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Regulation of long non-coding RNAs XIST and ROR induced by homeodomain protein TGIF2LX in colorectal cancer

1 Department of Biology, School of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran
3 Department of Genetic, Islamic Azad University, Varamin-Pishva Branch, Varamin, Iran

Date of Submission24-Jul-2020
Date of Decision20-Dec-2020
Date of Acceptance25-Dec-2020
Date of Web Publication02-Jun-2021

Correspondence Address:
Abolfazl Akbari,
Colorectal Research Center, Iran University of Medical Sciences, Tehran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_869_20

 > Abstract 

Background: Homeodomain protein transforming growth factor beta-induced factor 2 like, X-linked (TGIF2 LX) has been demonstrated to act as a transcription factor and regulate cancer cell proliferation. Long non-coding RNAs (lncRNAs) are well known as molecular regulators of colorectal cancer (CRC). Our aim was to evaluate the clinical and biological significance of TGIF2 LX and its effect on lncRNAs regulator of reprogramming (ROR) and X-inactive specific transcript (XIST) expression in CRC cells.
Materials and Methods: Thirty-six CRC tissues and 22 adjacent normal colorectal tissues were subjected to RNA extraction and analysis of TGIF2 LX gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). The human SW1116 cell line was transfected with cDNA for the TGIF2 LX gene. Microscopic analysis, reverse transcriptase PCR, and western blotting were used for confirming at transcriptional and translational levels. Methyl thiazolyl tetrazolium and colony formation assays were applied for evaluating the in vitro cell viability and colony-forming ability, respectively. LncRNA expression analysis was carried out using qRT-PCR.
Results: The results showed that the expression levels of TGIF2 LX were significantly downregulated in CRC tissues compared to adjacent normal tissues (P = 0.032). Furthermore, the overexpression of TGIF2 LX could reduce the CRC cell line proliferation. The gene expression analysis revealed a significantly reduced level of lncRNA ROR and lncRNA XIST in TGIF2 LX-transfected SW1116 cells compared to nontransfected cells.
Conclusion: Our findings provided evidence of molecular mechanisms by which TGIF2 LX may interact with lncRNAs ROR and XSIST to regulate CRC development by acting as a tumor suppressor. Thus, this protein may potentially be a promising option for CRC gene-based therapeutic strategies.

Keywords: Colorectal cancer, homeodomain protein, HOX gene, long non-coding RNA, transforming growth factor beta-induced factor 2 like, X-linked

How to cite this URL:
Tabarestani FO, Akbari A, Karizi SZ, Sotoodehnejadnematalahi F. Regulation of long non-coding RNAs XIST and ROR induced by homeodomain protein TGIF2LX in colorectal cancer. J Can Res Ther [Epub ahead of print] [cited 2022 Jul 4]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=317459

 > Introduction Top

Homeobox genes (HOX), including a large group of transcription factors, are featured by a sequence of 183 nucleotides encoding a hoemodomain of 61 amino acid that binds to DNA and Polycomb proteins, activating or repressing specific genes.[1] These genes characteristically are categorized in four clusters, including 13 paralogous groups according to position within the corresponding locus and the similarity of conserved homeodomains and homeobox sequences.[1] The Hox genes have been demonstrated to be implicated in the regulation of developmental processes. These development-associated genes also have frequently been revealed to contribute to the genetic and epigenetic regulation of cancers so that the expression of specific HOX genes is dysregulated in a range of malignancies, including colon and lung cancer, based on a tumor type- and site-dependent manner.[1],[2],[3] Mutation or abnormal expression of HOX genes is responsible for developmental disorders. In addition, changes in the levels and activation of certain HOX genes have been associated with the development of cancer.[4] The complexity of Hox gene regulation and corresponding signaling pathways has commonly been investigated by several researchers.[5] Moreover, increasing evidence indicates numerous conserved noncoding RNA (ncRNA) sequences mapped in HOX loci. In this regard, a number of ncRNAs located in the HOX gene network have been served as responsible for chromatin modification and epigenetic events.[6],[7],[8]

