Tumor Biol. DOI 10.1007/s13277-015-3465-4
RESEARCH ARTICLE
Long noncoding RNA CCHE1 promotes cervical cancer cell proliferation via upregulating PCNA Meng Yang 1 & Xu Zhai 2 & Bairong Xia 1 & Yanying Wang 3 & Ge Lou 1
Received: 8 March 2015 / Accepted: 15 April 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Long noncoding RNAs (lncRNAs) have been shown to play important roles in carcinogenesis and progression. However, the roles and functional mechanisms of lncRNAs in cervical cancer remain largely unknown. In this study, we found that cervical carcinoma high-expressed lncRNA 1 (lncRNA-CCHE1) was significantly upregulated in cervical cancer tissues. The higher expression of CCHE1 was significantly correlated with large tumor size, advanced Federation of Gynecology and Obstetrics stage, uterine corpus invasion, and poor survival. Gain-of-function and loss-offunction experiments demonstrated that CCHE1 overexpression promotes the proliferation of cervical cancer cell. By contrast, the depletion of CCHE1 inhibits the proliferation of cervical cancer cells. RNA pull-down assays confirmed that CCHE1 physically associates with proliferating cell nuclear antigen (PCNA) messenger RNA, consequently enhances the expression of PCNA. The expression of CCHE1 and PCNA is significantly correlated in cervical cancer tissues. The depletion of PCNA abolishes the effects of CCHE1 on the Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3465-4) contains supplementary material, which is available to authorized users. * Ge Lou [email protected] 1
Department of Gynecology, The Affiliated Tumor Hospital, Harbin Medical University, Baojian Road No. 6, Nangang District Harbin 150081, China
2
Department of Anesthesia, The Affiliated Tumor Hospital, Harbin Medical University, Baojian Road No. 6, Nangang District Harbin 150081, China
3
Department of Pathology, The Affiliated Tumor Hospital, Harbin Medical University, Baojian Road No. 6, Nangang District Harbin 150081, China
proliferation of cervical cancer cells. Taken together, these findings indicate that CCHE1 plays a pivotal role in cervical cancer cell proliferation via increasing PCNA expression and serves as a potential prognostic biomarker and therapeutic target in human cervical cancer. Keywords Long noncoding RNA . Cervical cancer . Proliferation . PCNA
Introduction Cervical cancer is the third most frequent cancer and fourth most frequent cause of cancer death among females worldwide [1–3]. 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 [7–10]. 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 [11–15]. However, in cervical cancer, there are only preliminary studies of some known lncRNAs, such as HOTAIR, MEG3, MALAT1, GAS5, and EBIC [16–20]. Whether other lncRNAs, particular unknown lncRNAs also contribute to the pathogenesis of cervical cancer and the underlying molecular mechanisms require further exploration [21].
Tumor Biol.
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.
(reverse); β-actin: 5′-GGGAAATCGTGCGTGACATTAA G-3′ (forward) and 5′-TGTGTTGGCGTACAGGTCTTTG3′ (reverse); and 18S rRNA: 5′-ACACGGACAGGATTGA CAGA-3′ (forward) and 5′-GGACATCTAAGGGCATCA CA-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′-GGGGTACCACCTGCCC TCCAGCCACTGCC-3′ (forward) and 5′-GCTCTAGAGT GGAGGAGGGGAGTATTGTTTTCTGAG-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′-CGAGGGCGAGCATGTTTGTTGTTTA3′ 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
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′-AAGGTCCCAGGATACT CGC-3′ (forward) and 5′-GTGTCGTGGACTGGCAAAAT3′ (reverse); PCNA: 5′-GCCATATTGGAGATGCTGT-3′ (forward) and 5′-TGAGTGTCACCGTTGAAGA-3′
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
Tumor Biol.
in 20 % methanol. The experiments were performed in triplicate three times.
secondary antibody (Cell Signaling Technology, Boston, USA) and visualized with enhanced chemiluminescence.
RNA pull-down assay
Statistical analysis
CCHE1 were in vitro transcribed from vector pSPT19CCHE1 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.
