6.1 Promotion of the migration and invasion of CCA cells

Studies from different groups have confirmed that the dysregulation of lncRNA expression is closely related to the biological behavior of CCA cells. Upregulation of these lncRNAs, including KCNQ1OT1 [48], LMCD1-AS1 [37], TP73-AS1 [22], SNHG1 [49], SOX2-OT [39], LINC01410 [50], ASAP1-IT1 [51], DANCR [52], FLVCR1-AS1 [53], NNT-AS1 [54], RMRP [55], CCAT2 [56], HOTAIR [57], LINC01296 [58], LOXL1-AS1 [59], NEAT1 [60], PVT1 [43], HEIH [61], LINC01061 [62], may promote the migration and invasion of CCA cells, . On the other hand, the downregulation of several lncRNAs plays the same role in CCA cells including LINC01714 [63], FENDRR [64], MEG3 [65], and MIR22HG [66]. Similarly, in studies of lncRNAs in iCCA, NEF [67], LFAR1 [46], SNHG3 [68], UCA1 [44], CRNDE [69] and CCAT2 [70], and can also facilitate the migration and invasion of iCCA cells (Table 1).

Epithelial-to-mesenchymal transition (EMT) is a fundamental process regulating the transformation of cells from an epithelial to a mesenchymal state. Primary epithelial tumor cells are transformed into mesenchymal cells with migration ability through the third type of EMT which governs tumor invasiveness, metastasis, and prognosis. During EMT, epithelial cadherin (E-cadherin) and claudins are downregulated while neural cadherin (N-cadherin), vimentin, and fibronectin are upregulated [71]. The E/N-cadherin switch has been identified as an essential step in manipulating cell invasion and metastasis in CCA. Zhang et al [60] found that the knockdown of NEAT1 significantly downregulated the levels of Vimentin and N-cadherin but increased E-cadherin expression in CCA cells. NEAT1 suppresses the expression of E-cadherin partly by binding EZH2, enabling hypermethylation of the E-cadherin promoter. Snail is a major transcription factor governing EMT, and snail-induced EMT has been validated to accelerate cancer metastasis through the induction of immunosuppression [72]. Silencing SPRY4-IT1 in CCA cells can increase the expression levels of E-cadherin and decrease the expression levels of Snail and vimentin [20]. The matrix metalloprotease (MMP) family is capable of activating TGF-β by inducing proteolysis to promote EMT, and MMP-9 has been confirmed to enhance the invasion capacity of cancer cells by damaging the histological barrier around the extracellular matrix [73]. Experiments have proved that the overexpression of the lncRNAs NNT-AS1 and PANDAR in CCA could enhance the expression of N-cadherin, MMP2 and MMP9, and decreased the expression of E-cadherin [54, 74].

Oxygen and nutrients supplied by the vasculature are essential for the function and survival of aberrantly proliferating cancer cells. To expand malignant lesions, incipient neoplasms tend to develop angiogenic ability. Moreover, angiogenesis can assist cancer cells in obtaining invasion and migration abilities. The E2F8 mutantin in extraembryonic trophoblastic cells is a proven key factor involved in the structural abnormalities of the placental vascular network [75]. Wang et al [76] discovered that medium derived from lncRNA SNHG6 silenced CCA cells could suppress Human Umbilical Vein Endothelial Cells (HUVECs) tube formation, and SNHG6 exerted its biological role mainly by targeting E2F8. Therefore, these suggest that lncRNAs may have an important role in the metastasis of cholangiocarcinoma cells by regulating angiogenesis.

Current studies have shown that the aggressive behaviors of CCA are regulated by lncRNAs and can be reflected in the high invasiveness and migration of CCA cells, activation of EMT, and angiogenesis. Additionally, the extracellular matrix (ECM) component of solid tumors has been implicated in metastatic progression. Altered macromolecular substrate activity in the ECM of cancer triggers pathological ECM remodeling and facilitates metastatic dissemination [77]. It will be a novel approach to explore the regulation of CCA cell migration and invasion by lncRNAs from the perspective of ECM because compared with the direct study of cell lines, it could better simulate the in vivo tumor microenvironment.

