| Nasopharyngeal carcinoma(NPC) is a type of cancer common in Southern China and Southeast Asia,especially those of Cantonese origin.Clinically, metastasis is frequently found,and cervical lymphadenopathy is often the only clinical manifestation of NPC patients.Radiotherapy has been the main treatment of choice for this cancer.NPC carcinogenesis is a multistep process whose progression can be measured by an increase in stage or by the accumulation of genetic changes in tumor cells,and its etiologies are associated with Epstein-Barr virus(EBV) infection, environmental factors and genetic susceptibility.The results of epidemiology showed NPC had a family history,which suggested genetic susceptibility might play an important role in the pathogenesis of NPC.Consistently high frequencies of genetic loss are observed on chromosomes 3p,9p and 14q.However,the molecular mechanisms of NPC pathogenesis are still unknown.Several researches demonstrated consistent,non-random and significant loss of heterozygosity(LOH) on 3p was an important feature of NPC,with a especially high incidence of LOH on 3p14-21(FHIT),3p21.3-22 and 3p25-26(VHL).Additionally, such genetic changes of 3p have taken place before EBV infection and malignant transformation of nasopharyngeal epithelial cells.Therefore,to find out unknown NPC-related genes in this region is very important to understand the early molecular mechanisms of NPC pathogenesis.MicroRNAs(miRNA) are a recently discovered family of small,19-25nt, noncoding,single-stranded molecules in cells that can negatively regulate the expression of target genes with their complementary sequence.They are believed to be critical in cell development,differentiation and communication.Specific roles include the regulation of cell proliferation,developmental timing,cell differentiation and apoptosis,haematopoieses,fat metabolism,insulin secretion and human tumorigenesis.Lately,it has been estimated that more than 50%of annotated human miRNA genes are located in cancer associated genomic regions(CAGR),including fragile chromosomal regions that are susceptible to amplification,deletion or translocation during the course of tumor development.Recent evidence indicates that miRNAs can function as tumour suppressors and oncogenes,and they are therefore referred to as "oncomirs",such as let-7,miR-21,miR-17-92 cluster,miR-143 and miR-145,miR-372 and miR-373,miR-26a,which have been estimated to be involved in development and progression of many cancers.Hsa-miR-26a(miR-26a) was first cloned from Hela cells,with 21nt.Its expression has no tissue specificity,and its function is unknown.Notably,the mature sequence of miR-26a is present of 3p23-21.31(MDR2:D3S1768-D3S1767),which are fragile chromosomal regions susceptible to LOH in many cancers.Therefore, miR-26a is speculated to be involved in development and progression of cancers, such as lung carcinoma,thyroid carcinoma,leiomyosarcoma uteri,breast cancer and colon carcinoma.Results of Iorio found that miR-26a was progressively up-regulated in breast cancers with estrogen receptor positive(ER+),progesterone receptor positive(PR+),and high proliferation index as compared with ER-,PR- and low proliferation index,and a reduced miR-26a expression might be associated with a poor prognosis.Then,the findings of Shi and Yanaihara proved that miR-26a as well as its hairpin precursor sequence were not expressed or expressed at low levels in lung cancer cell lines,correlated with their location in regions of LOH in lung tumors and cell lines.Moreover,the investigation of Xi indicated that the expression level of miR-26a was increased by nearly 2.27-fold in HCT-116(wt-p53) cells in contrast with HCT-116(null-p53),down-regulation of wt-p53 via small interfering RNA reduced the expression of miR-26a,and its expression was increased in HCT-116(wt-p53) cells in response to the increasing expression of wt-p53 after 5-Fu exposure.They supposed p53 as a transcription factor regulated the expression of miR-26a.Visone indicated a significant decrease in miR-26a was detected in ATC (anaplastic thyroid carcinomas) in comparison to normal thyroid tissue.The overexpression of miR-26a in human ATC-derived cell lines suggested a critical role of miR-26a downregulation in thyroid carcinogenesis,since a cell growth inhibition was achieved,and they predicted miR-26a was HMGA gene regulators.Based on the research review of miR-26a and its potential significance in the molecular mechnisms of NPC,the identification of its expression characteristics in