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Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer
YGYNO-975445; No. of pages: 8; 4C: Gynecologic Oncology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer Ling-Min Liao a,1, Xiao-Ying Sun b,1, An-Wen Liu c, Jian-Bing Wu c, Xiao-Ling Cheng d, Jia-Xin Lin e, Min Zheng e, Long Huang c,⁎ a
Department of Ultrasound, The Second Affiliated Hospital of Nanchang University, Nanchang, China Department of Gynecology and Obstetrics, Central Hospital Attached to Shenyang Medical College, Shenyang, China c Department of Oncology, The Second Affiliated Hospital of Nanchang University, Nanchang, China d Department of Medical Imaging, Women And Children Health Institute Futian, Shenzhen, China e Department of Gynecology, Cancer Center, Sun Yat-sen University, Guangzhou, China b
H I G H L I G H T S • LncRNA XLOC_010588 expression was downregulated in cervical cancer tissues. • Low expression of XLOC_010588 was an independent predictor for overall survival. • Ectopic expression of XLOC_010588 inhibited cervical cancer cell proliferation through downregulation of c-Myc.
a r t i c l e
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Article history: Received 12 November 2013 Accepted 16 March 2014 Available online xxxx Keywords: Cervical cancer Long noncoding RNA XLOC_010588 c-Myc
a b s t r a c t Objective. The identification and investigation of cancer-associated long non-coding RNAs are important for understanding the molecular biology of cancer. The aim of the present study was to examine the expression pattern of lncRNA XLOC_010588 in cervical cancer and to evaluate its biological role and clinical significance in tumor progression. Methods. We examined the expression of XLOC_010588 in 218 cervical cancer tissues and matched 218 adjacent normal tissues using real-time qRT-PCR. Over-expression and RNA interference approaches were used to investigate the biological functions of XLOC_010588. The effect of XLOC_010588 on proliferation was evaluated by MTT and BrdU assays. Western blot assays were used to investigate the molecular mechanism by which XLOC_010588 increases cervical cancer cell proliferation. Results. The results showed that XLOC_010588 expression in cervical cancer was significantly downregulated. Decreased XLOC_010588 expression was correlated with FIGO stage, tumor size and SCC-Ag. Moreover, cervical cancer patients with XLOC_010588 lower expression have shown poorer prognosis. Multivariate Cox regression analyses showed that XLOC_010588 expression served as an independent predictor for overall survival. Ectopic expression of XLOC_010588 inhibited the proliferation of HeLa and SiHa cells. By contrast, knockdown of XLOC_010588 promoted the growth of HCC94 cells. Western blot assays confirmed that XLOC_010588 physically associates with c-Myc, consequently decreasing the expression of c-Myc. The expression of XLOC_010588 and c-Myc is strongly correlated in cervical cancer tissues. Conclusion. These results suggested that XLOC_010588 plays a pivotal role in cervical cancer cell proliferation via decreasing c-Myc expression and implicated the potential application of XLOC_010588 in cervical cancer therapy. © 2014 Elsevier Inc. All rights reserved.
Introduction ⁎ Corresponding author at: Department of Oncology, The Second Affiliated Hospital of Nanchang University, 1 Minde Road, Nanchang, Jiangxi, China. Fax: +86 20 87343627. E-mail address: [email protected] (L. Huang). 1 Ling-Min Liao and Xiao-Ying Sun contributed equally to this work and should be regarded as joint first authors.
Although the incidence of cervical cancer has been greatly reduced with the application of pap smear screening, cervical cancer is still the third most commonly diagnosed cancer in women worldwide, with estimated 529,800 new cases diagnosed annually [1]. Moreover, higher
http://dx.doi.org/10.1016/j.ygyno.2014.03.555 0090-8258/© 2014 Elsevier Inc. All rights reserved.
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
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incidence rate and mortality rate are observed in developing countries versus developed countries [2]. Therefore, it is still urgent for us to find new and effective therapeutic strategies for cervical cancer. Recent genome sequencing studies have revealed that the human genome is comprised of less than 2% protein coding genes, and more than 90% of the genome is transcribed as non-coding RNAs [3,4]. These ncRNAs are classified into two groups depending on the nucleotide size. Those under 200 nucleotides in length are referred to as small ncRNAs, including the microRNAs (approximately 18–25 nucleotides in length) which have been intensively studied [5]. However, the long non-coding RNAs that consist of more than 200 nucleotides have relevant limited investigations [6]. The lncRNA LOC285194 is 1950 nt in length, located in chr13 (start: 44974183; end: 44980091; − strand), consisting of three exons. LOC285194 located on the downstream side of TGFb-stimulated clone22 (TSC-22) gene and is transcripted from the antisense direction of it. TSC-22 was originally identified as a TGF immediate early response gene in mouse osteoblasts [7]. Several investigations have postulated an important role for TSC-22 in embryogenesis [8–10]. Other studies have suggested a role for TSC-22 as tumor suppressor [11,12]. TSC-22 has DNA binding properties and may act as a transcriptional repressor [13]. In our previous study, we identified the lncRNA XLOC_010588 low expression in hepatocellular carcinoma by a microarray fabricated in house. However, little is known about the expression and impact of XLOC_010588 in other cancers. We examined the expression of lncRNA XLOC_010588 in gastric cancer, colon cancer, breast cancer, cervical cancer and ovarian cancer using real time qRT-PCR. The results revealed that XLOC_010588 expression was significantly down-regulated in cervical cancer and served as adverse prognostic factors. To better understand the role of XLOC_010588 in cervical cancer development, we investigated the expression pattern of lncRNA XLOC_010588 in cervical cancer tissues and cells and studied its biological role and clinical significance in tumor progression. Materials and methods Patients and sample collection Two hundred and eighteen cervical cancer tumors and 218 matched adjacent normal tissues were obtained from Sun Yat-sen University Cancer Center (SYSUCC) (Guangzhou, China) between January 2005 and October 2009. All patients recruited in this study were not subjected to preoperative radiotherapy and/or