Long non-coding RNAs (lncRNAs), as a well-known group of ncRNAs, are involved in the regulation of several physiological and of pathological processes. LncRNAs have been identified to function as critical effectors in cancer development and progression.[9],[8],[9],[10],[11] More recently, Hox molecular signaling networks associated with ncRNAs have been identified in several cancers.[12],[13] Among ncRNAs, lncRNAs and microRNAs (miRNAs) have more been characterized as biological players in cancer initiation and development.[14] Chromatin modification has been identified as a distinctive posttranscriptional event by which Hox genes may regulate cancer-associated ncRNAs. In addition, investigations revealed a cross-talking between Hox genes and ncRNAs in vitro and in vivo. For example, lncRNA HOTAIR encoded within the HOXC locus and HOXD genes is reciprocally involved in the epigenetic regulation of various developmental processes.[13],[15]

Furthermore, Hox genes are found in clusters that also contain genes for ncRNAs transcription. Interestingly, some functional studies revealed that Hox cluster-embedded ncRNAs preferentially target Hox mRNAs. Moreover, the presumed Hox target genes are predominantly situated on the 3′ side of each Hox ncRNA locus.[16],[17] These results suggest that lncRNA-mediated regulation seems to recapitulate interactions at other levels of gene expression, hence makes cancer signaling networks more complicated. It has been indicated that HOX-related lncRNAs may mostly be involved in cancer development by regulating gene expression and chromatin modification.[18],[19] The association between of Hox genes with specific domains and ncRNA signaling network helps to more fine gene regulation and pattern the identity of developmental events.

The three-amino acid loop extension (TALE) superclass of Hox proteins consists of members that contains three extra residues between helix 1 and helix 2 of the homeodomain. Some TALE homeodomain proteins function as cofactors for the HOX proteins. TG-interacting factors (TGIFs) comprise a family of TALE-homeodomain proteins that contain several members. Four TGIFs in human are TGIF, TGIF2, transforming growth factor beta-induced factor 2 like, X-linked (TGIF2 LX), and TGIF2 LY/X (TGIF2 like on X or Y chromosomes). Previous studies on cultured cells demonstrated that TGIF2 acts as a transcriptional repressor and antagonizes transforming growth factor-β (TGF-β) -activated gene expression.[20] Moreover, the TGIF2 gene was found to be amplified and overexpressed in various cancer cell lines.[21],[22] However, whether ncRNAs could play role as a mediator needs to be apparent.

Cancer stem cells (CSCs) are characterized as main responsible for cancer development, progression, and metastasis as well as chemoresistance and recurrence. A line of evidence indicates that lncRNAs contribute to CSC signaling pathways, in that way involving in tumorigenesis.[23],[24] One scenario for the regulatory roles of ncRNAs in cancer genetics may be due to their function as mediators in the cross-talk between various cancer signaling pathways in CSCs. Given the identified functions of TGIF2 LX in cancer development programs, its biological role needs to be clear in carcinogenesis. On the other hand, understanding the underlying molecular mechanisms of cancers involving ncRNA in the regulation of Hox genes, or mutually, may provide helpful insight into cancer development. This study was conducted to evaluate the biological role of TGIF2 LX and its effect on the possible lncRNA targets regulator of reprogramming (ROR) and X-inactive specific transcript (XIST) in colorectal cancer (CRC) cells.

 > Materials and Methods Top

Patients and samples

The study was approved by the Ethic Committee of Iran University of Medical Sciences (Ethical code: IR. IUMS. REC 97-4-75-13690). Thirty-six CRC tissues and 22 adjacent normal colorectal tissues (5 mg) were collected in the tubes containing RNAlater® RNA Stabilization Reagent (Thermo Fisher Scientific, Germany) and stored at −80°C until use. The tissue homogenization was performed to maximize RNA yield. Total RNA was extracted from all tissue specimens by RNX Plus RNA extraction Kit (SinaClon, Iran, Tehran).

