| BackgroundNasopharyngeal carcinoma (NPC), Commonly known as "Guangdong tumor", is one of the most common cancers in Southern China, especially in Guangdong province. Intensity modulated radiotherapy as the primary treatment is very efficient for patients with early NPC. But early detection of NPC is difficult in clinic because of its hiding onset and high metastatic potential. And nearly75%of the patients were reported late stages with local lymph nodes metastases or/and distant metastasis at first diagnosis. Once recurrence or metastasis occurs after treatment, prognosis is poor, which make it the leading cause of death. Therefore, identifying sensitive and convenient biomarkers for NPC early diagnosis, therapeutic options and prognosis prediction remains urgent and significant.The major etiological factors of NPC include genetic susceptibility, Epstein-Barr virus (EBV) infection and environmental factors, but there has been no efficient biomarkers for NPC in the clinical setting. In consideration of close association between NPC and Epstein-Barr virus (EBV) infection, EBV serology, including IgA antibodies against viral capsid antigen (VCA-IgA), early antigen (EA-IgA) and plasma EBV DNA, have been found to be useful markers in clinical practice and correlate well with disease status. However, the serological tests have limited sensitivity and specificity in monitoring disease progress and detecting recurrent diseases. Human leukocyte antigen (HLA) is considered to be genetic factors associated with nasopharyngeal carcinoma. HLA was first reported in the1970’s among Chinese Singaporeans nasopharyngeal carcinoma associated with nasopharyngeal carcinoma;"Nature" magazine reported in1990in the HLA region is recessive gene susceptibility to nasopharyngeal carcinoma. Since then, different research teams have also reported the relationship between HLA loci and incidence of nasopharyngeal carcinoma, confirmed the HLA is the most representative in nasopharyngeal carcinoma genetic factors.Other relevant proteins and its coding genetic markers of nasopharyngeal carcinoma, all stop at experimental or theoretical step only.With the deepening of the research, in addition to the cancer associated protein and its coding gene, the non-coding genes, especially a class of small non-coding RNAs(microRNAs, miRNAs), is found to be also closely related with the genesis and development of cancer. MiRNAs are a recently discovered class of small non-coding RNAs of18-25nucleotides in length that modulate gene expression post-transcriptionally by targeting the3’untranslated region (3’UTR) of its target mRNA. Recent evidences have shown that about50%of the human miRNAs are located in cancer-associated genomic regions. Their abnormal expression plays a key role in the hallmarks of cancer, such as cell proliferation, differentiation, apoptosis, migration, invasion, metastasis and angiogenesis. These miRNAs have been confirmed to downregulate the expression of many cancer-related genes and function as oncogenes and tumor suppressor genes. By comparing the tumor tissue and normal control, the abnormal expression of miRNAs were detected in most tumor tissue that can distinguish between tumor and normal tissue, and molecular classification of tumor. Some miRNAs were confirmed can be used to predict the prognosis and treatment sensitivity. In2008, circulating miRNAs were first reported to be stable existence without degradation after long-term preservation and multiple freezing and thawing. And the expression of circulating miRNAs has also significant differences in cancer patients compared with normal people, opened the prelude of research for circulating miRNAs as tumor biomarkers. Based on the demand of clinical application, tumor markers must be easy to test and good repeatability. Compared with tissue miRNA profiling, circulating miRNAs show a better application prospect. Circulating miRNAs studies provide new idea and method for cancer early diagnosis, prognosis, response to treatments and follow-up monitoring.Circulating miRNA as cancer molecular markers has advantages of stability, convenient and efficient. Multiple studies have already shown that circulating miRNAs are high stable in serum and plasma samples which