chemotherapy and were diagnosed as infiltrating carcinoma by pathology. The tumor stage was classified by two experienced gynecological oncologists according to the International Federation of Gynecology and Obstetrics (FIGO) staging system for cervical cancer. Clinical and pathological variables analyzed are shown in Table 1. All patients were regularly followed up, with a mean observation period of 42 months (range: 2–55 months). Tissue specimens were immediately stored at 4 °C for 24 h in RNA Later (Ambion Inc.), then at − 80 °C liquid nitrogen until the extraction of total RNA. Total RNA from fresh cervical cancer tissues was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. The study was approved by the medical ethics committee of Sun Yat-Sen University Cancer Center. Quantitative real-time reverse transcriptase PCR The quality and concentration of RNA were determined using a Nanodrop 1000 (Thermo, Wilmington, DE, USA). Then, 1 μg total RNA was reversely transcribed from each sample to synthesis cDNA by the RT reagent Kit (Promega, USA, A3500) according to the manufacturer's instructions with minor modification. Real-time PCR was performed to determine the relative expression level of target genes by the SYBR Green qRT-PCR kit (Promega, USA, A6001) on the CFX96 Real Time PCR Detection System (Bio-Rad). The level of XLOC_010588 expression
in each sample was normalized to the respective GAPDH expression level. The primers used were as follows: XLOC_010588 sense, 5′-TGTG AAGAGGAGAACATAAAAGG-3′ and antisense, 5′-AAGCAAGATAATAC AGTGGCGA-3′; c-Myc sense, 5′-CGCGCTG AGTATAAAAGCCG-3′ and antisense, 5′-CTATTCGCTCCGGATCTCCC-3′; and GAPDH sense, 5′-CTCCTC CTGTTCGACAGTCAGC-3′ and antisense, 5′-CCCAATACGACCAAATCC GTT-3′. The amplification profile was 95 °C for 5 min, followed by 42 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 60 s. The specificity of each PCR reaction was confirmed by melting curve analyses. The comparative Ct method (ΔΔCt) was used for quantification of the gene expression. The relative expression amount of XLOC_010588 to GAPDH was calculated using the equation 2−ΔΔCt, where ΔCt = Ct XLOC_010588 − Ct GAPDH. To minimize experimental variability, each sample was analyzed in triplicate and the mean expression level was calculated. Cell culture Cervical cancer cell lines (HeLa, SiHa and HCC94) were grown in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin– streptomycin. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Plasmid construction A XLOC_010588 expression construct was generated by sub-cloning the PCR amplified full length human XLOC_010588 cDNA into the pReceiver plasmid. Transfection of plasmids into HeLa and SiHa cells Table 1 The association of XLOC_010588 expression with clinicopathological features in cervical cancer. Clinicopathological
Number of case
XLOC_010588 expression Low
%
High
P value
%
Age ≤35 N35
91 127
42 67
19.3 30.7
49 60
22.5 27.5
0.336
Histology Squamous Adenocarcinoma
190 28
94 15
43.1 6.9
96 13
44 6
0.686
FIGO stage IB1 IB2-IIA IIb
155 42 21
53 36 20
24.3 16.5 9.1
102 6 1
46.8 2.8 0.5
b0.0001
Differentiation Well Moderate Poor
11 134 70
8 64 36
3.7 29.8 16.7
3 70 34
1.4 32.6 15.8
0.273
SCC-Ag (μg/L) ≤4 N4
168 25
78 19
40.4 9.9
90 6
46.6 3.1
0.006
Tumor diameter (cm) ≤4 158 N4 60
66 43
30.3 19.7
92 17
42.2 7.8
b0.0001
Depth of cervical invasion b2/3 111 ≥2/3 101
50 56
23.6 26.4
61 45
28.8 21.2
0.130
Uterine corpus invasion Negative 69 Positive 146
33 74
15.3 34.4
36 72
16.8 33.5
0.696
Lymphoid node Negative Positive
80 29
36.7 13.3
91 18
41.7 8.3
0.070
171 47
Pearson chi-square. Bold indicates significant values.
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
L.-M. Liao et al. / Gynecologic Oncology xxx (2014) xxx–xxx
C
20 18 16 14 12 10 8 6 4 2 0
Relative expression of XLC_010588O
Relative expression of XLOC_010588
A
T
Relative expression of XLO_010588C
B
3
20 18 16 14 12 10 8 6 4 2 0 ≤ 4 cm
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> 4 cm
D
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Fig. 1. The XLOC_010588 expression levels were detected in 218 cervical cancer samples and adjacent normal tissues by qRT-PCR. (A) XLOC_010588 expression was significantly downregulated in cancerous tissues compared with that in corresponding normal tissues. (B) XLOC_010588 expression was significantly lower in tumors of higher FIGO stage than that in tumors of lower FIGO stage. (C) XLOC_010588 expression was significantly lower in tumor size N 4 cm group than that in tumor size ≤ 4 cm group. (D) Receiver operating characteristic (ROC) curve analysis was employed to determine whether XLOC_010588 are really good candidates to discriminate tumor tissues from nontumorous tissues. Expression levels are normalized to GAPDH. Error bars represent SE.
was carried out using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer's instructions.
Overall survival rate (%)e
A 100 90 80 70 60 50 40 30 20 10 0
siRNA transfection XLOC_010588 -Low XLOC_010588 -High
P = 0.004 0
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Well differentiated cervical carcinoma HCC94 cells were transfected with 25 nM non-targeting siRNA control, or synthetic siRNAs against XLOC_010588 (Guangzhou RiboBio Co., Ltd.) for 48 h using Lipofectamine transfection reagent (Invitrogen) following the manufacturer's protocol. Following transfection, the cells were harvested and XLOC_010588 knockdown was confirmed by qRT-PCR.
Months MTT assays
Disease free survival (%)
B 100 90 80 70 60 50 40 30 20 10 0
Cells (2000 cells/well) were seeded in 96-well plates and stained at specified time points with 100 ml sterile MTT dye (0.5 mg/ml, Sigma) for 4 h at 37 °C. The culture medium was removed and 150 ml of dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) was added. The absorbance was measured at 570 nm, with 655 nm as the reference wavelength. All experiments were performed in triplicate.
XLOC_010588 -Low XLOC_010588 -High
P < 0.001 0
10
20
30
40
50
60
Months Fig. 2. Kaplan–Meier survival curve analysis shows that patients with lower expression of XLOC_010588 showed decreased (A) overall survival and (B) disease free survival compared with patients with lower expression of XLOC_010588. P-value was calculated by log-rank test.
BrdU labeling and immunofluorescence Cells were grown on coverslips (Fisher, Pittsburgh, PA, USA) and incubated with BrdU for 1 h and stained with anti-BrdU antibody (Upstate, Temecula, CA, USA), according to the manufacturer's instructions. Gray level images were acquired under a laser scanning microscope (Axioskop 2 plus, Carl Zeiss Co. Ltd., Jena, Germany).
