, regulating the metastasis associated lung

adenocarcinoma transcript 1 (MALAT-1) was suggested by Li et al. [25]. According to the authors,

the miR-200 family members were present in a high concentration in EEC, melanoma, and some

ovarian cancers. They showed that miR-200c levels were higher in most EEC specimens than in healthy

tissues, while MALAT-1 levels were much lower. However, they also found that miR-200c was directly

bound to MALAT-1 and miR-200c, both of which showed cross-repression, while TGF- β increased

MALAT-1 expression, working as an inhibitor of miR-200c. The interruption of the repressive effect of

miR-200c over MALAT-1 decreased the invasive capacity of EEC cells; the epithelial to mesenchymal

cell transition (EMT) markers’ expression was altered, at least in vitro [25]. However, the abnormal

expression of the lncRNA MALAT-1 or miR-200 family members were shown to facilitate EMT in

various human cancers. The details of the regulatory mechanism of MALAT1 and miR-200 remain

largely unknown.

lncRNAs can be a precursor for miRNAs. For example, H19 is the gene precursor of a ubiquitous

lncRNAs that show a negative or limiting regulation regarding body weight and cell proliferation. H19

is the RNA precursor of miR-675 [26]. Other miRNAs have been associated with tumor progression:

Vennin et al. showed that miR-675 enhanced tumor initiation, progression, and metastasis of breast

cancer cells by downregulating the c-Cbl and Cbl-b gene coding for the CBL protein [26].

This protein is an E3 ubiquitin-protein ligase involved in cell signaling [26]. The expression

pattern and biological functions of the nuclear-enriched abundant transcript 1 (NEAT1) was also

related to EEC. The levels of NEAT1 were up-regulated in ECC tissues and cell lines [27]. Moreover,

the lncRNA cancer susceptibility candidate 2 (CASC2) was identified as a potential tumor suppressor

in EC [28].

The ovarian adenocarcinoma amplified lncRNA (OVAL) is composed of three exons of

1489 nucleotides in length. OVAL was thought to encode various expressed sequence tags (ESTs) and

mRNAs, but recently an alternative first exon isoform has been identified, leading to the transcription

of the lncRNA OVAL [29]. The OVAL is over-expressed in Type I EC and in serous ovarian cancer

(OC). Type II EC, on the other hand, is four times less likely to show upregulation of the OVAL gene;

p53-regulated genes are upregulated in EC tissue samples with high expression of OVAL [29,30].

The carcinogenetic effect of estrogens in type I EC is well-documented [31]. Endometrial cancer

and breast cancer (BC) show a similar sensitivity to estrogens. According to Bhan et al., estradiol

induces lncRNA HOTAIR expression, as well as estrogen receptors and general transcription factors

of RNA polymerase II [32]. Thus, HOTAIR is not only abnormally expressed in BC, but in EC as

well [33]. According to He et al., HOTAIR upregulation was found in nearly three-quarters of EC

samples (63 out of 87), only 20% of control endometria tested positive for this lncRNA (4 out of 30).

Moreover, differentiated tumor samples (G3 grade) showed higher expression levels than Grade 1

samples. HOTAIR gene upregulation could be detected in about 50% of hyperplastic endometriums

(5 out of 12). HOTAIR is also associated with metastatic disease. High levels of HOTAIR expression

correlates with metastases and decreased patient survival in EC [34]. The lncRNA steroid receptor

RNA activator (SRA) regulates gene expression induced by steroid receptors [35]. SRA is upregulated

in BC and in other steroid-responsive tumor tissues, such as ovarian carcinoma [36]. In EC, high levels

of SRA expression are found with no relation to histological tumor grade. In reference tissue samples

of normal endometria, SRA expression is low and this may imply an early role in tumorigenesis [35].

Curiously, according to Lanz et al., SRA-transgenic mice did not develop any tumors, even with SRA

over-expression [37].

