Upregulation of cervical carcinoma expressed PCNA regulatory long non-coding RNA promotes esophageal squamous cell carcinoma progression.

Cervical carcinoma expressed PCNA regulatory long non-coding (lnc)RNA (CCEPR) has recently been reported to play oncogenic roles in some common types of human cancer. However, the clinical significance of CCEPR mRNA expression levels in esophageal squamous cell carcinoma (ESCC) and the exact function of CCEPR in regulating the malignant phenotypes of ESCC cells have not been previously investigated. In the present study, CCEPR mRNA expression level was upregulated in ESCC tissues and cell lines, and overexpression of CCEPR was associated with advanced TNM stage, lymph node metastasis, and poor prognosis in ESCC. In vitro experiments showed that silencing CCEPR mRNA expression levels significantly suppressed the proliferation, migration, and invasion of ESCC cells, while inducing ESCC cell apoptosis. Furthermore, inhibition of CCEPR decreased the protein expression levels of matrix metalloproteinase (MMP)2 and MMP9 and inhibited epithelial-mesenchymal transition in ESCC cells. In conclusion, the results showed that CCEPR plays an oncogenic role in ESCC and suggests that CCEPR could be used as a potential therapeutic target for ESCC treatment. Copyright: © Wang et al.

Introduction

Esophageal cancer is one of the most common cancers, and esophageal squamous cell carcinoma (ESCC) is the major type of esophageal cancer (1–3). Due to late diagnosis, recurrence and metastasis, the overall survival time of patients with ESCC is not satisfactory (1,4). Therefore it is important to identify the molecular mechanisms and develop novel strategies for ESCC therapy (5,6). Recently, some oncogenes have been reported to be responsible for the growth and metastasis of ESCC, and some of them have been suggested to be potential therapeutic targets (7,8). For instance, microRNA (miR)-130b promotes the progression of ESCC by targeting SASH1, and is suggested to be a therapeutic target for ESCC (7).

Long non-coding (lnc)RNAs, a type of non-coding RNA containing >200 nucleotides, have been demonstrated to play crucial roles in different physiological and pathological processes, such as cell viability, proliferation, migration, invasion, apoptosis, and tumorigenesis (9,10). Furthermore, some lncRNAs are upregulated or downregulated and have promotor or suppressor functions in malignant tumors, including ESCC (11–13). For example, lncRNA TINCR was higher in ESCC tissues compared with that in adjacent normal tissues, and knockdown of lncRNA TINCR expression inhibits the proliferation, migration and invasion of ESCC cells (13). In addition, lncRNA MEG3 induces ESCC cell apoptosis via endoplasmic reticulum stress (14).

Cervical carcinoma expressed PCNA regulatory lncRNA (CCEPR) is localized to chromosome 10q21.1 and has been frequently upregulated in several common types of cancer, such as gastric, liver, cervical, lung, colorectal and bladder cancers (15–20). Overexpression of CCEPR predicts a poor prognosis for cervical cancer (19). Furthermore, several studies have shown that CCEPR plays oncogenic roles by regulating tumor cell proliferation, apoptosis, migration and invasion (16,17). For example, Liao et al (16) showed that CCEPR promoted the proliferation, metastasis and invasion of non-small lung cancer cells, while Peng et al (17) found that knockdown of CCEPR using shRNA significantly induced growth arrest and cell apoptosis in hepatocellular carcinoma. However, to the best of our knowledge, the expression and exact role of CCEPR in ESCC have not been previously reported.

Therefore, the aim of the present study was to investigate the expression level of CCEPR in ESCC. In addition, to determine the function of CCEPR in regulating the malignant phenotypes of ESCC cells in vitro.

Materials and methods

Tissue collection

ESCC and adjacent non-tumor tissues (at least 3 cm from the primary tumor) were collected from 56 patients with ESCC who underwent surgical resection from September 2011 to April 2013. Patients with ESCC who received preoperative chemo- or radiotherapy were excluded from the study. All of the tissues were stored at −80°C until further use. The present study was approved by the Ethics Committee of the First People's Hospital of Chenzhou City (Chenzhou, China). Written informed consent was provided by all the participants prior to the study. The follow-up time was 5 years from surgical resection.