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
HL HeLa
7 6 5 4 3 2 1 0
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Relative MTT (OD)
Relative expression of XLOC_010588
A
Relative expression of XLOC_010588
L.-M. Liao et al. / Gynecologic Oncology xxx (2014) xxx–xxx
Relative expression of XLOC_010588
4
0
1
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Days
3
Days
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6
0
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Fig. 3. XLOC_010588 inhibited the proliferation of cervical carcinoma cells in vitro. (A) HeLa and SiHa cells were transiently transfected with pReceive-XLOC_010588 or control vector for 48 h. HCC94 cells were transfected with XLOC_010588 siRNA or control non-targeting siRNA for 48 h. qRT-PCR analysis was performed to examine XLOC_010588 RNA levels in HeLa, SiHa cells (two Left panels), and HCC94 cells (right panel). (B) HeLa and SiHa cells were transfected with pReceive-XLOC_010588 or pReceive plasmid for 5 days. HCC94 cells were transfected with XLOC_010588 siRNA or control siRNA for 5 days. Cell viability was measured using the MTT cell growth assay. The results shown are from three independent experiments and reported as the mean ± SD. *, P b 0.05; **, P b 0.01,***, P b 0.001.
Western blotting The western blotting analysis was performed according to standard methods, as previously described [14], using anti-c-Myc antibodies (Cell Signaling Technology, Danvers, MA, USA). The polyvinylidene fluoride membranes were stripped and re-blotted with an anti-GAPDH monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) as a loading control.
PCR. XLOC_010588 expression was significantly down-regulated in cancerous tissues compared with corresponding normal tissues (0.472 ± 0.086, P b 0.0001) (Fig. 1A). Furthermore, the results revealed a significant association between XLOC_010588 down-regulation and advanced FIGO stage (Fig. 1B), tumor size (Fig. 1C). ROC curve analysis showed that the XLOC_010588 expression is a good candidate to discriminate tumor tissues from nontumorous tissues (sensitivity: 84.4%, specificity: 86.7%). The areas under the ROC curve (AUC) are 0.918 (95% CI: 0.889–0.942, P b 0.0001) (Fig. 1D).
Statistical analysis Statistical analysis was carried out with SPSS software package (IBM, standard version 18.0). The gene expression levels of XLOC_010588 in tumor were compared with normal adjacent mucosa by Wilcoxon test. The relationship between XLOC_010588 expression and clinicopathological characteristics was assessed using Pearson's χ2 test. Survival curves were estimated by the Kaplan–Meier method. The logrank test was used to estimate the statistical differences between survival curves. Cox proportional hazards analysis was performed to calculate the hazard ratio (HR) and the 95% confidence interval (CI) to evaluate the association between XLOC_010588 expression and survival. Multivariate survival analysis was carried out for all of the parameters that were significant in the univariate analysis using the Cox regression model. A two-sided P value of b0.05 was considered statistically significant. Results
Correlations between the expression of XLOC_010588 and the clinicopathological factors in cervical cancer To identify the clinical relevance of XLOC_010588 expression in cervical cancer, correlation between XLOC_010588 expression and clinicopathological parameters such as age, histology, FIGO stage, tumor differentiation, SCC-Ag, tumor size, depth of cervical invasion, uterine corpus invasion and lymph-node metastasis was examined. The 218 human cervical cancer tissues were further classified into high — XLOC_010588 group (n = 109) and low — XLOC_010588 group (n = 109) according to the median XLOC_010588 expression level. The results showed that decreased XLOC_010588 expression was significantly correlated with FIGO stage (P b 0.0001), tumor size (P b 0.0001) and SCC-Ag (μg/L) (P = 0.006), but not with other clinical characteristics (Table 1). Association between XLOC_010588 expression and prognosis of cervical cancer patients
XLOC_010588 was lowly expressed in cervical cancer The XLOC_010588 expression levels were detected in 218 cervical cancer samples and adjacent, histologically normal tissues by qRT-
Overall survival curves and disease free survival curves in highXLOC_010588 group and low-XLOC_010588 group were shown in Fig. 2A, B. As was expected, patients with XLOC_010588 lower expression
Fig. 4. 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay shown XLOC_010588 inhibited the proliferation of cervical carcinoma cells. (A) HeLa and (B) SiHa cells were transiently transfected with pReceive-XLOC_010588 or control vector for 48 h. (C) HCC94 cells were transfected with XLOC_010588 siRNA or control non-targeting siRNA for 48 h. The cells were fixed, subjected to BrdU staining and visualized under a fluorescence microscope. Data were obtained from three independent experiments and showed similar results. Red: BrdU; Blue: DAPI. (D) Quantification of BrdU-incorporating cells in BrdU incorporation assay. The results shown are from three independent experiments and reported as the mean ± SD. *, P b 0.05; **, P b 0.01,***; P b 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
L.-M. Liao et al. / Gynecologic Oncology xxx (2014) xxx–xxx
BrdU
Merge
HeLa
*
pReceiver
pReceiver-XLOC_010588
100 90 80 70 60 50 40 30 20 10 0
SiHa
**
pReceiver
pReceiver-XLOC_010588
BrdU positive cell (%)
100 90 80 70 60 50 40 30 20 10 0
BrdU positive cell (%)
D
BrdU positive cell (%)
XLOC_010588 siRNA
Control siRNA
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pReceiver-XLOC_010588
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HCC94
pReceiver
HeLa
A
pReceiver-XLOC_010588
pReceiver
DAPI
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100 90 80 70 60 50 40 30 20 10 0
HCC94 **
Control-siRNA
XLOC_010588-siRNA
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
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Relative mRNA expressionA of c-MycM
A 1.2 1.0
pReceive-XLOC_010588
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HCC94 C-Myc
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B1.2
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R2 = 0.696 P < 0.0001
0
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HCC94
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CT (XLOC_010588)- CT(GAPDH)
Fig. 5. XLOC_010588 downregulates the expression of c-Myc through interacting c-Myc mRNA. c-Myc mRNA levels after the transfection of pReceive-XLOC_010588 or pReceive-control into HeLa (A) and SiHa (B) cells. (C) c-Myc mRNA levels after the transfection of XLOC_010588 siRNA or control siRNA into HCC cells. (D) c-Myc protein levels after the transfection of pReceive-XLOC_010588 or pReceive-control into HeLa and SiHa cells and transfection of XLOC_010588 siRNA or control siRNA into HCC cells. The numbers under panel of the c-Myc protein expression are quantification analyses of pReceive-XLOC_010588/pReceive-control or XLOC_010588 siRNA/control siRNA ratio by Image J. (E) The XLOC_010588 and c-Myc expression levels were significantly correlated in 50 cervical cancer tissues. The XLOC_010588 and c-Myc expression level in these tissues were measured by real-time qRT-PCR, and the respective ΔCt values (both XLOC_010588 and c-Myc were normalized to GAPDH) were subjected to a Pearson correlation analysis (r2 = 0.696, P b 0.0001). Data are presented as the mean ± SD based on at least three independent experiments. **P b 0.01, ***P b 0.001.
