Original Article Lowered Levels of Microrna-129 and Potential
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Int J Clin Exp Pathol 2017;10(7):7511-7527 www.ijcep.com /ISSN:1936-2625/IJCEP0052925 Original Article Lowered levels of microRNA-129 and potential signaling pathways in papillary thyroid carcinoma: a determination of microRNA sequencing in 507 patients and bioinformatics analysis Liang Liang1#, Yihuan Luo1#, Xia Yang3, Rui Zhang3, Hanlin Wang3, Hong Yang2, Yun He2, Gang Chen3, Wei Ma3*, Junqiang Chen1* Departments of 1Gastrointestinal Surgery, 2Ultrasonography, 3Pathology, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region, P. R. China. #Equal contributors and co-first authors. *Equal contributors. Received March 14, 2017; Accepted May 26, 2017; Epub July 1, 2017; Published July 15, 2017 Abstract: Papillary thyroid carcinoma (PTC) is one of the most common endocrine system malignancies. However, the mechanism of tumor development is unclear. microRNA-129-5p is a microRNA that plays an important role in the development of tumors. The main purpose of our article is to find the potential target genes of microRNA-129 and their pathways based on gene array, sequencing and bioinformatics studies. We obtained microRNA-129 expres- sion and clinical associations in the TCGA database. In addition, we found a microRNA-129-related chip GSE19933, which is overexpressing microR-129-5p in thyroid cancer cell lines. The down-regulated gene is considered to be a potential target gene for microRNA-129. The target genes were predicted through 12 online tools. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of all down-regulated and predicted target genes. Furthermore, protein-protein interactions (PPI) were also analyzed for all potential genes. Finally, with intersecting down-regulated genes by overexpressed microRNA-129 and predicted target genes, the 889 genes are mainly enriched in the calcium signaling pathway, cGMP-PKG signaling pathway, ErbB signaling pathway and Proteoglycans in cancer, etc. The role of ten hub genes is particularly prominent in PPI analysis. These genes are differentially expressed in the thyroid by immunohistochemistry. We confirmed that microRNA-129 may play a major role in PTC through the above pathways, but more experiments are still needed to prove our results. Keywords: microRNA-129, papillary thyroid carcinoma, TCGA, target gene, signaling pathways Introduction importance in clinical settings [5-8]. Recently, a class of non-coding single-stranded RNA mole- Thyroid cancer is a common disease and its cule coded by an endogenous gene, microRNAs diagnosis remains challenging. The symptoms (miRNAs) has been reported to be evidently of a palpation, examination by ultrasound, related to the incidence and progress of many detection of endocrine hormone, fine needle cancers, including PTC [9-13]. Hence, miRNAs aspiration (FNA) and pathological observation possess the potential to be trustworthy bio- play crucial parts in the diagnosis of thyroid car- markers in PTC. However, only a small number cinomas. However, the specificity and sensitivi- of miRNAs have been studied in PTC. ty are all required to be improved [1-4]. Papillary thyroid carcinoma (PTC) is the most common MicroRNA-129 is one of the miRNAs whose cancer originating from the thyroid. Although clinical role and function remain largely the prognosis of PTC is commonly good with a unknown in PTC. To the best of our knowledge, high 5-year survival rate, some patients have only two research groups have performed rele- poorer prognosis. An early diagnosis is of great vant studies on microRNA-129 in thyroid can- Down-regulated microRNA-129 in PTC Figure 1. Clinical value of microRNA-129 in papillary thyroid carcinoma based on microRNA sequencing data from TCGA. Expression levels of microRNA-129-1 (A) and microRNA-129-2 (B). Receiver Operating Characteristic (ROCs) curve of microRNA-129-1 (C) and microRNA-129-2 (D). NT: non-tumorous tissue, T: tumor. AUC: area under the ROC curve. cer. Brest et al. [14] found that histone deacety- type. Besides, only one target gene, RET, was lase inhibitors (HDACi)s, trichostatin A and identified. vorinostat, could enhance microRNA-129-5p expression in cultured cell lines of BCPAP, TPC- Therefore, in the present study, the expression 1, 8505C, and CAL62, as well as in primary cul- level of microRNA-129 was analyzed based on tures of PTC cells. Moreover, microRNA-129 the microRNA sequencing data from the Cancer was adequate to induce cell death and accen- Genome Atlas (TCGA) Research Network data- tuate the anti-proliferative effects of other can- bases, which has recently published a molecu- cer drugs. However, no clinical value or molecu- lar signature based on of 507 PTC and 59 lar target genes of microRNA-129 in PTC were matched non-cancerous adjacent tissues with identified in the study [14]. Duan et al. [15] regard to genomic, transcriptomic and pro- found that microRNA-129-5p was apparently teomic characteristics, as well as clinicopatho- down-regulated in medullary thyroid carcino- logical features including survival status [16, mas. And microRNA-129-5p could suppress 17]. Subsequently, the potential target genes of the RET proto-oncogene expression by directly microRNA-129 were gathered by both microar- binding its 3’-untranslated regions. However, ray data and predicting platforms. Finally, a the study by Duan et al. solely focused on med- comprehensive functional annotation and vali- ullary subtype of thyroid carcinoma, without dation of the proteins were also performed mentioning the more frequent papillary sub- using different in silico tools. 