Cell culture and transfection

The human Het-1A esophageal epithelial cell line, and four human ESCC cell lines, TE-9, ECA109, KYSE150 and EC9706, were obtained from the Cell Bank of the Chinese Academy of Sciences. The cell lines were cultured in DMEM with 10% FBS (both from Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2. For cell transfection, the TE-9 and EC9706 cells were transfected with 100 nM CCEPR short hairpin (sh)RNA1 (cat. no. n352462), CCEPR shRNA2 (cat. no. n352463), or negative control (NC) shRNA (cat. no. 4457289) using Lipofectamine® 2000 (all from Thermo Fisher Scientific, Inc.). Reverse transcription-quantitative PCR (RT-qPCR) was performed to examine the mRNA expression level of CCEPR, 48 h following transfection.

RT-qPCR

TRIzol® reagent (Thermo Fisher Scientific, Inc.) was used to extract total RNA from tissues and cell lines. The total RNA was then reversed transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.). The mRNA expression levels were then determined using a SYBR® Green Real-time PCR Master Mix (Toyobo Life Science) on a Roche 480 Real-Time PCR system (Roche Diagnostics). The following thermocycling conditions were used: Initial denaturation at 95°C for 3 min, followed by 40 cycles at 95°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. The relative expression was determined with the 2−ΔΔCq method (21). The primer sequences used were as follows: CCEPR forward, 5′-AAGGTCCCAGGATACTCGC-3′ and reverse, 5′-GTGTCGTGGACTGGCAAAAT-3′; GAPDH forward, 5′-CTGGGCTACACTGAGCACC-3′ and reverse, 5′-AAGTGGTCGTTGAGGGCAATG-3′.

Cell Counting Kit-8 (CCK-8) assays

Transfected TE-9 and EC9706 cells (5,000 cells/well) were seeded in 96-well plates, then cultured for 0, 24, 48 and 72 h. Next, 10 µl CCK-8 solution (Thermo Fisher Scientific, Inc.) was added to the cells and incubated at 37°C for 2 h, according to the manufacturer's instructions. The absorbance (450 nm) was determined using a microplate reader (Bio-Rad Laboratories, Inc.).

Colony formation assay

Transfected TE-9 and EC9706 cells (300 cells/well in 6-well plates) were cultured in DMEM supplemented with 10% FBS for 2 weeks. Then, the cells were stained with a 0.5% crystal violet solution at room temperature for 5 min. The colony numbers (containing >50 cells) were counted.

Cell apoptosis assay

Transfected TE-9 and EC9706 cells were washed with Dulbecco's phosphate (DPBS; Thermo Fisher Scientific, Inc.) and fixed in 75% ethanol at 4°C for 3 h. After washing with PBS, twice, the cells were stained with both FITC Annexin V and propidium iodide (both from BD Biosciences) at room temperature for 30 min. The cell apoptosis rate was examined using a FACScan flow cytometer and analyzed using the Accuri C6 system software v1.0 (both from BD Biosciences).

Cell migration assay

For the cell migration assay, transfected TE-9 and EC9706 cells in 12-well plates (5×105 cells/well) were cultured at 37°C, to ~95% confluence. A 100 µ sterile pipette tip was used to scratch the cells to generate a wound. The cells were washed with DPBS and were added to each well with serum-free DMEM. At this time point (indicated as 0 h), images of the wound were obtained, using a light microscope (×40). Then, the cells were incubated at 37°C for 24 h, and images of the wound were obtained again, under a light microscope (×40). Wound closure rate is calculated based on wound area relative to the original size using ImageJ (version 1.8; National Institutes of Health).

Cell invasion assay

Matrigel-coated Transwell chambers (BD Biosciences, USA) were used to perform the cell invasion assay. The precoating was performed at room temperature for 30 min. In brief, transfected TE-9 and EC9706 cells (50,000 cells/well) in 400 µl serum-free DMEM were added to the upper chamber. Next, 300 µl DMEM with 10% FBS was added to the lower chamber. After incubation at 37°C for 24 h, the invading cells were stained with a 0.1% crystal violet solution at room temperature for 10 min. Then, images were obtained using a light microscope (×200).

Western blot analysis

Total protein was extracted from the tissues and cells using a RIPA buffer (Thermo Fisher Scientific, Inc.). The protein concentrations were determined with a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). The proteins were separated using 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). After blocking with 5% skimmed milk overnight at 4°C, the membranes were incubated with the primary antibodies against caspase-3 (cat. no. ab13847; 1:500), Bcl2 (cat. no. ab32124; 1:200), Bax (cat. no. ab32503; 1:500), matrix metalloproteinase [(MMP)2; cat. no. ab97779; 1:200], MMP9 (cat. no. ab38898; 1:500), N-cadherin (cat. no. ab76057; 1:500), vimentin (cat. no. ab45939; 1:500), E-cadherin (cat. no. ab227639; 1:200), and GAPDH (cat. no. ab9485; 1:500) at room temperature for 5 h and then with the HRP-conjugated goat anti-rabbit secondary antibody (cat. no. ab6721; 1:10,000) at room temperature for 2 h (all antibodies were obtained from Abcam). A western lightning™ chemiluminescence reagent plus kit (Thermo Fisher Scientific, Inc.) was used to visualize the bands, while the ImageJ software v1.48 (National Institutes of Health) was used to determine the protein expression level.

