Abstract

Long non-coding RNAs (lncRNAs) are known to be potential factors in promoting tumor progression. However, the function and mechanism of lncRNA XIST in non-small cell lung cancer (NSCLC) remains poorly understood. The expression levels of lncRNA XIST in NSCLC tissues and cell lines were detected with real-time PCR, and the correlation of the expression level of XIST with histopathological characteristics and prognosis was analyzed. The biological function of lncRNA XIST was validated through assays in vivo and in vitro. The expression of lncRNA XIST was significantly up-regulated in NSCLC tissues. In addition, overexpression of XIST was positively correlated with the advanced clinical status of tumors, as well as poor overall survival and DFS. A tumor suppressive effect was presented via functional knockdown of lncRNA XIST. Up-regulation of XIST enhanced the proliferation, migration, and invasion ability of NSCLC cells both in vivo and in vitro. Mechanically, it was indicated that XIST could serve as an endogenous competitive RNA modulating miR-744, leading to the miR-744/RING1 signaling pathway inhibition and Wnt/β-catenin signaling pathway activation. Taken together, it was confirmed here that XIST overexpression is associated with tumor progression phenotype and the newly discovered XIST/miR-744/RING1 axis, which could serve as a potential biomarker and therapeutic target for NSCLC.

Introduction

Lung cancer is the most common form of cancer resulting in fatality in males, the 1-year survival rate of which is only 30%, while the 5-year survival rate is only 8% [1]. NSCLC accounts for 85% of the total incidence of lung cancer. In order to increase the currently dismal 5-year overall survival (OS) rate and reduce lung cancer mortality, it is extremely urgent to uncover the underlying mechanism of lung cancer progression, optimize existing therapeutics and screen for novel treatment targets [2].

LncRNAs are defined as non-coding transcripts longer than 200 nts [3]. Up to now, it has been reported that the function of lncRNAs includes regulating gene expression level via chromatin modification, modulating transcription (post-transcription), and translation process [4,5]. LncRNAs can modulate tumor suppression, and therefore could serve as new biomarkers and therapeutic targets [6]. It was reported recently that abnormal lncRNAs expression is involved in the development and progression of various tumors, including NSCLC [7,8].

LncRNA XIST, a specific expression product of inactivated X chromosome XIST gene, regulates X chromosome inactivation in mammals [9]. It was shown in recent studies that XIST overexpression is involved in the pathophysiology of many cancers, including hepatocellular adenocarcinoma, colorectal cancer, and osteosarcoma [10]. Nevertheless, it remains unknown whether XIST is related to the biological behavior and pathogenesis mechanism of NSCLC.

The present study investigated the function of XIST-miRNA in the regulation of NSCLC tumor cell growth, and uncovered its role in modulating the WNT/β-catenin signaling pathway. In summary, we are the first to provide evidence for a XIST-miR-744/RING1-Wnt/β-catenin signal axis in NSCLC, that could act as a potential therapeutic target in the treatment of lung cancer patients.

Materials and methods

Analysis with GEO database

The expression profiles of XIST in NSCLC were detected with two databases in GEO, GSE99870 (plasma samples) and GSE121090 (samples of lung squamous cell carcinomas and adenocarcinomas). R 3.4.3 software was applied for data analysis. The expression levels of XIST in NSCLC tissues and normal tissues were comparatively analyzed through a standardized process with the method described below.

Clinical samples

About 96 pairs of NSCLC tumor tissues and non-tumor tissues from patients undergoing digestive tract surgery during the corresponding period in Affiliated Zhongshan Hospital of Dalian University from 2012 to 2016 were collected. Patients had not been given tumor specific treatment prior to the diagnosis. The project has been approved by the Affiliated Zhongshan Hospital of Dalian University Ethics Committee and informed consents were given to all patients.

Cell culture and transfection

Human NSCLC cell lines A549, H1299, H23, H522, H460, H1650, and 95D (ATCC Company) were cultured in DMEM medium containing 10% calf serum at 37°C and 5% CO2. Liposome 2000 (Invitrogen, U.S.A.) was applied for cell transfection in accordance with the manufacturer’s instructions. MiR-744 simulators, miR-744 inhibitors, miRNA controls, targetted siRNA (siRNA-XIST, siRNA-RING1) and small interfering negative control RNA (siRNA-NC) were purchased from GenePharma Technology (Shanghai, China). XIST overexpression plasmid (pcDNA3.1-XIST, Genearray Biotechnology, Shanghai, China). The transfection was performed in accordance with the manufacturer’s instructions, and the successfully transfected subclones were called H522-XIST-cDNA cells and H23-XIST-cDNA cells, respectively. Cells were harvested at the designated time post-transfection for further study.

qRT-PCR

TRIzol kit (Invitrogen, U.S.A.) was applied to extract total RNA from tissues or cell lines in accordance with the kit operation guidelines. The first strand of DNA was extracted with reverse transcriptase. ABI 7500 real-time PCR system fluorescent dye method was performed for qRT-PCR. The fold changes were calculated with 2−ΔΔCT method.