6.2 Proliferation and anti-apoptosis effects on CCA cells

Unrestricted proliferation is one of the most fundamental characteristics of cancer cells. Most of the abnormally expressed lncRNAs in cholangiocarcinoma have been proven to be closely related to cell proliferation. (Table 1) For example, lncRNA TUG1 has been identified to be upregulated in CCA. When TUG1 was depleted, the protein level of PCNA in CCA cells was downregulated, followed by the inhibition of cell proliferation [78]. Proliferating cell nuclear antigen (PCNA) is widely known as an important factor in the metabolism of nucleic acids and plays a critical role in many aspects of DNA replication [79]. In addition, lncRNA FENDRR can inhibit the proliferation of CCA cells through the suppression of survivin [64]. Survivin is an evolutionarily conserved protein that is essential for cell division, and is generally expressed only in actively proliferating cells [80].

Cancer is regarded as a scenario where little apoptosis occurs, leading to an abundance of long-living malignant cells [81]. Some of the upregulated lncRNAs mentioned above are also closely related to the inhibition of CCA cell apoptosis, while overexpression of those downregulated lncRNAs can accelerate apoptosis. Among the 14 mammalian caspases discovered so far, caspase-3 and caspase-9 play a key role in the regulation of apoptosis. Additionally, Bcl-2 family proteins are regarded as central regulators of programmed cell death [82]. LncRNA UCA1 acts as an anti-apoptotic factor in CCA, and apoptosis-associated factors (caspase-3, and -9) are both increased followed by the knockdown of UCA1. Subsequent studies also confirmed that downregulated UCA1 increased apoptosis by activating the expression of Bax and suppressing Bcl-2 expression [83].

Attenuated apoptosis and unrestricted proliferation are fundamental characteristics of cancer cells. Numerous lncRNAs have been proven to regulate the expression of classic proliferation- and apoptosis-related proteins, thus participating in the biological characteristics of CCA cells.

6.3 Enhancement of CCA cells stemness

Genomic instability and the unique microenvironment of tumors have contributed to the discovery of small subpopulations of cancer cells with a high capacity for self-renewal and differentiation, and the ability to initiate tumorigenesis, which are referred to as cancer stem cells (CSCs) [84]. Emerging evidence indicates that lncRNAs are key positive regulators of the CSC subpopulation and contribute to self-renewal, drug resistance, and EMT in diverse cancers [85]. Wang et al [86] found that lncTCF7 activates TCF7 expression locally (in cis) by recruiting the SWI/SNF complex, an evolutionally conserved multi-subunit complex that can regulate gene expression, and TCF7 expression induces the Wnt signaling pathway to initiate self-renewal of the liver CSCs. Overexpression of lnc-PKD2-2-3 in CCA cells significantly increased CSC marker expression (CD44, CD133, and OCT4) and improved sphere formation efficiency, indicating that lnc-PKD2-2-3 may promote CCA stemness and potentially serve as a marker for CSCs in CCA [87]. In addition, lncRNA RMRP is highly expressed in CCA, and cancer stem cell-related markers Bmi1 and CD24 were found to be significantly decreased after RMRP knockdown, but surprisingly, the expression of CD44 remained unchanged [55]. There is a need for additional research on the role of RMRP as a CCA cell stemness promoter. However, this sole study in CCA did not determine the role of lncRNAs in cholangiocyte CSC self-renewal.

Inhibitor of differentiation 3 (ID3), which is highly expressed in human iCCA tissues compared with matched normal tissues, was found essential for stemness maintenance in iCCA and could serve as a promising biomarker in predicting iCCA patient responses to adjuvant chemotherapeutics [88]. Interestingly, Sanzo et al [89] demonstrated that the expression level of ID3 was regulated by lncRNA H19 in a leukemia cell line. Cancer stem cells (CSCs) tend to be responsible for chemoresistance. As a result, probing the role of lncRNAs in CSCs will contribute to possible therapeutic strategies.

7.1 Direct interaction with functional proteins

In breast cancer cells, lncRNA EPIC1 was identified to directly interact with the 148-220 aa region of the MYC protein through its 129-283 nt sequence, and then specifically regulate the occupancy of MYC on target genes to promote breast cancer [47]. Li et al [90] confirmed that EPIC1 was highly expressed in CCA tissues and plays an oncogenic role during CCA development by directly targeting the MYC protein.