Statistical analysis

The data are expressed as the mean ± SD and the experiments were repeated 3 times. SPSS v19.0 (IBM Corp.) was used for statistical analysis. A one-way ANOVA followed by Tukey's post hoc test was used to analyze the differences between more than two groups, while an unpaired Student's t-test was used to analyze the differences between 2 groups. Based on the median expression value (2.372) of CCEPR, the patients were divided into a high and a low CCEPR expression level group. The associations between CCEPR mRNA expression levels and the clinicopathological characteristics of patients with ESCC were analyzed using a χ2 test. The survival analysis was performed using Kaplan-Meier analysis and log-rank test. P<0.05 was considered to indicate a statistically significant difference.

Results

Upregulation of CCEPR is associated with ESCC progression

In the present study, the mRNA expression levels of CCEPR in ESCC tissues was determined using RT-qPCR. As shown in Fig. 1A, the expression levels of CCEPR were significantly higher in ESCC tissues compared with that in adjacent non-tumor tissues. In addition, the CCEPR mRNA expression level was also significantly higher in ESCC cell lines compared with that in the Het-1A cell line (Fig. 1B).

Figure 1.

Upregulation of CCEPR is associated with ESCC progression. CCEPR mRNA expression levels were higher in (A) ESCC tissues and (B) ESCC cell lines compared with that in adjacent non-tumor tissues and Het-1A cells, respectively. (C) Patients with ESCC and high CCEPR mRNA expression levels had shorter survival times compared with that in patients with low CCEPR mRNA expression levels. **P<0.01 vs. adjacent. ##P<0.01 vs. Het-1A. CCEPR, cervical carcinoma expressed PCNA regulatory lncRNA; ESCC, esophageal squamous cell carcinoma.

We hypothesized that the increased mRNA expression of CCEPR might be involved in the development and progression of ESCC. To investigate this hypothesis, the clinical significance of CCEPR mRNA expression in ESCC was determined. Based on the median expression value (2.372) of CCEPR, the patients were divided into a high and a low CCEPR expression level groups. As shown in Table I, a χ2 test data indicated that high CCEPR expression was significantly associated with advanced TNM stage, as well as lymph node metastasis, suggesting that overexpression of CCEPR may play an important role in ESCC progression. Subsequently, the overall survival time was significantly shorter for patients with ESCC and high CCEPR mRNA expression levels compared with that in patients with low CCEPR expression levels (Fig. 1C). Thus, upregulation of CCEPR may predict poor prognosis in ESCC.

Table I.

Association between cervical carcinoma expressed PCNA regulatory lncRNA mRNA expression level and clinicopathological characteristics in patients with esophageal squamous cell carcinoma.

		CCEPR mRNA expression level
Characteristics	Number (n=56)	High (n=28)	Low (n=28)	P-value
Age, years				0.778
<55	19	9	10
≥55	37	19	18
Sex				0.577
Male	36	17	19
Female	20	11	9
Grade				0.064
Well and moderate	42	18	24
Poor	14	10	4
Lymph node metastasis				0.032[a]
Negative	30	11	19
Positive	26	17	9
TNM stage				0.029[a]
I–II	34	13	21
III–IV	22	15	7

P<0.05.

Knockdown of CCEPR expression suppresses ESCC cell proliferation

Then, the function of CCEPR in regulating ESCC cells in vitro was investigated. As CCEPR was significantly upregulated in the ESCC cell lines, and the TE-9 and EC9706 cell lines showed the highest mRNA expression levels of CCEPR, the TE-9 and EC9706 cell lines were transfected with two shRNAs to downregulate CCEPR expression. CCEPR was downregulated in the CCEPR shRNA 1 and shRNA 2 groups compared with that in the NC shRNA group, in both cell lines (Fig. 2A and B). A CCK-8 assay was then performed to determine the effects of CCEPR downregulation on ESCC cell proliferation and the results showed that the number of TE-9 and EC9706 cells was significantly lower in the CCEPR shRNA groups compared with that in the NC shRNA group (Fig. 2C and D). Thus, CCEPR may promote ESCC cell proliferation.

