Gene microarray analysis of the circular RNAs expression profile in human gastric cancer

  • Authors:
    • Yonghua Shen
    • Juanjuan Zhang
    • Ziyi Fu
    • Bin Zhang
    • Min Chen
    • Xiufeng Ling
    • Xiaoping Zou
  • View Affiliations

  • Published online on: April 26, 2018     https://doi.org/10.3892/ol.2018.8590
  • Pages: 9965-9972
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Abstract

Human gastric cancer is a common malignant neoplasm of the digestive system and represents a threat to human health worldwide. The mechanisms underlying gastric cancer germination and development are not yet fully understood. Circular RNAs (circRNAs) serve crucial roles in various physiological and pathological processes, particularly cancer. However, few studies have focused on the mechanisms involving circRNAs in gastric cancer. Therefore the present study set out to identify the differentially expressed circRNAs in gastric cancer. Three specimens of gastric cancer and normal gastric tissue were selected and circRNA microarray analysis was performed to detect the differentially expressed circRNAs. The changes in circRNAs were confirmed by reverse transcription‑quantitative polymerase chain reaction analysis. A total of 347 upregulated and 603 downregulated circRNAs (fold‑change, >2.0) were identified in gastric cancer compared with the normal gastric tissue. A total of 20 selected circRNAs were dysregulated during gastric cancer, which suggests their potential role in gastric cancer. The present study identified circRNAs in the expression profile of human gastric cancer that were potentially involved in the underlying molecular mechanisms of its development.

Introduction

Human gastric cancer is the fourth most common malignancy and the second leading cause of cancer-related deaths worldwide (1). Adenocarcinoma is the most common pathological type of gastric cancer, while lymphoma, carcinoid, and sarcoma constitute <5% of the pathology (2). The pathogenesis of gastric cancer is complicated owing to the interaction of multiple factors, including Helicobacter pylori infection, environment, and heredity. The environmental factors play critical roles in the pathogenesis of gastric cancer; the major risk factors include smoking and diet (3). However, determining the molecular markers of gastric cancer is yet a great challenge.

Recently, the identification and characterization of circular RNA (circRNA) have revolutionized the field of RNA. CircRNAs have gained increasing attention in deciphering the complicated mechanisms underlying the malignant processes such as tumorigenesis, multidrug resistance, invasion, and metastasis. Although circRNAs have been reported as early as 20 years ago (4), they were mostly misinterpreted as splicing artefacts or gene rearrangements. Following high-throughput RNA sequencing and bioinformatics, thousands of different circRNAs have been rediscovered in the recent several years (58). Preliminary data revealed that circRNAs were abundantly expressed and evolutionarily conserved across the eukaryotes and functioned as miRNA sponges (5,6,9). Cdr1as (also known as ciRS-7), as the maximally studied circRNA, was reported as the miR-7 sponge or inhibitor (5,6).

ciRS-7 was highly expressed in a wide variety of cancer cell lines, and ciRS-7/miR-7 network suggested a therapeutic potential for carcinoma. This network might regulate the majority of the cancer pathways such as p21-activated kinase 1 (Pak1) (10), epidermal growth factor receptor (EGFR) (11), activated cdc42-associated kinase 1 (Ack1) (12), and phosphoinositide 3-kinase catalytic subunit delta (PIK3CD) (13). Increasing number of evidence indicated miR-7 as a potential tumor suppressor in several human cancers. Xiong et al reported that miR-7 selectively induced growth suppression and apoptosis of non-small cell lung cancer (NSCLC) by targeting B-cell lymphoma-2 (BCL-2) in vitro (14). Similarly, miR-7 was confirmed as a novel miRNA exhibiting tumor suppression function in colon cancer (15). Although the majority of reports supported the tumor-enhancing effect of circRNAs, the converse was also reported. ciR-ITCH demonstrated an inhibitory effect on esophageal squamous cell carcinoma, acting as a sponge of miR-7, miR-17, and miR-214 via the regulation of the Wnt/β-catenin pathway (16). Altogether, these results suggested that the relationship between circRNA and cancer was complicated and precise mechanisms needed further investigation.

In this study, we presented the circRNAs' expression profile in normal gastric tissue and gastric adenocarcinoma through microarray technology in order to explore the function of circRNAs in gastric cancer for early diagnosis and treatment of cancer.

