Tumor Biol. DOI 10.1007/s13277-014-2807-y
RESEARCH ARTICLE
Plasma long noncoding RNA protected by exosomes as a potential stable biomarker for gastric cancer Qier Li & Yongfu Shao & Xinjun Zhang & Tuo Zheng & Min Miao & Lijun Qin & Bojun Wang & Guoliang Ye & Bingxiu Xiao & Junming Guo
Received: 24 September 2014 / Accepted: 3 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Long intergenic non-protein-coding RNA 152 (LINC00152) is one of the long noncoding RNAs (lncRNAs) abnormally expressed in gastric cancer tissues. However, its value in the diagnosis of gastric cancer is unclear. The aim of this study is to evaluate the clinical significance of plasma LINC00152 as a biomarker in the screening of gastric cancer and to explore the possible mechanism underling its stable existence in blood. We analyzed the levels of plasma LINC00152 in patients with gastric cancer and gastric epithelial dysplasia and healthy controls using quantitative reverse transcription polymerase chain reaction and then confirmed by sequencing. We also compared its levels in paired preoperative and postoperative plasma samples. In addition, we compared the levels of LINC00152 in plasma and in exosomes, which were extracted from the same plasma and confirmed by transmission electron microscopy. The levels of plasma LINC00152 were significantly elevated in gastric cancer patients compared with healthy controls. The sensitivity and specificity of plasma LINC00152 in the diagnosis of gastric cancer were 48.1 and 85.2 %, respectively. There were no significant differences of LINC00152 levels between gastric epithelial dysplasia patients and healthy controls. LINC00152 levels in preoperative plasma samples were lower than those in postoperative ones. There were also no differences between LINC00152 levels in plasma and in exosomes. All these results suggested that LINC00152 can be detected in Q. Li : X. Zhang : T. Zheng : M. Miao : L. Qin : B. Wang : G. Ye (*) The Affiliated Hospital of Ningbo University School of Medicine, Ningbo 315010, China e-mail: [email protected] Y. Shao : B. Xiao (*) : J. Guo Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Biochemistry and Molecular Biology, Ningbo University School of Medicine, Ningbo 315211, China e-mail: [email protected]
plasma, and one of the possible mechanisms of its stable existence in blood was protected by exosomes. It has the possibility to be applied in gastric cancer diagnosis as a novel blood-based biomarker. Keywords Long noncoding RNA . LINC00152 . Gastric cancer . Plasma . Tumor marker . Exosome
Introduction Gastric cancer (GC) is one of the most common malignant tumors. Despite that the mortality of GC is ranking second in all cancers worldwide, the prognosis of early cancer is optimistic [1]. Therefore, early detection of GC is of particular significance to improve the survival rate. At present, the diagnosis of GC is mainly relied on endoscopy, which is not only invasive but also low efficient for detecting early GC. The examination of tumor markers in peripheral blood is an ideal method for screening early patients with GC. Therefore, looking for the sensitive and specific tumor markers is highly needed as well as one of the main targets of cancer research. Long noncoding RNAs (lncRNAs) are one type of nonprotein-coding transcripts and longer than 200 nucleotides (nt) [2]. They regulate the expression of associated genes at transcriptional, posttranscriptional, and epigenetic levels [3]. So far, accumulating studies have demonstrated that some lncRNAs function as tumor suppressor genes or oncogenes and are deregulated in numerous types of cancers, including GC [4–8]. For example, H19 and SUMO1 pseudogene 3 (SUMO1P3) are upregulated in GC, while GC-associated transcript 1 (GACAT1) is downregulated [4–8]. More importantly, it is feasible to detect cancer-related lncRNAs in body fluids, such as blood, saliva, and urine [9–11]. These facts imply that lncRNAs have potentiality to be used as novel tumor markers.
Tumor Biol.
Our previous study has demonstrated that long intergenic non-protein-coding RNA 152 (LINC00152) is one of the GCrelated lncRNAs, and its expression level in gastric carcinoma was significantly higher than in nontumorous tissues [12]. LINC00152 is a new long intergenic noncoding RNA (lincRNA) whose gene is located at Ch2p11.2. Its transcript length is 828 nt. In the present study, we investigated the levels of LINC00152 in plasma of GC patients, gastric epithelial dysplasia (GED) patients, and healthy volunteers. Our results show that the levels of plasma LINC00152 were significantly elevated in GC patients compared with healthy controls, supporting that blood based-LINC00152 may be a novel tumor biomarker for GC screening and diagnosis. We further found that exosome is a factor for stabling lncRNAs in blood.