Cell counting reagent-8 experiment

NSCLC cells were inoculated in 96-well cell culture dishes and cultured overnight at 37°C with a density of 4000 cells/well. Cell viability was quantitatively determined by cell counting reagent-8 (CCK-8, Japan). Briefly, on the day of measuring the growth rate of experimental cells, 100 µl fresh culture medium containing 10% CCK-8 was used to replace the same volume of the culture medium, which were cultured at 37°C for 1 h, and then 450 nm absorbance was detected by enzyme labeling instrument.

Transwell assay

The migration assay of 2 × 104 cells was carried out in an uncoated Transwell chamber without serum medium. Similarly, the invasion assay was carried out with 2 × 104 cells suspending in serum-free medium in a Transwell chamber, the upper chamber of which was covered with artificial basement membrane. Non-migrating or non-invasive cells were taken out with cotton swabs, stained with crystal violet, and counted under an inverted microscope.

Western blot

All proteins were lysed with 10% SDS/PAGE and transferred onto PVDF membrane, which were sealed in blocking solution for 90 min at room temperature, and then added into the culture solution containing antibodies RING1 (1:1000, Abcam, UK), β-catenin (1:1000, Abcam, U.K.), c-myc (1:1000, Abcam, U.K.), cyclin D1 (1:800, Abcam, U.K.), E-cadherin (1:1000, Abca, U.K.) or PDH (1:1000, Abcam, Abca, U.K.) and kept overnight. They were rinsed with hot water and incubated with secondary antibodies, which were labeled by horseradish peroxidase (HRP). The expression of protein was detected with an enhanced chemiluminescence method and chemiluminescence membrane method. The band intensity was quantitated with the laboratory image acquisition and analysis software.

Luciferase reporter assay

We cloned them into luciferase vector and quickly transfected the reporter plasmid with miR-744 analog and/or pcDNA3.1-XIST gene into H1299 and A549 cells. The activity of the enzyme was detected with the Dual luciferase reporter gene assay system (Promega) after 48-h incubation.

β-Catenin reporter assay

An XIST expression plasmid or siRNA and a β-catenin reporter pTopFlash were co-transfected into cells cultured in 24-well plates using Lipofectamine 2000 reagent (Invitrogen). pRLTK (Renilla luciferase) was co-transfected as an internal control. 48 h after transfection, the luciferase reporter assay was performed using the Dual luciferase reporter assay system (Promega).

Construction of transplanted tumor model

Animal work was conducted in the Key Laboratory of Biomarker High Throughput Screening and Target Translation of Breast and Gastrointestinal Tumor, Affiliated Zhongshan Hospital of Dalian University. BALB/c nude mice of 4–6 weeks old were raised in an environment without pathogens in accordance with the relevant regulations of the Animal Care Committee, which were fed with Sterile food, and water was not limited. Bilateral ribs of nude mice were subcutaneously injected with 1 × 106 transfected miRNA-744, and extracellular matrix of allogenic cells of the control to construct the transplanted tumor model. The tumors were taken out after 25 days of initial treatment, and the mice were euthanized. During the whole period, the tumor size was measured every five days, and the mean value was taken. The data were analyzed with variance analysis. The present study was carried out in accordance with the guide for the Care and Use of Laboratory Animals of National Institutes of Health. The experiments were approved by the animal experimental ethics committee of Affiliated Zhongshan Hospital of Dalian University.

Statistical analysis

Statistical analysis was performed with SPSS 18.0. Data were presented as mean ± S.D. from three independent experiments. Student’s t-test and variance analysis were applied for data analysis. The survival curve was plotted with Kaplan–Meier method and analyzed with logarithmic rank test. A value P<0.05 was statistically significant.

Results

Overexpression of lncRNA XIST in NSCLC tissues

To determine the prognostic value of lncRNA XIST expression in NSCLC patients, two independent microarray datasets were selected from the GEO database for statistical analysis. It was indicated that the median expression of XIST in tumors was significantly higher than that in normal tissues in both independent databases (Figure 1A,B). It was confirmed via Kaplan–Meier survival curve analysis and logarithmic rank test that relative to those with high XIST expression, patients with low XIST expression had higher OS (P=0.0264) and DFS (P=0.0227, Figure 1C,D). The data suggest that XIST is highly expressed in NSCLC patients, and is associated with poor prognosis.

Overexpression of XIST in NSCLC

Figure 1
Overexpression of XIST in NSCLC

(A,B) XIST expression in dataset GSE99870 and GSE121090. (C,D) Kaplan–Meier analyses indicated OS and DFS were shorter in high XIST expression group compared with XIST low expression group. **P<0.01, ***P<0.001 vs normal tissues.

Figure 1
Overexpression of XIST in NSCLC

(A,B) XIST expression in dataset GSE99870 and GSE121090. (C,D) Kaplan–Meier analyses indicated OS and DFS were shorter in high XIST expression group compared with XIST low expression group. **P<0.01, ***P<0.001 vs normal tissues.

The association of lncRNA XIST expression with clinicopathological features in NSCLC.

