HCMV-miR-UL148D Overexpression Promotes Cell Proliferation, Invasion And Metastasis Of Nasopharyngeal Carcinoma

BackgroundNasopharyngeal carcinoma (NPC) is one of the commonest malignancies in Southern China and Southeast Asia. Unlike other head and neck malignancies, NPC is notorious for its geographic pattern, familial aggreation of incidence and marked tendency to highly metastatic nature. The major etiological factors of NPC include Epstein-Barr virus(EBV) infection,chemical carcinogens and genetic susceptibility. NPC development is a complicated process with multistage and multimechanism,but the molecular mechanism of its pathogenesis is not yet fully understood.MiRNAs (miRNAs) are regarded as a novel gene regulation factor in tumorigenesis. Up to now, the research of the miRNAs which are related with nasopharyngeal carcinoma are focus on EBV-coding miRNAs and the miRNAs which are produced by organisms. Previous studies found that the human cytomegalovirus (HCMV) has a close relationship with the inflammatory diseases and malignancies,including human malignant gliomas, Kaposi’s sarcoma, EBV positive Hodgkin lymphoma, cervical cancer, prostate cancer and so on. These evidences implicated that the HCMV may play an important role in the pathogenesis of tumor cells and regulate the surrounding microenvironment.Based on our preliminary study, it was shown that human cytomegalovirus has a close relationship with some candidiate miRNAs, such as mir-20a\20b, mir-93, mir-17-5p, which are related with both NPC and TGF-PR2. Combined with the results of miRNA gene microarray and TargetScan online prediction, we think that the miRNA HCMV-miR-UL148D may has the same fuction with these candidiate miRNAs. Then we tested the NPC and CNP tissues to confirmed that HCMV-miR-UL148D was highly expressed in the carcinoma tissues.At the same time, it has been approved by the bioinformatics and the report of luciferase system that TGF-βR2 is the target of HCMV-miR-UL148D, and TGF-PR2 had a lower expression in NPC cell lines. All these analyses suggest that HCMV-miR-UL148D is a candidate oncogene to promote NPC metastasis.Hunman cytomegalovirus (HCMV), a member of β-herpesvirus family, is a common human pathogen infecting approximately 70～100% of world’s population. It remains latent for lifetime in its host after primary infection, during this period it may developed immune evasion strategies to avoid destruction by the various arms of the immune system.It can cause clinical symptoms and death when infect the immunocompromised or immunosuppression patients.A number of recent evidence suggests that HCMV involve in stimulate cell cycle, induce mutation, induction of angiogenesis, promote cells invasive and affect the virus replication, genetic expression, protein trafficking.HCMV has recently been implicated that may be specifically associated with some human malignancies,such as breast,colon,and prostate cancers as well as glioblastoma. Sufficient evidence exists to implicate a important role of HCMV in oncogenesis via HCMV-mediated effects of cells in the tumor microenvironment and in the tumor cells themselves.MicroRNAs (miRNA) are a class of small non-coding RNAs of 19-25 nucleotides in length, are mostly transcribed from a pre-miRNA which is about 70～100nt in length, and then undergo further processing by the ribonucleases Dicer. The mature miRNA targets the 3’UTR of its target mRNA, and induces mRNA degradation. MiRNAs are ubiquitous in animals,plants and viruses.Its involve in biological development, proliferation, differentiation and apoptosis,dysregμlation of miRNAs appears to cause a crucial diseaes,like cancer.Transforming growth factor β (TGF-β) has multifunctions, can widely participated in various pathophysiological process of mammals,this growth factor is involved in cell differentiation,wound healing,embryonic development,the formation of extracellular matrix,bone remodelling,immune regulation,the development of the nervous system and malignancies.Increasing evidence implicated that TGF-β pathway play a important role in human malignancies,and the TGF-βR1 and TGF-βR2 play a key role in this signaling pathway. Growing datas have revealed the the relationship between development and metastasis in lung cancer.uterine fibroids,prostate cancer and multiple myeloma.Now,some researchs have reported the TGF-PR2 was downregulated in nasopharyngeal carcinoma,which implied that the TGF-βR2 may be a protect factors in NPC.Here we based on the above findings and our preliminary study to validate the expression of HCMV-miR-UL148D by Real-time PCR and ISH method in NPC and CNP tissues,explicit the effects of HCMV-miR-UL148D overexpression on invasion and metastasis in vitro.Identify its possible role in NPC pathogenesis,and elucidate the molecular mechanisms by Real-time PCR and Western Blotting after overexpression of HCMV-miR-UL148D.Materials and Methods1. Cell cultureThe human NPC cell lines 5-8F,6-10B, CNE1 and CNE2 were culture in RPMI-1640 supplemented with 10% fetal bovin serum.NP69 cell (an immortalized nasopharyngeal epithelial cell) was cultured in Keratinocyte-SFM supplemented with bovine pituitary extract.293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM).All cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37℃.2. Clinical specimensPrimary NPC biopsy specimens and normal biopsies of thenaspharynx were obtained from Zhongshan city people’s Hospital (Sun yat-sen University,Guangzhou, China).None of the NPC patients had received preoperative radiation or chemotherapy before diagnosis.3. Poly (A) Real-time PCRTotal