The expression of lncRNA and their role in defining EEC subgroups and their clinical

aggressiveness must be defined further. Xu et al. suggested that six lncRNAs may be the

main regulators of endometrial carcinogenesis [38]. Xu measured a total of 172 lncRNAs and

188 mRNAs and reported that they were expressed differently in type I EC and the normal control

samples [38]. Mathematical analysis with the gene ontology (GO) pathway analysis and the

lncRNA and mRNA co-expression networks indicated that six lncRNAs (KIAA0087, RP11-501O2,

FAM212B-AS1, LOC102723552, RP11-140I24 and RP11-600K151) could be the main regulators of

endometrial carcinogenesis [38].

Another study drew a parallel between EEC and liver cancer (LC) in relation to lncRNA expression.

Jiang et al. isolated 1931 expressed lncRNAs and gave them importance as to tumor initiation with

a mathematical integrative analysis [39]. A clustering of lncRNA expression helped define three groups

according to the authors: (i) basal-like, (ii) luminal-like and (iii) Catenin, β-1 (CTNNB1)-enriched

subgroups. The basal-like subgroup had a much higher number of tumors with higher pathological

grade (p < 0.0001), and TNM stage (p = 0.01); in the luminal-like subgroup, progesterone (PGR) and

estrogen receptor (ESR1) genes were not downregulated as in the EEC basal-like subgroup. The lncRNA

profile of the CTNNB1-enriched EEC subgroup was almost equal to that of the CTNNB1-enriched LC

subgroup [39].

Liu et al. showed that the lncRNA Taurine Upregulated Gene 1 (TUG1) was involved in EC

tumorigenesis due to the inhibition of miR-299 and miR-34a-5p [39]. The relationship between

lncRNA-TUG1 and EC was studied in 104 EC specimens, and corresponding control tissues.

lncRNA-TUG1 expression in cancer samples was higher than that in adjacent normal tissues.

The authors were able to show that lncRNA-TUG1 helps the evolution and progression of EC through

the inhibition of miR-299 and miR-34a-5p [40].

In a large study, 30,586 lncRNAs and 26,109 transcripts (fold change > 2.0) were found in 45

tested EC samples [40]. In particular, compared with normal tissues, 4010 lncRNA were upregulated,

and 3350 of them were downregulated. Among these lncRNAs, 3 were upregulated and 4 were

downregulated. Pathway analysis revealed that 24 pathways were correlated to the upregulated

transcripts [41]. Chen et al. showed that miR-93 was expressed at high levels in EC samples compared

to normal endometrial samples [42].

Zhai et al. looked into the expression profiles of lncRNAs and coding genes in three paired EC and

adjacent healthy tissues [43]. There was significant a difference in lncRNA and coding gene expression

between EC and their adjacent healthy non-tumor tissues; 53 lncRNAs (p value < 0.05) were differently

expressed in EC, compared with controls. ASLNC04080 was the most upregulated lncRNA in 22 out

of 24 samples and in HEC-1-B cell line [43]. Li et al. measured the levels of 26 lincRNAs in 176 pairs

of EC and adjacent healthy biopsy samples in two separate regional Chinese populations [44]. They

found that a lincRNA, LINC00672, was abnormally downregulated during the development of EC.

LINC00672 is a p53-targeting lincRNA, acting along with heterogeneous nuclear ribonucleoproteins as

a suppressive cofactor that reinforces p53-mediated suppression of the LIM and SH3 domain protein

(LASP)-1; this mechanism could possibly be associated with increased tumor aggressiveness [44].

Sun et al. looked into lncRNAs using support vector machine and random forest methods [45].

All expressed lncRNAs were ranked according to the standardized drop in probable prediction accuracy.

An odds analysis for predictive performance was performed, adding one lncRNA at a time starting

with the top two lncRNAs; of the ranked list, five lncRNAs were the optimal mix for diagnostic accuracy.

When choosing more than five lncRNAs, there was a downward trend in predictive performance.

