Involvement of long non-coding RNAs in the progression of esophageal cancer

2.1 Promoting proliferation

The growth of normal cells depends on the regulation of growth factors, but EC cells can maintain their ability to continue to proliferate with little or no growth factors, Chen et al. [26] found that the expression of lncRNA SBF2-AS1 was significantly up-regulated in ESCC cells, and the proliferation ability of ESCC was significantly reduced after silencing lncRNA SBF2-AS1. Therefore, lncRNA SBF2-AS1 is considered as a new biomarker in ESCC and potential therapeutic target. Liu et al. [27] found that lncRNA DUXAP8 can promote the proliferation of EC cells and may be an important regulator of EC. In order to determine the biological role of lncRNA DLX6-AS1 in ESCC cells, Tian et al. [28] carried out in vitro functional loss experiments. The transfection of lncRNA DLX6-AS1 targeting oligonucleotides (si-dlx6-as1) was found to hinder the expression of lncRNA DLX6-AS1 in EC9706 and KYSE-520 cells Compared with negative control group, cell counting kit-8 (CCK-8) assay and colony formation analysis showed that the silencing of lncRNA DLX6-AS1 could significantly inhibit the proliferation of ESCC cells and reduce the number of colony formation, respectively. These results suggested that lncRNA DLX6-AS1 could accelerate the proliferation of ESCC cells in vitro, suggesting that lncRNA DLX6-AS played an important role in ESCC. Wu et al. [18] demonstrated that lncRNA CASC9 promoted the proliferation of EC cells by recruiting enhancer of zeste homolog 2 (EZH2) and subsequently changes H3K27me3 level to negatively regulate programmed cell death 4 (PDCD4) expression for the first time. As carcinogenic genes, lncRNA SBF2-AS1, lncRNA DUXAP8, lncRNA DLX6-AS1 and lncRNA CASC9 may be biomarker with diagnostic or prognostic value in ESCC.

2.2 Inhibiting proliferation

LncRNA LET, also known as NPTN intron transcript 1, is 2606 nucleotides in length and located on chromosome 15q24.1. As the latest identified lncRNA, lncRNA LET is under-expressed in several cancers and used as a tumor inhibitor [29, 30]. Chen et al. [31] investigated the expression, clinical significance and biological role of microRNAs-548k and lncRNA LET in ESCC. Bioinformatics analyses and cell experiments showed that lncRNA LET could inhibit the proliferation of ESCC cells, was a direct target of microRNA-548k, and mediated the carcinogenic effect of microRNA-548k in ESCC. These data indicated that the regulation axis of microRNA-LET could be used as a potential biomarker and therapeutic target in ESCC.

2.3 Inhibiting apoptosis

Apoptosis is a process of cell death caused by endogenous and exogenous factors triggering the intracellular death program. The infinite growth of EC cells is related to the inhibition of apoptosis, so the apoptosis disorder is closely related to the occurrence of EC. Fan et al. [32] confirmed that lncRNA SNHG6 significantly inhibited ESCC apoptosis. The overexpression of lncRNA PLncRNA-1 was related to the stage of advanced tumors and lymph node metastasis, and it could be a potential prognostic marker and therapeutic target of ESCC. Wang et al. [33] confirmed that lncRNA PLncRNA-1 could inhibit the apoptosis of ESCC cells. In a study by Tian et al. [28], the authors examined the expression and biological role of lncRNA DLX6-AS1 in EC and found that the down-regulation of DLX6-AS1 could accelerate the apoptosis of ESCC cells and affect the expression of apoptosis-related protein B-cell lymphoma-2 (Bcl-2). Moreover, bioinformatics predictions showed that DLX6-AS1 had a binding site of microRNA-99a/100, while microRNA-99a/100 had a mammalian target of rapamycin (mTOR) targeting site. Another study confirmed that microRNA-99a/100 promoted apoptosis by targeting mTOR in human ESCC. [34]. Yoon et al. [35] reported that lncRNA LUCAT1 promoted tumorigenesis by controlling ubiquitination and stability of DNA methyltransferase 1 in ESCC. In conclusion, these data suggested that lncRNA DLX6-AS1 and lncRNA lucat1 acted as an oncogene by inhibiting the apoptosis of EC cells.

2.4 Inducing apoptosis

Contrary to lncRNA and lncRNA LUCAT1, some lncRNAs can induce apoptosis in EC. Huang et al. [36] detected the expression level of lncRNA MEG3 in 28 cases of ESCC and adjacent tissues, and found that the expression of maternally expressed gene 3 (MEG3) in cancer tissues was significantly reduced. Further studies showed that MEG3 could induce the apoptosis of cancer cells. Lv et al. [37] reported that the expression of lncRNA MEG3 was down-regulated in ESCC, which could activate p53 and induce the apoptosis of cancer cells. It can be seen that lncRNA MEG3 is involved in regulating the apoptosis of EC cells.