The relationship of the clinicopathological features of NSCLC and the XIST expression level was investigated here to determine the significance of XIST in clinical practice. XIST expression was positively correlated with CEA level (P=0.003), tumor size (P=0.041), distant metastasis (P=0.022), TNM staging (P=0.036), whereas no significant correlation was found regarding age, sex, location of tumors, and lymph node metastasis (Table 1). To clarify the risk factors associated with OS in NSCLC patients, univariate and multivariate analyses were performed to determine whether XIST was an independent risk factor for poor prognosis. It was shown with univariate Cox regression analysis that XIST expression level, CEA level and TNM staging were significantly correlated with OS (Table 2). Furthermore, it was shown with multivariate Cox regression analysis that XIST expression level and TNM staging were independent predictive factors for OS in NSCLC patients (Table 2). It was suggested via these data that the high expression of XIST could be a predictive factor in terms of diagnosis and prognosis in NSCLC patients.

Table 1
Correlations between XIST expression and clinicopathologic features in 96 NSCLC patients
Clinicopathological feature Total Expression of XIST P-value (χ2 test) 
  High (n=48) Low (n=48)  
Age (years)     
<65 48 23 (47.90) 25 (52.10) 0.713 
≥65 48 25 (52.10) 23 (47.90)  
Sex     
Male 51 27 (52.94) 24 (47.06) 0.564 
Female 45 21 (46.67) 24 (53.33)  
CEA level     
≤5 ng/ml 45 30 (66.67) 15 (33.33) 0.003 
>5 ng/ml 51 18 (35.29) 33 (64.71)  
Lymph node metastasis     
Yes 41 23 (56.10) 18 (43.90) 0.375 
No 55 25 (45.45) 30 (54.55)  
Tumor size     
≤5 cm 57 33 (57.89) 24 (42.11) 0.041 
>5 cm 39 15 (38.46) 24 (61.54)  
Distant metastasis     
Yes 80 44 (55.00) 36 (45.00) 0.022 
No 16 4 (25.00) 12 (75.00)  
TNM stage     
Stage I/II 51 30 (58.82) 21 (41.18) 0.036 
Stage III/IV 45 18 (40.00) 27 (60.00)  
Clinicopathological feature Total Expression of XIST P-value (χ2 test) 
  High (n=48) Low (n=48)  
Age (years)     
<65 48 23 (47.90) 25 (52.10) 0.713 
≥65 48 25 (52.10) 23 (47.90)  
Sex     
Male 51 27 (52.94) 24 (47.06) 0.564 
Female 45 21 (46.67) 24 (53.33)  
CEA level     
≤5 ng/ml 45 30 (66.67) 15 (33.33) 0.003 
>5 ng/ml 51 18 (35.29) 33 (64.71)  
Lymph node metastasis     
Yes 41 23 (56.10) 18 (43.90) 0.375 
No 55 25 (45.45) 30 (54.55)  
Tumor size     
≤5 cm 57 33 (57.89) 24 (42.11) 0.041 
>5 cm 39 15 (38.46) 24 (61.54)  
Distant metastasis     
Yes 80 44 (55.00) 36 (45.00) 0.022 
No 16 4 (25.00) 12 (75.00)  
TNM stage     
Stage I/II 51 30 (58.82) 21 (41.18) 0.036 
Stage III/IV 45 18 (40.00) 27 (60.00)  
Table 2
Univariate and multivariate analyses of prognostic parameters for survival in NSCLC patients
Prognostic parameter Univariate analysis P-value Multivariate analysis P-value 
 HR 95% CI  HR 95% CI  
Expression of XIST       
(Low vs high) 1.867 1.074–3.581 0.020 1.914 1.083–3.416 0.026 
Age       
(<65 vs ≥65) 1.547 0.891–2.763 0.264 
Sex       
(Male vs female) 1.218 0.597–1.842 0.782 
Tumor size       
(≤5 vs >5 cm) 2.326 1.175–4.128 0.004 1.703 0.794–3.225 0.131 
CEA level       
(≤5 vs >5 ng/ml) 1.604 0.893–2.836 0.123 
Tumor location       
(Rectum vs colon) 1.207 0.716–2.146 0.567    
TNM stage       
(I vs II vs III vs IV) 2.284 1.659–3.442 0.000 2.192 1.604–3.126 0.000 
Prognostic parameter Univariate analysis P-value Multivariate analysis P-value 
 HR 95% CI  HR 95% CI  
Expression of XIST       
(Low vs high) 1.867 1.074–3.581 0.020 1.914 1.083–3.416 0.026 
Age       
(<65 vs ≥65) 1.547 0.891–2.763 0.264 
Sex       
(Male vs female) 1.218 0.597–1.842 0.782 
Tumor size       
(≤5 vs >5 cm) 2.326 1.175–4.128 0.004 1.703 0.794–3.225 0.131 
CEA level       
(≤5 vs >5 ng/ml) 1.604 0.893–2.836 0.123 
Tumor location       
(Rectum vs colon) 1.207 0.716–2.146 0.567    
TNM stage       
(I vs II vs III vs IV) 2.284 1.659–3.442 0.000 2.192 1.604–3.126 0.000 

LncRNA XIST promotes proliferation and invasion of NSCLC cells

While examining the expression of XIST in NSCLC cells, we found relatively high levels of XIST in H1299 and A549 cells and low expression in H522 and H23 cells. In further experiments, we knocked down XIST in H1299 and A549 cells and overexpressed XIST in H522 and A549 cells (Figure 2B,C). It was confirmed via CCK-8 and Transwell assays that compared with those of the control group, the proliferation and invasion ability of NSCLC cells transfected with siRNA-XIST (H1299, A549, H522, and H23) were significantly reduced (Figure 2D,F), whereas the proliferation and invasion of H522 and H23 cells transfected with pcDNA3.1-XIST were significantly enhanced (Figure 2E,G). Taken together, XIST promotes the proliferation and invasion of NSCLC cells.