Figure 2.

Knockdown of CCEPR mRNA expression level suppresses ESCC cell proliferation. CCEPR was downregulated in the (A) TE-9 and (B) EC9706 cell lines transfected with CCEPR shRNA1 or shRNA2 compared with that in cells transfected with NC shRNA. The proliferation of (C) TE-9 and (D) EC9706 cells in the CCEPR shRNA1 and −2 groups was decreased compared with that in the NC shRNA group. **P<0.01 vs. NC shRNA. CCEPR, cervical carcinoma expressed PCNA regulatory lncRNA; NC, negative control; sh, short hairpin; OD, optical density.

Knockdown of CCEPR induces cell apoptosis

The reduced cell proliferation caused by CCEPR downregulation may be due to increased cell apoptosis. Thus, flow cytometry was performed to examine the rate of cell apoptosis in each experimental group. As shown in Fig. 3A and B, apoptosis of both the ESCC cell lines transfected with 2 types of shRNA was significantly increased following silencing of CCEPR mRNA expression compared with that in the NC group. Subsequently, the protein expression levels of several key apoptotic-related genes, including pro-apoptotic Bax and caspase-3 and anti-apoptotic Bcl2 were investigated. Knockdown of CCEPR significantly increased the protein expression levels of Bax and caspase-3, while the protein expression levels of Bcl2 was decreased in both ESCC cell lines transfected with shRNA (Fig. 3C and D).

Figure 3.

Knockdown of CCEPR induces ESCC cell apoptosis. Apoptosis of (A) TE-9 and (B) EC9706 cells in the CCEPR shRNA1 and −2 groups was increased compared with that in the NC shRNA group. Western blot analysis was performed to examine the protein expression levels of Bax, caspase-3 and Bcl2 in (C) TE-9 and (D) EC9706 cell lines in the CCEPR shRNA1 and −2 groups and in the NC shRNA group. **P<0.01 vs. NC shRNA. CCEPR, cervical carcinoma expressed PCNA regulatory lncRNA; NC, negative control; sh, short hairpin.

Inhibition of CCEPR suppresses ESCC cell migration, invasion and epithelial-mesenchymal transition (EMT)

The effects of CCEPR knockdown on the migratory and invasive abilities of ESCC cells was subsequently investigated. The results from the wound healing assay indicated that the migration of TE-9 and EC9706 cells was significantly inhibited following downregulation of CCEPR (Fig. 4A and B). Similarly, silencing CCEPR expression also downregulated the invasive abilities of both ESCC cell lines (Fig. 4C and D). The protein expression levels of MMP2 and MMP9 were then examined using western blot analysis. As shown in Fig. 5A and B, downregulation of CCEPR significantly decreased the protein expression levels of MMP2 and MMP9. Then, the effects of CCEPR downregulation on EMT in both the ESCC cell lines was investigated and the results showed that silencing CCEPR increased the protein expression levels of E-cadherin, while the protein expression levels of N-cadherin and vimentin were decreased, indicating that EMT was reduced (Fig. 5C and D). Therefore, CCEPR may promote EMT in ESCC cell lines.

Figure 4.

Inhibition of CCEPR suppresses the migration and invasion ability of ESCC cells. The (A and B) migration and (C and D) invasion of TE-9 and EC9706 cells in the CCEPR shRNA1 and −2 groups were decreased compared with that in the NC shRNA group. **P<0.01 vs. NC shRNA. CCEPR, cervical carcinoma expressed PCNA regulatory lncRNA; NC, negative control; sh, short hairpin.

Figure 5.

Inhibition of CCEPR suppresses the protein expression level of MMP2 and MMP9 as well as EMT in ESCC cells. The protein levels of MMP2 and MMP9 in the (A) TE-9 and (B) EC9706 cell lines in the CCEPR shRNA1 and −2 groups were decreased compared with that in the NC shRNA group. Western blot analysis was performed to examine the protein expression levels of E-cadherin, N-cadherin and vimentin in the (C) TE-9 and (D) EC9706 cell lines in the CCEPR shRNA1 and −2 groups and in the NC shRNA group. **P<0.01 vs. NC shRNA. CCEPR, cervical carcinoma expressed PCNA regulatory lncRNA; NC, negative control; sh, short hairpin; MMP, matrix metalloproteinase.