Materials and methods Plasma sample collection All plasma samples were obtained from the Affiliated Hospital of Ningbo University School of Medicine and Ningbo Yinzhou People’s Hospital, China, from June 2012 to December 2013. Every research subject signed a prior informed consent that was approved by the Human Research Ethics Committee from Ningbo University. The relevant clinical data of all participants were available. Tumors were staged basing on the tumor-node-metastasis (TNM) staging system [13]. Paired plasma samples (preoperative and postoperative) were obtained from 79 GC patients, and the postoperative ones were collected 10 days after surgery. Another 31 plasma samples were from patients with GED, one type of premalignant lesion. All patients were diagnosed by histological examination. Histological grade was evaluated according to the National Comprehensive Cancer Network clinical practice guideline of oncology (V.1.2011). Control samples were obtained from 81 healthy volunteers without any cancers and other health problems. Immediately obtained, the fresh peripheral blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes followed by centrifugation (3000×g for 10 min). Then, the plasma samples were stored at −80 °C until use. Finally, to further remove cell debris, the plasma samples were centrifuged again (2000×g for 20 min followed by 10,000×g for 20 min) after thawing.
extracted using TRIzol Reagent (Invitrogen). The quality of RNA samples was assessed by a UV spectrophotometer (BioRad, Hercules, CA, USA), and the samples whose 260/280nm absorbance ratio was between 1.8 and 2.0 were considered qualified. Finally, the extracted RNA was dissolved in 10 μl diethylpyrocarbonate-treated water and then was mixed with 1 μl oligo(dT) 15 primer, 1 μl random primer, 4 μl GoScript 5× reaction buffer, 2 μl MgCl2, 1 μl nucleotide mix, 0.5 μl recombinant RNasin ribonuclease inhibitor, and 1 μl GoScript reverse transcriptase to synthesize cDNA according to the manufacturer’s instructions of GoScript Reverse Transcription (RT) System (Promega, Madison, WI, USA). Quantitative polymerase chain reaction The levels of LINC00152 in plasma were quantified by quantitative polymerase chain reaction (PCR) using GoTaq qPCR master mix (Promega) on an Mx3005P QPCR System (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. All experiments were repeated twice with negative control. Annealing temperature was set at 56 °C. The product length is 201 nt. The sequences of primers for LINC00152 were as follows: forward primer, 5′-CTCCAG CACCTCTACCTGTTG-3′; reverse primer, 5′-GGACAAGG GATTAAGACACACA-3′. The levels of LINC00152 were determined by the ΔΔCt method using glyceraldehyde-3phosphate dehydrogenase (GAPDH) as a control. The sequences of primers for GAPDH were as follows: forward primer, 5′-ACCCACTCCTCCACCTTTGAC-3′; reverse primer, 5′-TGTTGCTGTAGCCAAATTCGTT-3′. qRT-PCR product sequencing The quantitative RT (qRT)-PCR products of plasma LINC00152 were purified using a UNIQ-10 PCR Product Purification Kit and then cloned into the pUCm-T vector (Invitrogen) following the manufacturer’s instructions. Finally, sequencing was conducted by Invitrogen, Co., Ltd. Exosome isolation A total of 10 plasma samples were randomly divided into two equal parts (300 μl each). One part was used to extract total RNA; the other was used to isolate exosomes. Exosomes were isolated using Total Exosome Isolation Reagent (Invitrogen) following the manufacturer’s instructions. Observed exosome under transmission electron microscopy
Total RNA extraction and reverse transcription Total RNA in plasma was extracted using TRIzol LS Reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer’s instructions, while the total RNA in exosomes was
Isolated exosomes were first resuspended in phosphate buffer saline (PBS) buffer, and then, 10 μl was absorbed onto Cu grids for 5 min. Then, the grids were dyed using 1 % of uranyl acetate for 1 min at room temperature. Transmission electron
Tumor Biol. Fig. 1 Plasma LINC00152 levels in GC patients are significantly higher than those in healthy controls. The relative level of LINC00152 was determined by qRT-PCR with the ΔCt method. Smaller ΔCt value indicates higher level. a Increased levels of plasma LINC00152 in GC patients compared to healthy controls (P
RESEARCH ARTICLE
Plasma long noncoding RNA protected by exosomes as a potential stable biomarker for gastric cancer Qier Li & Yongfu Shao & Xinjun Zhang & Tuo Zheng & Min Miao & Lijun Qin & Bojun Wang & Guoliang Ye & Bingxiu Xiao & Junming Guo
