Clinical Significance of Upregulation of mir-196 a-5 p in Gastric Cancer and Enriched KEGG Pathway Analysis of Target Genes

Gastric cancer (GC) is the fifth most common cancer but the third leading cause of cancer death (Fock, 2014), and the common characteristics of GC is high incidence rates, poorly early diagnosis rates and high mortality rates. Epidemiology data indicated that the current trend of GC is severe worldwide, particular in Asia (Katanoda et al., 2013; Liu et al., 2013; Koessler et al., 2014). The major reason of which is associated with lack of specificity of symptoms for early diagnosis and treatment. For example, GC is diagnosed at an early stage in less than 10% of cases in Europe (Saika and Sobue, 2013). The funding


Introduction
Gastric cancer (GC) is the fifth most common cancer but the third leading cause of cancer death (Fock, 2014), and the common characteristics of GC is high incidence rates, poorly early diagnosis rates and high mortality rates.Epidemiology data indicated that the current trend of GC is severe worldwide, particular in Asia (Katanoda et al., 2013;Liu et al., 2013;Koessler et al., 2014).The major reason of which is associated with lack of specificity of symptoms for early diagnosis and treatment.For example, GC is diagnosed at an early stage in less than 10% of cases in Europe (Saika and Sobue, 2013).The funding genes or oncogenic genes in cancer tumorigenesis as well as in gastric cancer (Shenouda and Alahari, 2009;Bartels and Tsongalis, 2010;Cortes-Sempere and Ibanez de Caceres, 2011).Mir-196a is a newly reported miRNA biomarkers relevant to multiple cancers, such as breast cancer (Lee et al., 2014), lung cancer (Liu et al., 2012), cervical cancer (Gocze et al., 2013), renal cancer (Du et al., 2014a), head and neck cancer (Christensen et al., 2010), hepatocellular cancer (Hao et al., 2013), pancreatic cancer (Slater et al., 2014), colorectal cancer (Zhan et al., 2011;Du et al., 2014b) and early gastric cancer (Zheng et al., 2014).
In previous study, mir-196a-5p was discovered over expressed in gastric cancer cell lines and gastric cancer tissues (Sun et al., 2012;Tsai et al., 2014).But the clinical significance of mir-196a-5p in cancers of previous reports was different, probably, which may be caused by different representatives of different samples.So expanded study will clarify its clinical significance and understand its roles in gastric cancer, and will make it useful for GC cancer molecular biomarkers discovery.In present study, we decided to validate the expression of mir-196a-5p in seven gastric cancer cell lines comparing with GES-1 cell line, and detect the expression of mir-196a-5p in gastric cancer tissues comparing with adjacent non-cancerous tissues of 58 cases comes from a high gastric cancer incidental rate area.In addition, bioinformatics analysis of its target genes and enriched KEGG pathways will be execute to understand its roles in cancer tumorigenesis, particularly in the case of gastric cancer.

Cell lines
Seven gastric cancer cell lines, AGS, SGC-7901, MKN-45, MKN-28, MGC-803, BGC-823, HGC-27 and GES-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and Cancer Institute and Hospital, Chinese Academy of Medical Sciences (CAMS) (Beijing, China).All the gastric cancer cell lines were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum in a humidified cell incubator having an atmosphere of 5% CO2 at 37˚C.Exponentially growing cells were used for experiments.MiRNA PCR primers were purchased from GeneCopoeia™ Inc.