Materials and methods

Patient samples

The present study was approved by the Research Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University, Medical School, and all patients provided informed consent before the samples were collected. Gastric cancer was confirmed by histopathological diagnosis. Finally, 15 patients (eight men, seven women; mean age 64.1 years, range 48–81) were enrolled and 15 pairs of gastric carcinoma tissues and normal para-carcinoma samples were collected. All samples were rapidly frozen in liquid nitrogen and stored at −80°C for subsequent investigation. For circRNA microarray analysis, a total of 3 gastric carcinoma and 3 normal para-carcinoma gastric tissues (control) were randomly selected for the study.

RNA extraction

Total RNA was extracted from the frozen tissue block that was homogenized (IKA Werke GmbH & Co. KG, Staufen, Staufen, Germany) and resuspended in TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Then, the total RNA was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Additionally, the RNA integrity was assessed by denaturing agarose gel electrophoresis.

RNA labeling and array hybridization

The sample labeling and microarray hybridization were performed by KangChen Biotech (Shanghai, China). Briefly, the circRNA was treated with RNase R (Epicenter Biotechnologies, USA) to remove the linear RNAs. Each sample was amplified and transcribed into fluorescent cRNA using a random priming method (Arraystar Super RNA Labeling kit; Arraystar Inc., Rockville, MD, USA). Subsequently, these labeled cRNAs were purified by RNeasy Mini kit (cat. no. 74106, Qiagen GmbH, Hilden, Germany Germany), and the concentration and specific activity were measured by NanoDrop (NanoDrop Technologies). Then, 1 µg of each labeled cRNA was fragmented by adding 5 µl of 10X blocking agent and 1 µl of 25X fragmentation buffer, followed by heating at 60°C for 30 min. Then, 25 µl of 2X hybridization buffer was added to dilute the labeled cRNA, and 50 µl of the hybridization solution was dispensed into the gasket slide and assembled on the circRNA expression microarray slide. These slides were incubated for 17 h at 65°C in a hybridization oven (Agilent). The hybridized arrays were washed, fixed, and scanned using the Axon GenePix 4000B microarray scanner (Molecular Devices, LLC, Sunnyvale, CA, USA).

Microarray analysis

The Arraystar Human Circular RNA Microarray V2.0 (Arraystar, Inc.) was designed for the purpose of profiling both circRNAs and protein-coding RNAs in the human genome. The differentially expressed circRNAs were identified through fold-change filtering and standard Student's t-test. The circRNAs are exhibiting a fold-change ≥2.0 and a P-value <0.05 were selected as significantly differentially expressed circRNAs.

Quantitative reverse transcription PCR

cDNA samples were prepared from total RNA of gastric tissues by reverse transcription. In total, 20 circRNAs were analyzed by SYBR Green I dye-based detection with specific primer sequences. The primer sequences were shown in Table I. The 2−ΔΔCq method was performed in 18 samples and applied for the quantification of the relative expression of circRNAs that was normalized against the expression of the housekeeping gene, GAPDH.

Table I.

Primer sequences for reverse transcription-polymerase chain reaction.

Table I.

Primer sequences for reverse transcription-polymerase chain reaction.

