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Differential Expression Profiles of Long Noncoding RNA and Mrna in Colorectal Cancer Tissues from Patients with Lung Metastasis

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Differential Expression Profiles of Long Noncoding RNA and Mrna in Colorectal Cancer Tissues from Patients with Lung Metastasis 5666 MOLECULAR MEDICINE REPORTS 17: 5666-5675, 2018 Differential expression profiles of long noncoding RNA and mRNA in colorectal cancer tissues from patients with lung metastasis RUO SHU1,2*, YU XU1,3*, YAN TIAN2,3, YUJIAN ZENG1,3, LIANG SUN2,3, FANGYOU GONG2,3, YI LEI2,3, KUNHUA WANG1-3 and HUAYOU LUO1-3 1Department of Gastrointestinal and Hernia Surgery, The First Affiliated Hospital of Kunming Medical University; 2Kunming Engineering Technology Center of Digestive Disease; 3Yunnan Institute of Digestive Disease, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China Received January 19, 2017; Accepted November 21, 2017 DOI: 10.3892/mmr.2018.8576 Abstract. Lungs are the most common extra-abdominal lung metastasis, while the downregulated mRNA were site of metastasis of colorectal cancer (CRC), in which long associated with the phosphoinositide 3-kinase/Akt signaling noncoding RNA (lncRNA) may serve a role. In the present pathway. The results of the present study suggested that study, a high-throughput microarray assay was performed differentially expressed lncRNA may be associated with to detect lncRNA expression and identify novel targets lung metastasis and may provide insights into the biology and for further study of lung metastasis in CRC. In the CRC prevention of lung metastasis. tissues from patients with lung metastasis, 7,632 lncRNA (3,574 upregulated and 4,058 downregulated) and 6,185 mRNA Introduction (3,394 upregulated and 2,791 downregulated) were detected to be differentially expressed with a fold change ≥2 and Colorectal cancer (CRC) is one of the most common malig- P<0.05 compared with the CRC tissues without metastasis. A nant tumors and the third leading cause of cancer-associated total of six differentially regulated lncRNA were confirmed by mortality worldwide (1). Currently, resection and chemotherapy reverse transcription-quantitative polymerase chain reaction are commonly used to improve the outcomes in patients with in 20 pairs of CRC samples. Furthermore, gene ontology CRC; however, metastasis is an obstacle to successful treatment and pathway analysis were conducted to predict the possible of CRC (2). Among patients with CRC, ~50% eventually develop roles of the identified mRNA. The upregulated mRNA distant metastases, resulting in poor outcomes even when primary were associated with cell division (biological processes), tumors are resectable (3). Distant metastasis is the predominant protein kinase B binding (molecular functions) and cellular cause of death in patients with CRC (4). Lungs are the most components. The downregulated mRNA were associated common extra-abdominal site of metastasis (5). Diagnosis of with cell adhesion, platelet-derived growth factor binding lung metastasis in patients with CRC remains dependent on and membrane components. Pathway analysis determined radiology and biopsy (6,7). Radiology-guided biopsy has demon- that the upregulated mRNA were associated with the Wnt strated limited sensitivity (8). Therefore, investigation of novel signaling pathway in the CRC tissues from patients with biomarkers and molecular mechanisms underlying metastatic progression in CRC is necessary for improving the survival of patients with CRC, and may be beneficial for earlier diagnosis and treatment of patients with lung metastasis. Only ~2% of human genome sequences encode Correspondence to: Dr Huayou Luo, Department of proteins, while the remainder is divided into two groups Gastrointestinal and Hernia Surgery, The First Affiliated Hospital according the length of the sequence: Short noncoding RNA of Kunming Medical University, 295 Xichang Road, Kunming, Yunnan 650032, P.R. China (<200 nucleotides); and long noncoding RNA (lncRNA; E-mail: [email protected] >200 nucleotides) (9,10). lncRNA regulate gene expression on transcriptional or post-transcriptional levels (11,12). Dr Kunhua Wang, Yunnan Institute of Digestive Disease, The First Currently, an increasing number of studies have demonstrated Affiliated Hospital of Kunming Medical University, 295 Xichang that lncRNA serve important roles in cancer progression Road, Kunming, Yunnan 650032, P.R. China E-mail: [email protected] and metastasis as well as cellular processes, including cell proliferation and apoptosis (13-15). Furthermore, it has been *Contributed equally reported that many lncRNA serve roles in the regulation of CRC (16). However, biological and pathological functions Key words: colorectal cancer, lung metastasis, long noncoding of the majority of lncRNA in patients with CRC and lung RNA, biological process, molecular function, cellular component metastasis remain to be elucidated. Therefore, in the present study, differentially expressed lncRNA and mRNA in tissues from patients with CRC and SHU et al: ALTERED lncRNA AND