are non-invasive and simple to obtain. Expression of circulating miRNAs is frequently dysregulated in cancer and it seem to be tissue-specific. Deregulation of circulating miRNAs is earlier than the changes of coding genes and proteins, and it happened in the early tumorigenesis and before cancer metastasis. A study comparing nasopharyngeal carcinoma and normal serum miRNAs expression, found four serum miRNAs (miR-17, miR-20a, miR-29c and miR-223) can better distinguish between NPC and normal controls, diagnostic sensitivity and specificity can be above95%. Another study found that four serum miRNAs (miR-22, miR-572, miR-638and miR-638) with TNM stages can more accurately predict the prognosis of nasopharyngeal carcinoma, and significantly associated with survival. Due to the differences between individuals, experiment methods and evaluation criterion, even the same kind of disease and the same scope of miRNAs, each laboratory screening out the dysregulated miRNAs are also different. It is difficult to define a normal circulating miRNAs expression range, circulating micrornas become true tumor markers also faces many problems. In order to avoid individual differences, our laboratory screened five candidate miRNAs according to reports in past literatures, detected plasma micrornas as pairing testing before and after treatment in patients with nasopharyngeal carcinoma. Our results showed good effect in diagnosis and predicting prognosis of NPC.Extracellular miRNAs in cancer signal path network play a very important role, but the question of what mechanism protects circulating miRNAs from degradation remains obscure. Recent studies have revealed that miRNAs may be protected either in microvesicles(MVs) or in protein complexes. Circulating miRNAs in thought mainly secreted into the bloodstream by various cells with MVs or protein complexes form, transfer biological information between cells. Microvesicle is a kind of molecules of lipid bilayer membrane surrounding the cystic structure, contain protein, lipid, and all kinds of RNA, divided into microparticle, exosome and other small sac bubble. Study of in vitro cultured cells secrete MVs showed MVs is unitary, mainly exosomes around50nm in diameter. Exosomes containing miRNAs enable intercellular and interorgan communication in the body, suggesting a new mechanism of cell-cell communication. In fact, cancer cells also could release exosomes containing miRNAs into the peripheral blood, which lead to the desregulated expression of plasma miRNAs profiling.Although miRNA signatures for NPC have been established, elucidation of the role of deregulated miRNAs in NPC carcinogenesis remains in the early stage of development. And there has few report about plasma miRNA profiling of NPC. Based on our preliminary experiments, we screened plasma miRNA levels in patients with NPC by the miRNA chip and RT-qPCR method. And then we tracked dynamic changes of these dysregulated miRNAs at different time points after treatment. we will investigate the clinical value of these deregulated miRNAs in NPC development.Materials and Methods1. Clinical dataFrom December2009to December2013, the286NPC patients and50control included the patients of chronic suppurative otitis media and chronic sinusitis were recruited from Department of Otolaryngology-Head and Neck Surgery of Nanfang Hospital. No patient received chemotherapy or radiotherapy before diagnosis. Pathologists confirmed the histopathological diagnosis for each specimen. The histopathological types of NPC included nonkeratinizing undifferentiated carcinoma and keratinizing undifferentiated carcinoma. Tumors were staged according to the Chinese2008staging system for NPC. The treatment was according to the Guidelines in Oncology-v.2.2008NCCN. The follow-up of all patients were record, and the therapeutic effect was determined by a video laryngoscope examination and an imageological diagnosis.2. Blood specimen collection and preservationWhole blood (6-8ml) before treatment was drawn into EDTA-containing tubes. The blood samples after treatment were drawn at3month,6month,12month,24month,36month after chemoradiotherapy. Informed consent was obtained from each patient, and the research protocols were approved by the Ethics