For comparisons, Wilcoxon signed-rank test, Pearson Chisquare 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
RESEARCH ARTICLE
Long noncoding RNA CCHE1 promotes cervical cancer cell proliferation via upregulating PCNA Meng Yang 1 & Xu Zhai 2 & Bairong Xia 1 & Yanying Wang 3 & Ge Lou 1
Received: 8 March 2015 / Accepted: 15 April 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Long noncoding RNAs (lncRNAs) have been shown to play important roles in carcinogenesis and progression. However, the roles and functional mechanisms of lncRNAs in cervical cancer remain largely unknown. In this study, we found that cervical carcinoma high-expressed lncRNA 1 (lncRNA-CCHE1) was significantly upregulated in cervical cancer tissues. The higher expression of CCHE1 was significantly correlated with large tumor size, advanced Federation of Gynecology and Obstetrics stage, uterine corpus invasion, and poor survival. Gain-of-function and loss-offunction experiments demonstrated that CCHE1 overexpression promotes the proliferation of cervical cancer cell. By contrast, the depletion of CCHE1 inhibits the proliferation of cervical cancer cells. RNA pull-down assays confirmed that CCHE1 physically associates with proliferating cell nuclear antigen (PCNA) messenger RNA, consequently enhances the expression of PCNA. The expression of CCHE1 and PCNA is significantly correlated in cervical cancer tissues. The depletion of PCNA abolishes the effects of CCHE1 on the Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3465-4) contains supplementary material, which is available to authorized users. * Ge Lou [email protected] 1
Department of Gynecology, The Affiliated Tumor Hospital, Harbin Medical University, Baojian Road No. 6, Nangang District Harbin 150081, China
2
Department of Anesthesia, The Affiliated Tumor Hospital, Harbin Medical University, Baojian Road No. 6, Nangang District Harbin 150081, China
3
Department of Pathology, The Affiliated Tumor Hospital, Harbin Medical University, Baojian Road No. 6, Nangang District Harbin 150081, China
proliferation of cervical cancer cells. Taken together, these findings indicate that CCHE1 plays a pivotal role in cervical cancer cell proliferation via increasing PCNA expression and serves as a potential prognostic biomarker and therapeutic target in human cervical cancer. Keywords Long noncoding RNA . Cervical cancer . Proliferation . PCNA
Introduction Cervical cancer is the third most frequent cancer and fourth most frequent cause of cancer death among females worldwide [1–3]. 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 [7–10]. 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 [11–15]. However, in cervical cancer, there are only preliminary studies of some known lncRNAs, such as HOTAIR, MEG3, MALAT1, GAS5, and EBIC [16–20]. Whether other lncRNAs, particular unknown lncRNAs also contribute to the pathogenesis of cervical cancer and the underlying molecular mechanisms require further exploration [21].
Tumor Biol.
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.
(reverse); β-actin: 5′-GGGAAATCGTGCGTGACATTAA G-3′ (forward) and 5′-TGTGTTGGCGTACAGGTCTTTG3′ (reverse); and 18S rRNA: 5′-ACACGGACAGGATTGA CAGA-3′ (forward) and 5′-GGACATCTAAGGGCATCA CA-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′-GGGGTACCACCTGCCC TCCAGCCACTGCC-3′ (forward) and 5′-GCTCTAGAGT GGAGGAGGGGAGTATTGTTTTCTGAG-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′-CGAGGGCGAGCATGTTTGTTGTTTA3′ 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
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′-AAGGTCCCAGGATACT CGC-3′ (forward) and 5′-GTGTCGTGGACTGGCAAAAT3′ (reverse); PCNA: 5′-GCCATATTGGAGATGCTGT-3′ (forward) and 5′-TGAGTGTCACCGTTGAAGA-3′
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
Tumor Biol.
in 20 % methanol. The experiments were performed in triplicate three times.
secondary antibody (Cell Signaling Technology, Boston, USA) and visualized with enhanced chemiluminescence.
RNA pull-down assay
Statistical analysis
CCHE1 were in vitro transcribed from vector pSPT19CCHE1 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.
For comparisons, Wilcoxon signed-rank test, Pearson Chisquare 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