Cell culture

Human colon adenocarcinoma cell line, SW1116, was obtained from the National Cell Bank of Iran associated with the Pasteur Institute (Tehran, Iran). The cell line was cultured in RPMI-1640 medium (Gibco Invitrogen, USA) containing 5% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco Invitrogen, USA), 2 mM glutamax (Gibco Invitrogen, USA), 100 units/ml penicillin, and 50 μg/ml streptomycin (Gibco Invitrogen, USA) in culture flask 25 cm2 (SPL, Life Science, South Korea). The cell culture condition was followed at 37°C in a humidified 95% atmosphere, 5% CO2 atmosphere incubator. Cell viability was examined using trypan blue exclusion test and found in all flasks to contain more than 90% viable cells [Figure 1]a.
Figure 1: Microscopic evaluation for transforming growth factor beta-induced factor 2 like, X-linked expression. Green fluorescent protein-transforming growth factor beta-induced factor 2 like X-linked gene expression was detected with fluorescent microscopy to validate the transfection efficiency. Continuous culture of un-manipulated colon adenocarcinoma SW1116 cells (a). SW1116 cells transfected with green fluorescent protein or mock control (b) and transforming growth factor beta-induced factor 2 like, X-linked-green fluorescent protein (c), three weeks post-transfection under a fluorescent microscope (×40). The single-cell transfected with TGIFLX-green fluorescent protein (d) that shows transforming growth factor beta-induced factor 2 like, X-linked gene expresses in the nucleus (×400)

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Transfection of SW1116 cells with transforming growth factor beta-induced factor 2 like, X-linked

The subcultured SW1116 cell was transfected with either plasmid pEGFP with or without (mock constructs) TGIF2 LX cDNA sequence using FuGENE®6 Transfection Reagent (Promega, Madison, WI, US) consistent with the manufacturer's directions. The transfected cells were examined by fluorescence stereoscopic microscopy (Olympus, Tokyo, Japan) 48 and 72 h after transfection to confirm green fluorescent protein (GFP) emission as an internal control.

Stable cell line generation and evaluation

For the establishment of SW-1116 cell line permanently expressing TGIF2 LX, among 72 h posttransfected cell lines, the highly GFP-expressing cells were isolated. Since the used vectors encode neomycin (G418) for selected mammalian cells, the cells were continuously subcultured for 21 days in the presence of 750 μg/mL G418 (Sigma-Aldrich, Belgium) to selection, as described previously [Figure 1]b, [Figure 1]c, [Figure 1]d.[25]

Expression analysis of transforming growth factor beta-induced factor 2 like, X-linked gene by reverse transcriptase–polymerase chain reaction

Total RNA was extracted using RNX Plus Solution (SinaClon, Iran) according to the manufacturer's instructions and stored at −70°C. cDNA synthesis was completed using 0.5 μg of RNA and stored at −20°C. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed to analyze/confirm the expression of TGIF2 LX gene in the transfected cells, as described previously.[26] The PCR products were electrophoresed using gel agarose 1.5% [Figure 2].
Figure 2: Gene expression analysis of transforming growth factor beta-induced factor 2 like, X-linked. The gene expression of transforming growth factor beta-induced factor 2 like, X-linked in three tumor groups (SW1116, SW1116-transforming growth factor beta-induced factor 2 like, X-linked-green fluorescent protein and SW1116-green fluorescent protein) were confirmed by real-time reverse transcriptase-polymerase chain reaction using the GAPDH (190bp) as a housekeeping control gene. Polymerase chain reaction products were resolved on a 2% agarose gel stained with gel red

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Examination of transforming growth factor beta-induced factor 2 like, X-linked expression at the translational level

Protein expression was evaluated by western blot technique, as previously explained.[25] Briefly, cell lysate was isolated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transported to a nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked for 1 h with 3% bovine serum albumin and incubated with polyclonal rabbit anti-TGIF2 LX antiserum raised against the C-terminus (sc-459, Santa Cruz Biotechnology, Santa Cruz, CA, USA) (2 h, at room temperature). After that, the membranes were incubated with goat horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody, followed by development with 4-chloro-1-naphthol (Immun-Blot, Bio-Rad Laboratories, Hercules, CA, USA), as previously described [Figure 3].
Figure 3: Western blotting for validating transforming growth factor beta-induced factor 2 like, X-linked protein expression. Detection of transforming growth factor beta-induced factor 2 like, X-linked knockdown by small interfering RNA using western blotting technique. The trasfected cells were treated with two small interfering RNA duplexes to transforming growth factor beta-induced factor 2 like, X-linked, or with a nonsilencing control small interfering RNA and protein prepared at 72 h posttransfection. Protein lysates were analyzed for transforming growth factor beta-induced factor 2 like, X-linked expression by western blotting technique

