Effection Of Epstein-Barr Virus Latent Membrane Protein 1 On Oncomirs Expression Profile In Nasopharyngeal Carcinoma Cell Line CNE1

Background and objective:The incidence of nasopharyngeal carcinoma (NPC) is most prevalent in Southeast Asia, particularly in Southern China. NPC is characterized by regional distribution and EBV-associated. LMP1 is a viral oncogene expressed in nasopharyngeal carcinoma cells, which exhibite latency type II of EBV infection. LMP1 can promote epithelial cells transformation through hijacking cell signaling pathway such as NF-κB, p38/MAPK and JNK signaling to activate the transcription of downstream genes. The activated protein expression profile are widely involed in cell growth, proliferation, apoptosis, invasion and metastasis. Meanwhile, LMP1 can regulate the expression of miRNAs in lymphoma that exhibte latency typeⅢof EBV infection.This regulation can keep the latent infection of EBV and change the biological characters of tumor cells.miRNAs is a novel class of small(-22 nucleotide) non-protein-coding RNA molecular, which function as post-transcriptional gene regulator. miRNAs recruit other factors to assemble a complex named with miRISC(miRNA induced silencing complex), which suppress translation or promote degration of target mRNAs. miRNAs regulate diverse biological process such as development, differetiation, proliferation, apoptosis and metabolism. Differential expression profile study by highthrough screening through miRNA microarray to find differentially expressed miRNA, especially oncomiRs, is a good way to elucidate the globle molecular mechanism of development and progression of tumor, and also a good index in typing and prognosis of cancer. The targets of differentially expressed miRNA can be predicted by various predicting software to comprehend their biological function in cells. Recently, one group found the differentially expressed miRNAs between nasopharyngeal carcinoma tissue and adjacent normal tissue. Then the cell signal pathway mediated by their targets were predicted in silicon to elucidate their biological effects. Various signal pathway, which includes several pathway associated with tumoric biological characters, were interferred by their targets and were involved in cell growth, proliferation, apoptosis, metastasis and angiogenesis. Function of miRNA can be studied in model cells by transfecting with miRNA mimics and antagomiRs (miRNA inhibitors) to induced the function of miRNA positively or inversely. The results from bioinformatics can also be verified by transfecting assay. The efficacy of transfection can be validation by targets detection. Simultaneously, the detailed mechanism of miRNA functioning can be indicated by targets assay.In order to explore the regulating effection of LMP1 on oncomiRs expression in CNE1 cell line and ultimate effection on biological characters in nasopharyngeal carcinoma. We investigate the differential oncomiRs expression profile between nasopharyngeal carcinoma cell line CNE1 and it's steady EBV-LMP1-transfected cell line CNE1-LMP1. Among the differentially expressed oncomiRs, the miRNAs play a key role in cellular biological function were picked out by bioinformatical assay. AntagomiRs were used to block the selected miRNAs to investigate it's role in biological characters of nasopharyngeal cells. It's expression in nasophayngeal carcinoma tissue were detected and the association with expression of LMP1 were assessed. Both of them were evaluated their association with clinopathological characters of nasopharyngeal carcinoma to explore the significance of their expression in nasopharyngeal carcinoma tissue.Then detect the most differentially expressed miRNA in tissues of nasopharyngeal carcinoma, evaluate the association between it's expression value and LMP1 expression and their expression with clincopathological characters. And transfection knockdown probe into model cell to block the miRNA to elucidate its prelimilary function.Methods:1. A membrane-based microRNA array that targets 132 of the most well studied oncomiRs were used to detect the expression profile of CNE1 and CNE1-LMP1. And then real time qRT-PCR assay verified the expression data of the most differentiatedly expressed(changed over 2 folds) miRNAs. The data of miRNA array and qRT-PCR were analysised by correlation analysis to verify the reliability of miRNA array assay.2. The KEGG (kyoto encyclopedia of genes and genomes) pathway employed by the differentially expressed oncomiRs to execute their biological function were predicted by DIANA-mirPath, a online functional prediction tool of miRNA.3. Cell cycle, proliferation, apoptosis, cell migration and invasion in CNE1-LMP1 cells were evaluated after knockdown of hsa-miR-19b. STAT3 signaling pathway and SOCS1 (a target gene of hsa-miR-19b, also suppressor of STAT3 signaling) were also detected to elucidate the preliminary function and fundamental mechanism of