Introduction

Cervical cancer is the third most frequent cancer and fourth most frequent cause of cancer death among females worldwide [13]. Despite efforts using pap smear screening and other diagnostic techniques, the overall survival of cervical cancer patients remains poor [4, 5]. Therefore, further revealing the molecular mechanisms that contribute to the development and progression of cervical cancer is urgent for developing effective therapy [6].

Recently, various genome-wide sequencings have found that the majority of the human genome is transcribed, but less than 2 % of the genome represents protein-coding genes [710]. Among these transcribed noncoding transcripts, long noncoding RNAs (lncRNAs), which are longer than 200 nucleotides with no protein-coding potential, have been shown to be deregulated in many disease states and have multiple functions in a wide array of cellular biological processes [1115]. However, in cervical cancer, there are only preliminary studies of some known lncRNAs, such as HOTAIR, MEG3, MALAT1, GAS5, and EBIC [1620]. Whether other lncRNAs, particular unknown lncRNAs also contribute to the pathogenesis of cervical cancer and the underlying molecular mechanisms require further exploration [21].

In a primary screen of differently expressed lncRNAs in cervical cancer, we found that a novel lncRNA-Cervical Cancer High-Expressed lncRNA 1 (lncRNA-CCHE1, GenBank number AK055418, located in an intergenic region on chromatin 10) was significantly highly expressed in cervical cancer tissues compared with normal tissues. In this study, we further investigated the expression pattern of CCHE1, its clinical significance, and its biological functions in cervical cancer. Our results demonstrate that CCHE1 is upregulated in cervical cancer tissues, and is associated with larger tumor size, advanced Federation of Gynecology and Obstetrics (FIGO) stage, higher SCC-Ag level, uterine corpus invasion, and poor prognosis of cervical cancer patients. CCHE1 upregulates expression of proliferating cell nuclear antigen (PCNA) through binding PCNA messenger RNA (mRNA) and promotes the proliferation of cervical cancer cells.

Materials and methods

Tissue samples

A total of 182 cervical cancer tissues and their pair-matched adjacent normal tissues were obtained with informed consent from patients who underwent radical resections at The Affiliated Tumor Hospital, Harbin Medical University, Harbin, China. This study was performed with the approval of the Research Ethics Committee of Harbin Medical University.

Cell cultures

The human cervical cancer cell lines SiHa and HeLa were obtained from the American Type Culture Collection (ATCC, VA, USA). The cells were grown in DMEM medium supplemented with 10 % fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA) and maintained in a humidified 37 °C incubator with a 5 % CO2 atmosphere.

RNA extraction and quantitative reverse transcription-polymerase chain reaction

Total RNAs were extracted using the Trizol reagent (Takara, Dalian, China). First-strand complementary DNA (cDNA) was generated using the PrimeScript™ RT Reagent Kit (Takara). Real-time PCR was performed using the standard SYBR-Green PCR Kit protocol on ABI 7500 (Applied Biosystems, Foster City, CA, USA). For each sample, gene expression was normalized to the respective 18S ribosomal RNA (rRNA) expression level. The primer sequences used were as follows: CCHE1: 5′-AAGGTCCCAGGATACTCGC-3′ (forward) and 5′-GTGTCGTGGACTGGCAAAAT-3′ (reverse); PCNA: 5′-GCCATATTGGAGATGCTGT-3′ (forward) and 5′-TGAGTGTCACCGTTGAAGA-3′ (reverse); β-actin: 5′-GGGAAATCGTGCGTGACATTAAG-3′ (forward) and 5′-TGTGTTGGCGTACAGGTCTTTG-3′ (reverse); and 18S rRNA: 5′-ACACGGACAGGATTGACAGA-3′ (forward) and 5′-GGACATCTAAGGGCATCACA-3′ (reverse). The real-time PCR reactions were performed in triplicate. The relative RNA expression was calculated using the comparative Ct method.

Vectors construction

Total RNAs were extracted from HeLa cells using the Trizol reagent (Takara). The extracted total RNA was treated with Recombinant DNase I to remove genomic DNA. First-strand cDNA was generated using the PrimeScript™ RT Reagent Kit (Takara). The cDNA encoding CCHE1 was PCR amplified by the Pfu Ultra II Fusion HS DNA Polymerase (Stratagene, Agilent Technologies, Palo Alto, CA, USA) and subcloned into the Kpn I and Xba I sites of pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), named pcDNA3.1-CCHE1. The primers used were as follows: 5′-GGGGTACCACCTGCCCTCCAGCCACTGCC-3′ (forward) and 5′-GCTCTAGAGTGGAGGAGGGGAGTATTGTTTTCTGAG-3′ (reverse). pcDNA3.1-CCHE1 was double digested with Kpn I and Xba I, and the lncRNA fragment was subcloned into pSPT19, named pSPT19-CCHE1.