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Knockdown of the transforming growth factor beta-induced factor 2 like, X-linked gene by small interfering RNA

SW1116 cells in exponential phase were seeded in a 24-well plate (6 × 105 cells/well) with RPMI1640 medium containing 5% (v/v) FBS. After overnight growing the cells, the small interfering RNA (siRNA) transfection process was conducted using 2.5 μl X-treme-GENE siRNA Transfection Reagent (Roche Diagnostics, Grenzach-Wyhlen, Germany) and 0.5 μg of the respective siRNA, according to the manufacturer's instructions. After total RNA extraction and cDNA synthesis, quantitative reverse transcription PCR (RT-qPCR) was executed by RealQ Plus 2x Master Mix Green (Ampliqon A/S, Stenhuggervej, Odense, Denmark). For detecting TGIF2 LX gene, PCR reactions were performed using ABI real-time PCR thermocycle using the following conditions: 95°C for 1 min and 95°C for 5 min, followed by 35 cycles at 95°C for 15 s and 61°C for 1 min. The results were normalized against housekeeping GAPDH gene expression.

Colony formation assay

The soft agar colony formation assay was performed using SW-48 cells transfected with TGIF2 LX by standard techniques (Rafehi et al., 2011). Briefly, SW1116 cells expressing TGIF2 LX were trypsinized, and 2 × 104 cells were plated in 6-well plates. The cell colonies were cultured for 10 days and fixed with methanol and stained using 0.5% crystal violet for 45 min. Using a microscope, colonies were evaluated and those with more than 50 cells were counted. Colony formation of SW1116-TGX cells was similar to that observed in the control of SW1116 cells.

Methyl thiazolyl tetrazolium assay

To evaluate the growth inhibitory effect of TGIF2 LX, all the three (untransfected SW1116, pEGFP-N1-SW1116, and pEGFP-TGIF2 LX-SW1116) cells were seeded in 96-well flat-bottom microtitration plates (SPL, Life Science, South Korea) at a density of 3 × 103 cells/well (200 μL media/well). After 48 h, the medium was replaced by 100 μL methyl thiazolyl tetrazolium (MTT) (Sigma-Aldrich, Belgium). Following 3 h incubation at 37°C, 150 μL dimethyl sulfoxide (Sigma-Aldrich, Belgium) was added to each well. Later than gently mixing for 20 min, the absorbance of the converted dye was read at a wavelength of 570 nm using a spectrophotometric microplate reader (BioTek Elx 808).

The expression analysis of lncRNAs

The expression levels of lnc-XIST and lnc-ROR were evaluated using a quantitative real-time PCR (qRT-PCR) technique by specific primer sequences [Table 1]. All reactions were carried out in duplicate and the mean threshold cycle (Ct) was achieved using cycle threshold settings. The relative expression levels of lncRNAs were normalized to that of housekeeping gene GAPDH as an internal control. The data were analyzed using the equation: log10 (2ΔΔCt), in which ΔΔCt = Cttarget lncRNA – Ctinternal control.
Table 1: The oligonucleotide sequences used as primers

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Statistical analysis

The statistical analysis of lncRNAs expression was completed by SPSS software version 21.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed using the Mann–Whitney U-test. P < 0.05 was considered as the level of significance for a statistical test.

 > Results Top

Gene expression levels of transforming growth factor beta-induced factor 2 like, X-linked in colorectal cancer specimens

To investigate the possible biological role of TGIF2 LX in the CRC, we first analyzed the expression levels of TGIF2 LX in CRC samples by qRT-PCR. The mean of relative expression was 0.85 ± 0.02 for cancer and 1.34 ± 0.043 for adjacent normal tissue. Our analysis showed that TGIF2 LX was significantly downregulated in tumor samples compared to adjacent normal tissues with a fold change − 2.11 (±0.084). Notably, of 36 CRC samples, downregulation of TGIF2 LX was shown in 27 (75%) samples. In 4 (11%) tumor tissues, the TGIF2 LX expression was similar to normal tissues. Then, the cancer samples with a high significant downregulation(≥-1.5 fold change) of TGIF2 LX expression were subjected to investigate correlations between TGIF2 LX expression and clinicopathological features in CRC patients. Further analysis showed no correlation between gene expression and clinicopathological features of CRC patients, including age, gender, lymphovascular infiltration, and differentiation (P > 0.05). Furthermore, the expression pattern of TGIF2 LX gene was not significantly different between various clinical stages of CRC (P > 0.05). However, an in-depth analysis found a negative correlation between TGIF2 LX gene expression and tumor size (P < 0.05).