GeneDirectionPrimer sequence
hsa_circ_0001017Forward AGTGCGAAGTAATCTATGCCAGC
Reverse AGCCATTCTTTGCTGGGCTC
hsa_circ_0001772Forward GCCAGAGGAGGAGCAGCTTTA
Reverse GCTCTTCATCTGACAAATCCGAC
hsa_circ_0002346Forward GTGCAAACCAGTTTTCGGCG
Reverse TCCAGTTCTCATCTTGTTGGCA
hsa_circ_0000072Forward TTGGCAGCAAATGGAGTTCGT
Reverse GTGCCTGCCACCATTTCCTTA
hsa_circ_0003221Forward GATGCGGGGCAATGCACTA
Reverse ACCAGTACCCAGGTGAGTCTT
hsa_circ_0001865Forward GCTCCACAGACTTCCCAGAGT
Reverse GGCAAGTTCCAACGTCTCCT
hsa_circ_0003441Forward ACCACAGTTCTTGGTGGTGAAG
Reverse TGACTTTGTCTGGAGAGCTTGTG
hsa_circ_0008285Forward GCTGTTAACGGGAAAGGTTGAA
Reverse GCGTCTGTTGAAGTCGTGGA
hsa_circ_0023923Forward AGCACATCAAAGCTGCCCAA
Reverse TGCACTGAATTAAGTCTCCCCA
hsa_circ_0000347Forward GAAAAAGAACCAATGCAAAGAAGGT
Reverse GCACTGAATTAAGTCTCTGCAACT
hsa_circ_0046881Forward AAGTCAGGCAGCTTTGCTGG
Reverse CACAGTTGGTTAGCCACAGC
hsa_circ_0023940Forward ATGCTCCTGTTCAAAGATGCCA
Reverse TTTGAAGACCACCACCCAACT
hsa_circ_0023891Forward CCTGCTACTACACCAACAGGC
Reverse ACTGAATTAAGTCTGTGCTCCTGA
hsa_circ_0002433Forward TGAGCGTTTTATTCAGTATTTGGCT
Reverse GCACTGAATTAAGTCTTGCAATCCA
hsa_circ_0050278Forward AAGCCAGACCTGATCACTTGTC
Reverse TGTCAATGGTCCCTGTGGGT
hsa_circ_0000154Forward ACCAACGTTGAGCAAGATGC
Reverse TTCTCCAGTGTCATTCCAACAGA
hsa_circ_0075048Forward GGCCACATCGACAACTCCAT
Reverse GCTCGTTCACACTTGTTGATGC
hsa_circ_0001824Forward TGCATCAGCTCCAGGGCAAT
Reverse TTGAAAGAAATGTGGCATGTGAGA
hsa_circ_0000835Forward CAGCATGGTCATGGAGGATGG
Reverse ATGCTTGATGCCTATTGCCACT
hsa_circ_0000825Forward GAAAAGCGCGCTAAAGCTGA
Reverse TCCATCTCAGCACGGAGTTCA
hsa_circ_0009109Forward ATCTGGCTCAGATGACACCAA
Reverse TATGTTTGCTCGGTGCCCTG
hsa_circ_0087855Forward ACTTCCACACCTGCATCCAT
Reverse TGCTTTCACCTGTCAGTTGCT
hsa_circ_0001747Forward GACAAGCTGGTGTTGAAGGGT
Reverse AGCAGGCCTTTCGAGCTTTAG
hsa_circ_0009061Forward CCAAGCATCAGGTGTGGAGG
Reverse TCTCTGTACTCTACTGTGCGGT
hsa_circ_0000997Forward TGCACCACTGGATGTTGTTTACT
Reverse GTGGTCTCCACCTGTTTTGGAT
hsa_circ_0001073Forward ACTTGTTCCAACTCAAGTGCTATAC
Reverse GTAGCAAAACAATGCCGCCG
hsa_circ_0000085Forward TTTGGCAGACTTTTACCTGGTG
Reverse TGGATTGCTGCTTAAGCTTCCT
hsa_circ_0088021Forward GCTGAACAGGTGCCTGAACT
Reverse CAATTCCAGGTCTGCTGCCG
hsa_circ_0020353Forward GCAGACTCCTGCAAGTTCCC
Reverse GTGCTTATCCACAAGGGCCA
Statistical analysis

The two groups were compared by the standard Student's t-test for the evaluation of the microarray analysis. The results were considered statistically significant at a P-value <0.05. The false discovery rate (FDR) was calculated to correct the P-value. Fold-change ≥2.0 and P-value <0.05 were used to identify the differentially expressed circRNAs.

Results

circRNA expression profiles in gastric cancer tissues relative to adjacent normal gastric tissues

Randomly, 3 gastric cancer and 3 adjacent normal gastric tissues were selected for a standard circRNA microarray independently. The circRNA expression patterns between gastric cancer and the adjacent normal gastric tissues were found to be significantly different. After scanning and normalization, a total of 950 circRNAs were found to be differentially expressed in the microarray (fold-change in expression ≥2.0, P<0.05), consisting of 347 upregulated circRNAs and 603 downregulated circRNAs (data not shown). The hierarchical clustering of circRNA expression described the variation in the expression between the groups of gastric cancer and normal gastric tissues (Fig. 1). Furthermore, the variation in circRNA expression among the samples was assessed by Scatter-plot visualization (Fig. 2).

Annotation of differentially expressed circRNAs in gastric cancer tissues

The general data, including the chromosome location, length distribution and functional classification of these differentially expressed circRNAs were summarized. Fig. 3A and B demonstrated that the up- and downregulated circRNAs were located in human chromosomes. The length data displayed two peaks that were distributed among these dysregulated circRNAs in ≤400 bp and 1,601–25,600 bp (Fig. 3C). Fig. 3D revealed the relationship between the mentioned circRNAs and their molecular targets including exonic, intronic, antisense and intragenic. Nevertheless, the exonic targets occupied the vast majority of all types of functional classification.