Small interfering RNA synthesis and transfection

Small interfering RNAs (siRNAs) specifically targeting CCHE1 were synthesized by Invitrogen. The siRNA sequences were 5′-CGAGGGCGAGCATGTTTGTTGTTTA-3′ for CCHE1. siRNAs specifically targeting PCNA were purchased from Invitrogen (siRNA s10134). Transfections were performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. The transfected cells were harvested 48 h after transfection.

Cell proliferation assay

A total of approximately 5.0 × 103 cervical cancer cells were plated in 96-well plates. Cell proliferation was assessed using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. The cell proliferation curves were plotted using the absorbance at each time point. Ethynyl deoxyuridine (EdU) immunofluorescence staining was performed with an EdU Kit (Roche, Mannheim, Germany). All experiments were performed in triplicate.

Colony formation assay

Approximately 2000 cells were seeded per well for six-well plates and were grown for 10 days with normal medium. Colonies were fixed and stained with 0.5 % crystal violet solution in 20 % methanol. The experiments were performed in triplicate three times.

RNA pull-down assay

CCHE1 were in vitro transcribed from vector pSPT19-CCHE1 and biotin-labeled with the Biotin RNA Labeling Mix (Roche) and SP6 RNA polymerase (Roche), treated with RNase-free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). One milligram of whole-cell lysates from HeLa cells were incubated with 3 μg of purified biotin-labeled CCHE1 for 1 h at 25 °C; complexes were isolated with streptavidin agarose beads (Invitrogen). The RNA present in the pull-down material was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis.

Western blotting

Total cell lysates were prepared in a 1× sodium dodecyl sulfate buffer. Identical quantities of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. After incubation with antibodies specific for PCNA (Abcam, Hong Kong, China) or β-actin (Sigma-Aldrich, Saint Louis, MO, USA), the blots were incubated with goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology, Boston, USA) and visualized with enhanced chemiluminescence.

Statistical analysis

For comparisons, Wilcoxon signed-rank test, Pearson Chi-square test, Log-rank test, Student’s t test, and Pearson correlation analysis were performed as indicated. All P values were obtained using the SPSS 18.0 software package (SPSS, Chicago, IL, USA). Differences were defined as statistically significant for P values <0.05.

Results

CCHE1 is upregulated in cervical cancer tissues and predicts poor prognosis of cervical cancer patients

We first used ORF Finder from the National Center for Biotechnology Information to predict coding potential of CCHE1. Analysis of the sequences by ORF Finder failed to predict a protein of more than 53-amino acids. Moreover, CCHE1 does not contain Kozak consensus sequence. In addition, we used txCdsPredict from UCSC to calculate its coding potential. The txCdsPredict score of CCHE1 is 272, which further supports that CCHE1 is a long noncoding RNA.

The expression level of CCHE1 was examined using qRT-PCR in 182 pairs of cervical cancer tissues and pair-matched adjacent normal tissues. CCHE1 expression was significantly upregulated in cervical cancer tissues compared with corresponding normal tissues (P < 0.001, Fig. 1a). Next, we analyzed the correlation between the expression of CCHE1 and clinicopathological characteristics of the 182 cervical cancer patients. The results showed that CCHE1 expression was upregulated in cervical cancer tissues with larger tumor size (Fig. 1b). The 182 cervical cancer tissues were classified into two groups according to the median expression level of CCHE1. Correlation regression analysis showed that increased CCHE1 expression was significantly correlated with larger tumor size (P < 0.001), advanced FIGO stage (P = 0.002), higher SCC-Ag (P = 0.004), and uterine corpus invasion (P = 0.030) (Table 1).

Fig. 1
figure 1

Aberrant expression of CCHE1 in clinical samples and its association with patients’ prognosis. a CCHE1 expression in human cervical cancer tissues and pair-matched adjacent normal tissues. b CCHE1 expression in tumor size <4 cm group and tumor size ≥4 cm group. For (a) and (b), the expression level of CCHE1 was analyzed by qRT-PCR. The horizontal lines in the box plots represent the medians, the boxes represent the interquartile range, and the whiskers represent the 2.5th and 97.5th percentiles. The significant differences between samples were analyzed using the Wilcoxon signed-rank test. c, d Kaplan-Meier analyses of correlations between the CCHE1 expression level and c overall survival and d recurrence-free survival of 182 cervical cancer patients are shown. The median expression level was used as the cutoff

Table 1 Association between clinicopathological characteristics and CCHE1 expression

We further examined whether the CCHE1 expression level was correlated with the outcome of cervical cancer patients. Kaplan-Meier survival estimates showed that high CCHE1 expression in cervical cancer tissues is significantly associated with poorer overall survival (P = 0.002) and recurrence-free survival (P = 0.004) (Fig. 1c, d). These results indicate that CCHE1 may play a pivotal role in the pathogenesis of cervical cancer and malignant outcomes of patients with cervical cancer.