Phosphorylation of the threonine 58 (Thr58) site on MYC protein resulted in its degradation through the ubiquitin-proteasome pathway. In human breast cancer cells, lncRNA PVCT1 can increase the level of MYC protein by blocking the phosphorylation of the Thr58 site on MYC [91]. In the human genome, MYC is located only 53 kb upstream of PVT1, and both of them have been reported to play important roles in CCA [43, 92]. Therefore, whether there is a direct interaction between MYC and PVT1 in CCA remains to be confirmed in subsequent studies.

LINC01714, which is downregulated in CCA cells, was found to interact with the forkhead (FH) DNA-binding domain of the FOXO3 protein through its 1- to 195-nt fragment. Moreover, the overexpression of LINC01714 significantly decreased the phosphorylation level of FOXO3-Ser318, and in comparison to CCA patients with low LINC01714-FOXO3 levels, those with high LINC01714-FOXO3 levels had better overall survival [63].

Both MYC and FOXO3 are widely-studied functional proteins in cancer. However, it is still unclear which domains of MYC can directly interact with those of EPIC1 and whether EPIC1 is involved in MYC occupancy on target genes in CCA.

7.2 ceRNA regulatory network

In 2011, Salmena et al [93] first proposed the well-known competing endogenous RNA (ceRNA) hypothesis, which states that RNAs can cross-talk with each other and manipulate biological functions independently of protein translation. Studies have demonstrated that lncRNAs can function as ceRNAs to compete with mRNAs for binding to miRNAs like sponges and further regulate gene expression [94]. Furthermore, this mechanism has been widely demonstrated to play an important role in the carcinogenesis and development of numerous cancers, including cholangiocarcinoma (Figure 2).

miR-203, a well-described miRNA, has been shown to play a critical role in tumor development [95]. Gu et al [96] found that lncRNA NNT-AS1 could sponge miR-203 and downregulate the expression of miR-203 in CCA cells. The proliferation and EMT process of CCA cells were notably accelerated by NNT-AS1 overexpression, but after the miR-203 mimic was transfected into CCA cells, both proliferation and EMT were inhibited. This study also corroborated that NNT-AS1 overexpression promoted the activation of the PI3K/AKT and ERK1/2 signaling pathways by downregulating miR-203. However, whether the interaction between miR-203 and ZEB1/IGF1R, two potential candidate targets of miR-203, is involved in the cancer-promoting effect of NNT-AS1 remains to be further confirmed [96]. In addition, lncRNA SNHG1, as a ceRNA for miR-140, can promote TLR4 expression and activate the NF-κB signaling pathway, thus regulating the growth and tumorigenesis of CCA [97]. LncRNA UCA1 was confirmed to directly bind to miR-122 and negatively regulate the expression of miR-122 in iCCA cells. In addition, miR-122 mimics inhibited the promoting effects of UCA1 on iCCA cell proliferation and invasion [44]. In another study, UCA1 was considered to be an activator of the AKT/GSK3β/CCND1 signaling pathway in CCA [83]. Therefore, whether miR-122 is involved in the activation of this pathway remains to be explored.

Currently, cancer is considered a special metabolic disease, and abnormal glutamine metabolism has proven to be a critical hallmark of cancer by participating in carcinogenesis [98]. Sirt3, a member of the Sirtuin family, was identified as a positive regulator of glutamate dehydrogenase (GDH). The upregulated lncRNA TUG1 significantly increased Sirt3 and GDH expression by sponging miR-145 in iCCA cells, resulting in the enhancement of glutamine metabolism [99]. In cholangiocarcinoma, in addition to miR-203, lncRNA NNT-AS1 could also sponge miR-142-5P and miR-485, and then target HMGA2 and BCL9, respectively, to play a tumor-promoting role; suggesting that there may be a complex ceRNA regulatory network in the development of CCA [54, 100].

