| BackgroundCancer of the larynx is one of the most common types of head and neck cancer. Laryngeal cancer is the second most common cancer of the respiratory tract and squamous cell carcinoma accounts for about95%of all laryngeal cancer. With the continuous industrialization in China, the prevalence of laryngeal cancer has gradually increased by about25%. Laryngeal cancer often occurs in middle-aged and elderly men, with an estimated incidence rate of5.1/100,000cases in males worldwide in the year2008and the mortality in laryngeal cancer for males is2.2/100,000. Although new surgical approach, chemotherapy drugs, and advanced radiotherapy have been used in the treatment of laryngeal cancer in the past30years, the overall survival of patients with laryngeal cancer does not improve, only about50%. The survival rate of advanced laryngeal cancer is only30to40%.It is generally believed that the occurrence of laryngeal cancer is related to tobacco use, alcohol consumption, air pollution, and occupational factors. With the rapid development of molecular biology, the occurrence and development of laryngeal carcinoma has been proved to be a multi-gene, multi-step process. However, the molecular carcinogenesis is still unclear. So it is hard to improve the survival rate of laryngeal cancer using molecular methods. microRNAs (miRNAs) are19to25nucleotides non-coding RNAs that incompletely or completely bind the3’UTR(3’untranslated region) of multiple target mRNAs, enhancing their degradation and inhibiting translation. miRNAs possess normal biological functions, such as regulation of development, cell metabolism, proliferation, differentiation and apoptosis. miRNAs are negative regulators of gene expression and play a crucial role in the regulatory network of the genome. miRNAs have been found to regulate over30%of mRNAs, although they account for about only2%of total number of genes.More and more studies have shown that the specific miRNAs can function as an oncogene or tumor suppressor, and dysregulation of miRNAs have been linked to the aetiology, progression, invasion, metastasis and angiogenesis of cancer. In addition, approximately50%of all annotated human miRNA genes are located in fragile sites or areas of the genome that are associated with cancer.Some studies have shown that miRNAs may play a critical role in carcinogenesis and progression of head and neck cancer, suggesting that miRNAs may have a potential clinical value of early diagnosis, treatment and prognosis in laryngeal cancer. miRNAs research could help to investigate the occurrence and development mechanism of laryngeal cancer, look for novel diagnostic biomarkers and therapeutic targets.However, the laryngeal cancer miRNAs expression profile is still not clear, and biological function of miRNAs in laryngeal cancer needs further investigation. Most of the current studies of head and neck cancer miRNA expression profiles involved different sites including oral cavity, pharynx and larynx. To avoid any bias derived from all head and neck cancer together, it is necessary to perform research specific for laryngeal cancer.High expression of miRNAs in tumors may function as oncogene to promote tumor cell proliferation, invasion and metastasis; low expression of miRNAs can be considered to be tumor suppressor gene. However, the biological function of differentially expressed miRNAs must be confirmed through in vivo and in vitro experiments. There are only a small number of miRNAs which have been studied in-depth about their functions. The biological function of miRNAs in laryngeal cancer is still unknown and needs further study.In this study, the6th generation miRCURYTM LNA microarray which covered all human microRNAs in the latest miRbase database were used to screen the differentially expressed miRNAs in laryngeal cancer. The results of microarray were validated by quantitative real-time PCR and the effect of miR-125a (also known as miR-125a-5p) on proliferation of laryngeal cancer cell line was studied. The aim of this study is to provide clues for further study of the relationship between miRNAs and laryngeal cancer development, and to provide molecular markers and more effective targets in laryngeal cancer, in order to ultimately improve the rate of early diagnosis and therapeutic effect of laryngeal cancer.Part I Screening differentially expressed miRNAs of laryngeal cancer using microarray technologyAim:To investigate and establish the differential miRNAs expression profiling between laryngeal cancer and adjacent normal laryngeal mucosa using microarray chip.Method:The laryngeal cancer tissues and blood samples of patients with laryngeal cancer in the Guangdong General Hospital were collected from2010and the laryngeal cancer specimen bank was setup. We established and improved standard operating procedures of specimen collection, processing and storage. We also setup an information management system.A total of ten pairs of laryngeal cancer and adjacent normal tissue in the Guangdong General Hospital laryngeal cancer specimen bank were randomly selected and detected using the latest miRCURYTM LNA microarray(Exiqon). After extraction of total RNA and quality control, the RNA was processed for fluorescent labeling, hybridization, washing, scanning and signal digitizing. The scanned image was input to GenePix Pro6.0(Axon) software for coordinates adjustment and data extraction. Significantly differentially expressed miRNAs were filtered and confirmed by volcano plot. Cluster analysis was performed by MEV software (v4.6, TIGR). Furthermore, the microarray data were analyzed using Significance Analysis of Microarrays software (SAM, http://www-stat.stanford.edu/-tibs/sam/). The false discovery rate (FDR) was set to be0.05.Result:The result of total RNA quality identification showed that