7512 Int J Clin Exp Pathol 2017;10(7):7511-7527 Down-regulated microRNA-129 in PTC cally by TCGA data portal (https://gdc-portal.nci.nih. gov/). The expressions of microRNAs were log2 trans- formed and records were considered as censored wh- en the expression level was less than one. Student’s t test was performed examine the difference of microR- NA-129-1 and microRNA- 129-2 between cancerous tissues and their non-can- cerous counterparts. Rec- eiver operating characteris- tic (ROC) was drawn to evalu- ate the diagnostic values of microRNA-129-1 and microR- NA-129-2. Kaplan-Meier an- alysis and univariate Cox proportional hazards regres- sion model were used to assess the prognostic value of microRNA. Overall survival (OS), relapse free survival (RFS) and metastasis free survival data were obtained from PROGmiRV2-Pan Can- cer miRNA Prognostics Da- tabase (http://xvm145.jef- ferson.edu/progmir/index. php). SPSS 22.0 (SPSS Inc., Chicago, IL, USA) was used for the statistics and P<0.05 was regarded as being significant. Potential target genes of microRNA-129 Figure 2. Correlation between microRNA-129 level and overall survival in pap- illary thyroid carcinoma based on microRNA sequencing data from TCGA. Ex- To collect the potential target pression levels of microRNA-129-1 (A) and microRNA-129-2 (B) were divided according to the median level. The analysis was performed by “PROGmiR” genes of microRNA-129, we (http://www.compbio.iupui.edu/progmir). combined two parts of genes together. Materials and methods The first part was the differentially expressed genes (DEGs) post pre-microRNA-129 overex- Clinical significance of microRNA-129 in papil- pression from Gene Expression Omnibus (GEO) lary thyroid carcinoma based on microRNA database. After searching for “miR-129 OR sequencing data microRNA-129” in thyroid cancer in GEO, we The expression levels of microRNA-129-1 and obtained the database of GSE19933. In the microRNA-129-2, as well as the clinical data of study [14], three independent experiments papillary thyroid carcinoma patients and non- were performed in dye-swap with TCP1 PTC tumorous thyroid tissues were provided publi- cells: miRNA-129-5p versus miR-Neg. The 7513 Int J Clin Exp Pathol 2017;10(7):7511-7527 Down-regulated microRNA-129 in PTC The second part was the pre- dicted target genes achieved from 12 different predicting tools, including Targetscan miRWalk, miRMap, Microt4, miRanda, mirbridge, miRDB, miRNAMap, PITA, Pictar2, RNA22, and RNAhybrid. The genes co-predicted by at le- ast six databases were se- lected. Finally, the overlapped genes from the two parts were con- sidered as potential target genes of microRNA-129 in PTC. Functional annotation of the target genes of microR- NA-129 To evaluate the function of microRNA-129 target genes in PTC, we analyzed the Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation throu- gh DAVID (http://david.abcc. ncifcrf.gov/). The GO terms with a modified Fisher Exact P-value less than 0.01 and the KEGG pathways with P-value less than 0.05 were Figure 3. Correlation between microRNA-129 level and relapse free survival in chosen for next analysis. The papillary thyroid carcinoma based on microRNA sequencing data from TCGA. GO enrichment analysis was Expression levels of microRNA-129-1 (A) and microRNA-129-2 (B) were divid- visualized by software Cy- ed according to the median level. The analysis was performed by “PROGmiR” (http://www.compbio.iupui.edu/progmir). toscape v3.5.0. Further, the protein-protein interactions network of genes was con- experiments included a negative pre-miRNA as ducted through STRING v10.0 (http://string. control and a synthetic pre-miRNA-129-5p as embl.de/). experimental group. TCP1 papillary thyroid car- cinoma cells were transfected with 10 nM of a Validation of the protein expression level of synthetic pre-miRNA-129-5p or a negative pre- potential targets miRNA using Lipofectamine RNAiMAX reagent. RNA samples were harvested at 24 and 48 To further confirm the relationship between hours post-transfection. The results of three microRNA-129 and some of the potential target experiments were merged and the down- genes. The hub genes from the most signifi- expressed genes with a log(FC) value less than cantly enriched pathway in KEGG analysis were -0.5 were gathered as the genes which were selected randomly.