The top five lncRNAs (FLJ27354, RP11-275I14.4, VIM-AS1, CTB-51J22.1 and RP11-229P13.20) were

selected as optimal predictive lncRNA biomarkers of uterine corpus EC (UCEC) progression. Only

one lncRNAs (FLJ27354) was clearly associated with disease progression, the other top four lncRNA

markers (RP11-275I14.4, VIM-AS1, CTB-51J22.1 and RP11-229P13.20) were silent. Using these top five

markers led to a high discriminatory performance in distinguishing advanced stages from early stages

with 78% prediction accuracy, 96.6% sensitivity and 76.6% specificity. These lncRNAs could have

a functional role in the progression of EC initiating, and in the promotion of important cancer-related

processes [45].

4. Regulation of Cell Growth by Long Non-Coding RNAs in Endometrioid Endometrial

Adenocarcinoma

lcnRNA H19 levels were significantly higher in EC tissues than in para-tumoral samples [45].

The expression pattern of H19 in EC tissues was addressed by quantitative polymerase chain reaction

(Q-PCR), and its function characterized in the EC cell line through the inhibition of expression with

small interfering RNAs (siRNAs) [45]. Lowering H19 levels did not affect the growth of HEC-1-B EC

cells, but it did suppress their capability to migrate and invade. Furthermore, H19 downregulation

decreased the transcription factor Snail, associated with E-cadherin downregulation, destabilization

of the adherent junction, and cellular polarization, and increased matrix metalloproteinase (MMP)

expression, with no effect on vimentin levels, indicating at least a partial reversion of EMT [46]. H19

was able to enhance EC aggressiveness by modulating the EMT process [47].

The lncRNA BANCR promotes EC cell replication and tumor invasion, by regulating Matrix

MetalloProteinase-1 (MMP1) and MMP2, and via extracellular signal-regulated kinase-1 (ERK)–MAPK

signaling pathways. BANCR was highly expressed in type 1 EC tissues, promoting EC cell proliferation,

migration, and invasion by activating the ERK–MAPK signaling pathway that regulates MMP2 and

MMP1 expressions [48]. For this reason, BANCR could become a prognostic marker and important

drug target in type 1 EC. MEG3 and two signaling molecules, Notch1 and Hes1, were detected in

both EC tissues and cell lines by RT-PCR and western blot analysis, all of them showing significant

expression. Lentiviral vectors with whole MEG3 transcripts or Short (or small) hairpin RNA (shRNA)

targeting MEG3 (shMEG3) was transinfected to evaluate cell proliferation. MEG3 dysregulation was

studied in xenograft models (established by subcutaneous implantation), and tumor growth was

compared. Important downregulation of MEG3 was observed in EC samples compared to controls,

while levels of Notch1 and Hes1 were found to be significantly upregulated. Cell proliferation was

greatly inhibited by MEG3 over-expression, while the opposite was observed in MEG3 knockout

cells. Interestingly enough, MEG3 changes could be reversed by Notch1 regulators. Moreover,

over-expression of MEG3 was strongly associated with repressed in vivo growth, along with Notch

signaling inhibition. Downregulated MEG3 was an important anti-proliferative factor in EC, by

repressing the Notch signaling pathway [49].

Cisplatin-resistant Ishikawa cells (a human EC cell line) showed high autophagy activity

compared with non-cisplatin-resistant parent Ishikawa cells. After lncRNA profiling, HOTAIR was

identified as the culprit: it was downregulated both in cisplatin-resistant Ishikawa cells and parental

Ishikawa cells treated with cisplatin. RNA interference on HOTAIR reduced the proliferation of

cisplatin-resistant Ishikawa cells and increased autophagy in cisplatin-resistant Ishikawa cells with or

without cisplatin treatment. Furthermore, beclin-1, multidrug resistance (MDR), and P-glycoprotein

(P-gp) expression was mediated by lncRNA HOTAIR. It was concluded that HOTAIR can regulate the

cisplatin resistance of human EC cells, regulating autophagy by influencing Beclin-1, MDR, and P-gp

expression [30].