2.5 Affecting cell cycle

Abnormal regulation of tumor cell proliferation is the result of disordered control of cell cycle correction points. This disorder is secondary to abnormalities in genes that regulate cell cycle correction points or mutations or abnormal expression of oncogenes or tumor suppressor genes that encode proteins in the transmembrane signal transduction pathway that control cell proliferation. Lu et al. [38] reported that the knockout of lncRNA BC032469 in TE13 and ECA109 cells could induce cell cycle arrest at the G0/G1 phase. Cell cycle analysis showed that inhibiting the expression of lncRNA CASC9 resulted in cell cycle arrest in G1 phase of cancer cells KYSE450 and KYSE150 and decrease in the proportion of S phase cells [18]. In view of the fact that lncRNA MALAT1 significantly promotes ESCC cell growth in vitro and in vivo, Hu et al. [39] confirmed that inhibiting lncRNA MALAT1 might activate ATM-CHK2 pathway in EC cells and ultimately led to G2/M stagnation in EC. There are growing evidence that HOXA transcripts at the distal tip (HOTTIP) of the 5'end are dysfunctional in various cancers. Chen et al. [26] found that lncRNA SBF2-AS1 could be related to the cell cycle of EC and could affect the G2 phase transition of ECA109 cells through reducing the expression of cyclin-dependent kinase inhibitor 1A (CDKN1A). It can be seen from the above research that lncRNAs mediate the abnormal cycle of EC cells.

3.1 Promoting invasion and metastasis

Tumor cell invasion and metastasis is a complex process, which is affected by many factors, such as microenvironment, host cells, genes, signal molecules and so on. Liu et al. [40] found that lncRNA AK001797 could regulate ESCC cell growth and cell cycle by activating murine double minute 2 (MDM2)/p53 signal, significantly increased the proportion of S-phase ESCC cells and reduced the proportion of G2/M phase ESCC cells. Pan et al. [41] studied the expression and function of lncRNA CASC9 in ESCC. They found that the expression of lncRNA CASC9 was significantly up-regulated in ESCC tissues. In addition, the knockout of lncRNA CASC9 significantly inhibited the migration and invasion of ESCC cells, suggesting that lncRNA CASC9 might be a new marker of poor prognosis of EC and a potential therapeutic target for intervention of EC. Tian et al. [28] conducted the experiments to explore the effect of lncRNA DLX6-AS1 on the invasion of ESCC cells. Transwell invasion experiment showed that the number of invasive cells in the lncRNA DLX6-AS1 knockout group was lower than that in the control group in ESCC cell lines (EC9706 and KYSE-520). Western blot analysis showed that the levels of mTOR, Bcl-2 and matrix metalloproteinase-2 (MMP-2) in ESCC cell lines were lower than those in the control group. Therefore, these data suggested that lncRNA DLX6-AS1 could promote the invasion of ESCC cells. In order to study the biological function of lncRNA SNHG6 in ESCC, Zhang et al. [19] introduced si-SNHG6 into EC109 and EC1 cells to inhibit the expression of lncRNA SNHG6. The results showed that the expression of small nuclear RNA host gene 6 (SNHG6) significantly decreased after transfection with si-SNHG6-1 and si-SNHG6-2. The silencing efficiency in EC109 was 75.4% and 77.3%, respectively, and in EC1 was 80.8% and 79.4%, respectively. The transwell assay result showed that lncRNA SNHG6 could enhance the migration ability of EC109 and EC1 cells. Compared with the si-NC group, the numbers of migrating cells in the si-SNHG6-1 and si-SNHG6-2 groups were significantly decreased. At the same time, compared with the si-NC group, the invasive ability of the si-SNHG6-1 and si-SNHG6-2 groups was significantly inhibited. These results suggested that lncRNA SNHG6 might play a carcinogenic role in ESCC. Lu et al. [38] used real-time quantitative reverse transcription-polymerase chain reaction to detect the specific differential expression of lncRNA BC032469 in ESCC and evaluated the role of lncRNA BC032469 in the occurrence and development of EC by silencing and overexpressing lncRNA in vitro and in vivo. The results showed that the expression level of lncRNA BC032469 in ESCC was higher than that in corresponding non-cancerous tissues, while knockout of low lncRNA BC032469 inhibited the migration and invasion of EC cells. Western blotting analysis showed that lncRNA BC032469 could regulate the expression of human telomerase reverse transcriptase (hTERT), which is very important for cell invasion and metastasis. In addition, the restored expression of hTERT protein could weaken the inhibition of lncRNA BC032469 on ESCC cells. In conclusion, these results showed that lncRNA-BC032469 was a carcinogenic lncRNA that promoted cancer progression. Yoon et al. [35] transfected KYSE-30 cells and HCE-4 cells with si-LUCAT1, and then determined wound healing and invasion. It was noteworthy that si-LUCAT1 significantly inhibited wound closure compared with cells transfected with si-NC. The invasion of KYSE-30 cells and HCE-4 cells was also significantly reduced by transfection with lncRNA LUCAT1 small interfering RNA (si-LUCAT1). These effects of LUCAT1 siRNA were significantly blocked by pcDNA-LUCAT1 transfection. Meanwhile, transfection of si-LUCAT1 reduced the expression of N-cadherin, snail and Zinc finger E-box binding homeobox-1 (ZEB1), while the expression of E-cadherin increased. Overexpression of lncRNA LUCAT1 could reverse these changes suggesting that lncRNA LUCAT1 was involved in the invasion and migration of EC cells. Further studies have shown that this was related to the ubiquitination of DNA methyltransferase 1 (DNMT1) regulated by lncRNA LUCAT1.