Researchers from Harbin Medical University and Miami University jointly constructed a comprehensive database LnCeVar (http://www.bio-bigdata.net/LnCeVar/) aimed at providing a lncRNA-associated ceRNA regulatory network for investigating the functions and mechanisms of personalized genomic variations. This database is based on the published literature and high-throughput datasets including TCGA, COSMIC, and 1000 Genomes Project, and demonstrates the potential for the generation of biomarkers that may be conducive to a better understanding of individual disease pathology [101]. Zhang et al [102] obtained RNA-sequencing datasets of lncRNAs, miRNAs, and mRNAs in CCA and relevant clinical information from The Cancer Genome Atlas (TCGA) database. After that, differentially expressed RNAs were identified and selected to construct a ceRNA network that contained 28 molecules and 47 interactions. In this ceRNA network, seven lncRNAs were identified and expected to become new research targets. However, the authors only utilized a small sample size (36 tumor samples and 9 non-tumor samples) which could generate false-positive findings. In another related study, Xu et al [103] constructed an iCCA-related ceRNA regulatory network that included 340 lncRNA-miRNA-mRNA regulatory relationships based on the NCBI GEO database. Next, a core regulatory pathway RP11-328K4.1-hsa-miR-27a-3p-PROS1 was identified and subsequently validated to be a tumor suppressor pathway in iCCA.

With the application of specific biochemical and computational approaches, a large number of circular RNAs (circRNAs) have been identified in eukaryotes. CircRNAs are covalently closed noncoding RNA molecules generated by a process named back-splicing, and are implicated in various diseases, including cancer, by acting as microRNA or protein sponges [104]. However, few studies have utilized circRNA-containing whole-transcriptome sequencing technology, which allows accurate examination of the transcriptomic landscape of CCA. Chu et al [105] explored the molecular mechanisms of CCA by performing circRNA-containing whole-transcriptome sequencing and validation with the TCGA database. In this study, miR-144-3p was identified to be located in the center of the ceRNA network and might play an important role in the pathogenesis of CCA. Future mechanistic studies should determine the role of miR-144-3p in CCA and which noncoding RNAs function as sponges. According to the latest studies, we summarized some of the ceRNA regulatory pathways in Table 2.

7.3 Epigenetic modification of gene expression

Polycomb repressive complex 2 (PRC2) is an extremely conserved protein complex capable of silencing gene expression by methylating lysine 27 on histone H3. Histone methyltransferase enhancer of zeste homolog 2 (EZH2) is a catalytic subunit of PRC2 [106]. LncRNA SNHG1 can act as an oncogenic molecule of CCA by binding to EZH2, followed by epigenetically suppressing the transcription of CDKN1A in the nucleus [49]. CDKN1A, as a target of the tumor suppressor p53, encodes a potent cyclin-dependent kinase inhibitor and induces the apoptosis of cancer cells [107]. The FBP1 gene encodes a rate-limiting gluconeogenic enzyme, the loss of which can accelerate cancer progression by enhancing aerobic glycolysis, and the antineoplastic role of FBP1 has been verified in multiple cancers including CCA [108]. Additionally, upregulated DANCR can promote CCA progression through the epigenetically transcriptional inactivation of FBP1 [52]. In addition, NEAT1 and PVT1 are also able to recruit EZH2 and increase H3K27me3 at the promoters of E-cadherin and ANGPTL4, respectively, in CCA [43, 60] (Figure 3A).

A study by Tsai et al [109] showed that lincRNA HOTAIR could serve as a scaffold for at least two distinct histone modification complexes. In cholangiocarcinoma, Xu et al [20] discovered that lncRNA SPRY4-IT1, as a ceRNA for miR-101-3p, could bind to miR-101-3p and release its inhibition of EZH2 mRNA, leading to increased EZH2 protein levels. Subsequently, SPRY4-IT1 recruited EZH2 and DNMT1 to the KLF2 promoter region, and recruited EZH2 and LSD1 to the LATS2 promoter region, and repressed their transcription. Subsequent experiments determined that the oncogenic function of SPRY4-IT1 was partly dependent on inhibiting KLF2 and LATS2 expression [20]. Therefore, a lncRNA may play a role in promoting cancer by mediating multiple ways of epigenetic modifications.