Clinical samples
58 gastric cancer samples were obtained during surgery and used after obtaining informed consent.All patients underwent curative resection of the primary tumor at WuWei city tumor Hospital from the year of 2010 to 2012 (WuWei, China, a high incidental rate area with gastric cancer (Li et al., 2004;ZHANG et al., 2009).All patients had a clear histological diagnosis of gastric cancer, based on the clinicopathologic criteria.All data, including age, sex, histological grade, depth, lymph node metastasis, Local invasion, Depth of tumor invasion, lymph node metastasis, lymphatic invasion, Venous invasion, borrmann type and clinical stage were obtained from clinical and pathologic records.No patients received neoadjuvant chemotherapy or radiotherapy before surgery and adjuvant radiotherapy after surgery.Resected cancerous tissues (T) and paired noncancerous tissues (N) were immediately cut and stored in, frozen in liquid nitrogen, and kept at-80˚C until RNA extraction.
Written informed consent was obtained from each patient for his or her participation in the study.The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the ethics committee of First Hospital of Lanzhou University.

Total RNA isolation and quality analysis
Total RNA of gastric cancer cell lines and frozen tissues of gastric cancer were extracted using RNeasy mini kit (QIAGEN, Hilgen, Germany) according to the manufacturer's instructions.Concentrations and purity of the RNA samples were assayed by electrophoresis and spectrophotometric methods.

MiRNA quantification by qRT-PCR
MiRNA quantification by real-time qRT-PCR.SYBR green qRT-PCR assay was used for miRNA quantification.In brief, 40 ng of total RNA containing miRNA was polyadenylated by poly(A) polymerase and was reversely transcripted to cDNA using miScript Reverse Transcription kit according to the manufacturer's instructions (GeneCopoeia™, Rockville, USA).miScript SYBR Green PCR kit was used and miscript Universal primer was provided by the manufacturer (GeneCopoeia™, Rockville, USA), qRT-PCR was performed in BIORAD CFX96 Real-time PCR system.Each reaction was performed in a final volume of 10 μl containing 2 μl of cDNA, 0.5 mM of each primer and 1X SYBR Green PCR Master mix (GeneCopoeia™, Rockville, USA).The amplification program was: denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 10 sec, 60˚C for 30 sec and 72˚C for 30 sec, in which fluorescence was acquired.At the end of the PCR cycles, melting curve analyses were performed as well as electrophoresis of the products on 2.5% agarose gels in order to validate the specific generation of the expected PCR product.Each sample was run in triplicates for analysis.The expression levels of miRNAs were normalized to RNU6B.Relative gene expression was calculated as 2-(CTmiRNA-CTRNU6B RNA).

MiRNA Targeted Gene Prediction and KEGG Pathway
Analyses by miRWalk and DAVID 6.7 We utilized a miRNA target gene prediction database miRWalk (Dweep et al., 2011) to select predicted and validated targets, and to analysis enriched KEGG pathways by bioinformatics tool DAVID 6.7.The miRWalk prediction database integrated 10 bioinformatics Target Prediction Tools: DIANA-mT, miRanda, miRDB, miRWalk, RNAhybrid, PICTAR4, PICTAR5, PITA, RNA22 and TargetScan.Enriched KEGG pathway analyses of mir-196a-5p targeted genes were performed by bioinformatics tool DAVID 6.7.Prediction Databases Support Number were at least 5, P value of Fisher Test were P<0.05.DOI:http://dx.doi.org/10.7314/APJCP.2015.16.5.1781 Clinical Significance of mir-196a-5p in Gastric Cancer andEnriched KEGG Pathway Analysis of its Target Genes MiRNA Targeted Gene Prediction and KEGG Pathway Analyses by mirfocus 3.0 We utilized a miRNA target gene prediction database mirfocus 3.0 (http://mirfocus.org/index.php) to select validated targets of mir-196a-5p to analysis its enriched KEGG pathways and to annotate the molecular function of the miRNA targeted genes.The mirfocus 3.0 integrated 5 bioinformatical Target Prediction Tools: MiRanda, MirTarget2, PicTar, microT and TargetScanS, and the experimental validated Target Tools include miRecords, miR2Disease, TarBase and miRTarBase.Enriched KEGG pathway of mir-196a-5p targeted genes also were performed by mirfocus 3.0.Prediction Databases Support Number was 3, P value of Fisher Test were P<0.05.