hsa-miR-19b.4.46 cases of nasopharyngeal carcinoma were enrolled in the study before radiotherapy. LMP1 protein detected by immunohistochemisty and hsa-miR-19b by real time qRT-PCR. The correlation between these two index were assessed. hsa-miR-19b detected by real time qRT-PCR. The correlation between LMP1 and hsa-miR-19b expression were assessed. Meanwhile, evaluate the correlation between LMP1 or hsa-miR-19b and clinicopathological characters.Results:1. The oncomiRs expression profile of CNE1 cells shows as below. Among the restricted 132 miRNAs,21 were detectable in CNE1. There is 4 miRNAs with higher expression levels(expression levels higher than internal reference RUN48); And 17 with lower expression levels(expression levels lower than internal reference RUN48).2. The oncomiRs expression profile of CNE1-LMP1 cells shows as below. Among the restricted 132 miRNAs,30 were detectable in CNE1. There is 7 miRNAs with higher expression levels(expression levels higher than internal reference RUN48). And 23 with lower expression levels(expression levels lower than internal reference RUN48).3. The differential expression profile between CNE1 and CNE1-LMP1. Among the restricted 132 miRNAs,30 were detectable in CNE1-LMP1 and 21 in CNE1. There is 21 shared miRNAs, among which 7 miRNAs'expression level (hsa-miR-19b, hsa-miR-17-3p, hsa-miR-22, hsa-miR-149, hsa-miR-150, hsa-miR-188 and hsa-miR-205) elevated over two folds. Among the 9 specifically expressed miRNAs in CNE1-LMP1, hsa-miR-122a has a highest expression level surpass the internal control sample. Altogether, CNE1-LMP1 showed higher total miRNAs levels.4. qRT-PCR verify the expression detected by miRNA array. The expression differentiation of 7 most differentiatedly expressed miRNAs confirmed by the real qRT-PCR. The results show that they are closely correlated(r=0.970, P=0.000).5. DIANA-miPath predict the pathway and function of differentially expressed miRNAsThe software predicts that the total targets involved in cell signal pathway is 95. Among 8 differentially expressed miRNAs, miR-17 has 44 targets, miR-19b has 53 and the other 6 miRNAs has only 4. From this, we included that miR-17 and miR-19b contribute to most of the biological function through signal pathway. The two major miRNAs involved in various pathway. Among them, the signal pathway which closely associated with tumor includes enviromental signal transduction, cell motibility, cell growth and death, cell communication and tumor associated pathway. Taking the previous expression data together(miR-19b shared the higher expression level and changed times), we pick out miR-19b for further functional assay.6. The preliminary function of hsa-miR-19b in CNE1-LMP1After knockdown the hsa-miR-19b, cell proliferation decreased (5pmol group cell proliferation inhibit rate is 20.45%, llpmol group is 44.50%); the percentage of cells in G1 stage increased, cell cycle is blocked in G1 stage; And the apoptosis increased. Meanwhile, the expression of it's target protein, SOCS1 increased; lead to lower phosphorylation level of STAT3.7. The in situ expression of LMP1 and hsa-miR-19b and their correlation with clinopathological charactersThe positive expression rate of LMP1 protein is 60.95%(28/46). The relative expression value of hsa-miR-19b is 68.27±69.00. Both LMP1 and hsa-miR-19b were associated with stage and lymphatic metastasis(P<0.05).The expression of LMP1 were associated with the expression of hsa-miR-19b (r=0.390, P<0.05).Conclusions:1. oncomiRs are differentially expressed between CNE1 cells and CNE1-LMP1 cells.2. LMP1 can regulate the expression of oncomiRs in nasopharyngeal carcinoma cell line and elevate the total miRNAs level through it's transcription-activating function.3. Regulating the expression of miRNAs would be another important pathway employed by LMP1 to function as a viral oncogene.4. Among 8 differentially expressed miRNAs, hsa-miR-17 and has-miR-17 contribute most to the cell signal pathway, among which signal transduction, cell motibility, cell growth and death, cell communication and tumor associated pathway were closely associated with the development and progression of tumor.5. Hsa-miR-19b can promote cell cycle and proliferation, inhibit apoptosis. The possible mechanism employed in these process may be constructively activate the STAT3 signaling. In which hsa-miR-19b inhibit the translation of it's target gene SOCS1, and ultimately abolished the blockage of the phosphorylation of STAT3. Hsa-miR-19b can also promote cell migration and ivasion in vitro.6. LMP1 and hsa-miR-19b showed expression in low differentiated squamous cell carcinoma of nsaopharynx epithelia.7. LMP1 would be involved in expression regulation of hsa-miR-19b in low differentiated squamous cell carcinoma of nsaopharynx epithelia.8. LMP1 and hsa-miR-19b can functionate as a diagnosis molecular marker in low differentiated squamous cell carcinoma of nsaopharynx epithelia.