mRNA IN CRC TISSUES FROM PATIENTS WITH LUNG METASTASIS 5667 with or without lung metastasis were investigated in order to Data analysis. Scanned images were imported to the Feature identify novel diagnostic and prognostic markers for patients Extraction software (version 11.0.1.1) for raw data extraction with CRC and lung metastasis. (Agilent Technologies, Inc.). When comparing two groups of profile differences (such as metastasis vs. non‑metastasis), Materials and methods the fold change (i.e., the ratio of group averages) between the groups of each lncRNA was computed. A t-test was used to Ethical approval. All samples were collected from patients determine the differences between two groups. The lncRNA in the Department of Gastrointestinal Surgery, The First demonstrating fold changes ≥2 and P<0.05 compared with the Affiliated Hospital of Kunming Medical University (Kunming, control were selected as differentially expressed. China) between January 2013 and December 2015. The project was reviewed and approved by the Ethics Committee of The Reverse transcription‑quantitative polymerase chain reaction First Affiliated Hospital of Kunming Medical University. (RT‑qPCR) validation. A total of 20 pairs of primary CRC All patients included in the present study provided written samples obtained from patients with CRC + m and patients informed consent prior to the surgery. with CRC - m were used for validation of the results. Following extraction of total RNA, the RNA were reverse-transcribed Samples. A total of 6 primary CRC samples were obtained to cDNA using 5x HiScript® II qRT SuperMix II (R223-01, from 3 male patients with CRC and lung metastasis Vazyme, Piscataway, NJ, USA) according to the manufac- (CRC + m) (age, 66.33±7.37 years) and 3 male patients with turer's protocol. Subsequently, qPCR was performed in 10 µl CRC without metastasis (CRC - m) (age, 64.67±11.93 years). reactions, including 0.5 µl PCR forward primer (10 µM), 0.5 µl All patients were histologically confirmed to have colorectal PCR reverse primer (10 µM), 2 µl cDNA, 5 µl 2x QuantiFast® cancer and did not receive any other forms of therapy at the SYBR® Green PCR Master Mix (204054, Qiagen, Inc.) and time of enrollment. At the time of surgery, all tissue samples 2 µl double distilled water. The following thermocycling were immediately frozen in liquid nitrogen and stored conditions were used for qPCR: Initial denaturation at 95˚C at ‑80˚C for further use. for 10 min; followed by final extension of 40 cycles of 95˚C for 10 sec and 60˚C for 60 sec. β-actin served as a reference RNA extraction and quality control. Total RNA was extracted gene. Relative expression levels of each lncRNA was calcu- from each sample using a homogenizer and TRIzol regent lated using the 2-ΔΔCq method (17). The statistical significance (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according of the difference was estimated by a t-test using SPSS v20.0 to the manufacturer's protocol. Subsequently, the quantity and (IBM Corp., Armonk, NY, USA); P<0.05 was considered to quality of purified RNA were assessed by NanoDrop ND‑1000 indicate a statistically significant difference. The primers used (Thermo Fisher Scientific, Inc.). The integrity of RNA was in the present study were as follows: HOXA distal transcript assessed by electrophoresis on a denaturing 1.5% agarose gel antisense RNA (HOTTIP) forward (F), 5'-CCT AAA GCC and staining with ethidium bromide for 30 min at 60˚C. ACG CTT CTT TG-3' and reverse (R), 5'-TGC AGG CTG GAG ATC CTAC T-3'; RP11-79H23.3 F, 5'-GCA AGG AGA GTA ATG Microarray analysis. Sample labeling and human lncRNA CTG GA-3' and R, 5'-CAA TGA GGA TGA GAA GAG GTC-3'; array hybridization (Human LncRNA Expression Microarray urothelial cancer associated 1 (UCA1) F, 5'-GTC AAC GGA v4.0, Arraystar, Inc., Rockville, MD, USA) were performed TTT GGT CTG TAT T-3' and R, 5'-AGT CTT CTG GGT GGC according to the manufacturer's protocol. Total RNA were AGT GAT-3'; metastasis asisassociated lung adenocarcinoma digested with RNase R (Epicentre; Illumina, Inc., San transcript1 (MALAT1) F, 5'-GGT AAC GAT GGT GTC GAG Diego, CA, USA) to remove linear RNA and enrich circular GTC-3' and R, 5'-CCA GCA TTA CAG TTC TTG AAC ATG-3'; (circ)RNA. The enriched circRNA were amplified and LOC100507661 F, 5 -CT CGG ATC CTA CCA TCA TGG CTC transcribed into fluorescent lncRNA using random primers ACT GCA ACC TC-3' and R, 5'-CCC TCT AGC GCC GCT (Arraystar Super RNA Labeling kit; Arraystar, Inc.). Labeled TTT TAT GCA TCA AAA ATA AAG GTG-3'; maternally lncRNA were purified by RNeasy Mini Kit (Qiagen, Inc., expressed 3 (MEG3) F, 5'-CTG CCC ATC TAC ACC TCA Valencia, CA, USA). The concentration and specific activity of CG-3' and R, 5'-CTC TCC GCC GTC TGC GCT AGG GGC T-3'; the labeled lncRNA (pmol Cy3/µg lncRNA) were measured by and β-actin F, 5'-AGC ACA GAG CCT CGC CTT TG-3' and R, NanoDrop ND-1000. A total of 1 µg of each labeled lncRNA 5'-CTT CTG ACC CAT GCC CAC CA-3'. was fragmented by adding 5 µl 10X blocking agent and 1 µl 25X fragmentation buffer (Arraystar Super RNA Labeling Gene ontology (GO) analysis.
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