have shown significantly poorer overall survival (P = 0.004) and disease free survival (P b 0.001) than those with lower XLOC_010588 expression. Univariate and multivariate analyses show that XLOC_010588 expression is an independent predictor for overall survival As shown in Supplemental Table 1, univariate analysis identified four prognostic factors: FIGO stage (IB2-IIB vs. IB1), depth of cervical invasion (≥ 2/3 vs. b2/3), lymph-node (positive vs. negative) and XLOC_010588 expression (high vs low). Other clinicopathological characteristics, such as age, histology, differentiation, SCC-Ag, tumor size and uterine corpus invasion, were not statistically significant prognosis factors. When it comes to multivariate, Cox regression model revealed that only FIGO stage (P = 0.005, HR = 1.920, 95% CI = 1.215–3.032), lymph-node metastasis (P = 0.019, HR = 2.436, 95% CI = 1.159–5.120) and XLOC_010588 expression level (P = 0.015, HR = 0.401, 95% CI = 0.193–0.836) served as independent prognostic factors for poor overall survival.
pReceive (control vector), and cell viability was examined using the MTT cell growth assay. Over-expression of XLOC_010588, as confirmed by qRT-PCR (Fig. 3A, left two panels), significantly inhibited the growth of HeLa or SiHa cells compared with control (Fig. 3B, left two panels). To study whether down-regulation of XLOC_010588 promotes cervical cancer cell proliferation, XLOC_010588 was knocked down by siRNA in the well-differentiated HCC94 cells which have high levels of endogenous XLOC_010588 (Fig. 3A, right panel). siRNA-XLOC_010588 transfection decreased the growth of HCC94 cells (Fig. 3B, right panel). These results indicate that XLOC_010588 inhibits the proliferation of cervical cancer cells in vitro. The mechanism underlying the inhibition of cellular proliferation by XLOC_010588 was investigated by a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. As shown in Fig. 4A–C, upregulation of XLOC_010588 in HeLa or SiHa cell lines significantly decreased the percentage of BrdU-positive cells. By contrast, downregulation of XLOC_010588 in HCC94 cell lines significantly increased the percentage of BrdU-positive cells. Quantification of BrdU-incorporating cells in HeLa, SiHa and HCC94 was shown in Fig. 4D.
XLOC_010588 inhibits cell proliferation in cervical carcinoma cell lines in vitro
XLOC_010588 downregulates the expression of c-Myc
Based on the result that decreased XLOC_010588 expression was significantly correlated with tumor size (P b 0.0001) in cervical cancer, we hypothesized that XLOC_010588 is involved in the regulation of cervical cancer proliferation. We tested this hypothesis in cell lines HeLa and SiHa, which express very low levels of XLOC_010588. HeLa or SiHa cells were transiently transfected with pReceive-XLOC_010588 or
As shown in previous studies, c-Myc is regarded as an oncoprotein and is upregulated in cervical cancer [15–20]. Moreover, we found that XLOC_010588 could inhibit cervical cancer cell proliferation in current study. To understand the molecular mechanism by which XLOC_010588 inhibits cervical cancer cell proliferation, we examined whether c-Myc was a downstream XLOC_010588 effector that mediates
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
L.-M. Liao et al. / Gynecologic Oncology xxx (2014) xxx–xxx
the function of XLOC_010588 in cervical cancer. As depicted in Fig. 5A–D, enhanced XLOC_010588 expression significantly decreased the mRNA and protein levels of c-Myc in HeLa, SiHa and HCC94 cells, while the inhibition of XLOC_010588 expression significantly increased the mRNA and protein levels of c-Myc in HeLa, SiHa and HCC94 cells. Because XLOC_010588 downregulates the expression of c-Myc, we investigated whether a correlation exists between c-Myc expression and the XLOC_010588 level in cervical cancer tissues. We performed a correlation analysis of the c-Myc mRNA and XLOC_010588 expression levels in the same cervical cancer tissues. A significant correlation was found between c-Myc mRNA and XLOC_010588 (r 2 = 0.696, P b 0.0001, Fig. 5E), supporting the role of XLOC_010588 in the expression of c-Myc.
Discussion LncRNAs have been recently implicated as having oncogenic [21] and tumor suppressor roles [22]. In this study, we found low expression of XLOC_010588 in cervical cancer tissues compared to that in adjacent normal tissues. To our knowledge, it is the first time to show that lncRNA XLOC_010588 expression was downregulated in cervical cancer tumor tissues than that in the adjacent normal tissues. Besides, we showed that decreased XLOC_010588 expression was correlated with advanced FIGO stage. It is more important that we also confirmed that XLOC_010588 expression was an independent predictor for overall survival. These results suggested that lncRNA XLOC_010588 might be involved in the development and progression of cervical cancer. Recently, a number of LncRNAs have been implicated in various types of cancer. The LncRNA MALAT-1 has been found to promote cell motility in lung adenocarcinoma cells [23]. HOTAIR has been identified to overexpression in a variety of cancers such as breast, colon, pancreatic and lung cancers [24–27], and enhanced HOTAIR expression in patients has been correlated with breast and colon cancer metastases [28–30]. PRNCR1 have been identified for their involvement in prostate carcinogens [31]. Although evidence of carcinogenicity of these lncRNAs is strong, the molecular mechanism by which tumor development and metastasis are promoted is not fully understood. The detailed mechanisms by which XLOC_010588 inhibits tumor growth remain to be determined. In this study, we found that XLOC_010588 downregulates the expression of c-Myc. A correlation between c-Myc mRNA and XLOC_010588 expression in clinical cervical cancer tissues supports the role of XLOC_010588 in c-Myc expression. The c-Myc gene was found to be the cellular homolog of retroviral v-Myc oncogene and recognized as an important regulator of proliferation, growth, differentiation and apoptosis [32]. Expression of the c-Myc oncogene or its protein product is elevated in virtually all types of malignant diseases [33]. Therefore, the deregulation of Myc expression is a major event in cancer pathogenesis or progression [34]. Groups of genes involved in cell cycle regulation, metabolism, ribosome biogenesis, protein synthesis and mitochondrial function are overrepresented in the c-Myc target gene network. In BrdU incorporation assay of this study, we found upregulation of XLOC_010588 in cervical cancer cells significantly inhibited the DNA replication. By contrast, downregulation of XLOC_010588 in cervical cancer cells significantly promoted the DNA replication. Therefore, we speculated that XLOC_010588 could bind c-Myc mRNA and decrease its expression, thereby inducing cell cycle arrest and promoting cell proliferation in cervical cancer. However, further studies are needed to establish what role XLOC_010588 has in the regulation of the critical transcriptional factor c-Myc. In conclusion, for the first time, we indicate that XLOC_010588 is a prognosis factor for cervical cancer which could decrease c-Myc expression, and when upregulated, it inhibits the growth of cervical cancer. These findings indicate that XLOC_010588 is an important molecular marker for determining prognosis and has the potential to be an important target for cervical cancer therapy.
7
Conflict of interest statement The authors declare that they have no competing interests.