Transforming growth factor beta-induced factor 2 like, X-linked expression in transfected colon adenocarcinoma cells

The expression of TGIF2 LX-GFP in SW-1116 cells was established using microscopic validation of GFP emission. The quantitative transfection efficiency of the cells was approximately 80% [Figure 1]a, [Figure 1]b, [Figure 1]c. The expression of TGIF2 LX at the gene and protein levels was confirmed by real-time PCR [Figure 2] and western blotting assay, respectively. Microscopic examination revealed that the TGIF2 LX-GFP was mainly localized into the nucleus [Figure 1]d, suggesting this protein as a probable transcription factor.

Inhibition effect of transforming growth factor beta-induced factor 2 like, X-linked on the colony formation of SW1116 cells

The results of colony formation assay revealed that the colony-forming capacity in TGIF2 LX-SW1116 cells was noticeably lower than the vehicle-treated cells.

In addition, colony-forming efficiency of normal SW1116 cells was significantly higher than that of TGIF2 LX SW1116 cells [Figure 4].
Figure 4: Colony formation assay showed that TGIF2LX silencing enhanced cell growth and colony formation in colorectal cancer SW1116 cells. Colony formation was evaluated in non-transfected SW1116 cells (a), pEGFP-N1-SW1116 cells (b), and pEGFP-TGIF2LX-SW1116 cells (c) (magnification × 20). The results revealed that colony formation was significantly decreased by TGIF2LX overexpression in pEGFP-TGIF2LX-SW1116. Values were expressed as mean ± Standard error of the mean (n = 3). Data were analyzed by one-way ANOVA test. *P value < 0.05 was considered as significant level

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Overexpression of transforming growth factor beta-induced factor 2 like, X-linked reduced cell proliferation

MTT assay was used for evaluating the TGIF2 LX effect on SW1116 cell proliferation. The results of the test demonstrated that CRC cells expressing the high TGIF2 LX presented a significant cell growth compared to nontransfected SW1116 cells [Figure 5]; P < 0.05].
Figure 5: Methyl thiazolyl tetrazolium assay for evaluating the cell viability. Quantitative analysis by methyl thiazolyl tetrazolium assay of SW1116 cell viability showed that the overexpressin of transforming growth factor beta-induced factor 2 like, X-linked decreased the proliferation and growth capability

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Knockdown of the transforming growth factor beta-induced factor 2 like, X-linked gene by small interfering RNA

To validate the effect of TGIF2 LX on the lncRNAs expression level as well as the pathological process of CRC, we knocked down TGIF2 LX to examine the changes of gene expression and cell proliferation and colony formation of cancer cells. The results obtained from RT-qPCR analysis following siRNA knockdown, 72 h after transfection, showed a significant decrease in TGIF2 LX mRNA expression level. Successful knockdown of TGIF2 LX was also observed using the siRNA as seen by the decreased levels of the protein [Figure 4]. As well, the cell assays confirmed that knockdown of the TGIF2 LX gene results in an increased cell proliferation.

Overexpression of transforming growth factor beta-induced factor 2 like, X-linked downregulates the expression of lncRNA regulator of reprogramming and X-inactive specific transcript

To evaluate the effect of TGIF2 LX on CRC-associated lncRNAs expression, we analyzed the relative expression of lncRNAs ROR and XIST in SW1116 cells overexpressing the homeoprotien. Our functional investigations showed that the overexpression of TGIF2 LX can change the expression levels of lncRNA ROR and lncRNA XIST in CRC cell line SW1116. The gene expression analysis by qRT-PCR revealed a significantly reduced level of lncRNA ROR and lncRNA XIST in TGIF2 LX-transfected SW1116 cells compared to nontransfected cells.