XIST enhanced proliferative and invasive abilities of NSCLC cells

Figure 2
XIST enhanced proliferative and invasive abilities of NSCLC cells

(A) The expression levels of XIST, miR-744, and RING1 were detected by qRT-PCR in NSCLC cells. (B,C) XIST overexpression/knockdown was confirmed by qRT-PCR in NSCLC cells. (D–G) Effects of XIST overexpression/knockdown on proliferation and invasion were detected by CCK-8 and transwell assay. *P<0.05, **P<0.01, ***P<0.001 vs si-XIST group.

Figure 2
XIST enhanced proliferative and invasive abilities of NSCLC cells

(A) The expression levels of XIST, miR-744, and RING1 were detected by qRT-PCR in NSCLC cells. (B,C) XIST overexpression/knockdown was confirmed by qRT-PCR in NSCLC cells. (D–G) Effects of XIST overexpression/knockdown on proliferation and invasion were detected by CCK-8 and transwell assay. *P<0.05, **P<0.01, ***P<0.001 vs si-XIST group.

LncRNA XIST interacts directly with miR-744 in NSCLC cells

It was reported recently that XIST can interact with miRNAs as competitive endogenous RNAs and regulate their biological functions. We used starBase 2.0 online software to search for complementary bases paired miRNAs with XIST. Amongst the potential miRNAs, the expression of miR-744 was most significantly down-regulated in NSCLC cells (Figure 3A). Moreover, the expression of miR-744 decreased significantly in NSCLC transfected with pcDNA3.1-XIST, while it increased significantly in NSCLC transfected with siRNA-XIST, whereas no significant change was found regarding miR-193b-3p (Figure 3B,C). We further constructed WT XIST and Mut XIST plasmids transcript with encoding firefly luciferase (Figure 3D). Overexpressed miR-744 significantly inhibited WT XIST luciferase reporter activity, but had no significant effect on XIST mutant (Figure 3E). In addition, we observed a negative correlation between XIST and miR-744 in NSCLC tissues (P=0.024, R = −0.5236, Figure 3F). Taken together, we assumed that lncRNA XIST serves as a ceRNA in NSCLC, which could down-regulate the expression of miR-744.

XIST is a ceRNA of miR-744

Figure 3
XIST is a ceRNA of miR-744

(A,B) MiR-744 expression was examined by qRT-PCR in 20 NSCLC tissues. (B,C) MiR-744 expression was examined in pcDNA3.1-XIST or siRNA-XIST cells. (D) Bioinformatic analysis was performed to predict potentially target of XIST. (E) Luciferase activity in H1299 and A549 co-transfected with miR-744 mimics and luciferase reporters containing WT or Mut XIST transcript. (F) Negative correlation was found between XIST and miR-744. *P<0.05, **P<0.01, ***P<0.001.

Figure 3
XIST is a ceRNA of miR-744

(A,B) MiR-744 expression was examined by qRT-PCR in 20 NSCLC tissues. (B,C) MiR-744 expression was examined in pcDNA3.1-XIST or siRNA-XIST cells. (D) Bioinformatic analysis was performed to predict potentially target of XIST. (E) Luciferase activity in H1299 and A549 co-transfected with miR-744 mimics and luciferase reporters containing WT or Mut XIST transcript. (F) Negative correlation was found between XIST and miR-744. *P<0.05, **P<0.01, ***P<0.001.

LncRNA XIST regulates NSCLC cell proliferation and invasion through miR-744

MiR-744 mimics were transfected into NSCLC cells stably co-transfected with XIST cDNA or XIST-Mut cDNA or a negative control. CCK-8 assays revealed that the increased proliferation rate induced by XIST cDNA was significantly reversed by miR-744 mimics (Figure 4A,B). Moreover, overexpression of miR-744 inhibited the enhanced invasive ability of NSCLC cells by XIST cDNA (Figure 4C,D). Furthermore, CCK8 or transwell assays demonstrated that XIST-Mut cDNA did not affect cell proliferation and invasion compared with the control group (Figure 4E–G). Taken together, these data indicated that XIST promotes NSCLC cell proliferation and invasion through miR-744.

MiR-744 mimics restored the enhanced proliferative effect on NSCLC cells induced by XIST overexpression

Figure 4
MiR-744 mimics restored the enhanced proliferative effect on NSCLC cells induced by XIST overexpression

(A,B) The enhanced proliferative effect of XIST overexpression was restored by miR-744 mimics. (C,D) Cell invasion induced by XIST overexpression was abolished by miR-744 mimics. (E–G) The reduced proliferation and invasion of H522 and H23 cells induced by miR-744 mimics cannot be reversed in XIST-Mut cDNA group. *P<0.05, **P<0.01, ***P<0.001.