Received: 24 September 2014 / Accepted: 3 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Long intergenic non-protein-coding RNA 152 (LINC00152) is one of the long noncoding RNAs (lncRNAs) abnormally expressed in gastric cancer tissues. However, its value in the diagnosis of gastric cancer is unclear. The aim of this study is to evaluate the clinical significance of plasma LINC00152 as a biomarker in the screening of gastric cancer and to explore the possible mechanism underling its stable existence in blood. We analyzed the levels of plasma LINC00152 in patients with gastric cancer and gastric epithelial dysplasia and healthy controls using quantitative reverse transcription polymerase chain reaction and then confirmed by sequencing. We also compared its levels in paired preoperative and postoperative plasma samples. In addition, we compared the levels of LINC00152 in plasma and in exosomes, which were extracted from the same plasma and confirmed by transmission electron microscopy. The levels of plasma LINC00152 were significantly elevated in gastric cancer patients compared with healthy controls. The sensitivity and specificity of plasma LINC00152 in the diagnosis of gastric cancer were 48.1 and 85.2 %, respectively. There were no significant differences of LINC00152 levels between gastric epithelial dysplasia patients and healthy controls. LINC00152 levels in preoperative plasma samples were lower than those in postoperative ones. There were also no differences between LINC00152 levels in plasma and in exosomes. All these results suggested that LINC00152 can be detected in Q. Li : X. Zhang : T. Zheng : M. Miao : L. Qin : B. Wang : G. Ye (*) The Affiliated Hospital of Ningbo University School of Medicine, Ningbo 315010, China e-mail: [email protected] Y. Shao : B. Xiao (*) : J. Guo Zhejiang Provincial Key Laboratory of Pathophysiology, Department of Biochemistry and Molecular Biology, Ningbo University School of Medicine, Ningbo 315211, China e-mail: [email protected]
plasma, and one of the possible mechanisms of its stable existence in blood was protected by exosomes. It has the possibility to be applied in gastric cancer diagnosis as a novel blood-based biomarker. Keywords Long noncoding RNA . LINC00152 . Gastric cancer . Plasma . Tumor marker . Exosome
Introduction Gastric cancer (GC) is one of the most common malignant tumors. Despite that the mortality of GC is ranking second in all cancers worldwide, the prognosis of early cancer is optimistic [1]. Therefore, early detection of GC is of particular significance to improve the survival rate. At present, the diagnosis of GC is mainly relied on endoscopy, which is not only invasive but also low efficient for detecting early GC. The examination of tumor markers in peripheral blood is an ideal method for screening early patients with GC. Therefore, looking for the sensitive and specific tumor markers is highly needed as well as one of the main targets of cancer research. Long noncoding RNAs (lncRNAs) are one type of nonprotein-coding transcripts and longer than 200 nucleotides (nt) [2]. They regulate the expression of associated genes at transcriptional, posttranscriptional, and epigenetic levels [3]. So far, accumulating studies have demonstrated that some lncRNAs function as tumor suppressor genes or oncogenes and are deregulated in numerous types of cancers, including GC [4–8]. For example, H19 and SUMO1 pseudogene 3 (SUMO1P3) are upregulated in GC, while GC-associated transcript 1 (GACAT1) is downregulated [4–8]. More importantly, it is feasible to detect cancer-related lncRNAs in body fluids, such as blood, saliva, and urine [9–11]. These facts imply that lncRNAs have potentiality to be used as novel tumor markers.
Tumor Biol.
Our previous study has demonstrated that long intergenic non-protein-coding RNA 152 (LINC00152) is one of the GCrelated lncRNAs, and its expression level in gastric carcinoma was significantly higher than in nontumorous tissues [12]. LINC00152 is a new long intergenic noncoding RNA (lincRNA) whose gene is located at Ch2p11.2. Its transcript length is 828 nt. In the present study, we investigated the levels of LINC00152 in plasma of GC patients, gastric epithelial dysplasia (GED) patients, and healthy volunteers. Our results show that the levels of plasma LINC00152 were significantly elevated in GC patients compared with healthy controls, supporting that blood based-LINC00152 may be a novel tumor biomarker for GC screening and diagnosis. We further found that exosome is a factor for stabling lncRNAs in blood.