The tumor suppressor candidate 7 (TUSC7) is an antisense lncRNA: if downregulated, it acts

as a possible tumor suppressor in several cancers. In the study by Shang et al., the low expression

of TUSC7 was associated with high pathological stages of EC, which revealed that TUSC7 might be

involved in the initiation and progression of EC. Moreover, the expression of TUSC7 in EC samples

and EC cell lines resistant to cisplatin CDDP, and Taxol was lower than that in sensitive EC tissues and

cell lines, which indicated that the TUSC7 expression level was positively related to the response of EC

patients to chemotherapy with CDDP and Taxol [50].

Li et al. showed that the expression and function of NEAT1 in EEC was elevated in ECC samples

and cell lines, with a high expression levels of NEAT1 being associated with enhanced cell growth,

colony formation ability, and invasive and migratory ability in pGCMV-NEAT1, while limiting the

expression of NEAT1 in HEC-59 cells by siNEAT1 transfection showed the opposite effects [27].

Guo et al. demonstrated that the growth arrest-specific 5 (GAS5) functions as a significant tumor

suppressing lncRNA in EC. Inhibiting the expression of miR-103, GAS5 enhances the expression of

the PTEN gene leading to cancer cell apoptosis. GAS5 downregulation could represent an important

factor in the pathogenesis of EC [51].

Moreover, the lncRNA Fer-1-like protein 4 (FER1L4) might suppress EC cell proliferation.

A plasmid containing FER1L4 was transfected into HEC-50 cells, showing low levels of FER1L

expression. Western blot analysis was used to determine PTEN expression and Akt phosphorylation.

FER1L4 was significantly downregulated in EC tissues compared to controls. This was positively

correlated with decreased PTEN expression. Therefore, FER1L4 could promote PTEN expression

and inhibit Akt phosphorylation. A significant decrease of cell proliferation was observed in FER1L4

overexpressing cells, with cell cycle arrest at G0/G1 phase and enhanced apoptosis [52].

5. Long Intergenic Non-Protein Coding RNA, Regulator of Reprogramming and Endometrioid

Endometrial Adenocarcinoma

Ample evidence indicates that linc- Regulator of Reprogramming (ROR) has a significant role in

tumor initiation and progression. In tumorigenesis, linc-ROR might act as an oncogene [53]. In general,

linc-ROR can be easily associated with cell proliferation, differentiation, apoptosis, invasion, and

metastasis in many human cancers. However, the molecules that act as mechanisms that mediate

linc-ROR’s effects must still be clearly identified before linc-ROR can be used in tumor treatment. Many

studies have shown the importance of linc-ROR as a tumor marker. Marked upregulation of linc-ROR

can be observed in various cancers, like BC [54,55], pancreatic cancer (PC) [56,57], hepatocellular

cancer (LC) [58,59], EC [60,61], and nasopharyngeal carcinoma [62]; furthermore, linc-ROR works as

a tumor suppressor in glioma [53]. Although linc-ROR is over-expressed in various cancers, the target

genes are variable and depend on the cell type.

In the paper by Chen et al., RNA was isolated from malignant and adjacent non-affected

endometrial tissue from six patients with low-grade, type I EC. Subsequently, Illumina paired-end RNA

sequencing was performed, to determine differential transcriptome expression patterns. Linc RNAs

were specifically analyzed. LINC00958 was upregulated, and four lincRNAs including LINC01480,

LINC00645, LINC00891 and LINC00702 demonstrated exquisite specificity for malignant endometrium

compared to normal endometrium, while also distinguishing EC from OC and Cervical Cancers

(CC) [62].

Zhou et al. studied a linc-RoR which was possibly implicated in the expression regulation

of core stem cell transcription factors (TFs) as ceRNA [60]. Adenovirus vectors carrying green

fluorescent protein (GFP) were transfected into E-Twenty-six (ETs) TFs to evaluate the effect of

down- or upregulation of miR-145, linc-RoR or Dicer. Expression of linc-RoR and core stem cell

TFs was associated with cell differentiation of the ET TF, whereas miR-145 expression increased

after ET differentiation. A higher expression of miR-145 successfully led to the downregulation of

linc-RoR and core TFs. The opposite was achieved by knocking down miR-145 expression. The effects

of miR-145 could be nullified by increasing the expression of linc-RoR in ETs or mutated targeted

sequences in linc-RoR. Reduced Dicer expression were able to enhance the expression of linc-RoR

and core TFs. The linc-RoR acted as a miR-145 inhibitor on the differentiation of ETs in endometrial

carcinogenesis [60].