NPC tissues and cell lines will help understand the mechanisms by which miR-26a contributes to origin and progression of NPC.Furthermore,the implication of the role of miR-26a in NPC might indicate a miR signature associated with NPC.We made some researches to elucidate the characteristics and regulatory mechanisms of miR-26a as follows:1.Identification of expression characteristics of miR-26a in NPC tissues and cell linesQuantitive real-time PCR was used to detect the expression of miR-26a in NPC-derived cell lines(5-8F,6-10B,CNE1,CNE2,C666-1 and HONE1) and immortalized nasopharyngeal epithelial cells NP69.The results of one way ANOVA ananlysis showed that the expression of miR-26a in 7 cell lines was significantly different from each other(F=111.846,P＜0.001).And its expression in each NPC cell line was significantly different from each other(P＜0.05).The results of 2? method showed the expression of miR-26a was significantly reduced in 5-8F,CNE1, CNE2,C666-1 and HONE1 as compared with NP69,but with a significantly upregulated expression in 6-10B.In situ hybridization was used to detect the expression of miR-26a in NPC tissues and chronic nasopharyngitis tissues.The results indicated there were no positive signals in NPC tissues,however,strongly positive signals were manifested in the pseudostratified ciliated columnar epithelium in nasopharynx of chronic nasopharyngitis.2.Bioinformatic prediction of miR-26a targets and bioinformatic analysis of target genesThe following three bioinformatics algorithms were utilized to predict miR-26a target sites:miRBase,Pictar and TargetScan.Afterwards,miRBase predicted 1114 targets of miR-26a,TargetScan predicted 536 targets and Pictar predicted 501 targets. In contrast to these results,the intersection of 3 algorithms included 56 targets.We used the Gene Ontology(GO) enrichment analysis to find biological functions that were most significantly affected by multiple targets.Moreover,the results indicated the main biological functions of 56 target genes included transferase activity,metal ion binding,transcription factor activity,receptor activity,cell cycle,Ras protein signal transduction,positive regulation of I-κB kinase/NF-κB cascade,cell proliferation,cell apoptosis,cell differentiation,cell adehesion and cell motility.All of these biological functions were associated with traits that were the hallmarks of the malignant phenotype,with a focus on tumor-host interactions.Accordingly,the disorder of miR-26a expression might lead to the changes of pathways and multiple cell regulatory mechanisms in NPC.3.Effects of miR-26a overexpression on biological behaviors of 5-8F cellsApproximately 194bp miR-26a gene segments containing the flanking sequence of miR-26a hairpin were amplified from genomic locus and cloned downstream of the human H1 polⅢpromoter in a plVTHM vector containing EF1-GFP.And then NPC-derived cell line 5-8F was transfected with the vector plVTHM/miR-26a and then subjected to FACS analysis for GFP expression.Lastly,a NPC-derived cell line 5-8F-plVTHM/miR-26a was established,with stable overexpression of miR-26a.5-8F-plVTHM/miR-26a cells showed a significantly reduced proliferation compared with 5-8F and 5-8F-plVTHM cells as determined by in vitro MTT assay (F=5.146,P＜0.001).In addition,5-8F-plVTHM/miR-26a cells had a significant reduction in their ability to form colonies in plate as compared with 5-8F and 5-8F-plVTHM(F=38.862,P＜0.001).However,the cell cycle distribution detected by flow cytometry is not significantly different from each other.These results indicated overexpression of miR-26a partially reduced proliferatin of 5-8F cells.The results of in vitro invasion assay showed that 5-8F-plVTHM/miR-26a cells had significantly reduced invasiveness as compared with 5-8F and 5-8F-plVTHM cells(F=109.334,P＜0.001).Furthermore,5-8F-plVTHM/miR-26a cells also had significantly reduced motility as compared with 5-8F and 5-8F-plVTHM cells by use of Coming Transwell Inserts(F=53.881,P＜0.001).All of these results showed overexpression of miR-26a partially led to a downregulated migration and invasion of 5-8F cells. Conclusions1.The expression of miR-26a is abnormal in NPC-derived cell lines and NPC tissues.2.Overexpression of miR-26a in vitro results in a significant reduce of cell proliferation,motility and invasion in NPC-derived cell lines 5-8F.3.Bioinformatic analysis predicts miR-26a might be involved in origin and progression of NPC as target gene regulators,for instance,HMGA1,HMGA2, CCNE1 and so on.
|
PDF Full Text Request