Figure 4
MiR-744 mimics restored the enhanced proliferative effect on NSCLC cells induced by XIST overexpression

(A,B) The enhanced proliferative effect of XIST overexpression was restored by miR-744 mimics. (C,D) Cell invasion induced by XIST overexpression was abolished by miR-744 mimics. (E–G) The reduced proliferation and invasion of H522 and H23 cells induced by miR-744 mimics cannot be reversed in XIST-Mut cDNA group. *P<0.05, **P<0.01, ***P<0.001.

LncRNA XIST regulates RING1 expression via competitively binding to miR-744

Two bioinformatics software programs (microRNA.org and PicTar) were interrogated to identify potential downstream target genes of LncRNA XIST. Interestingly, RING1 was predicted to be a direct target. RING1 is a member of RING finger family, which has been shown to play a critical function in tumorigenesis [11]. RING1 3′-UTR fragment containing the WT or Mut miR-744 binding site was inserted into the luciferase ORF (Figure 5A). RING1 3′-UTR is a direct target of miR-744 in NSCLC cells (Figure 5B). RING1 level was reduced after XIST knockdown in H1299 and A549 cells, while miR-744 inhibitor reversed this effect (Figure 5C). In addition, the expression of RING1 and the luciferase activity of the RING1 3′-UTR was enhanced with XIST overexpression but not by XIST mutant, which was blocked by expression of miR-744 mimics (Figure 5D–F). Finally, we found that RING1 expression was positively correlated with XIST expression (R = 0.5107, P=0.003, Figure 5G), and negatively correlated with miR-744 expression (R = −0.4827, P=0.004, Figure 5H). Collectively, XIST regulates RING1 expression via competitively binding with miR-744, thereby promoting the initiation and development of NSCLC.

XIST regulated RING1 expression by directly targeting miR-744

Figure 5
XIST regulated RING1 expression by directly targeting miR-744

(A) Bioinformatic analysis found the putative RING1 binding sites. (B) Luciferase activities were detected after co-transfection. (C) RING1 expression reduced by XIST knockdown was rescued by miR-744 inhibitors. (D) Up-regulation of RING1 induced by XIST or XIST mutant was abrogated by miR-744 mimics. (E) Reduced luciferase activity of pmirGLO-RING1-3′-UTR after XIST knockdown was restored by miR-744 inhibition. (F) Increased luciferase activity of pmirGLO-RING1-3′-UTR after XIST overexpression was abolished by miR-744. (G–H) Correlation between XIST and RING1 expression, as well as RING1 and miR-744 in 20 NSCLC tissues. *P<0.05, **P<0.01, ***P<0.001.

Figure 5
XIST regulated RING1 expression by directly targeting miR-744

(A) Bioinformatic analysis found the putative RING1 binding sites. (B) Luciferase activities were detected after co-transfection. (C) RING1 expression reduced by XIST knockdown was rescued by miR-744 inhibitors. (D) Up-regulation of RING1 induced by XIST or XIST mutant was abrogated by miR-744 mimics. (E) Reduced luciferase activity of pmirGLO-RING1-3′-UTR after XIST knockdown was restored by miR-744 inhibition. (F) Increased luciferase activity of pmirGLO-RING1-3′-UTR after XIST overexpression was abolished by miR-744. (G–H) Correlation between XIST and RING1 expression, as well as RING1 and miR-744 in 20 NSCLC tissues. *P<0.05, **P<0.01, ***P<0.001.

LncRNA XIST promotes the progression of malignant NSCLC through Wnt/β-catenin signaling pathway

Western blot results have shown that RING1 plays a critical role in H522 and H23 cells via activating Wnt/β-catenin signaling pathway (Figure 6A). XIST knockdown significantly reduced β-catenin reporter activity, while XIST overexpression increased the activity (Figure 6B). Consistently, the expression levels of β-catenin and several key members of Wnt/β-catenin pathway, such as c-myc and cyclin D1, were remarkably reduced, whereas the expression of E-cadherin increased significantly in NSCLC after XIST knockdown (Figure 6C). Moreover, the opposite results were found after XIST overexpression (Figure 6D). In addition, si-RING1 transfection in NSCLC cells could hinder the activation of Wnt/β-catenin pathway induced by XIST cDNA treatment (Figure 6E). It was concluded that XIST promotes the progression of NSCLC tumors through RING1-Wnt/β-catenin signaling pathway.

XIST activate the Wnt/β-catenin signaling pathway through RING1

Figure 6
XIST activate the Wnt/β-catenin signaling pathway through RING1

(A) The effect of RING1 knockdown on Wnt/β-catenin signaling. (B) β-catenin reporter assay in NSCLC cell lines following XIST knockdown/overexpression. (C) The effects of XIST knockdown/overexpression on Wnt/β-catenin signaling pathway. (D,E) Wnt/β-catenin signaling pathway promoted by XIST overexpression was restored by RING1 inhibition. *P<0.05, **P<0.01, ***P<0.001.