6. Micro IRNA in Endometrioid Endometrial Adenocarcinoma

MicroRNA-93 is derived from a paralog (miR-106b-25) of the miR-17-92 cluster and has

been implicated in tumor initiation and progression of breast, colorectal, hepatic, lung, ovarian,

and pancreatic cancer.

The article by Chen et al. demonstrated miR-93 overexpression in EC tissue samples as compared

with normal endometrium. According to Chen et al., miR-93 overexpression promotes cell migration

and invasion, by downregulation of E-cadherin while N-cadherin expression in EC cells seems

significantly enhanced [42].

miRNA-205 is probably a key regulator of EC gene expression. Higher levels of miRNA-205

expression were measured in EC with invasion limited to less than half of the myometrium and

initial EC. A better survival rate was associated with higher levels of miRNA-205 using Kaplan–Maier

analysis (p = 0.034); miRNA-205 could thus be used as a prognostic marker [63]. Levels of miR-944

were analyzed in 68 cancerous and 20 normal endometrial samples: miR-944 was highly overexpressed

in EC tissues compared to normal controls. Furthermore, increased levels of miR-944 were observed

in EC cell lines. miR-944 expression was associated with the EC stages and pathology results of the

International Federation of Gynecology and Obstetrics (FIGO) [64].

Using small-RNA sequencing and microarrays, significant differences in Small Non-Coding

RNAs (sncRNA) expression patterns between normal, hyperplastic and neoplastic endometrium could

be easily identified. The authors propose a sncRNA signature (129 microRNAs, 2 of which were

previously unknown, 10 piRNAs and 3 snoRNAs) of neoplastic transformation. This new sncRNA

signature probably reflects events leading to EC development [65,66].

Micro RNA as a Marker and Promoter of Tumor Aggression in EEC

Can miRNA evaluation accurately predict the risk of lymph node extension in EC patients?

Eighty-six matched EC cases evenly distributed between lymph node-positive and lymph

node-negative cases, were analyzed in the study by Ahsen et al. [67]. A predictive matrix was

generated using genomic miRNA expression in EC node-positive patients. An independent set of 28

other tumor samples was similarly characterized and was used in a test cohort. A predictive signature

of miRNA expression was generated and used to predict the metastatic status of the independent

test cohort. Using 18 miRNAs, 100% accuracy was obtained with the control group. The EC group

showed 90% accuracy in the node-positive cases, and 80% in node-negative cases (false discovery rate,

FDR = 6.25%) [67]. The Dicer1 endoribonuclease plays a critical role in miRNA biogenesis. Bahubeshi

et al. showed that Dicer1 dysfunction leads to the enrichment of tumor stemness and tumor aggression

both in vitro and in vivo [68]. Loss of Dicer1-induced abnormal expression of the miRNA let-7 family,

which comprises well-known tumor suppressors, thus regulating stemness in EC cells [68].

Hu showed that silencing nc886, a ncRNA, leads to apoptosis of human EC cells in vitro.

In addition, tests results showed higher level of nc886 in the late phase of human EC tissue, slightly

more than in the early phase of EC but always much higher than in normal endometrial tissue. nc886

inhibition led to increased protein levels of phosphorylated PKR (p-PKR) and caspase-3, NF-kB and

vascular endothelial growth factor (VEGF) were on the opposite decreased. The rate of apoptosis

in the nc886 inhibition group was increased and cell proliferation was much slower as compared to

controls [69].