The SETDB1 protein belongs to the SET-domain protein methyltransferase family and is involved in the trimethylation of H3K9 and silencing gene expression [110]. Emerging evidence shows that the aberrant overexpression of SETDB1 is closely related to the development of cancer, such as melanoma, breast cancer, and mesothelioma [111-113]. In a study by Qin et al [64], lncRNA FENDRR expression levels were significantly decreased in CCA, and FENDRR was capable of enriching SETDB1 in CCA cells. The overexpression of FENDRR inhibited the expression of survivin, a well-studied oncogene, via SETDB1-mediated H3K9 methylation to suppress the proliferation, migration, and invasion of CCA cells (Figure 3B) [64].

Abnormally expressed lncRNAs in CCA have been proven to mediate multiple epigenetic modifications of genes by serving as scaffolds for histone modification complexes. With the silencing of target gene expression, CCA development is promoted. DNA methylation mediated by lncRNAs has been discovered in hepatocellular carcinoma and colorectal cancer [114, 115]. There are still other patterns of epigenetic modifications mediated by lncRNAs in CCA urging further exploration.

7.4 Synergism and antagonism with host genes

Previous studies have demonstrated that the function of lncRNAs is partly dependent on their genomic location, and lncRNAs have been proposed to play essential roles in modulating the expression of nearby loci [116]. LncRNA AFAP1-AS1 is derived from the antisense DNA strand in the AFAP1 gene locus [117]. A recent study showed that AFAP1-AS1 was significantly upregulated in CCA and could promote the growth and metastasis of CCA cells. However, the knockdown of AFAP1-AS1 increased AFAP1 protein and mRNA levels [118]. The AFAP1 protein monitors actin filament integrity and functions as an adaptor protein linking members of the Src family and other signaling proteins involved in actin filaments [119]. Therefore, we deduced that the metastatic effects of AFAP1-AS1 on CCA cells may be mediated by altered AFAP1 protein levels.

The lncRNA CPS1-IT1 and its host gene CPS1 were observed to be highly co-expressed in iCCA tissue and associated with a poor iCCA phenotype. The high expression of CPS1 was observed not only at the mRNA level, but also at the protein level. Furthermore, CPS1 and CPS1‑IT1 knockdown inhibited iCCA cell proliferation and accelerated cell apoptosis [120]. CPS1 protein is traditionally regarded as the first key enzyme in the urea cycle, which occurs only in the liver and can convert toxic ammonia into less toxic urea [121]. Due to the accumulation of ammonia, the translation of ODC mRNA, the polyamine biosynthetic rate-limiting enzyme, decreased significantly, thus inhibiting polyamine biosynthesis and cell proliferation [122]. Cancer progression is frequently accompanied by rearrangements of metabolic pathways. Perhaps to meet the abnormal metabolic demands of CCA cells, the urea cycle is likely to be accelerated in CCA.

The upregulation of lncRNA SOX2-OT can promote the progression of CCA by activating the PI3K/AKT signaling pathway. In addition, the positive regulatory effects of SOX2-OT on SOX2 have been verified in CCA cells [39]. Since the SOX2 gene is embedded in the intronic region of SOX2-OT, SOX2-OT can regulate the expression of SOX2 to affect the biological behaviors and stemness of various cancers, such as breast cancer, osteosarcoma, and esophageal squamous cell carcinoma [123-126]. In addition, previous studies have shown that the expression of SOX2 was associated with aggressive behavior and poor overall survival in iCCA [127]. However, the specific mechanism by which SOX2-OT activates the transcription of SOX2 remains unclear. Synergism and antagonism between lncRNAs and their host genes are still just macro conceptions, and how lncRNAs specifically regulate the expression of host genes and how they interact with each other remains unknown.

