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Original Article a Comprehensive Investigation Using Meta-Analysis

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Original Article a Comprehensive Investigation Using Meta-Analysis Am J Transl Res 2018;10(8):2246-2263 www.ajtr.org /ISSN:1943-8141/AJTR0078040 Original Article A comprehensive investigation using meta-analysis and bioinformatics on miR-34a-5p expression and its potential role in head and neck squamous cell carcinoma Jixi Li1,2, Kang Liu1,2, Tingting Zhang1,2, Zhendong Yang1,2, Rensheng Wang1,2, Gang Chen3, Min Kang1,2 1Department of Radiation Oncology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi, P. R China; 2Guangxi Tumor Radiation Therapy Clinical Medical Research Center, Nanning 530021, Guangxi, P. R China; 3Department of Pathology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, P. R. China Received April 17, 2018; Accepted July 14, 2018; Epub August 15, 2018; Published August 30, 2018 Abstract: HNSCC is the sixth most common cancer worldwide and is characterized as an aggressive, malignant tu- mor. MiR-34a-5p (miR-34a) expression has been strongly linked to HNSCC development. However, the exact target gene of miRNA-34a-as well as its biological and mechanistic pathways-are unclear. It is critical that the clinical value of HNSCC receive further study. We conducted a continuous variable, meta-analysis from data found in the cancer literature as well as that provided by the Cancer Genome Atlas (TCGA) to estimate miR-34a expression in HNSCC. Next, TCGA database, microarray, and MiRWalk were all used to predict the target gene of miR-34a in HNSCC. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to explore the underlying molecular mechanism of miR-34a in HNSCC. Finally, we used Spearman’s analysis and survival curves to identify the roles of related target genes involved in significant pathways during the development of HNSCC. Expression levels of miR-34a in HNSCC were significant lower than those in normal tissues (P<0.05) and summary receiver operating characteristic (sROC) was 0.72. The collective results obtained from KEGG and GO in- dicated that miR-34a may be involved in the development of HNSCC via known cancer pathways, including the p53 and/or PI3K-Akt signalling pathways. Our results suggested miR-34a has potential use as a novel, non-invasive and highly sensitive biomarker for diagnostic in HNSCC. Finally, it likely plays an essential role in the deterioration and ultimate tumorigenesis of HNSCC through determined cancer pathways. Keywords: miR-34a-5p, HNSCC, meta-analysis, bioinformatics, TCGA Introduction Head and neck squamous cell carcinoma (HNSCC) is a malignant cancer that develops in MicroRNAs (miRNAS) include such family mem- the squamous epithelium of the mouth, phar- bers as miR-34, which itself has several iso- ynx (nasopharynx, oropharynx, hypopharynx), forms (miR-34a, miR-34b, and miR-34c). Of and larynx. HNSCC is the sixth most common these, miR-34b and miR-34c share a common cancer worldwide and is characterized as an original transcript, while miR-34a is encoded aggressive, malignant tumor. Alarmingly, it has by its own transcript [1]. Recently, work has focused on the unique miR-34a-5p (miR-34a), now become the fifth leading cause of cancer- which has become an oncological research related deaths worldwide [12]. Approximately hotspot [2]. Moreover, miR-34a is encoded in 50,000 new cases of HNSCC are reported each the chromosomal location 1p36.22, and is year, resulting in the deaths of 13000 people associated with various tumors [3, 4]. As a con- annually in the United States alone [13]. Ad- sistent tumor suppressor, it has been shown to vances in surgical treatment as well as radio- regulate tumor cell apoptosis [5, 6]. Previous therapeutic and chemotherapeutic approaches studies have shown that miR-34a expression have significantly increased the survival rate for is related to multiple cancer types, including the disease. However, the overall survival rate colorectal, osteosarcoma, lung, breast, and for HNSCC has remained stagnant over the pancreatic [7-11]. past 20 years. A comprehensive investigation on miR-34a-5p expression and its role in HNSCC Materials and methods Literature search and screen- ing A search was conducted on both English and Chinese me- dical literature using PubMed, Science Direct, Web of Scien- ce, Wiley Online Library, EMBA- SE, the China National Know- ledge Infrastructure Database (CNKI), the Chinese Biomedi- cal Literature Database (CBM), the China Science and Tech- nology Journal Database (VIP), the Wanfang Database. All publications related to miR- Figure 1. The flow chart of the search and selection of docu- 34a expression and HNSCC ments. up to February 20, 2018 were included. The retrieval strate- gy for this study was as fol- lows: (SCC OR hnscc OR “squa- Table 1. The characteristics of literature with inclusion and exclu- mous cell carcinoma” OR “squ- sion