LncRNA HOTAIR is a 2158 NT long lncRNA in the human genome. It is located between HoxC11 and HoxC12 genes on chromosome 12 and regulates the Hox gene family [42]. It has been reported that lncRNA HOTAIR can also inhibit the expression of WNT inhibitory factor 1 (WIF-1) in ESCC by binding with polycomb repressive complex 2 (PRC2) complex, and then activate histone and Wnt/β-catenin pathways in H3K27 promoter region [43]. Chen et al. [15] also reported that the expression of lncRNA HOTAIR was significantly higher in EC than in adjacent non-cancerous tissues band multivariate analysis showed that the expression of lncRNA HOTAIR was an independent prognostic factor for lymph node metastasis. It can be seen from the above that lncRNA AK001797, lncRNA CASC9, lncRNA DLX6-AS1 and other lncRNAs promote the invasion and metastasis of EC cells through different mechanisms.

3.2 Inhibiting invasion and metastasis

Metastasis and spread are one of the reasons why EC is difficult to cure, and certain lncRNAs can effectively inhibit the invasion and metastasis of EC cells. Zhang et al. [44] showed that antisense lncRNA EZR-AS1 was positively correlated with ezrin (EZR) expression in ESCC tissues and cell lines. LncRNA EZR-AS1 promotes cell migration by up-regulating EZR expression. In mechanism, antisense lncRNA EZR-AS1 forms a complex with RNA polymerase II to activate EZR transcription. In addition, lncRNA EZR-AS1 can also recruit SET and MYND domain-containing protein 3 (SMYD3) to binding sites in GC-rich regions downstream of EZR promoter to promote their binding. The interaction between lncRNA EZR-AS1 and SMYD3 can further enhance the transcription and expression of EZR, suggesting that lncRNA EZR-AS1, as a member of RNA polymerase complex, plays an important role in inhibiting the invasion of EC cells by inhibiting SMYD3-dependent H3K4 methylation. These results showed that lncRNAs inhibited EC cells invasion and metastasis through affecting related gene expression.

3.3 Epithelial-mesenchymal transition (EMT)

EMT plays an important role in the invasion of various types of cancer by transforming adherent and polarized epithelial cells into invasive and active mesenchymal cells [45]. Transcription factors, such as Twist and Snail in EMT, can increase the expression level of interstitial markers and decrease the expression of epithelial markers. The rupture of tight junctions can lead to the loss of epithelial markers and the acquisition of mesenchymal markers [46].The expression level of lncRNA FAL1 in ESCC cell lines was found to increase abnormally. Knockout of lncRNA FAL1 inhibited cell invasion and EMT by affecting related genes, while overexpression of lncRNA FAL1 had the opposite effect, suggesting that lncRNA FAL1 could promote the invasion of EC cells [47]. Huang et al. [48] found that lncRNA H19 might be involved in the regulation of EMT marker expression in EC cell lines. Lin et al. [49] studied the role of lncRNA HOTTIP in ESCC and observed that lncRNA HOTTIP was up-regulated in ESCC and promoted cell metastasis in vivo and in vitro. Interestingly, as a molecular sponge, lncRNA HOTTIP has the binding site of microRNA-30b, which can regulate the level of microRNA-30b in nucleus and cytoplasm, thus mediating the inhibition of HOXA13. In addition, lncRNA HOTTIP upregulates Snail 1 through competitively binding to microRNA-30b, and then promotes EMT and invasion. Therefore, one of the pathways of lncRNAs mediated the development of EC is to affect the EMT process.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

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CONSENT FOR PUBLICATION

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AVAILABILITY OF DATA AND MATERIALS

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COMPETING INTERESTS

The authors declare that they have no competing interests.

FUNDING

This study was supported by the Natural Science Foundation of Henan Province of China (202300410460); Henan Province Medical Science and Technology Research Project Joint Construction Project (Grant No. LHGJ20190003, LHGJ20190055); the National Natural Science Foundation of China (Grant No. 31670895).

AUTHORS' CONTRIBUTIONS

WHX, YYZ and ZBS contributed substantially to the conception of the review. WHX, LFL and ZJZ searched for document materials and extract information. WHX, WBW, YKZ and ZRF wrote the original draft. JZ, QCK and YKZ revised the manuscript. All authors read and approved the final manuscript.