7.5 Cancer-related signaling pathways regulated by lncRNAs

7.5.6 NF-κB signaling pathway

Nuclear factor-κB (NF-κB) is an important and evolutionarily conserved transcription factor involved in coordinating immune and inflammatory responses [145]. In recent years, numerous studies [146] have confirmed that the NF-κB pathway is constitutively activated in various cancers, which leads to the occurrence and progression of cancer via the upregulation of multiple antiapoptotic and oncogenic genes. The Toll-like receptor (TLR) superfamily is one of the most common NF-κB-activating signals [146]. Overexpression of lncRNA SNHG1 in CCA accelerated the expression of TLR4 and activated the NF-κB pathway, thus regulating the growth and tumorigenesis of CCA [97]. Lu et al [147] confirmed that the downregulation of lncRNA MEG3 in CCA cells induced the overexpression of miR-361-5p, resulting in the inhibition of TRAF3 expression. The miR-361-5p inhibitor significantly repressed the expression of the p-p65 protein, a key member of the NF-κB family, and this effect was eliminated by TRAF3 knockout. Under normal circumstances, NF-κB-inducing kinase (NIK) is continuously degraded by a TRAF3-dependent E3 ubiquitin ligase. As a result of TRAF3 degradation, NIK becomes stabilized, leading to the activation of the noncanonical NF-κB pathway [148]. Interestingly, the downregulated lncRNA-NEF induced high expression of RUNX1 in iCCA [67]. However, RUNX1, a member of the Runt-related transcription factor 1 family, has been proven to serve as an attenuator of the NF-κB signaling pathway in gastric cancer and myeloid tumors [149, 150], indicating that the NF-κB signaling pathway may play a dual role in cancer, including CCA.

The activation of diverse cancer-related signaling pathways plays a decisive role in the progression of CCA. However, the involvement of lncRNAs in the Hedgehog pathway has not been well described in CCA. Interestingly, in other kinds of cancers, activation of the Hedgehog pathway mediated by lncRNAs leads to modulation of cancer stem cell properties and glucose metabolism reprogramming [151, 152]. In Figure 4, we illustrated these pathways regulated by lncRNAs in CCA.

8.1 Correlation between lncRNAs and clinicopathological features of CCA patients

Several lncRNAs were demonstrated to be statistically correlated with the clinicopathological features of CCA patients, such as TNM stage, tumor size, postoperative recurrence, lymph node invasion, and tumor differentiation. For example, patients with overexpression of TP73-AS1 had larger tumor size (P = 0.008) and more advanced TNM stage (P = 0.026) compared with the low TP73-AS1 expression group patients [22]. It is a common view that tumor size >5 cm is associated with microscopic vascular invasion and higher tumor grade, which implies a worse prognosis [153]. It is worth noting that according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual, the periductal invasion was removed from the T4 category due to a lack of recent data on the prognostic effect, which indicates that the TNM stage of CCA patients’ needs to be carefully classified in future studies [154]. Interestingly, lnc-PKD2-2-3 levels were significantly associated with abnormal CEA levels (P = 0.024) and poor differentiation (P = 0.002), and the upregulation of lncRNA CPS1-IT1 was associated with higher levels of CA19-9 (P = 0.044) [87, 120]. To provide a more comprehensive understanding, we summarized the dysregulated lncRNAs and their correlation with the clinicopathological features of CCA in Table 3. None of the lncRNAs were significantly associated with patient age or gender.

8.2 Diagnostic and prognostic value of lncRNAs

Compared with mRNAs and microRNAs, lncRNAs are expressed in a more cell-type- and tissue-specific manner, and therefore, lncRNAs may have more advantages as biomarkers for the diagnosis and prognosis of CCA [1]. As mentioned above, many studies have demonstrated that upregulated lncRNAs are related to aggressive tumor phenotypes and unfavorable prognosis, suggesting that lncRNAs could function as potential molecular biomarkers of the prognosis of CCA patients [155]. For instance, higher expression levels of SPRY4-IT1 indicated worse progression-free survival (PFS) and overall survival (OS) after radical surgery in CCA patients [20]. Cox regression analyses confirmed that SPRY4-IT1 could be regarded as an independent predictor of adverse PFS (P = 0.018) and OS (P = 0.027) in CCA patients [20]. Other lncRNAs associated with PFS and OS of CCA patients are summarized in Table 3.