The inhibition of the enhancer of zeste homolog 2 (EZH2), (a histone-lysine N-methyltransferase

enzyme), and the reactivation of tumor suppressor miRNAs might lead to a functional cancer therapy

regime strategy. Ihira et al. observed that EZH2-suppressed miRNA let 7b and miR-361, two possible

tumor suppressors, with consequent EC cell proliferation and invasion, and negated their stem-cell-like

properties. In EC cells, EZH2 induced and worked together with the transcription factor Yin Yang

1 (YY1) to suppress miR-361, which in turn upregulated Twist, a target of miR-361. Treating EC

cells with GSK343, a targeted EZH2 inhibitor, had the same effect of a siRNA-mediated EZH2

knockdown, upregulating miR-361 and downregulating Twist expression. Combining GSK343 with

5-AZA-2’-deoxycytidine suppressed cell proliferation with an apparent synergic effect and blocked

invasion in vitro, lowering the tumor size and weight in EC cell grafted mice. Worse patient outcomes

could be predicted: a quantitative real-time PCR (Q-RT-PCR) analysis of 24 primary EC tissues showed

that lower let-7b and miR-361 levels were dire prognostic indicators. Results were confirmed on a large

patient data set from The Cancer Genome Atlas [70]. EZH2 might drive EC progression by regulating

miR-361–Twist signaling and indicate EZH2 inhibition as a possible anti-EC therapeutic strategy [68].

miRNA-93 overexpression increased cell migration and invasion, and downregulated E-cadherin

expression while increasing N-cadherin expression [42]. Chen et al. showed, by dual-luciferase reporter

assay, that miR-93 directly binds to t 30 untranslated region of Forkhead Box A1 (FOXA1), and that its

over-expression downregulated FOXA1 expression. On the other hand, miR-93 inhibitor transfection

upregulated FOXA1 expression at protein and mRNA levels. The conclusion was that miR-93 may

aid or initiate the process of EMT in EC cells by targeting FOXA1 [42]. An intriguing hypothesis by

Gong et al. was that the miRNA-194 downregulation (miRNA-194, is an inhibitor of EMT in several

cancers) is associated to a worse prognosis in human EC. Self-renewal factor Sox3 induces EMT at

gastrulation and is also involved in EMT in several cancers. Ectopic expression of microRNA-194 in EC

stem cells induced a MET by normalizing E-cadherin expression, decreasing vimentin expression, and

interfering with cell invasion in vitro. Moreover, overexpression of microRNA-194 inhibited EC stem

cell invasion or metastasis in vivo by the injection of adenovirus microRNA-194. This demonstrates

the new mechanism by which Sox3 contributes to EC stem cell invasion and suggests that repression

of Sox3 by miRNA-194 may have a therapeutic use in the suppression of EC metastasis. The cancer

stem cell marker CD133 might be the most significant surface marker of EC stem cell [69]. The effects

of miR-29b regulate via MAPK–ERK and PI3K–Akt signaling pathways the angiogenesis in EC by

targeting Vascular endothelial growth factor A (VEGFA). In the paper by Chen et al., EC tissues were

compared with the adjacent normal tissues and showed that miR-29b expression was downregulated,

while the mRNA and protein expression of VEGFA, ERK, Akt, mTOR and B-cell lymphoma 2 (Bcl-2)

were upregulated [71]. The left-right determination factor 2 (LEFTY2), considered a suppressor of

cell proliferation, tumor growth, and regulator of stem cell properties and embryonic differentiation,

seems to be a negative regulator of cancer cell programming.

LEFTY2 seems to downregulate MKi67 expression and focal adhesion kinase (FAK) activity, while

upregulating miR-200 and E-cadherin. It is thus a significant negative regulator of endometrial cell

growth and migration, probably under-expressed in EEC [72].