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Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
Contents lists available at ScienceDirect
Gynecologic Oncology journal homepage: www.elsevier.com/locate/ygyno
Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer Ling-Min Liao a,1, Xiao-Ying Sun b,1, An-Wen Liu c, Jian-Bing Wu c, Xiao-Ling Cheng d, Jia-Xin Lin e, Min Zheng e, Long Huang c,⁎ a
Department of Ultrasound, The Second Affiliated Hospital of Nanchang University, Nanchang, China Department of Gynecology and Obstetrics, Central Hospital Attached to Shenyang Medical College, Shenyang, China c Department of Oncology, The Second Affiliated Hospital of Nanchang University, Nanchang, China d Department of Medical Imaging, Women And Children Health Institute Futian, Shenzhen, China e Department of Gynecology, Cancer Center, Sun Yat-sen University, Guangzhou, China b
H I G H L I G H T S • LncRNA XLOC_010588 expression was downregulated in cervical cancer tissues. • Low expression of XLOC_010588 was an independent predictor for overall survival. • Ectopic expression of XLOC_010588 inhibited cervical cancer cell proliferation through downregulation of c-Myc.
a r t i c l e
i n f o
Article history: Received 12 November 2013 Accepted 16 March 2014 Available online xxxx Keywords: Cervical cancer Long noncoding RNA XLOC_010588 c-Myc
a b s t r a c t Objective. The identification and investigation of cancer-associated long non-coding RNAs are important for understanding the molecular biology of cancer. The aim of the present study was to examine the expression pattern of lncRNA XLOC_010588 in cervical cancer and to evaluate its biological role and clinical significance in tumor progression. Methods. We examined the expression of XLOC_010588 in 218 cervical cancer tissues and matched 218 adjacent normal tissues using real-time qRT-PCR. Over-expression and RNA interference approaches were used to investigate the biological functions of XLOC_010588. The effect of XLOC_010588 on proliferation was evaluated by MTT and BrdU assays. Western blot assays were used to investigate the molecular mechanism by which XLOC_010588 increases cervical cancer cell proliferation. Results. The results showed that XLOC_010588 expression in cervical cancer was significantly downregulated. Decreased XLOC_010588 expression was correlated with FIGO stage, tumor size and SCC-Ag. Moreover, cervical cancer patients with XLOC_010588 lower expression have shown poorer prognosis. Multivariate Cox regression analyses showed that XLOC_010588 expression served as an independent predictor for overall survival. Ectopic expression of XLOC_010588 inhibited the proliferation of HeLa and SiHa cells. By contrast, knockdown of XLOC_010588 promoted the growth of HCC94 cells. Western blot assays confirmed that XLOC_010588 physically associates with c-Myc, consequently decreasing the expression of c-Myc. The expression of XLOC_010588 and c-Myc is strongly correlated in cervical cancer tissues. Conclusion. These results suggested that XLOC_010588 plays a pivotal role in cervical cancer cell proliferation via decreasing c-Myc expression and implicated the potential application of XLOC_010588 in cervical cancer therapy. © 2014 Elsevier Inc. All rights reserved.
Introduction ⁎ Corresponding author at: Department of Oncology, The Second Affiliated Hospital of Nanchang University, 1 Minde Road, Nanchang, Jiangxi, China. Fax: +86 20 87343627. E-mail address: [email protected] (L. Huang). 1 Ling-Min Liao and Xiao-Ying Sun contributed equally to this work and should be regarded as joint first authors.
Although the incidence of cervical cancer has been greatly reduced with the application of pap smear screening, cervical cancer is still the third most commonly diagnosed cancer in women worldwide, with estimated 529,800 new cases diagnosed annually [1]. Moreover, higher
http://dx.doi.org/10.1016/j.ygyno.2014.03.555 0090-8258/© 2014 Elsevier Inc. All rights reserved.
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incidence rate and mortality rate are observed in developing countries versus developed countries [2]. Therefore, it is still urgent for us to find new and effective therapeutic strategies for cervical cancer. Recent genome sequencing studies have revealed that the human genome is comprised of less than 2% protein coding genes, and more than 90% of the genome is transcribed as non-coding RNAs [3,4]. These ncRNAs are classified into two groups depending on the nucleotide size. Those under 200 nucleotides in length are referred to as small ncRNAs, including the microRNAs (approximately 18–25 nucleotides in length) which have been intensively studied [5]. However, the long non-coding RNAs that consist of more than 200 nucleotides have relevant limited investigations [6]. The lncRNA LOC285194 is 1950 nt in length, located in chr13 (start: 44974183; end: 44980091; − strand), consisting of three exons. LOC285194 located on the downstream side of TGFb-stimulated clone22 (TSC-22) gene and is transcripted from the antisense direction of it. TSC-22 was originally identified as a TGF immediate early response gene in mouse osteoblasts [7]. Several investigations have postulated an important role for TSC-22 in embryogenesis [8–10]. Other studies have suggested a role for TSC-22 as tumor suppressor [11,12]. TSC-22 has DNA binding properties and may act as a transcriptional repressor [13]. In our previous study, we identified the lncRNA XLOC_010588 low expression in hepatocellular carcinoma by a microarray fabricated in house. However, little is known about the expression and impact of XLOC_010588 in other cancers. We examined the expression of lncRNA XLOC_010588 in gastric cancer, colon cancer, breast cancer, cervical cancer and ovarian cancer using real time qRT-PCR. The results revealed that XLOC_010588 expression was significantly down-regulated in cervical cancer and served as adverse prognostic factors. To better understand the role of XLOC_010588 in cervical cancer development, we investigated the expression pattern of lncRNA XLOC_010588 in cervical cancer tissues and cells and studied its biological role and clinical significance in tumor progression. Materials and methods Patients and sample collection Two hundred and eighteen cervical cancer tumors and 218 matched adjacent normal tissues were obtained from Sun Yat-sen University Cancer Center (SYSUCC) (Guangzhou, China) between January 2005 and October 2009. All patients recruited in this study were not subjected to preoperative radiotherapy and/or chemotherapy and were diagnosed as infiltrating carcinoma by pathology. The tumor stage was classified by two experienced gynecological oncologists according to the International Federation of Gynecology and Obstetrics (FIGO) staging system for cervical cancer. Clinical and pathological variables analyzed are shown in Table 1. All patients were regularly followed up, with a mean observation period of 42 months (range: 2–55 months). Tissue specimens were immediately stored at 4 °C for 24 h in RNA Later (Ambion Inc.), then at − 80 °C liquid nitrogen until the extraction of total RNA. Total RNA from fresh cervical cancer tissues was isolated using Trizol reagent (Invitrogen) according to the manufacturer's instructions. The study was approved by the medical ethics committee of Sun Yat-Sen University Cancer Center. Quantitative real-time reverse transcriptase PCR The quality and concentration of RNA were determined using a Nanodrop 1000 (Thermo, Wilmington, DE, USA). Then, 1 μg total RNA was reversely transcribed from each sample to synthesis cDNA by the RT reagent Kit (Promega, USA, A3500) according to the manufacturer's instructions with minor modification. Real-time PCR was performed to determine the relative expression level of target genes by the SYBR Green qRT-PCR kit (Promega, USA, A6001) on the CFX96 Real Time PCR Detection System (Bio-Rad). The level of XLOC_010588 expression