Committee of Nanfang Hospital and registered in ClinicalTrials.gov (No. NCT01171235). Blood samples were centrifuged to spin down the blood cells, and the supernatant was transferred into microcentrifuge tubes, followed by second centrifugation completely remove cellular components. Plasma was then aliquoted and stored at-80℃until use. Blood samples were processed and plasma was frozen within4h of the blood draw.3. miRNA microarray20NPC and10control plasma samples performed Micrornas chip analysis by KangChen Bio-tech Inc. Small RNA extracted from the plasma samples were labelled using the miRCURY Hy3/Hy5Power labelling kit and hybridised on the miRCURY LNA Array (Version16.0, Exiqon, Vedbaek, Denmark). The threshold value for differentially expressed miRNAs was a fold change>1.5with a value of P<0.05. The raw and normalised miRNA data were available through GEO accession number GSE48442. The candidate miRNAs were further filtered on the basis of expression levels.4. qRT-PCRTotal RNA was polyadenylated and reverse transcribed to cDNA. Real-time PCR was performed in duplicate measurements using SYBR Green PCR kit. The expression levels of candidate miRNAs were normalized to U6snRNA, miR-634and miR-1228, and were calculated utilizing the2-△△Ct method. H2O was used as the negative control.5. Cell cultureThe human NPC cell lines5-8F、6-10B、CNE1、CNE2and C666-1were cultured in RPMI-1640supplemented with10%FBS and1%penicillin-streptomycin. An immortalized nasopharyngeal epithelial cell NP69was cultured in KSFM supplemented with bovine pituitary extract. All cells were cultured in a humidified atmosphere of95%air and5%CO2at37℃.6. miRNA transfection The transfection was performed using LipofectamineTM2000reagent, and the working concentration of miRNA was20-80nmol in a12-well plate.7. Cell radiationWhen NPC cells and NP69cells were cultured after3-4generations, we exposed cells to a range of doses of IR (2,4,6and8Gy). The expression of miRNAs in cells was detected by Real-time PCR after radiation. Dead cells were dyed blue obviously by Trypan Blue Solution after radiation, while survival cells were colorless. Cell survival rate after radiation was calculated with artificial counting under a microscope. Cell survival rate(%)=number of living cells/(number of dead cells+number of living cells)×100%.8. Exosome extraction and purificationWhen the NPC cells reached80%confluence, they were cultured in RPMI1640without FBS for24-48h, and then the supernatants containing exosomes were harvested. The exosomes were purified by ladder high-speed centrifugation. At last collecting precipitation, soluble in1ml of PBS buffer, used for the next experiment.5. Statistical analysisSPSS16.0software was used for statistical analysis. Data were presented as mean±SEM of at least3independent experiments. Two-tailed Student’s t test was used for comparisons of2independent groups.3or more independent groups were analyzed by One-way ANOVA, with the LSD tests for multiple comparisons. Multivariate logistic regression model was established. Receiver-operating characteristics (ROC) curves were established to evaluate the diagnostic value of plasma miRNAs for differentiating between tumors and controls. The expression levels of miRNAs before and after treatment was compared with paired t test. Repeated measure analysis of variance (ANOVA) was used for plasma miRNA levels before and3,6and12months after treatment, with the LSD tests for multiple comparisons. P value of less than0.05was considered statistically significant.Results1.NPC plasma miRNAs screening and verificationWe identified a microRNA panel (downregulated:miR-9-3p, miR-92a-2-5p, miR-124-3p, miR-892b, miR-3676-3p; upregulated:miR-214-3p, miR-3135a, miR-4257) by microarray and qRT-PCR.