7. Conclusions

Abnormal expression of several sets of genes is probably the prime mover or a significant factor

in the progression of a cancer, and both mechanisms can be at work in sequence. Genetic alterations

include both mutational and epigenetic changes, such as methylation, acetylation, and phosphorylation

of nuclear chromatin [73–75]. Cellular genes are in charge of proliferation, apoptosis, and stem cell

differentiation; all of them can undergo epigenetic modifications in cancer [76,77]. The significance of

ncRNA is starting to be put under investigation, but more questions than answers are available for

now. Is ncRNA a possible marker of cancer risk? Can it be used as a biomarker for tumor diagnosis

and as an indicator of tumor progression or of a risk of tumor metastases? Can selective activation

or inhibition of RNA pathways help cure or control the aggressiveness of the disease? Are results

valid for all cancers? Do some of the promising preliminary results obtained in other conditions

apply to EC? Various information-carrying genes have shown important biological and regulatory

functions without expressing a translation of their information, such as classically the production of

proteins. In particular, lncRNAs show specific and altered expression patterns in EC compared to

normal endometrial tissue (Table 1, Figure 1). Some well-known factors associated with EEC show

an association with ncRNA expression. Levels of sex hormones are involved in the development of

EC. Hormones affect RNAs like lncRNAs SRA, H19 and HOTAIR, and their levels could play a role

in the carcinogenesis of EC. Estradiol is an important link between expression of these lncRNAs as it

correlates positively with upregulation of H19 and HOTAIR in EC [32,78]. Preliminary in vitro studies

showing the reduced growth of EEC cells indicate that a possible new therapeutic pathway for EEC is

possible. Study of the mechanisms of expression of RNA in EEC should be pursued, and their effect

on cancer growth clarified. lncRNAs could be useful clinical biomarkers of EEC for progression to

advanced stages. Hence, lncRNAs could be optimal biomarkers, useful to identify patients at high

risk for progression to advanced stage, and the profiling of lncRNAs could actually offer improved

diagnostic accuracy.

3. Competing Endogenous RNAs, miRNA, lncRNA Profiling Regulation and Cancer

7. Conclusions

Abnormal expression of several sets of genes is probably the prime mover or a significant factor in the progression of a cancer, and both mechanisms can be at work in sequence. Genetic alterations include both mutational and epigenetic changes, such as methylation, acetylation, and phosphorylation of nuclear chromatin ^[62][63][64]. Cellular genes are in charge of proliferation, apoptosis, and stem cell differentiation; all of them can undergo epigenetic modifications in cancer ^[65][66]. The significance of ncRNA is starting to be put under investigation, but more questions than answers are available for now. Is ncRNA a possible marker of cancer risk? Can it be used as a biomarker for tumor diagnosis and as an indicator of tumor progression or of a risk of tumor metastases? Can selective activation or inhibition of RNA pathways help cure or control the aggressiveness of the disease? Are results valid for all cancers? Do some of the promising preliminary results obtained in other conditions apply to EC? Various information-carrying genes have shown important biological and regulatory functions without expressing a translation of their information, such as classically the production of proteins. In particular, lncRNAs show specific and altered expression patterns in EC compared to normal endometrial tissue (Table 1, Figure 1). Some well-known factors associated with EEC show an association with ncRNA expression. Levels of sex hormones are involved in the development of EC. Hormones affect RNAs like lncRNAs SRA, H19 and HOTAIR, and their levels could play a role in the carcinogenesis of EC. Estradiol is an important link between expression of these lncRNAs as it correlates positively with upregulation of H19 and HOTAIR in EC ^[21][67]. Preliminary in vitro studies showing the reduced growth of EEC cells indicate that a possible new therapeutic pathway for EEC is possible. Study of the mechanisms of expression of RNA in EEC should be pursued, and their effect on cancer growth clarified. lncRNAs could be useful clinical biomarkers of EEC for progression to advanced stages. Hence, lncRNAs could be optimal biomarkers, useful to identify patients at high risk for progression to advanced stage, and the profiling of lncRNAs could actually offer improved diagnostic accuracy.

Figure 1. Differentially expressed long non-coding RNA (lncRNA), long intergenic non-coding RNAs (lincRNAs) lincRNA and micro RNAs (miRNA) in Endometrial Cancer (EC).