CCHE1 promotes the proliferation of cervical cancer cells

Based on the results that CCHE1 is overexpressed in cervical cancer and associated with large tumor size and poor prognosis of cervical cancer patients, we next investigated the biological function of CCHE1 in cervical cancer. We successfully enhanced CCHE1 expression in SiHa cells by transfecting a CCHE1 expression vector (pcDNA3.1-CCHE1) and inhibited CCHE1 expression in HeLa cells by transfecting CCHE1-specific siRNAs (Fig. Fig. S1; Fig. 2a, b). Cell Counting Kit-8 assays indicated that enhanced CCHE1 expression accelerated cell proliferation in SiHa cells (Fig. 2c); by contrast, the depletion of CCHE1 inhibited HeLa cell proliferation (Fig. 2d). Furthermore, EdU incorporation assays showed that upregulation of CCHE1 in SiHa cells had higher percentage of EdU-positive cells (Fig. 2e), while EdU-positive cells significantly decreased after CCHE1 knockdown in HeLa cells (Fig. 2f). The growth promoting effect of CCHE1 was further demonstrated by colony formation assays. Overexpression of CCHE1 significantly increased the number of colonies formed by SiHa cells (Fig. 2g). Additionally, the depletion of CCHE1 expression significantly decreased the number of colonies formed by HeLa cells (Fig. 2h). These results demonstrated that CCHE1 significantly promotes cervical cancer cell proliferation.

Fig. 2
figure 2

CCHE1 promotes cervical cancer cell proliferation. a CCHE1 expression levels after transfection of the CCHE1 expression vector (pcDNA3.1-CCHE1) or control vector into SiHa cells. b CCHE1 expression levels after transfection of CCHE1 siRNA or control siRNA into HeLa cells. c, d The effects of CCHE1 on SiHa and HeLa cells proliferation were determined by the CCK-8 assay, and the relative number of cells to 0 h is presented. e, f The effects of CCHE1 on SiHa and HeLa cell proliferation were assessed using EdU immunofluorescence staining. The blue color represents the nuclei, and the red color indicates EdU-positive nuclei. Scale bars = 100 μm. The graph on the right shows the percentage of EdU-positive cells. g The colony number of CCHE1 overexpressed SiHa cells per well in six-well plates. h The colony number of CCHE1 inhibited HeLa cells per well in six-well plates. For all panels, data are presented as the mean ± standard error based on at least three independent experiments. **P < 0.01; ***P < 0.001

CCHE1 physically associates with PCNA mRNA and upregulates the expression of PCNA

We next sought to investigate the mechanisms behind the effect of CCHE1 on cervical cancer cells proliferation. Recently, many reports have shown that lncRNAs could physically associate with mRNAs and increase their stability [22, 23]. To identify mRNA species bound by CCHE1, we used in vitro transcribed biotin-labeled CCHE1 to pull-down endogenous mRNAs associated with CCHE1. Biotinylated CCHE1 was incubated with HeLa cell lysates, followed by RNA extraction and detection by RT-qPCR. As shown in Fig. 3a, CCHE1 was selectively interacting with PCNA mRNA but not β-actin mRNA. Next, we test whether CCHE1 regulate the expression of PCNA. Our results showed that the overexpression of CCHE1 significantly increased the mRNA and protein level of PCNA in SiHa cells, while the depletion of CCHE1 significantly decreased the mRNA and protein level of PCNA in HeLa cells (Fig. 3b–e).

Fig. 3
figure 3

CCHE1 associated with PCNA mRNA and upregulated its expression. a HeLa cell lysates were incubated with biotin-labeled CCHE1; after pull-down, mRNA was extracted and assessed by qRT-PCR. Data are shown as a percentage of input RNA. Right, the representative electrophoresis images of RCP products. b PCNA mRNA levels after the transfection of pcDNA3.1 or pcDNA3.1-CCHE1 into SiHa cells. c PCNA mRNA levels after the transfection of control siRNA or CCHE1 siRNA into HeLa cells. d PCNA protein levels after the transfection of pcDNA3.1 or pcDNA3.1-CCHE1 into SiHa cells. e PCNA protein levels after the transfection of control siRNA or CCHE1 siRNA into HeLa cells. For (a)–(e), data are presented as the mean ± standard error based on at least three independent experiments. **P < 0.01; ***P < 0.001. f The correlation between CCHE1 transcript level and PCNA mRNA level was measured in 182 cervical cancer tissues. The respective ΔCt values (both CCHE1 and PCNA were normalized to 18S rRNA) were subjected to a Pearson correlation analysis (r = 0.663, P < 0.001, Pearson’s correlation)