In the iCCA-related ceRNA regulatory network constructed by Xu et al [103], ROC analysis based on the NCBI GEO database and the TCGA database revealed that lncRNA RP11-328K4.1, miRNA hsa-miR-27a-3p, and mRNA PROS1 could distinguish between iCCA tumor tissues and matched adjacent nontumor tissues, and particularly, hsa-miR-27a-3p might have significant diagnostic value in identifying iCCA of different clinical stages. Currently, serum levels of CA19-9 is the major tumor marker for iCCA diagnosis, but the sensitivity and specificity of CA19-9 are only 62% and 63%, respectively [13]. Ma et al [120] found that with increasing expression of lncRNA CPS1-IT1, the CA19-9 positive rate was greater (P = 0.044), indicating that CPS1‑IT1 may serve as a potential biomarker for the diagnosis of iCCA. Shi et al [156] examined the expression of the 12 candidate lncRNAs in perihilar cholangiocarcinoma patients and healthy controls, and found that the combination of PCAT1, MALAT1, and CPS1-IT1 presented a high sensitivity (85.5%) and specificity (93.2%) with an area under the ROC curve value (AUC) of 0.893. Therefore, plasma levels of these three lncRNAs might function as a signature for predicting perihilar cholangiocarcinoma [156].

8.3 Adjuvant effect of lncRNAs on chemotherapy

The combination of gemcitabine and cisplatin is the standard cytotoxic treatment for advanced/metastatic cholangiocarcinoma, but unfortunately, it confers a median survival of only approximately 1 year [8]. Shen et al. [63] found that LINC01714 knockdown dramatically increased the half-maximal inhibitory concentrations (IC50s) of gemcitabine in gemcitabine-treated CCA cells, while the overexpression of LINC01714 led to the opposite result. Subsequent experiments revealed that LINC01714 enhanced the cytotoxic effect of gemcitabine on CCA cells by modulating phosphorylated FOXO3-Ser318. This observation suggests that LINC01714 may act as a candidate for combinatorial chemotherapy with gemcitabine for CCA patients (Figure 5A).

Mutations in the chromatin modulator BRCA-1 associated protein-1 (BAP1) have been identified as the most frequent genetic alterations in CCA, occurring in 22%-24% of cases [157]. Parasramka et al [158] found that BAP1 expression could modulate CCA cell sensitivity to gemcitabine, or more specifically, sensitivity to gemcitabine was increased in low BAP1-expressing CCA cells than in high BAP1-expressing CCA cells. Moreover, lncRNA NEAT1 was identified to be enriched after BAP1 knockdown, and researchers further confirmed that NEAT1 could serve as a functional downstream target of BAP1 involved in drug responses [158] (Figure 5B). Previous preclinical data indicated that DZNep, an EZH2 inhibitor, could induce apoptosis and G1 phase cell cycle arrest in CCA cells, and compared with gemcitabine alone, the combination of DZNep and gemcitabine enhanced the induced apoptosis and G1 arrest [159]. However, in the study by Parasramka et al [158], the sensitivity to the combination of EZH2 inhibition and gemcitabine was reduced in low-BAP1 CCA cells compared with high-BAP1 CCA cells. Since the recruitment effect of NEAT1 on EZH2 has been confirmed in CCA, low BAP1-expressing cells may have a higher expression of NEAT-1 and EZH2, resulting in lower sensitivity to EZH2 inhibition [60]. Therefore, the expression levels of BAP1, NEAT1, and EZH2 should be evaluated in future studies to institute an accurate clinical treatment plan.

5-Fluorouracil (5-FU) is a first-line chemotherapeutic drug for CCA but its efficacy has been curbed with the development of drug resistance [160]. However, emerging evidence suggests that the proportion of cells with high expression of cancer stem cell (CSC) markers among the surviving cells is significantly increased in response to 5-Fu, resulting in drug resistance and disease relapse. This phenomenon has been discovered in various cancers, including esophageal adenocarcinoma [161], hepatocellular carcinoma [162], gastric cancer [163], and non-small cell lung cancer [164]. Lnc-PKD2-2-3 is one of the upregulated lncRNAs identified in CCA and is associated with an aggressive phenotype and poor prognosis. Qiu et al [87] proposed lnc-PKD2-2-3 as a novel CSC marker in CCA. With the increase in lnc-PKD2-2-3 levels in CCA cells, the expression of the apoptotic marker caspase-3, as well as the apoptosis rate of cells, were decreased after 5-FU treatment. Consequently, it may be feasible to utilize lnc-PKD2-2-3 as a target to eliminate 5-FU resistance in CCA patients (Figure 5C).