criteria amous cell cancer”) AND (oro- Researcher Year Country Cancer/normal Methods Sample pharyngeal OR oropharynx OR TCGA 2016 USA 485/44 qPCR Tissue “head and neck” OR “oral cav- Bhavna K. et al [14] 2012 USA 15/15 qPCR Tissue ity” OR nasopharynx OR hypo- Manikanda. et al [21] 2015 India 52/8 qPCR Tissue pharynx OR laryngopharynx Li W. et al [22] 2014 China 8/8 qPCR Tissue OR oral OR larynx OR laryngo- pharyngeal OR laryngeal OR pharyngeal OR laryngeal OR It has been reported that there are approxi- tongue OR tonsil OR tonsillar OR nose OR “nasal mately 50 miRNAs that are known to have sinus” OR lip OR cheek OR “nasal cavity” OR altered expression in HNSCC [14]. For instance, “paranasal sinuses” OR palatal OR buccal) AND Bhavna and colleagues [15] demonstrated that (miR-34a OR miRNA-34a OR microRNA-34a dysregulation of miR-34a expression was re- OR miR34a OR miRNA34a OR microRNA34a lated to the development of HNSCC. However, OR “miR 34a” OR “miRNA 34a” OR “microRNA the exact target gene of this miRNA-as well as 34a” OR miR-34a-5p OR miRNA-34a-5p OR its biological and mechanistic pathways-are microRNA-34a -5p). unclear. Given this, it is critical that the clinical value of HNSCC receive further study. Our meta-analysis results were evaluated by two independent researchers (JX Li and DN Here, we performed a continuous variable, Yao) who used the same multistep process. meta-analysis from data obtained from the First, the title and abstract were read to exclude cancer literature as well as the Cancer Geno- any irrelevant literature. The remaining studies me Atlas (TCGA). These data were used to eval- were then separately examined by the resear- uate miR-34a expression in HNSCC. In addi- chers. Finally, any controversial issues were tion, the potential diagnostic value of miR-34a addressed by a third reviewer. The study was was estimated using a diagnostic meta-analy- determined to be eligible if it met the following sis. In addition, the possible molecular mecha- inclusion criteria: (1) Clinical samples were ta- nisms for miR-34a in HNSCC were analyzed ken from HNSCC and normal tissue or cell lines; comprehensively using both the Kyoto Encyclo- (2) Reverse transcription quantitative PCR (RT- pedia of Genes and Genomes (KEGG) and Gene qPCR) was used to determine miR-34a expres- Ontology (GO) enrichment analyses. sion levels; and (3) Study provided sufficient 2247 Am J Transl Res 2018;10(8):2246-2263 A comprehensive investigation on miR-34a-5p expression and its role in HNSCC data to determine relevant descriptive statistics (mean and standard deviation, SD) as well as 95% confi- dence intervals (95% CI). A study was excluded if it met one of the following condi- tions: (1) It was a duplicate studies, (2) It had insuffi- ciently detailed data to cal- culate the needed descrip- tive and inferential statis- tics, and/or (3) It was a review, letter, case report, or conference article. If a study lacked sufficient data for our analysis, we made all attempts to contact the Figure 2. The scatter diagram of miR-34a expression levels in HNSCC incorpo- author for the original data- rate each study. set. Finally, we extracted pivotal information from all eligible studies, including author name, published ti- me, country, sample size, analysis method, and type of samples used. TCGA data mining The expression levels and clinical parameter data for miR-34a were extracted using the OncoLnc website (http://www.oncolnc.org/) [16]. Correlations between all tested clinical parame- ters and miR-34a expres- sion levels was determined using a commercially avail- able statistical package (SP- SS 22.0) (Nanning, Guang- xi Zhuang Autonomous Re- gion, China). Statistical analysis STATA 12.0 software was used to analyze the result- ing data culled from our meta-analysis as well as Figure 3. Forest plots evaluating the miR-34a expression between HNSCC and our TCGA data (Nanning, normal tissues based on selected studies and TCGA data. A. A fixed-effect mod- Guangxi Zhuang Autono- el of Forest plots; B. A random effects model of Forest plots. mous Region, China). We 2248 Am J Transl Res 2018;10(8):2246-2263 A comprehensive investigation on miR-34a-5p expression and its role in HNSCC all statistical analysis. All measurement data were shown using mean ± SD (X ± s) and t-tests were used for comparisons between groups. Rates for count data were given as percent- ages (%) and compared using χ2 tests. When deter- mining the diagnostic value of miR-34a, according to true positive (TP), false pos- itive (FP), false negative (FN) and true negative (TN), and then we use meta-Disc software to calculate the Specificity; Sensitivity; Po- sitive likelihood ratio (PLR); Negative likelihood ratio (NLR); Diagnostic Odds ra- tio (DOR) and summary re- ceiver operating character- istic (sROC) curve. More- over, all expression data of miR-34a, including TCGA and studies, have been normalized to log2.
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