Statistical analysis
Student's unpaired t-test was used to compare values of samples of 7 gastric cancer cell lines and samples of GES-1 gastric cell line.Differences between groups were estimated using the X 2 test.A probability level of 0.05 was chosen for statistical significance, and all statistical analyses were performed using the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).

Expression of mir-196a-5p in gastric cancer tissues comparing with adjacent non tumor tissues
After detection the expression of mir-196a-5p in gastric cancer cell lines, the levels of mir-196a-5p in 58 cancerous and corresponding non-cancerous tissues were also detected by qRT-PCR method.Results showed that the numbers of mir-196a-5p in high-expression group (T/ N>2) and low-expression group (T/N<0.5)amounts to 47 and 11 respectively, according to the median cancer (T)/ noncancerous (N) tissue ratio of mir-196a-5p expression (Figure 2, Table 1)..2015.16.5.1781 Clinical Significance of mir-196a-5p in Gastric Cancer andEnriched KEGG Pathway Analysis of its Target Genes KEGG pathway analyses of mir-196a-5p targeted genes by miRWalk and DAVID 6.7 In order to investigate the possible regulation mechanisms of mir-196a-5p in the process of gastric cancer, we utilized an online bioinformatics database miRWalk to select plausible targets and validated targets of this miRNA and to analysis Enriched KEGG pathways by DAVID 6.7.
Based on predicted targets of miRWalk, a total of 1081 target genes were searched as the target genes of mir-196a-5p.Enriched KEGG pathway analyses showed that the targeted genes which were regulated by mir-196a-5p were involved 31 pathways, and 13 of which were centralized in cancer associated terms, which were as follows: prostate cancer, chronic myeloid leukemia, acute myeloid leukemia, glioma, small cell lung cancer, melanogenesis, pancreatic cancer, TGF-beta signaling pathway, mTOR signaling pathway, chemokine signaling pathway, Melanoma, Pathways in cancer and p53 signaling pathway (Table 2, Figure 3).
Based on validated targets of miRWalk, a total of 209 target genes were searched as the target genes of mir-196a-5p.Enriched KEGG pathway analyses showed that the targeted genes which were regulated by mir-196a-5p were involved 14 pathways, and 10 of which were centralized in cancer associated terms, which were as follows: acute myeloid leukemia, apoptosis, renal cell carcinoma, prostate cancer, pancreatic cancer, chronic myeloid leukemia, toll-like receptor signaling pathway, small cell lung cancer, cell cycle and pathways in cancer (Table 3, Figure 4).KEGG pathway analyses of mir-196a-5p targeted genes by Mirfocus 3.0 In order to investigate the possible regulation mechanisms of mir-196a-5p in the process of gastric cancer, we utilized a bioinformatical database mirfocus 3.0 to select plausible targets of this miRNA.A total of 348 target genes including predicted and validated genes were choosed as the target genes of mir-196a-5p.
Results of enrichment KEGG pathway indicated that the targeted genes which were regulated by mir-196a-5p were involved 18 pathways, and 6 of which were centralized in cancer associated terms, which were as follows: MicroRNAs in cancer, transcriptional misregulation in cancer, prostate cancer, glioma, cell cycle and PI3K-Akt signaling pathway ((Table 4, Figure 5).