Figure 6
XIST activate the Wnt/β-catenin signaling pathway through RING1

(A) The effect of RING1 knockdown on Wnt/β-catenin signaling. (B) β-catenin reporter assay in NSCLC cell lines following XIST knockdown/overexpression. (C) The effects of XIST knockdown/overexpression on Wnt/β-catenin signaling pathway. (D,E) Wnt/β-catenin signaling pathway promoted by XIST overexpression was restored by RING1 inhibition. *P<0.05, **P<0.01, ***P<0.001.

LncRNA XIST promotes growth and metastasis of NSCLC via inhibiting the miR-744/RING1/Wnt/β-catenin axis in vivo

H522-XIST-cDNA cells and control cells were subcutaneously injected into nude mice to verify the effect of XIST on tumorigenesis and metastasis in vivo. Compared with the control group, the tumor size, weight, and liver metastasis in H522-XIST-cDNA group were significantly reduced. Interestingly, miR-744 agomir + H522-XIST-cDNA cells inhibited the tumor growth and reduced the numbers of liver metastasis (Figure 7A–E). RT-qPCR and western blot assays showed that up-regulation of XIST expression promoted RING1 expression and activated Wnt/β-catenin signaling pathway, which could be inhibited by miR-744 agomir (Figure 7F,G). Taken together, XIST plays a significant role in the growth and metastasis of NSCLC via inhibiting the miR-744/RING1/Wnt/β-catenin axis.

XIST promotes NSCLC growth and metastasis by inhibiting miR-744/RING1/Wnt/β-catenin pathway

Figure 7
XIST promotes NSCLC growth and metastasis by inhibiting miR-744/RING1/Wnt/β-catenin pathway

(A–E) Effect of XIST on tumorigenesis and metastasis in NSCLC. The tumors and livers were taken out 25 days after cell injection. (F,G) Effect of XIST on expression of RING1 and Wnt/β-catenin signaling pathway in NSCLC. (H) Schematic diagram that XIST promotes cell growth and migration via miR-744/RING1-mediated Wnt//β-catenin signaling in NSCLC. *P<0.05, **P<0.01, ***P<0.001.

Figure 7
XIST promotes NSCLC growth and metastasis by inhibiting miR-744/RING1/Wnt/β-catenin pathway

(A–E) Effect of XIST on tumorigenesis and metastasis in NSCLC. The tumors and livers were taken out 25 days after cell injection. (F,G) Effect of XIST on expression of RING1 and Wnt/β-catenin signaling pathway in NSCLC. (H) Schematic diagram that XIST promotes cell growth and migration via miR-744/RING1-mediated Wnt//β-catenin signaling in NSCLC. *P<0.05, **P<0.01, ***P<0.001.

Discussion

LncRNAs regulate many pathophysiological processes including tumorigenesis, which also participates in tumor proliferation, metastasis, drug resistance, and energy metabolism [12]. It was shown with multiple evidence that XIST plays a crucial role in the development of various human malignant tumors. For instance, XIST knockdown can inhibit the proliferation of tumor cells in gastric cancer, showing a tumor suppressive effect [13]. Yu et al. reported that Wnt/β-catenin signaling pathway is significantly activated with down-regulation of lncRNA-CRNDE, which would promote the proliferation and metastasis of NSCLC tumor cells through the miR-217/TCF7L2 axis [14]. Moreover, Li et al. suggested that XIST expression was an independent prognostic factor for cervical cancer [15]. It was confirmed that XIST was an oncogene or a risk factor of NSCLC [16]. They are consistent with previous reports. It was confirmed both in vivo and in vitro experiments that the survival and invasiveness of NSCLC were enhanced by XIST, suggesting that XIST is an oncogene of NSCLC.

Several LncRNAs were shown to be involved in regulating the malignant process of tumors via enhancing or inhibiting the Wnt/β-catenin signaling pathway [17]. It was reported by Wang et al. that LINC00968 could regulate NSCLC. Ma et al. reported that LINC00675 overexpression could promote the proliferation, migration, and invasion of cervical cancer cells via activating Wnt/β-catenin signaling pathway [18]. Wang et al. pointed out that ZEB2-AS1 increased the activity of Wnt/β-catenin signaling pathway via regulating the levels of VEGF, MMP9, and Axin2 in gastric cancer [19]. Sun et al. revealed that XIST shows carcinogenic gene activity via sponging miR-34a and affecting Wnt/β-catenin signaling pathway in colon cancer [20]. Hu et al. also reported that XIST promotes cell growth and metastasis by regulating miR-139-5p-mediated Wnt/β-catenin signaling pathway in bladder cancer [21]. Consistent with these reports, our data revealed that XIST could act as a ceRNA of miR-744 and regulate Wnt/β-catenin signaling pathway in NSCLC. Together, these studies indicate that XIST regulates the miRNA-mediated Wnt/B-catenin signaling pathway which might be a common mechanism of action for XIST in tumor development and progress. Here, we defined that miR-744 is a target of XIST in NSCLC. Further investigations are still needed to find out whether XIST could regulate other miRNAs, such as miR-34a and miR-139-5p.