Reverse transcription-PCR validation of some differentially expressed circRNAs

We set a threshold as log2 fold-change >5 in upregulated circRNAs, >3 in downregulated circRNAs as the previous study (17), and P-value <0.05, and found 29 upregulated (Table II) and 28 downregulated differentially expressed circRNAs (Table III). Next, we randomly selected 20 differentially expressed circRNAs, including 10 upregulated (102713, 100715, 100261, 100924, 104804, 104707, 102830, 102298, 100911, and 102293) and 10 downregulated circRNAs (103538, 104318, 103477, 104280, 100144, 104984, 001369, 103901, 100677, and 102464) for substantiation in the gastric tissue samples. The results of the microarray were in agreement with those of the real-time PCR; 6 selected upregulated circRNAs (Fig. 4A) and 5 selected downregulated circRNAs (Fig. 4B) were verified. However, the expressions of upregulated (Fig. 4C) and downregulated circRNAs (Fig. 4D) were not related to the expression of the host genes.

Table II.

Upregulated circRNAs between gastric cancer and normal gastric tissues.

Table II.

Upregulated circRNAs between gastric cancer and normal gastric tissues.

circRNAP-valueFCLog2 FCRegulationcircRNA typeChromStrandBest transcriptGene symbol
hsa_circRNA_104804<0.001117.2196.873UpExonicchr9uc004amv.3UBQLN1
hsa_circRNA_102678<0.001125.1196.967UpExonicchr2+uc002rpd.3CRIM1
hsa_circRNA_1002610.01141.3195.368UpExonicchr1uc001dex.4ANKRD13C
hsa_circRNA_1009270.00270.9196.148UpExonicchr11uc001pbl.3PICALM
hsa_circRNA_1009240.00188.2196.463UpExonicchr11uc001pbl.3PICALM
hsa_circRNA_100922<0.001101.1196.659UpExonicchr11uc001pbl.3PICALM
hsa_circRNA_1027130.01044.1195.463UpExonicchr2uc002rus.3SRBD1
hsa_circRNA_1024990.00856.5195.820UpExonicchr19+uc002npf.3ZNF85
hsa_circRNA_1023150.00752.2195.706UpExonicchr18+uc002ktp.3MIB1
hsa_circRNA_1009110.00471.3196.156UpExonicchr11uc001pbl.3PICALM
hsa_circRNA_1048500.01545.3195.502UpExonicchr9+uc011lwa.2RAD23B
hsa_circRNA_1009250.00275.2696.233UpExonicchr11uc001pbl.3PICALM
hsa_circRNA_1022930.01049.9195.641UpExonicchr18+uc002knq.2CCDC165
hsa_circRNA_102737<0.001133.6197.061UpExonicchr2uc002sbj.3XPO1
hsa_circRNA_1048680.02134.5195.109UpExonicchr9uc010muc.1KIAA0368
hsa_circRNA_1003810.01348.6195.603UpExonicchr1+uc001gev.3DCAF6
hsa_circRNA_104707<0.001120.0196.907UpExonicchr8uc003yvs.3PTK2
hsa_circRNA_104532<0.001132.2197.046UpExonicchr7+uc003wme.3RBM33
hsa_circRNA_1044920.01245.3195.502UpExonicchr7+uc003vqs.3MKLN1
hsa_circRNA_1046890.00953.7195.747UpExonicchr8uc003ysz.2ASAP1
hsa_circRNA_1003800.01056.2195.812UpExonicchr1+uc001gev.3DCAF6
hsa_circRNA_1040160.01154.2195.760UpExonicchr5+uc003mby.4ERGIC1
hsa_circRNA_1007150.02134.4195.105UpExonicchr10uc001lif.4CTBP2
hsa_circRNA_101270<0.001108.2196.757UpExonicchr13+uc001vib.4TDRD3
hsa_circRNA_1028300.01441.5195.375UpExonicchr2+uc002twg.3ACVR2A
hsa_circRNA_100241<0.001123.4196.947UpExonicchr1uc001cyx.1OMA1
hsa_circRNA_1040520.001103.3196.690UpExonicchr6+uc003mwi.3CDYL
hsa_circRNA_1022980.00387.3196.448UpExonicchr18uc002kod.1PPP4R1
hsa_circRNA_1000970.01144.6195.479UpExonicchr1+uc001bgi.2KDM1A

[i] FC, fold change; chrom, chromosome; circRNA, circular RNA.