(hereafter called miR-9, miR-92, miR-124, miR-892, miR-3676, miR-214, miR-3135, miR-4257)2. The diagnostic value of NPC plasma miRNAs and correlation with TNM and clinical stagesArea under the receiver operating characteristic curve (AUC) was used to evaluate diagnostic accuracy of plasma miRNAs. The result showed that miR-9and miR-124yielded AUC of78%. The AUC for the8miRNA panel was95.8%(sensitivity=91.1%and specificity=86.7%). The AUC for5miRNA panel(miR-9, miR-124, miR-3135, miR-3676, miR-4257) was95.3%(sensitivity=90.4%and specificity=86.7%). The satisfactory diagnostic performance of the5miRNA panel persisted regardless of NPC clinical stages (AUC for early stage was91.9%, sensitivity=84.4%and specificity=86.7%). Plasma miR-9was negatively correlated with T stage (F=3.992, P=0.003), with the increase of T stage, plasma miR-9expression quantity gradually decreases; Plasma miR-9, miR-92, miR-892showed a negative correlation with N stages (F=3.663, P=0.014; F=4.571, P=0.004; F=6.071, P=0.001). Eight kinds of plasma miRNAs expression quantity correlation with M stages were no statistical difference; Plasma miR-9(F=5.622, P=0.001), miR-92(F=4.638, P=0.004), miR-124(F=6.560, P<0.001), miR-892(F=3.102, P=0.029), miR-3676(F=3.225,P=0.025) and miR-4257(F=3.160, P=0.037) were negatively correlated with clinical stages.3. Dynamic changes of NPC plasma miRNA before and after treatmentThree months after treatment, miR-9(t=5.068, P<0.001),miR-124(t=2.833, P=0.006), miR-892(t=4.544, P<0.001) and miR-3676(t=3.463, P=0.001) were up-regulated compared with pri-treatment, while miR-3135was down-regulated (t=-2.229, P=0.029).3,6,12months after treatment, miR-9(F=4.256, P=0.018), miR-124(F=2.959, P=0.039), miR-892(F=6.289, P=0.001) and miR-3676(F=4.058, P=0.01) were also up-regulated. miR-3135was down-regulated (F=3.175, P=0.03). Other candidate miRNAs also showed the trend of reverse, but there were No statistical differences.4. Changes of NPC plasma miRNA at recurrence or metastasis8cases of patients occurred recurrence or metastasis. The plasma miR-9(t=-4.169, P=0.004) and miR-124(t=-4.675, P=0.002) were down-regulated further than pri-treatment in all8cases, miR-892(t=-2.886, P=0.023) and miR-3676(t=-2.459, P=0.044) were down-regulated in7cases. Other4miRNAs showed no obvious regularity.5. Expression of miRNAs in NPC cell lines before and after radiationmiR-9, miR-124and miR-892in all5NPC cell lines are lower expression than NP69. miR-3676was lower expression and miR-92was higher expression in4NPC cell lines. After radiation, miR-9, miR-124and miR-892were up-regulated compared with NP69. The other4miRNAs changed without consistency.6. miR-892b affect radiation survival rate of NPC cell linesBecause miR-892b showed the biggest change in NPC cell lines after radiation, we explored the effect of miR-892b on radiation survival rate of NPC cell lines.5-8F and6-1OB cells were transiently transfected with miR-892b mimic or miR-892b inbibitor, respectively. The results displayed that miR-892b overexpression inhibited cell survival rate in5-8F cells by53%(t=-9.824, P<0.001) and in6-10B cells by44%(t=-4.638, P=0.002) at48h post-radiation, and miR-892b downregulation promoted cell survival rate in5-8F cells by55%(t=4.411, P=0.002) and in6-1OB cells by65%(t=6.599, P<0.001) respectively.7. Expression of miRNAs in exosome of NPC cell supernatantThe exosomes were purified in5-8F and6-1OB cells supernatant by ladder high-speed centrifugation. The results displayed that miR-9, miR-92, miR-124and miR-892were relatively high expression in exosomes, and miR-214and miR-3135were relatively high expression in exosome free supernatant, while miR-3676and miR-4257were not detected in supernatant.Conclusions1. The plasma level of miR-9, miR-92, miR-124, miR-892, miR-3676, miR-214, miR-3135and miR-4257were dysregulation in NPC patients compared with healthy control.2. The plasma miRNA panel (miR-9, miR-124, miR-3135, miR-3676and miR-4257) provides a high satisfactory diagnostic accuracy of NPC, as well in early-stage NPC. These plasma miRNAs (miR-9, miR-124and miR-892) were correlated with TNM stages and clinical stages of NPC.3. The dynamic changes of Plasma miR-9, miR-124, miR-892and miR-3676levels after treatment and relapse or metastasis shows almost the same change trends with states of disease.4. The expression changes of miR-9, miR-124and miR-892in NPC cell lines after radiation shows consistent with NPC plasma. And miR-892can affect radiation survival rate of NPC cell lines, may be markers in radiation resistance of NPC. 5. miR-9, miR-92, miR-124and miR-892mainly exist in exosomes of cell supernatant, and miR-214and miR-3135mainly exist in protein complexes. |
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