in each sample was normalized to the respective GAPDH expression level. The primers used were as follows: XLOC_010588 sense, 5′-TGTG AAGAGGAGAACATAAAAGG-3′ and antisense, 5′-AAGCAAGATAATAC AGTGGCGA-3′; c-Myc sense, 5′-CGCGCTG AGTATAAAAGCCG-3′ and antisense, 5′-CTATTCGCTCCGGATCTCCC-3′; and GAPDH sense, 5′-CTCCTC CTGTTCGACAGTCAGC-3′ and antisense, 5′-CCCAATACGACCAAATCC GTT-3′. The amplification profile was 95 °C for 5 min, followed by 42 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 60 s. The specificity of each PCR reaction was confirmed by melting curve analyses. The comparative Ct method (ΔΔCt) was used for quantification of the gene expression. The relative expression amount of XLOC_010588 to GAPDH was calculated using the equation 2−ΔΔCt, where ΔCt = Ct XLOC_010588 − Ct GAPDH. To minimize experimental variability, each sample was analyzed in triplicate and the mean expression level was calculated. Cell culture Cervical cancer cell lines (HeLa, SiHa and HCC94) were grown in DMEM medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA) and 1% penicillin– streptomycin. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. Plasmid construction A XLOC_010588 expression construct was generated by sub-cloning the PCR amplified full length human XLOC_010588 cDNA into the pReceiver plasmid. Transfection of plasmids into HeLa and SiHa cells Table 1 The association of XLOC_010588 expression with clinicopathological features in cervical cancer. Clinicopathological
Number of case
XLOC_010588 expression Low
%
High
P value
%
Age ≤35 N35
91 127
42 67
19.3 30.7
49 60
22.5 27.5
0.336
Histology Squamous Adenocarcinoma
190 28
94 15
43.1 6.9
96 13
44 6
0.686
FIGO stage IB1 IB2-IIA IIb
155 42 21
53 36 20
24.3 16.5 9.1
102 6 1
46.8 2.8 0.5
b0.0001
Differentiation Well Moderate Poor
11 134 70
8 64 36
3.7 29.8 16.7
3 70 34
1.4 32.6 15.8
0.273
SCC-Ag (μg/L) ≤4 N4
168 25
78 19
40.4 9.9
90 6
46.6 3.1
0.006
Tumor diameter (cm) ≤4 158 N4 60
66 43
30.3 19.7
92 17
42.2 7.8
b0.0001
Depth of cervical invasion b2/3 111 ≥2/3 101
50 56
23.6 26.4
61 45
28.8 21.2
0.130
Uterine corpus invasion Negative 69 Positive 146
33 74
15.3 34.4
36 72
16.8 33.5
0.696
Lymphoid node Negative Positive
80 29
36.7 13.3
91 18
41.7 8.3
0.070
171 47
Pearson chi-square. Bold indicates significant values.
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
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C
20 18 16 14 12 10 8 6 4 2 0
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T
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20 18 16 14 12 10 8 6 4 2 0 ≤ 4 cm
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> 4 cm
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Fig. 1. The XLOC_010588 expression levels were detected in 218 cervical cancer samples and adjacent normal tissues by qRT-PCR. (A) XLOC_010588 expression was significantly downregulated in cancerous tissues compared with that in corresponding normal tissues. (B) XLOC_010588 expression was significantly lower in tumors of higher FIGO stage than that in tumors of lower FIGO stage. (C) XLOC_010588 expression was significantly lower in tumor size N 4 cm group than that in tumor size ≤ 4 cm group. (D) Receiver operating characteristic (ROC) curve analysis was employed to determine whether XLOC_010588 are really good candidates to discriminate tumor tissues from nontumorous tissues. Expression levels are normalized to GAPDH. Error bars represent SE.
was carried out using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer's instructions.
Overall survival rate (%)e
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siRNA transfection XLOC_010588 -Low XLOC_010588 -High
P = 0.004 0
10
20
30
40
50
60
Well differentiated cervical carcinoma HCC94 cells were transfected with 25 nM non-targeting siRNA control, or synthetic siRNAs against XLOC_010588 (Guangzhou RiboBio Co., Ltd.) for 48 h using Lipofectamine transfection reagent (Invitrogen) following the manufacturer's protocol. Following transfection, the cells were harvested and XLOC_010588 knockdown was confirmed by qRT-PCR.
Months MTT assays
Disease free survival (%)
B 100 90 80 70 60 50 40 30 20 10 0
Cells (2000 cells/well) were seeded in 96-well plates and stained at specified time points with 100 ml sterile MTT dye (0.5 mg/ml, Sigma) for 4 h at 37 °C. The culture medium was removed and 150 ml of dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) was added. The absorbance was measured at 570 nm, with 655 nm as the reference wavelength. All experiments were performed in triplicate.
XLOC_010588 -Low XLOC_010588 -High
P < 0.001 0
10
20
30
40
50
60
Months Fig. 2. Kaplan–Meier survival curve analysis shows that patients with lower expression of XLOC_010588 showed decreased (A) overall survival and (B) disease free survival compared with patients with lower expression of XLOC_010588. P-value was calculated by log-rank test.
BrdU labeling and immunofluorescence Cells were grown on coverslips (Fisher, Pittsburgh, PA, USA) and incubated with BrdU for 1 h and stained with anti-BrdU antibody (Upstate, Temecula, CA, USA), according to the manufacturer's instructions. Gray level images were acquired under a laser scanning microscope (Axioskop 2 plus, Carl Zeiss Co. Ltd., Jena, Germany).
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
HL HeLa
7 6 5 4 3 2 1 0
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Relative MTT (OD)
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Relative MTT (OD)
Relative expression of XLOC_010588
A
Relative expression of XLOC_010588
L.-M. Liao et al. / Gynecologic Oncology xxx (2014) xxx–xxx
Relative expression of XLOC_010588
4
0
1
2
Days
3
Days
4
5
6
0
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2
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4
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6
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Fig. 3. XLOC_010588 inhibited the proliferation of cervical carcinoma cells in vitro. (A) HeLa and SiHa cells were transiently transfected with pReceive-XLOC_010588 or control vector for 48 h. HCC94 cells were transfected with XLOC_010588 siRNA or control non-targeting siRNA for 48 h. qRT-PCR analysis was performed to examine XLOC_010588 RNA levels in HeLa, SiHa cells (two Left panels), and HCC94 cells (right panel). (B) HeLa and SiHa cells were transfected with pReceive-XLOC_010588 or pReceive plasmid for 5 days. HCC94 cells were transfected with XLOC_010588 siRNA or control siRNA for 5 days. Cell viability was measured using the MTT cell growth assay. The results shown are from three independent experiments and reported as the mean ± SD. *, P b 0.05; **, P b 0.01,***, P b 0.001.