Table III.

Downregulated circRNAs between gastric cancer and normal gastric tissues.

Table III.

Downregulated circRNAs between gastric cancer and normal gastric tissues.

circRNAP-valueFCLog2 FCRegulationcircRNA typechromStrand Best_transcriptGene symbol
hsa_circRNA_103538<0.001−40.819−5.351DownExonicchr3+uc003fpi.3MAP3K13
hsa_circRNA_1020160.038−8.032−3.005DownExonicchr17uc002heo.1SSH2
hsa_circRNA_1024640.006−17.362−4.117DownExonicchr19+uc002myp.3PKN1
hsa_circRNA_1001090.036−8.135−3.024DownExonicchr1+uc001bmt.1ARID1A
hsa_circRNA_1034770.002−20.819−4.379DownExonicchr3+uc003eqt.3EPHB1
hsa_circRNA_1008010.007−21.424−4.421DownExonicchr11+uc001mxq.4HSD17B12
hsa_circRNA_1049840.028−12.154−3.603DownExonicchrXuc004czk.2MAP3K15
hsa_circRNA_1049680.030−9.387−3.230DownExonicchr9+uc004coa.3EHMT1
hsa_circRNA_1015850.034−11.713−3.550DownExonicchr15uc010biv.1CELF6
hsa_circRNA_1025400.035−10.064−3.331DownExonicchr19+uc002ohk.3SIPA1L3
hsa_circRNA_1035680.038−9.790−3.291DownExonicchr3uc003fxp.2DLG1
hsa_circRNA_100144<0.001−57.519−5.845DownExonicchr1+uc001bui.3TXLNA
hsa_circRNA_1050410.005−27.519−4.782DownExonicchrXuc004flx.1G6PD
hsa_circRNA_103901<0.001−36.919−5.206DownExonicchr5uc003kfo.3LHFPL2
hsa_circRNA_1000610.004−19.228−4.265DownExonicchr1uc001aub.3DHRS3
hsa_circRNA_1040460.003−20.371−4.348DownExonicchr6+uc003mtz.3WRNIP1
hsa_circRNA_1043510.040−9.361−3.226DownExonicchr7uc011kbg.2GLI3
hsa_circRNA_1046010.035−12.922−3.691DownExonicchr8uc003xpe.3SLC20A2
hsa_circRNA_102489<0.001−35.219−5.138DownExonicchr19+uc002nkf.3UPF1
hsa_circRNA_1024710.041−8.065−3.011DownExonicchr19+uc002nfj.1MYO9B
hsa_circRNA_1042800.002−21.819−4.447DownExonicchr7uc003six.1PRKAR1B
hsa_circRNA_1016570.038−9.248−3.209DownExonicchr15+uc010urq.2IGF1R
hsa_circRNA_1043180.032−11.642−3.541DownExonicchr7uc003sti.3ANKMY2
hsa_circRNA_1022120.038−9.358−3.226DownExonicchr17uc002jwc.1USP36
hsa_circRNA_1007520.007−24.319−4.604DownExonicchr11uc001maq.2OR51B5
hsa_circRNA_1041900.002−23.098−4.529DownExonicchr6uc003qez.2HBS1L
hsa_circRNA_0013690.024−10.828−3.436DownAntisensechr12NM_000020ACVRL1
hsa_circRNA_1006770.005−21.387−4.418DownExonicchr10uc009xxl.3PCGF6

[i] FC, fold change; circRNA, circular RNA; chrom, chromosome.

Discussion

Human gastric cancer is one of the most commonly known malignancies all over the world. A large number of studies have shown that the occurrence of gastric cancer involves several molecular mechanisms. However, the precise biological process of gastric cancer is not yet clearly elucidated. Several circRNAs have been recently discovered constituting a new specific class of endogenous non-coding RNAs. Hsa_circ_001569 promoted colorectal cancer in cell proliferation and invasion as a sponge of miR-145 (18). On the contrary, hsa_circ_002059 was found to be significantly down-regulated in gastric cancer as a typical circRNA, and its expression level was correlated with tumor metastasis and TNM stage (19). Thus, circRNAs might play a major role in the occurrence and development of gastric cancer; however, our understanding about the correlation between circRNAs expression and gastric cancer remains controversial due to the limited number of studies. Hence, the expression profile of circRNAs in gastric cancer necessitates further exploration with respect to the potential mechanisms.