The results showed that the lncRNA ROR expression level was significantly downregulated by 2.8 times in SW1116-TGX cell lines with P < 0.001. Furthermore, it was found that the fold change of lncRNA XIST is − 1.9 in SW1116-TGX cells compared to nontransfected SW1116 cells (P < 0.05).

 > Discussion Top

A line of evidence has indicated that the aberrant expression of developmental Hox gene causes may contribute to tumorigenesis.[3],[4] The homeodomain proteins have been revealed to act as transcription factors to regulate the cell growth and proliferation. For example, human homeobox CDX1 can regulate the cell cycle by modulating several checkpoint proteins in intestinal cells.[25]

TGIF2 LX, as a homeodomain protein and a transcription factor, has confirmed to be lost in some cancer cell lines, including the CRC, indicating an important role in cellular homeostasis. In this regard, it has been investigated that TGIF2 LX could reduce proliferation and angiogenesis of colorectal tumors in vitro and in vivo. The findings indicated that homeobox protein TGIF2 LX may act as a tumor suppressor through regulating some oncogenic signaling pathways.[25] Recent studies demonstrated that human TGIF2 could reduce cancer cell proliferation and tumor size through regulating cancer cell signaling pathways. Another study indicated that overexpression of TGIF2 LX could downregulate BCL2 and upregulate BAX in cancer cells.[27] Further bioinformatic analyses revealed a downstream signaling network involved in the pathogenesis of CRC.[25] Given the potential role of TGIF2 LX in CRC tumorigenesis, we aimed to evaluate its effect on the expression of cancer-associated lncRNAs.

LncRNAs are characterized as the main group of ncRNA that play key roles as tumor suppressor genes or oncogenes in cancer development and progression. These RNA molecules have been shown to be involved in several genetic and epigenetic events in the cell.[11],[19] Growing evidences showed that ncRNAs, including lncRNAs, may be associated with Hox genes signaling pathways. Indeed, lncRNAs and Hox genes may be in a cross-talk in cancer underlying mechanisms.

These interplaying molecular events between Hox and lncRNAs have been shown to regulate the proliferation and invasion in cancer cells.[28] On the other hand, HOX-embedded lncRNAs have been more recently described in tumorigenesis. Even though cancer-associated lncRNAs have received a great deal attention recently, the biological function of HOX lncRNAs axis in the development of cancer is unclear.[18],[29] Our functional results showed that the overexpression of TGIF2 LX can significantly downregulate the expression levels of lncRNA ROR and lncRNA XIST in CRC cell line SW1116.

A line of evidence showed that ncRNAs play imperative roles in tumorigenesis and may be served as diagnostic biomarkers and therapeutic targets.[11] The expression level of lncRNA-ROR has been reported to increase in colorectal tumors in comparison with adjacent tissues, indicating an oncogenic function of lncRNA-ROR in the cancer progression and development. LncRNA-ROR may be involved in the regulation of cell proliferation and apoptosis, in vitro and in vivo.[30],[31] Another study has shown that lncRNA-ROR can regulate the translational level of tumor suppressor p53 and its downstream signaling pathways. In vitro and in vivo negative correlation between P53 protein and expression level of lncRNA-ROR indicated an underlying mechanism of lncRNA-ROR through regulating the p53 signaling in CRC.[30] On the other hand, several functional and experimental evidences have indicated TGIF protein as a co-repressor for TGF-β-activated Smads to regulate cell cycle-associated genes such as P15, P21, and P27.[32],[33] A recent study showed that the overexpression of TGIF2 LX can upregulate the Fragile histidine triad gene, indicating this protein plays an important role as a tumor suppressor in CRC.[20] In this regard, it has also been demonstrated that TGIF may exert its tumor-suppressive function in promyelocytic leukemia by modulating some TGF-β signaling-associated proteins to regulate P53.[34] Molecular interactions between cancer-associated communicator and mediators need to be further investigated. Recent investigations have shown that link-ROR is dysregulated in breast cancer, especially in the advanced stages. In adddition, the overexpression of this lncRNA is correlated with clinicopathological features, indicating an oncogenic role in the cancer development and progression.[35],[36] In this regard, TGF-β signaling pathway was suggested as a downstream target of linc-ROR, contributing to the induction of epithelial-to-mesenchymal transitions (EMT) in cancer progression.[31] Related findings have revealed that the function of linc-ROR is mediated by the TGF-β signaling pathway and cancer-associated biological processes.[31] Since the TGF-β has been demonstrated to be involved in cell reprogramming, more investigation of the biological role of TGIF2 LX as an upstream repressor of TGF-β may facilitate to realize how the versatile functions of TGF-β are regulated during CSC differentiation and proliferation. The functional studies considering cross-talk between lncRNAs and TGIF2 LX will ultimately improve molecular insights to develop therapeutic strategies.