Since CCHE1 could upregulate PCNA, we next examined whether CCHE1 is coexpressed with PCNA in human cervical cancer samples. We measured the expression levels of CCHE1 and PCNA in the same set of 182 cervical cancer tissues shown in Fig. 1a and then performed a correlation analysis of the PCNA mRNA and CCHE1 expression levels. A statistically significant correlation was found between PCNA mRNA and CCHE1 expression levels (r = 0.663, P < 0.001, Pearson’s correlation, Fig. 3f), supporting the role of CCHE1 in modulating PCNA.

The effect of CCHE1 on cervical cancer cells proliferation is dependent upon upregulation of PCNA

To test whether PCNA mediated the function of CCHE1 in cervical cancer cells proliferation, we inhibited PCNA in CCHE1-upregulated SiHa cells (Fig. 4a) and then used different functional assays to clarify the importance of PCNA in the enhancement of proliferation by CCHE1. Cell-Counting Kit-8 assays, EdU incorporation analyses and colony formation assays all indicated that the depletion of PCNA abolished the pro-proliferation role of CCHE1 on cervical cancer cells (Fig. 4b–d).

Fig. 4
figure 4

CCHE1-induced proliferation is dependent upon upregulation of PCNA. a The expression of PCNA in indicated SiHa cells after transfection of PCNA siRNA or control siRNA. b After the transfection of PCNA siRNA or control siRNA into indicated SiHa cells, the proliferation of the cells were determined by the CCK-8 assay, and the relative number of cells to 0 h is presented. c The proliferation of the cells was determined using EdU immunofluorescence staining. The blue color represents the nuclei, and the red color indicates EdU-positive nuclei. Scale bars = 100 μm. The graph on the right shows the percentage of EdU-positive cells. d The colony number of indicated cells per well in six-well plates. For all panels, data are presented as the mean ± standard error based on at least three independent experiments. **P < 0.01; ***P < 0.001

Discussion

In this study, we detected the expression of the novel long noncoding RNA CCHE1 in cervical cancer tissues and their adjacent normal tissues. We also identified the function of CCHE1 in cervical cancer cells by applying gain-of-function and loss-of-function approaches. Our results demonstrate that CCHE1 is upregulated in cervical cancer tissues in comparison with surrounding normal tissues and that CCHE1 upregulation is correlated with larger tumor size, advanced FIGO stage, higher SCC-Ag level, and uterine corpus invasion. High CCHE1 expression in cervical cancer tissues indicates poor recurrence free survival and overall survival of cervical cancer patients. Enhanced CCHE1 expression promotes the proliferation of cervical cancer cells, while the inhibition of CCHE1 inhibits cervical cancer cell proliferation. Therefore, our results demonstrated that CCHE1 functions as an oncogene in cervical cancer.

lncRNAs exert biological functions mainly via regulating expression of some important cancer associated genes [2426]. lncRNAs may regulate genes expression through directly binding proteins, microRNAs, or mRNAs, and altering the activity or/and expression of proteins, microRNAs, or mRNAs [2730]. In this study, we found that CCHE1 directly binds PCNA mRNA and then enhances the expression of PCNA mRNA and protein. As a proliferation marker, PCNA is exclusively expressed in proliferating cancer cells and could promote proliferation and tumorigenesis of cancer cells, including cervical cancer cells [3133]. In this study, a correlation between CCHE1 expression and PCNA mRNA level in clinical cervical cancer tissues supported the role of CCHE1 in PCNA expression. Our results also indicated that the pro-proliferation role of CCHE1 is dependent upon upregulation of PCNA. CCHE1 upregulates PCNA mRNA level through binding PCNA mRNA. But the underlying molecular mechanism of PCNA mRNA level upregulation is still unknown. Through binding mRNAs, lncRNAs can change the stability of corresponding mRNAs, and further their expressions, such as the BCAE1-antisense transcript and Wrap53 [23, 34]. Nevertheless, through binding mRNAs, other lncRNAs can regulate the translation of corresponding mRNAs, such as lincRNA-p21 and antisense Uchl1 [22, 35]. Whether CCHE1 enhances the stability of PCNA mRNA, promotes the translation of PCNA mRNA, or has both the functions requires further investigation.

Collectively, our studies indicate that CCHE1 is a prognosis factor for cervical cancer, which could physically associate with PCNA mRNA and upregulate expression of PCNA, and when upregulated, it promotes the proliferation of cervical cancer cells. These findings demonstrate that CCHE1 is an important cervical cancer biomarker for determining prognosis and could be an effective target for cervical cancer therapy.