RING1 possesses carcinogenic gene activity, which is overexpressed in various tumor tissues [11]. Xiong et al. demonstrated that down-regulation of RING1 could reduce the proliferation of HCC cells. Knockdown of RING1 may result in repressing of cell proliferative rate as well as the progression of G1/S cell cycle [21]. Shen et al. revealed that high RING1 expression is associated with poor prognosis in HCC patients [22]. Our study indicated that XIST acts as an inhibitory ceRNA of the feedback loop of miR-744/RING1 and promotes the progress of NSCLC. Additionally, down-regulation of RING1 expression can antagonize the inhibition of the expression of β-catenin, c-myc, and cyclin-D1 resulted from XIST overexpression.

In conclusion, it was shown in the present study that XIST is a carcinogenic lncRNA, which promotes the tumorigenesis and progression of NSCLC via competitively binding to miR-744, increasing RING1 expression and enhancing Wnt/β-catenin signaling pathway. A possible tumorigenic mechanism of XIST in NSLC was illustrated with the existing results, which indicates that XIST is a useful biomarker and a potential therapeutic target for NSLC.

Clinical perspectives

  • To achieve better therapeutic outcomes and improve the survival of NSCLC patients, it is essential to understand the underlying mechanisms.

  • The findings described here shed light on the functional role of XIST, which is up-regulated in NSCLC and critically regulates progression and metastasis by repressing ID1 in NSCLC cells.

  • These data may provide a new molecular mechanism for the treatment of NSCLC metastasis, which may be a promising new therapeutic target in NSCLC.

Author Contribution

J.W. carried out the experiments. H.C. and Z.D. carried out the experiments and drafted the manuscript. J.W. participated in the design of the study and performed the statistical analysis. G.W. conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

The work was supported by the Natural Science Foundation of Shandong Province [grant number ZR2014HL064] and Wu Jieping Medical Foundation [grant number 320.6750.18490].