Western blotting The western blotting analysis was performed according to standard methods, as previously described [14], using anti-c-Myc antibodies (Cell Signaling Technology, Danvers, MA, USA). The polyvinylidene fluoride membranes were stripped and re-blotted with an anti-GAPDH monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) as a loading control.
PCR. XLOC_010588 expression was significantly down-regulated in cancerous tissues compared with corresponding normal tissues (0.472 ± 0.086, P b 0.0001) (Fig. 1A). Furthermore, the results revealed a significant association between XLOC_010588 down-regulation and advanced FIGO stage (Fig. 1B), tumor size (Fig. 1C). ROC curve analysis showed that the XLOC_010588 expression is a good candidate to discriminate tumor tissues from nontumorous tissues (sensitivity: 84.4%, specificity: 86.7%). The areas under the ROC curve (AUC) are 0.918 (95% CI: 0.889–0.942, P b 0.0001) (Fig. 1D).
Statistical analysis Statistical analysis was carried out with SPSS software package (IBM, standard version 18.0). The gene expression levels of XLOC_010588 in tumor were compared with normal adjacent mucosa by Wilcoxon test. The relationship between XLOC_010588 expression and clinicopathological characteristics was assessed using Pearson's χ2 test. Survival curves were estimated by the Kaplan–Meier method. The logrank test was used to estimate the statistical differences between survival curves. Cox proportional hazards analysis was performed to calculate the hazard ratio (HR) and the 95% confidence interval (CI) to evaluate the association between XLOC_010588 expression and survival. Multivariate survival analysis was carried out for all of the parameters that were significant in the univariate analysis using the Cox regression model. A two-sided P value of b0.05 was considered statistically significant. Results
Correlations between the expression of XLOC_010588 and the clinicopathological factors in cervical cancer To identify the clinical relevance of XLOC_010588 expression in cervical cancer, correlation between XLOC_010588 expression and clinicopathological parameters such as age, histology, FIGO stage, tumor differentiation, SCC-Ag, tumor size, depth of cervical invasion, uterine corpus invasion and lymph-node metastasis was examined. The 218 human cervical cancer tissues were further classified into high — XLOC_010588 group (n = 109) and low — XLOC_010588 group (n = 109) according to the median XLOC_010588 expression level. The results showed that decreased XLOC_010588 expression was significantly correlated with FIGO stage (P b 0.0001), tumor size (P b 0.0001) and SCC-Ag (μg/L) (P = 0.006), but not with other clinical characteristics (Table 1). Association between XLOC_010588 expression and prognosis of cervical cancer patients
XLOC_010588 was lowly expressed in cervical cancer The XLOC_010588 expression levels were detected in 218 cervical cancer samples and adjacent, histologically normal tissues by qRT-
Overall survival curves and disease free survival curves in highXLOC_010588 group and low-XLOC_010588 group were shown in Fig. 2A, B. As was expected, patients with XLOC_010588 lower expression
Fig. 4. 5-Bromo-2′-deoxyuridine (BrdU) incorporation assay shown XLOC_010588 inhibited the proliferation of cervical carcinoma cells. (A) HeLa and (B) SiHa cells were transiently transfected with pReceive-XLOC_010588 or control vector for 48 h. (C) HCC94 cells were transfected with XLOC_010588 siRNA or control non-targeting siRNA for 48 h. The cells were fixed, subjected to BrdU staining and visualized under a fluorescence microscope. Data were obtained from three independent experiments and showed similar results. Red: BrdU; Blue: DAPI. (D) Quantification of BrdU-incorporating cells in BrdU incorporation assay. The results shown are from three independent experiments and reported as the mean ± SD. *, P b 0.05; **, P b 0.01,***; P b 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
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BrdU
Merge
HeLa
*
pReceiver
pReceiver-XLOC_010588
100 90 80 70 60 50 40 30 20 10 0
SiHa
**
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BrdU positive cell (%)
100 90 80 70 60 50 40 30 20 10 0
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BrdU positive cell (%)
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Control siRNA
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pReceiver-XLOC_010588
pReceiver
DAPI
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XLOC_010588-siRNA
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
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Relative mRNA expressionA of c-MycM
A 1.2 1.0
pReceive-XLOC_010588
0.8
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HCC94 C-Myc
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Relative mRNA expressionm of c-Myc
B1.2
20
15
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5
R2 = 0.696 P < 0.0001
0
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HCC94
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20
CT (XLOC_010588)- CT(GAPDH)
Fig. 5. XLOC_010588 downregulates the expression of c-Myc through interacting c-Myc mRNA. c-Myc mRNA levels after the transfection of pReceive-XLOC_010588 or pReceive-control into HeLa (A) and SiHa (B) cells. (C) c-Myc mRNA levels after the transfection of XLOC_010588 siRNA or control siRNA into HCC cells. (D) c-Myc protein levels after the transfection of pReceive-XLOC_010588 or pReceive-control into HeLa and SiHa cells and transfection of XLOC_010588 siRNA or control siRNA into HCC cells. The numbers under panel of the c-Myc protein expression are quantification analyses of pReceive-XLOC_010588/pReceive-control or XLOC_010588 siRNA/control siRNA ratio by Image J. (E) The XLOC_010588 and c-Myc expression levels were significantly correlated in 50 cervical cancer tissues. The XLOC_010588 and c-Myc expression level in these tissues were measured by real-time qRT-PCR, and the respective ΔCt values (both XLOC_010588 and c-Myc were normalized to GAPDH) were subjected to a Pearson correlation analysis (r2 = 0.696, P b 0.0001). Data are presented as the mean ± SD based on at least three independent experiments. **P b 0.01, ***P b 0.001.