LncRNA XIST has been reported to be upregulated in various malignancies including glioma and ovarian cancer and exerts an oncogenic function by promoting cell proliferation, invasion, and metastasis.[37],[38] One earlier study also demonstrated that lncRNA XIST promotes gastric cancer development and metastasis through regulating the expression of EZH2.[39] Nevertheless, one study showed that lncRNA XIST could act as a tumor suppressor in breast cancer.[40] It has been found that lncRNA XIST was upregulated in CRC tissues compared to adjacent normal tissues. In addition, lncRNA XIST overexpression was associated with tumor size, tumor stage, and lymph node metastasis.[41],[42] The expression of this lncRNA also has been shown to be associated with chemoresponses to 5FU and poor survival of CRC patients. The findings concluded from the incorporated approaches have indicated that lncRNA XIST may be served as a probable biomarker and therapeutic target in CRC patients.[43] LncRNA XIST has been confirmed to target and regulate PTEN by sponging miR-181a in hepatocellular carcinoma progression.[44] It also was found that this lncRNA could promote pancreatic cancer proliferation through miR-133a/EGFR.[45] Thus, depending on the tumor type and genomic background, lncRNA XIST may function as an oncogene in one cell/tissue whereas tumor suppressive. Functional studies demonstrated that lncRNA XIST promoted CRC cell proliferation and migration in vitro. Related findings revealed that lncRNA XIST is involved in the regulation of EMT, an important step in tumor progression and metastasis.[41] Furthermore, it has been reported that lncRNA XIST could affect the stemness of CRC cells and promote CSCs, a key source of cancer metastasis and progression.[41],[46] In addition, experimental and bioinformatics studies identified several miRNAs such as miR-200b-3p that might interact with lncRNA XIST.[41] It has been confirmed that miR-200b-3p could directly bind to the 3′-end of lncRNA XIST to regulate.[41] Similar to our results, a recent report indicated that lncRNA XIST functions as a molecular sponge for miR-139-5p in hepatocellular carcinoma. It has been found that the lncRNA XIST could indirectly regulate ZEB1 expression by way of sponging to miR-200b-3p in CRC cells. ZEB1 is an important regulator of EMT and cell invasion, stem-like properties, and stimulates tumor progression. We determined whether lncRNA XIST could modulate the transactivation of ZEB1 mRNA. Taken together, these data showed that lncRNA XIST functions as a ceRNA for miR-200b-3p to regulate ZEB1 expression.[41] Furthermore, lncRNA XIST has been confirmed to be used as a prognostic biomarker for CRC patients.[41] Based on our findings, it is concluded that the overexpression of TGIF2 LX may function by targeting several signaling pathways, including lncRNAs involved in the CRC stem cell proliferation. However, further studies to find direct targets are needed to realize the experiential effect. More functional and bioinformatic investigations on cross-talk between Hox gene TGIF2 LX and ncRNA networks can improve our insight into the role of Hox/lncRNA axis in CRC development.

 > Conclusion Top

Our findings provide support for the role of HOX gene TGIF2 LX in CRC, partly through regulating the lncRNA expression. Given the biological role of TGIF2 LX-signaling pathway, it is imperative to identify genes within the pathway that are most dysregulated in CRC and how dysregulated lncRNAs directly and indirectly influence those genes. Although the experimental data provide a basis for additional examination and targeting the key genes and lncRNAs, further animal and clinical data are essential to validate the results.


This study was supported by the grant number 97-4-75-13690 from Iran University of Medical Sciences.

Financial support and sponsorship

This study was supported by Iran University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.

 > References Top

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