Abbreviations

     
  • CEA

    carcinoembryonic antigen

  •  
  • DFS

    disease free survival

  •  
  • DMEM

    Dulbecco’s Modified Eagle Medium

  •  
  • CCK-8

    cell counting reagent-8

  •  
  • GEO

    Gene Expression Omnibus

  •  
  • HRP

    horseradish peroxidase

  •  
  • LncRNA

    Long non-coding RNA

  •  
  • NSCLC

    non-small cell lung cancer

  •  
  • OS

    overall survival

  •  
  • PVDF

    polyvinylidene difluoride

  •  
  • qRT-PCR

    Real-time polymerase chain reaction

  •  
  • XIST

    X-inactive specific transcript

References

References
1.
Bray
F.
,
Ferlay
J.
,
Soerjomataram
I.
,
Siegel
R.L.
,
Torre
L.A.
and
Jemal
A.
(
2018
)
Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries
.
CA Cancer J. Clin.
68
,
394
424
[PubMed]
2.
Chen
Z.
,
Fillmore
C.M.
,
Hammerman
P.S.
,
Kim
C.F.
and
Wong
K.K.
(
2014
)
Non-small-cell lung cancers: a heterogeneous set of diseases
.
Nat. Rev. Cancer
14
,
535
546
[PubMed]
3.
Schmitt
A.M.
and
Chang
H.Y.
(
2016
)
Long noncoding RNAs in cancer pathways
.
Cancer Cell
29
,
452
463
[PubMed]
4.
Yan
X.
,
Hu
Z.
,
Feng
Y.
,
Hu
X.
,
Yuan
J.
,
Zhao
S.D.
et al. .
(
2015
)
Comprehensive genomic characterization of long non-coding RNAs across human cancers
.
Cancer Cell
28
,
529
540
[PubMed]
5.
Yuan
J.H.
,
Yang
F.
,
Wang
F.
,
Ma
J.Z.
,
Guo
Y.J.
,
Tao
Q.F.
et al. .
(
2014
)
A long noncoding RNA activated by TGF-beta promotes the invasion-metastasis cascade in hepatocellular carcinoma
.
Cancer Cell
25
,
666
681
[PubMed]
6.
Battaglin
F.
,
Naseem
M.
,
Puccini
A.
and
Lenz
H.J.
(
2018
)
Molecular biomarkers in gastro-esophageal cancer: recent developments, current trends and future directions
.
Cancer Cell Int.
18
,
99
[PubMed]
7.
Zhang
Z.
,
Peng
Z.
,
Cao
J.
,
Wang
J.
,
Hao
Y.
,
Song
K.
et al. .
(
2019
)
Long noncoding RNA PXN-AS1-L promotes non-small cell lung cancer progression via regulating PXN
.
Cancer Cell Int.
19
,
20
[PubMed]
8.
Ponting
C.P.
,
Oliver
P.L.
and
Reik
W.
(
2009
)
Evolution and functions of long noncoding RNAs
.
Cell
136
,
629
641
[PubMed]
9.
Chen
D.L.
,
Chen
L.Z.
,
Lu
Y.X.
,
Zhang
D.S.
,
Zeng
Z.L.
,
Pan
Z.Z.
et al. .
(
2017
)
Long noncoding RNA XIST expedites metastasis and modulates epithelial-mesenchymal transition in colorectal cancer
.
Cell Death Dis.
8
,
e3011
[PubMed]
10.
Liu
X.
,
Ming
X.
,
Jing
W.
,
Luo
P.
,
Li
N.
,
Zhu
M.
et al. .
(
2018
)
Long non-coding RNA XIST predicts worse prognosis in digestive system tumors: a systemic review and meta-analysis
.
Biosci. Rep.
38
,
11.
Rui
X.
,
Xu
Y.
,
Jiang
X.
,
Ye
W.
,
Huang
Y.
and
Jiang
J.
(
2018
)
Long non-coding RNA C5orf66-AS1 promotes cell proliferation in cervical cancer by targeting miR-637/RING1 axis
.
Cell Death Dis.
9
,
1175
[PubMed]
12.
Li
J.
,
Huang
L.
,
Li
Z.
,
Zhong
X.
,
Tai
S.
,
Jiang
X.
et al. .
(
2019
)
Functions and roles of long noncoding RNA in cholangiocarcinoma
.
J. Cell. Physiol.
13.
Zhang
Q.
,
Chen
B.
,
Liu
P.
and
Yang
J.
(
2018
)
XIST promotes gastric cancer (GC) progression through TGF-beta1 via targeting miR-185
.
J. Cell. Biochem.
119
,
2787
2796
[PubMed]
14.
Zhu
L.
,
Yang
N.
,
Du
G.
,
Li
C.
,
Liu
G.
,
Liu
S.
et al. .
(
2018
)
LncRNA CRNDE promotes the epithelial-mesenchymal transition of hepatocellular carcinoma cells via enhancing the Wnt/beta-catenin signaling pathway
.
J. Cell. Biochem.
112
,
456
463
15.
Yu
B.
,
Ye
X.
,
Du
Q.
,
Zhu
B.
,
Zhai
Q.
and
Li
X.X.
(
2017
)
The Long Non-Coding RNA CRNDE Promotes Colorectal Carcinoma Progression by Competitively Binding miR-217 with TCF7L2 and Enhancing the Wnt/beta-Catenin Signaling Pathway
.
Cell. Physiol. Biochem.
41
,
2489
2502
[PubMed]
16.
Jiang
H.
,
Zhang
H.
,
Hu
X.
and
Li
W.
(
2018
)
Knockdown of long non-coding RNA XIST inhibits cell viability and invasion by regulating miR-137/PXN axis in non-small cell lung cancer
.
Int. J. Biol. Macromol.
111
,
623
631
[PubMed]
17.
Luo
Y.
,
Chen
J.J.
,
Lv
Q.
,
Qin
J.
,
Huang
Y.Z.
,
Yu
M.H.
et al. .
(
2019
)
Long non-coding RNA XIST promotes colorectal cancer progression by competitively binding miR-744 with SIRT1 and enhancing the Wnt/beta-catenin signaling pathway
.
Cancer Lett.
440-441
,
11
22
[PubMed]
18.
Ma
S.
,
Deng
X.
,
Yang
Y.
,
Zhang
Q.
,
Zhou
T.
and
Liu
Z.
(
2018
)
The lncRNA LINC00675 regulates cell proliferation, migration, and invasion by affecting Wnt/beta-catenin signaling in cervical cancer
.
Biomed. Pharmacother.
108
,
1686
1693
[PubMed]
19.
Wang
F.
,
Zhu
W.
,
Yang
R.
,
Xie
W.
and
Wang
D.
(
2019
)
LncRNA ZEB2-AS1 contributes to the tumorigenesis of gastric cancer via activating the Wnt/beta-catenin pathway
.
Mol. Cell. Biochem.
456
,
73
83
20.
Sun
N.
,
Zhang
G.
and
Liu
Y.
(
2018
)
Long non-coding RNA XIST sponges miR-744 to promotes colon cancer progression via Wnt/beta-catenin signaling pathway
.
Gene
665
,
141
148
[PubMed]
21.
Hu
Y
,
Deng
C
,
Zhang
H
,
Zhang
J
,
Peng
B
and
Hu
C
(
2017
)
Long non-coding RNA XIST promotes cell growth and metastasis through regulating miR-139-5p mediated Wnt/β-catenin signaling pathway in bladder cancer
.
Oncotarget
8
,
94554
94568
[PubMed]
22.
Xiong
Y.
,
Hu
B.
,
Wei
L.
,
Jiang
D.
and
Zhu
M.
(
2015
)
Upregulated expression of polycomb protein Ring1 contributes to poor prognosis and accelerated proliferation in human hepatocellular carcinoma
.
Tumour Biol.
36
,
9579
9588
[PubMed]
23.
Zhu
X.
,
Yan
M.
,
Luo
W.
,
Liu
W.
,
Ren
Y.
,
Bei
C.
et al. .
(
2017
)
Expression and clinical significance of PcG-associated protein RYBP in hepatocellular carcinoma
.
Oncol. Lett.
13
,
141
150
[PubMed]