have shown significantly poorer overall survival (P = 0.004) and disease free survival (P b 0.001) than those with lower XLOC_010588 expression. Univariate and multivariate analyses show that XLOC_010588 expression is an independent predictor for overall survival As shown in Supplemental Table 1, univariate analysis identified four prognostic factors: FIGO stage (IB2-IIB vs. IB1), depth of cervical invasion (≥ 2/3 vs. b2/3), lymph-node (positive vs. negative) and XLOC_010588 expression (high vs low). Other clinicopathological characteristics, such as age, histology, differentiation, SCC-Ag, tumor size and uterine corpus invasion, were not statistically significant prognosis factors. When it comes to multivariate, Cox regression model revealed that only FIGO stage (P = 0.005, HR = 1.920, 95% CI = 1.215–3.032), lymph-node metastasis (P = 0.019, HR = 2.436, 95% CI = 1.159–5.120) and XLOC_010588 expression level (P = 0.015, HR = 0.401, 95% CI = 0.193–0.836) served as independent prognostic factors for poor overall survival.
pReceive (control vector), and cell viability was examined using the MTT cell growth assay. Over-expression of XLOC_010588, as confirmed by qRT-PCR (Fig. 3A, left two panels), significantly inhibited the growth of HeLa or SiHa cells compared with control (Fig. 3B, left two panels). To study whether down-regulation of XLOC_010588 promotes cervical cancer cell proliferation, XLOC_010588 was knocked down by siRNA in the well-differentiated HCC94 cells which have high levels of endogenous XLOC_010588 (Fig. 3A, right panel). siRNA-XLOC_010588 transfection decreased the growth of HCC94 cells (Fig. 3B, right panel). These results indicate that XLOC_010588 inhibits the proliferation of cervical cancer cells in vitro. The mechanism underlying the inhibition of cellular proliferation by XLOC_010588 was investigated by a 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. As shown in Fig. 4A–C, upregulation of XLOC_010588 in HeLa or SiHa cell lines significantly decreased the percentage of BrdU-positive cells. By contrast, downregulation of XLOC_010588 in HCC94 cell lines significantly increased the percentage of BrdU-positive cells. Quantification of BrdU-incorporating cells in HeLa, SiHa and HCC94 was shown in Fig. 4D.
XLOC_010588 inhibits cell proliferation in cervical carcinoma cell lines in vitro
XLOC_010588 downregulates the expression of c-Myc
Based on the result that decreased XLOC_010588 expression was significantly correlated with tumor size (P b 0.0001) in cervical cancer, we hypothesized that XLOC_010588 is involved in the regulation of cervical cancer proliferation. We tested this hypothesis in cell lines HeLa and SiHa, which express very low levels of XLOC_010588. HeLa or SiHa cells were transiently transfected with pReceive-XLOC_010588 or
As shown in previous studies, c-Myc is regarded as an oncoprotein and is upregulated in cervical cancer [15–20]. Moreover, we found that XLOC_010588 could inhibit cervical cancer cell proliferation in current study. To understand the molecular mechanism by which XLOC_010588 inhibits cervical cancer cell proliferation, we examined whether c-Myc was a downstream XLOC_010588 effector that mediates
Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555
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the function of XLOC_010588 in cervical cancer. As depicted in Fig. 5A–D, enhanced XLOC_010588 expression significantly decreased the mRNA and protein levels of c-Myc in HeLa, SiHa and HCC94 cells, while the inhibition of XLOC_010588 expression significantly increased the mRNA and protein levels of c-Myc in HeLa, SiHa and HCC94 cells. Because XLOC_010588 downregulates the expression of c-Myc, we investigated whether a correlation exists between c-Myc expression and the XLOC_010588 level in cervical cancer tissues. We performed a correlation analysis of the c-Myc mRNA and XLOC_010588 expression levels in the same cervical cancer tissues. A significant correlation was found between c-Myc mRNA and XLOC_010588 (r 2 = 0.696, P b 0.0001, Fig. 5E), supporting the role of XLOC_010588 in the expression of c-Myc.
Discussion LncRNAs have been recently implicated as having oncogenic [21] and tumor suppressor roles [22]. In this study, we found low expression of XLOC_010588 in cervical cancer tissues compared to that in adjacent normal tissues. To our knowledge, it is the first time to show that lncRNA XLOC_010588 expression was downregulated in cervical cancer tumor tissues than that in the adjacent normal tissues. Besides, we showed that decreased XLOC_010588 expression was correlated with advanced FIGO stage. It is more important that we also confirmed that XLOC_010588 expression was an independent predictor for overall survival. These results suggested that lncRNA XLOC_010588 might be involved in the development and progression of cervical cancer. Recently, a number of LncRNAs have been implicated in various types of cancer. The LncRNA MALAT-1 has been found to promote cell motility in lung adenocarcinoma cells [23]. HOTAIR has been identified to overexpression in a variety of cancers such as breast, colon, pancreatic and lung cancers [24–27], and enhanced HOTAIR expression in patients has been correlated with breast and colon cancer metastases [28–30]. PRNCR1 have been identified for their involvement in prostate carcinogens [31]. Although evidence of carcinogenicity of these lncRNAs is strong, the molecular mechanism by which tumor development and metastasis are promoted is not fully understood. The detailed mechanisms by which XLOC_010588 inhibits tumor growth remain to be determined. In this study, we found that XLOC_010588 downregulates the expression of c-Myc. A correlation between c-Myc mRNA and XLOC_010588 expression in clinical cervical cancer tissues supports the role of XLOC_010588 in c-Myc expression. The c-Myc gene was found to be the cellular homolog of retroviral v-Myc oncogene and recognized as an important regulator of proliferation, growth, differentiation and apoptosis [32]. Expression of the c-Myc oncogene or its protein product is elevated in virtually all types of malignant diseases [33]. Therefore, the deregulation of Myc expression is a major event in cancer pathogenesis or progression [34]. Groups of genes involved in cell cycle regulation, metabolism, ribosome biogenesis, protein synthesis and mitochondrial function are overrepresented in the c-Myc target gene network. In BrdU incorporation assay of this study, we found upregulation of XLOC_010588 in cervical cancer cells significantly inhibited the DNA replication. By contrast, downregulation of XLOC_010588 in cervical cancer cells significantly promoted the DNA replication. Therefore, we speculated that XLOC_010588 could bind c-Myc mRNA and decrease its expression, thereby inducing cell cycle arrest and promoting cell proliferation in cervical cancer. However, further studies are needed to establish what role XLOC_010588 has in the regulation of the critical transcriptional factor c-Myc. In conclusion, for the first time, we indicate that XLOC_010588 is a prognosis factor for cervical cancer which could decrease c-Myc expression, and when upregulated, it inhibits the growth of cervical cancer. These findings indicate that XLOC_010588 is an important molecular marker for determining prognosis and has the potential to be an important target for cervical cancer therapy.
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Conflict of interest statement The authors declare that they have no competing interests.
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Please cite this article as: Liao L-M, et al, Low expression of long noncoding XLOC_010588 indicates a poor prognosis and promotes proliferation through upregulation of c-Myc in cervical cancer, Gynecol Oncol (2014), http://dx.doi.org/10.1016/j.ygyno.2014.03.555