The specific repeated pattern of chromosomal aberrations is not associated with tumorigenesis and progression in gastric cancer. The current study conformed to the conclusion considering that all the chromosomes can experience unequable changes (20). However, there are inconsistencies regarding the chromosomal location of dysregulated circRNAs. Our experimental results revealed that chromosomal abnormalities were mainly distributed on chromosomes 1, 2, 3, 6, 9, 11, and 17, while previous studies designated chromosomes 8, 12, 15, 17, and 20 in gastric cancer (21,22). This phenomenon might be attributed to the following: firstly, the experimental method to detect chromosomal dysregulation in the past was FISH technology, while currently gene microarray is employed. Secondly, the sample size in the current study was small due to the high cost of gene microarray technology. Finally, the inherent differences in gastric cancer, such as the degree of pathological differentiation, stages, and grades, might also result in the differential distribution of chromosomal abnormalities.

Here, we reverse transcription-PCR verified the microarray analysis results. Recent evidence demonstrated that circRNAs play a crucial role in fine-tuning the level of miRNA-mediated regulation of gene expression via miRNA sequestration (23,24). In addition, several of the predicted binding sites of circRNAs on miRNAs are functional and appear to be under less selective pressure as compared to the corresponding miRNA binding sites in mRNAs (25). However, in the current study, the expression of circRNAs did not correlate with the expression of the host genes, suggesting an independent regulation of transcription vs. circRNA formation. We will verify the findings in further studies with larger sample size. Combining with previous studies (26,27), we currently propose that the circRNA-miRNA-mRNA axis may be the putative mechanism promoting the growth of the tumor, although the specific effect might not be deduced. Thus, further studies are essential for an insight into the exact mechanism.

In conclusion, we reported the profile of differentially expressed circRNAs between normal gastric and gastric cancer tissues. The network of differentially expressed numerous circRNAs was constructed and they found to be involved in the development and metabolism of gastric cancer with our and previous studies (2729). Therefore, a further exploration of the biological processes and molecular mechanisms of the dysregulated circRNAs is imperative in order to clarify the pathogenesis of gastric cancer or provide a new therapeutic target via the regulation of the key circRNAs.

Acknowledgements

The authors would like to thank Dr Tianwei Gu for his linguistic assistance.

Funding

The present study was supported by the Outstanding Youth Project of Nanjing City (grant no. JQX14005) and the Medjaden Academy & Research Foundation for Young Scientists (Grant no. MJR20170029).

Availability of data and materials

All data generated or analyzed during the study are included in this published article.

Authors' contributions

XZ and XL designed the research. YS, JZ, ZF, BZ, MC, XL and XZ performed the experiments. YS, JZ, BZ and MC analyzed the data. YS wrote the paper and ZF, XL and XZ critically revised the manuscript for important intellectual content.

Ethics approval and consent to participate

The present study was approved by the Research Ethics Committee of the Affiliated Drum Tower Hospital of Nanjing University, Medical School (Nanjing, China) and all patients provided informed consent prior to their inclusion within the study.

Consent for publication

All patients provided written informed consent for the publication of their data.

Competing interests

The authors declare that they have no competing interests.

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June-2018
Volume 15 Issue 6

Print ISSN: 1792-1074
Online ISSN:1792-1082

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Spandidos Publications style
Shen Y, Zhang J, Fu Z, Zhang B, Chen M, Ling X and Zou X: Gene microarray analysis of the circular RNAs expression profile in human gastric cancer. Oncol Lett 15: 9965-9972, 2018
APA
Shen, Y., Zhang, J., Fu, Z., Zhang, B., Chen, M., Ling, X., & Zou, X. (2018). Gene microarray analysis of the circular RNAs expression profile in human gastric cancer. Oncology Letters, 15, 9965-9972. https://doi.org/10.3892/ol.2018.8590
MLA
Shen, Y., Zhang, J., Fu, Z., Zhang, B., Chen, M., Ling, X., Zou, X."Gene microarray analysis of the circular RNAs expression profile in human gastric cancer". Oncology Letters 15.6 (2018): 9965-9972.
Chicago
Shen, Y., Zhang, J., Fu, Z., Zhang, B., Chen, M., Ling, X., Zou, X."Gene microarray analysis of the circular RNAs expression profile in human gastric cancer". Oncology Letters 15, no. 6 (2018): 9965-9972. https://doi.org/10.3892/ol.2018.8590