the samples were high-quality without degradation and suitable for microarray experiments. Significantly differentially expressed microRNAs between laryngeal carcinoma and adjacent tissues were obtained using microarray and SAM software. Compared with those of adjacent normal tissue,11miRNAs were significantly up-regulated and114miRNAs were down-regulated in laryngeal cancer tissues. Down-regulated miRNAs accounted for the majority of all significantly differentially expressed miRNAs.Conclusion:1、There are significantly differentially expressed miRNAs between laryngeal carcinoma and adjacent normal tissues. These differentially expressed miRNAs may play an important role in the occurrence and development of laryngeal carcinoma and become the new molecular markers of laryngeal cancer in the future.2、A well developed laryngeal cancer bio bank and information management are critical for laryngeal cancer basic research. It can preserve precious medical resources and provide high-quality specimens for laryngeal cancer studies.Part Ⅱ Validation of differentially expressed miRNAs in laryngeal cancer using quantitative real-time PCR Aim:To further validate differentially expressed miRNAs in laryngeal cancer tissues using quantitative real-time polymerase chain reaction (qRT-PCR). Method:Thirty-two pairs of laryngeal squamous cell cancer tissues and adjacent normal laryngeal mucosa specimens were randomly selected from Guangdong General Hospital laryngeal cancer specimen bank. The stem-loop qRT-PCR experiments were performed on these samples. The expression levels of let-7f-5p, miR-10a-5p, miR-125a, miR-144-3p, miR-195-5p, miR-203in laryngeal cancer tissues and adjacent normal tissues were detected using U6as internal reference: extracting total RNA of specimens, reverse transcription, quantitative PCR amplification. Ct values were calculated by2-△△Ct relative quantification method.Result:Six down-regulated miRNAs:let-7f-5p, miR-10a-5p, miR-125a, miR-144-3p, miR-195-5p, miR-203were verified by quantitative real-time PCR analysis in32pairs of laryngeal cancer and adjacent normal laryngeal tissues. These six differentially expressed miRNAs were statistically significant (P<0.05). The dissolution curve of miRNAs quantitative PCR indicated that the specificity of PCR amplification.Conclusion:The results of microarray were accordant with those from qRT-PCR. The result of miRNAs microarray was confirmed to be true and reliable. By adding these down-regulated miRNAs might inhibit the occurrence and development of laryngeal cancer.Part Ⅲ The study of miR-125a on the proliferation of laryngeal cancer cell lineAim:miR-125a (also known as miRNA-125a or miR-125a-5p) was significantly down-regulated in both microarray and qRT-PCR experiments. We studied the effect of miR-125a on the proliferation of laryngeal cancer cell line in order to acquire a better understanding of the biological function of miR-125a in laryngeal cancer.Method:miR-125a-mimic and miR-125a-inhibitor were transfected into laryngeal cancer Hep2cell lines. The experiment was divided into three groups:negative control group (NC), miR-125a-mimic group, miR-125a-inhibitor group. The MTT assay, colony formation and soft agar colony formation experiments were performed to observe the effect of overexpression or inhibition of miR-125a on proliferation of laryngeal Hep2cell. BrdU and flow cytometry assay were performed to evaluate the influence of overexpression or suppression of miR-125a on the cell cycle of laryngeal cancer cell; The effect of miR-125a on the expression levels of cell cycle-related proteins were detected using western blot analysis. Result:1. The result of MTT assay showed that:the number of living cells was significantly reduced in miR-125a-mimic group compared with the control group (P<0.05), and the ability of proliferation was significantly inhibited; the number of living cells in miR-125a-inhibitor group compared with the control group was significantly increased (P<0.05).2. Colony formation assay revealed that:colony formation number in miR-125a-mimics group was significantly lower than the NC group (P<0.01); colony formation number in miR-125a-inhibitor group was significantly higher than NC group (P<0.01). Laryngeal Hep2cell proliferation was significantly inhibited by the overexpression of miR-125a.3. The result of soft agar colony formation experiment showed that:the formation number in the miR-125a-mimics group was significantly lower than the NC group (P<0.01), and the formation number of miR-125a-inhibitor group compared with NC group was significantly higher (P<0.01). It also suggested that miR-125a expression have a significant impact on the proliferation of laryngeal carcinoma cell.4. The result of BrdU incorporation experiment showed that:in the miR-125a-mimics group, the proportion of S phase cells was significantly lower than the NC group (P<0.05), and in the miR-125a-inhibitor group, the proportion of S phase cells was significantly higher than the NC group (P<0.05).5. Flow cytometry showed miR-125a could increase the proportion of G0/G1phase laryngeal cancer cells, decrease that of S phase and G2/M phase cells, indicating that G0/G1cell cycle arrest and the cell proliferation was inhibited.6. The expression of several cell cycle related proteins in laryngeal Hep2cell lines of miR-125a overexpression were detected and analyzed by western blot technique. The expression levels of cyclin Dl, cyclin E were found to be significantly down-regulated, p21and p27were significantly up-regulated.Conclusion:1、miR-125a may inhibit the proliferation of laryngeal cancer cells through cell cycle arrest at G0/G1phase. 2. miR-125a has the potential to become a novel therapeutic target against laryngeal cancer in the future. |
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