Discussion

A previous study has shown that overexpression of CCEPR predicts poor prognosis for cervical cancer, and could promote cervical cancer cell proliferation by upregulating proliferating cell nuclear antigen (19). To the best of our knowledge, however, the exact role of CCEPR in ESCC has not been previously reported. Therefore, the present study investigated the mRNA expression level of CCEPR in ESCC and it was found that CCEPR was upregulated in ESCC tissues and cell lines, and upregulation of CCEPR was associated with tumor progression and poor prognosis in ESCC. Furthermore, knockdown of CCEPR inhibited cell proliferation, migration, invasion, and EMT in ESCC cells, while inducing ESCC cell apoptosis.

In recent years, a large number of lncRNAs have been found to be increased or decreased in a variety of human malignancies, and some specific lncRNAs have been reported to play crucial roles during ESCC progression (22–25). For example, lncRNA SNHG6 was upregulated in ESCC tissues and cell lines and was associated with advanced TNM stage, lymph node metastasis, and shorter survival time in patients with ESCC, and SNHG6 promotes ESCC cell proliferation, migration and invasion (26). In addition, overexpression of lncRNA SNHG20 was found to promote ESCC cell growth and metastasis by regulating the ATM-JAK-PD-L1 signaling pathway (27). In the present study, CCEPR mRNA expression levels were significantly higher in ESCC tissues compared with that in adjacent non-tumor tissues. In addition, CCEPR mRNA expression was also higher in ESCC cell lines compared with that in esophageal epithelial Het-1A cell line. As no previous study has revealed the clinical significance of CCEPR mRNA expression in ESCC, a χ2 test was performed to investigate the clinical significance of CCEPR mRNA expression level in ESCC. Upregulation of CCEPR was significantly associated with advanced TNM stage, as well as lymph node metastasis in ESCC. Therefore, we hypothesized that overexpression of CCEPR may be involved in the malignant progression of ESCC. To further investigate this hypothesis, the association between CCEPR mRNA expression levels and prognosis in patients with ESCC was investigated and the results showed that patients with high CCEPR mRNA expression levels had a shorter survival time compared with than those with low CCEPR mRNA expression levels. These findings suggested that high expression of CCEPR predicts poor prognosis in patients with ESCC.

To further reveal the role of lncRNA CCEPR during ESCC progression, in vitro experiments were performed to identify the exact function of CCEPR in regulating ESCC cell proliferation, apoptosis, migration and invasion. As the TE-9 and EC9706 cell lines had the highest expression level of CCEPR, among the four examined ESCC cell lines, these were selected for the in vitro experiments. A total of 2 CCEPR-specific shRNAs were then used to transfect TE-9 and EC9706 cells to reduce the expression level of CCEPR. Knockdown of CCEPR significantly suppressed ESCC cell proliferation and colony formation abilities, while apoptosis was increased. Thus, CCEPR may promote ESCC cell growth. Subsequently, silencing CCEPR significantly inhibited ESCC cell migration and invasion. Thus, CCEPR may have promoting effects on ESCC metastasis. MMP2 and MMP9, two key matrix metalloproteinases, play key roles in cancer metastasis by promoting tumor cell migration and invasion (28,29). Consistent with the results from the cell migration and invasion assays, the protein expression levels of MMP2 and MMP9 were significantly reduced following CCEPR knockdown, suggesting that MMP2 and MMP9 may be involved in CCEPR-mediated ESCC cell metastasis. EMT is characterized by epithelial phenotype loss and mesenchymal phenotype acquisition (30). EMT has been demonstrated to play a promoting role during tumor metastasis, and inhibition of EMT could suppress the migratory and invasive abilities of various cancer cells such as cholangiocarcinoma and ovarian cancer (31,32). Furthermore, some lncRNAs have been reported to be associated with EMT in different types of cancer (33,34). For example, lncRNA SNHG6 promoted lung cancer cell migration and invasion by increasing EMT (33). E-cadherin, N-cadherin and vimentin are the most representative EMT markers (30). E-cadherin is an important epithelial marker, while N-cadherin and vimentin are two key mesenchymal markers (30). The present study showed that knockdown of CCEPR significantly increased the protein expression levels of E-cadherin and reduced the protein expression levels of N-cadherin and vimentin in ESCC cell lines, indicating that EMT was suppressed. These findings suggested that the inhibitory effects of CCEPR downregulation on ESCC metastasis in vitro may be by inhibiting EMT.

In conclusion, the findings in the present study indicated that upregulation of CCEPR was associated with tumor progression and poor prognosis in ESCC and that knockdown of CCEPR expression significantly suppressed the malignant phenotypes of 2 ESCC cell lines. Therefore, the lncRNA CCEPR may be used as a promising therapeutic target for ESCC treatment.