Discussion
Aberrantly expressed miRNAs are unique cancer biomarkers which will bring benefit to early diagnosis, treatment guidelines and prognosis estimation of cancer as well as mRNA and protein biomarkers.In recent years, more and more miRNAs were found up-regulated or down-regulated in gastric cancer, so that they were potential biomarkers which will supplement classic cancer biomarkers in clinical applications, such as miR-21 (Guo et al., 2013;Ma et al., 2013).But an ideal biomarker not only must be associated with cancer clinical significance, but also functional experiments of over expression or silence should prove it having great impact on cancer behavior.Mir-196a-5p was newly discovered promising gastric cancer biomarker, more studies should be carried out to fully understand its multiple roles and functions and to clarify any previous inconsistent clinical significance.This study employed 58 gastric cancer cases comes from WuWei city tumor Hospital from the year of 2010 to 2012 which belong to WuWei, Gan su, China, an area is famous for its high incidental rate with gastric cancer.So the clinical data is typical for clinical significance study, and is qualified for biomarker evaluation.
Clinical data analysis indicated that the numbers of mir-196a-5p in high-expression group (T/N>2) and low-expression group (T/N<0.5)amounts to 47 and 11 respectively.The mir-196a-5p high-expression group showed more extensive degree of lymph node metastasis and clinical stage than the high-expression group (P < 0.05; x2 test).However, no significant differences were observed among age, gender, depth of tumor invasion, venous invasion, tumor cell differentiation, tumor size, Borrmann type, and lymphatic invasion.
Previous study discovered that overexpression of miR-196a was significantly associated with tumor progression and poorer 5-year survival outcomes, and overexpression of miR-196a enhanced GC cell migration and invasion (Tsai et al., 2014).In another study (Sun et al., 2012), higher expression of miR-196a in gastric cancer tissues was reported associated with tumor size, higher clinical stage, and shorter overall survival, and downregulation of miR-196a expression significantly suppressed the cellcycle progression, proliferation, and colony formation of gastric cancer cells in vitro, and ectopic miR-196a expression significantly enhanced the development of tumors in nude mice.Result of our study is partly consistent with these studies.
Bioinformatics analysis of plausible targets and validated targets of miRNA and following analysis of KEGG pathway clustered by target genes is a promising way to give insights on potential biomarkers and key events involved in cancer tumorigenesis and relevant pathways.In recent years, this kind of combination analysis of any identified miRNA, its corresponding target  doi.org/10.7314/APJCP.2015.16.5.1781 Clinical Significance of mir-196a-5p in Gastric Cancer and Enriched KEGG Pathway Analysis of its Target Genes genes, enriched KEGG pathways, and even miRNA-mRNA network were performed in cancer studies (Satoh, 2012;Liu et al., 2014), particularly in cancer biomarker discoveries (Romero-Cordoba et al., 2012;Beta et al., 2013).In this study, based on miRWalk database combined with DAVID 6.7 tool and mirfocus 3.0, bioinformatics prediction and Enriched KEGG pathways analysis of miR-196a-5p indicated that both predicted target genes and validated target genes of miR-196a were clustered in cancer associated KEGG pathways.
In conclusion, our study fully reinforced the expressions of mir-196a-5p in gastric cancer lines and gastric cancer tissues were up regulated.These results, combined with bioinformatics analysis of Enriched KEGG pathways clustered by target genes provide ways to identify mir-196a-5p as new biomarkers in gastric cancer, and new light should be shed on its clinical significances of gastric cancer diagnosis and prognosis.

Figure 3 .
Figure 3.The 31 Enriched KEGG Pathways from Predicted Target Genes of mir-196a-5p which were Searched in MiRWalk Database and Analysed by DAVID 6.7

Figure 4 .Figure 5 .
Figure 4.The 14 Enriched KEGG Pathways from Validated Target Genes of mir-196a-5p which were Searched in miRWalk Database and analysed by DAVID 6.7

Table 1 . mir-196a-5p Level and Clinocopathologic Factors in Patients with Gastric Cancer
All clinicopathologic factors were analyzed in relation to mir-196a-5p level, The mir-196a-5p high-expression group showed more lymph node metastasis and clinical stage than the high-expression group (p<0.05;X 2 test).However, no significant differences were observed among age, gender, extensive degree of tumor cell differentiation, tumor size, depth of tumor invasion, venous invasion, borrmann type, and lymphatic invasion (Table1).
The expression of mir-196a-5p in all cases showed different levels between gastric cancer tissues and adjacent non-cancerous tissues.aP<0.05, lymph node metastasis; TNM stageClinical significances of mir-196a-5p in gastric cancer tissues comparing with adjacent non tumor tissues