RNA and miRNA were extracted from cells and tissues. The RNA was reversely transcribed into cDNA and PCR amplified using Taqman probes and primers.All samples were normalized to internal controls and fold changs were calculated through relative quantification (2-ΔΔCt).4. ISH detectionParaffin sections of specimens prepared for detection were stained by SP method, and the results of ISH were observed by light microscope and judged the new standard of c-erbB-2 immunohistochemica.5. Cell transfectionUsing LipofectamineTM 2000 reagent,and working concentration of miRNA was 100 nmol in a 6-well plate.Transfection the HCMV-miR-UL148D mimics into NPC cell lines.After 24h transfection,the cells were collected for follow study.6. Cell biology experiments(1) MTT assay:A total of 2×103 cells were seeded in a 96-well plate,then allowed to grow in normal medium for 3 days.For MTT assay,cells were incubated in 20μl of 5mg/ml solution of MTT at 37℃ for 4h and lysed in 200μl DMSO.The absorbance in each well was measured at 490 nm by a microplate reader.(2) Colony formation assay:200 cells were plated in 6-well plated and grown for 2 weeks.After 2 weeks,the cells were be collected,fixed with methanol,and stained with crystal violet.The number of colonies was counted under the microscope.(3) Invasion Chamber assay:Cells after 24h serum-free medium incubation were trypsinized,and 2.5×105 105 cells in 200μl of serum-free medium were added into the upper chamber.In the lower chamber,500μl of medium containing 10% fetal bovine serum were placed.The cells were allowed to migrate though the intermediate membrane for 24h at 37℃.Membranes were then fixed with paraformaldehyde, stained with 5% crystal violet,and quantified in microscopy.7. Bioinformatic predictWe used three database (miRBase, TargetScan and RNAhybrid) to predict the target gene of HCMV-miR-UL148D in NPC cells,and found that TGF-βR2 was identified as a target gene.8. Luciferase reporter assayAbout 2.5×105 293T cells were seeded in a 24-well plate and cotransfection pRL-SV40 plasmid to monitor the transfection efficiency.After 24h cotransfection, using the dual-luciferase reporter assay system detected the activity of Firefly luciferase and Renilla luciferase.Relative luciferase activity was normalized with Renilla luciferase activity and then compared with the pGL3 control.9. Western BlottingTotal protein was isolated and quantitated using BCA method.The protein lysates were separated by SDS-PAGE,and electrophoretically transferred to PVDF membrance.Then,the membrane was incubated with antibodies and detected by chemiluminescence.Results1. The expression of HCMV-miR-UL148D in NPC tissues(1) Poly (A) Real-time PCR:The Real-time PCR results displayed that HCMV-miR-UL148D were more higher expression in NPC tissues than chronic nasopharyngitis tissues,which had a significant difference(P=0.0001).(2) ISH:The ISH method directly show the HCMV-miR-UL148D distribution in the nasopharyngeal carcinoma tissues.2. The effects of HCMV-miR-UL148D on the NPC cells(1) Transient transfection:The transfection was preformed using LipofectamineTM 2000 reagent,and the HCMV-miR-UL148D’s working concentration of miRNA was 100 nmol in a 6-well plate.(2) Cell biology experiments① MTT assay:After overexpression HCMV-miR-UL148D mimics, the growth rate in CNEl cells was increased 1.312 times and 1.61 times in 6-10B cells,which had a significant difference(P<0.001).②Colony formation assay:The colony formation assay displayed that HCMV-miR-UL148D overexpression increased cell growth in CNE1 cells by 68.3%(P<0.001) and in 6-10B cells by 66.5%(P<0.001) at 72h post-transfection.③Boyden invasion assay:The invasion ability of CNE1 and 6-10B cell lines have increased 72.4%(P<0.001) and 63.2%(P=0.0011) after overexpression.④Transwell migration assay:The migration ability of CNE1 and 6-10B cell lines have increased 65.9%(P<0.001) and 54.8%(P<0.001) after overexpression.3. The molecular mechanisms of HCMV-miR-UL148D in NPC(1) Bioinformatics analysis:As the three online database predicted the TGF-βR2 to be a target gene of HCMV-miR-UL148D.And HCMV-miR-UL148D may play its role through combining its seed sequence with 3’UTR of TGF-PR2.(2) Luciferase activity assay:After cotransfection the miRNAs,the luciferase activities of HCMV-miR-UL148D was obviously decreased(P<0.05),which implied that the TGF-βR2 to be a target gene of HCMV-miR-UL148D.(3) The TGF-βR2’s expression in NPC cell lines:A panal of human NPC cell lines (CNE1, CNE2,5-8F,6-10B) was analyzed to quantitate the expression level of TGF-βR2.The result of RT-PCR showed that the expression of TGF-βR2 was downregulated in all cell lines,compared with the immortalized nontumorigenic cell line NP69(P=0.002).The result were similar with Western Blotting.(4)The effect of HCMV-miR-UL148D deregμlation on EMT-related markers:After overexpression the HCMV-miR-UL148D increased the expression of epithelial markers of E-cadherin,and downregulate the expression of mesenchymal markers Vimantin.Conclusions1. HCMV-miR-UL148D existed in NPC tissues, which were detected by Real-Time PCR and ISH methods,and it high expressed in NCP tissues.2. HCMV-miR-UL148D can promote the migration, invasion and metastasis ability of NPC cells in vitro after overexpression,which functions as a promoter gene in NPC.3. TGF-βR2 was the target gene of HCMV-miR-UL148D in NPC.And HCMV-miR-UL148D overexpression increased the expression of epithelial markers, and downregulate the expression of mesenchymal markers. Implied that the HCMV-miR-UL148D may throught the TGF-β signaling pathways to affect NPC.