Materials and methods Plasma sample collection All plasma samples were obtained from the Affiliated Hospital of Ningbo University School of Medicine and Ningbo Yinzhou People’s Hospital, China, from June 2012 to December 2013. Every research subject signed a prior informed consent that was approved by the Human Research Ethics Committee from Ningbo University. The relevant clinical data of all participants were available. Tumors were staged basing on the tumor-node-metastasis (TNM) staging system [13]. Paired plasma samples (preoperative and postoperative) were obtained from 79 GC patients, and the postoperative ones were collected 10 days after surgery. Another 31 plasma samples were from patients with GED, one type of premalignant lesion. All patients were diagnosed by histological examination. Histological grade was evaluated according to the National Comprehensive Cancer Network clinical practice guideline of oncology (V.1.2011). Control samples were obtained from 81 healthy volunteers without any cancers and other health problems. Immediately obtained, the fresh peripheral blood samples were collected in ethylenediaminetetraacetic acid (EDTA) tubes followed by centrifugation (3000×g for 10 min). Then, the plasma samples were stored at −80 °C until use. Finally, to further remove cell debris, the plasma samples were centrifuged again (2000×g for 20 min followed by 10,000×g for 20 min) after thawing.
extracted using TRIzol Reagent (Invitrogen). The quality of RNA samples was assessed by a UV spectrophotometer (BioRad, Hercules, CA, USA), and the samples whose 260/280nm absorbance ratio was between 1.8 and 2.0 were considered qualified. Finally, the extracted RNA was dissolved in 10 μl diethylpyrocarbonate-treated water and then was mixed with 1 μl oligo(dT) 15 primer, 1 μl random primer, 4 μl GoScript 5× reaction buffer, 2 μl MgCl2, 1 μl nucleotide mix, 0.5 μl recombinant RNasin ribonuclease inhibitor, and 1 μl GoScript reverse transcriptase to synthesize cDNA according to the manufacturer’s instructions of GoScript Reverse Transcription (RT) System (Promega, Madison, WI, USA). Quantitative polymerase chain reaction The levels of LINC00152 in plasma were quantified by quantitative polymerase chain reaction (PCR) using GoTaq qPCR master mix (Promega) on an Mx3005P QPCR System (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. All experiments were repeated twice with negative control. Annealing temperature was set at 56 °C. The product length is 201 nt. The sequences of primers for LINC00152 were as follows: forward primer, 5′-CTCCAG CACCTCTACCTGTTG-3′; reverse primer, 5′-GGACAAGG GATTAAGACACACA-3′. The levels of LINC00152 were determined by the ΔΔCt method using glyceraldehyde-3phosphate dehydrogenase (GAPDH) as a control. The sequences of primers for GAPDH were as follows: forward primer, 5′-ACCCACTCCTCCACCTTTGAC-3′; reverse primer, 5′-TGTTGCTGTAGCCAAATTCGTT-3′. qRT-PCR product sequencing The quantitative RT (qRT)-PCR products of plasma LINC00152 were purified using a UNIQ-10 PCR Product Purification Kit and then cloned into the pUCm-T vector (Invitrogen) following the manufacturer’s instructions. Finally, sequencing was conducted by Invitrogen, Co., Ltd. Exosome isolation A total of 10 plasma samples were randomly divided into two equal parts (300 μl each). One part was used to extract total RNA; the other was used to isolate exosomes. Exosomes were isolated using Total Exosome Isolation Reagent (Invitrogen) following the manufacturer’s instructions. Observed exosome under transmission electron microscopy
Total RNA extraction and reverse transcription Total RNA in plasma was extracted using TRIzol LS Reagent (Invitrogen, Karlsruhe, Germany) following the manufacturer’s instructions, while the total RNA in exosomes was
Isolated exosomes were first resuspended in phosphate buffer saline (PBS) buffer, and then, 10 μl was absorbed onto Cu grids for 5 min. Then, the grids were dyed using 1 % of uranyl acetate for 1 min at room temperature. Transmission electron
Tumor Biol. Fig. 1 Plasma LINC00152 levels in GC patients are significantly higher than those in healthy controls. The relative level of LINC00152 was determined by qRT-PCR with the ΔCt method. Smaller ΔCt value indicates higher level. a Increased levels of plasma LINC00152 in GC patients compared to healthy controls (P
