Bioinformatics Analysis On Genes Involved In Hypoxia Induced Autophagy In Primary Salivary Adenoid Cystic Carcinoma

Objective:Bioinformatics is an interdisciplinary field that develops methods and software tools for understanding biological data. As an interdisciplinary field of science, bioinformatics combines computer science, statistics, mathematics, and engineering to analyze and interpret biological data. Bioinformatics has become an important part of many areas of biology. In experimental molecular biology, bioinformatics techniques, such as image and signal processing, allow the extraction of informational results from large amounts of raw data. In the field of genetics and genomics, it aids in sequencing and annotating genomes and their observed mutations. It plays a role in the text mining of biological literature and the development of biological and gene ontologies to organize and query biological data. It also plays a role in the analysis of gene and protein expression and regulation. Bioinformatics tools aid in the comparison of genetic and genomic data and more generally in the understanding of evolutionary aspects of molecular biology. At a more integrative level, it helps analyze and catalogue the biological pathways and networks that are an important part of systems biology. In structural biology, it aids in the simulation and modeling of DNA, RNA, and protein structures as well as molecular interactions.Adenoid cystic carcinoma is a rare type of cancer that can exist in many different body sites. It most often occurs in the areas of the head and neck, in particular the salivary glands, however, it has also been reported in the breast, lacrimal gland of the eye, lung, brain, bartholin gland, trachea, and the paranasal sinuses. Salivary adenoid cystic carcinoma (SACC) is the third most common malignant salivary gland tumor overall (after mucoepidermoid carcinoma and polymorphous low grade adenocarcinoma). It represents 28% of malignant submandibular gland tumors, making it the single most common malignant salivary gland tumor in this region. Patients may survive for years with metastases resulting from the well-differentiation and slow growth of this tumor. It is reported that the disease specific survival was 77.3%,59.6%,44.9%,35.0% and 25.5% at 5,10,15,20 and 25 years, indicating the deaths from late-occurring metastatic disease.Autophagy is the basic catabolic mechanism that involves the cell degradation of unnecessary or dysfunctional cell components through the actions of lysosomes. The breakdown of the cell components promotes cell survival during the starvation by maintaining cell energy levels. Autophagy allows the degradation and the recycling of the cell components. During this process, the targeted cytoplasmic constituents are isolated from the rest of the cell within a double-membraned vesicle known as an autophagosome. The autophagosome then fuses with a lysosome and its cargo is degraded and recycled. There are three different forms of autophagy that are commonly described as macroautophagy, microautophagy and chaperone-mediated autophagy. In the context of diseases, autophagy has been seen as an adaptive response to the stress that promotes cell survival, whereas in other cases it appears to promote cell death and mortality.Recent studies show that hypoxia could induce autophagy and that Hypoxia-Inducible Factor 1 a, the product of HIF1A, plays a vital role in hypoxia-induced autophagy. However, the correlation between HIF1A expression and the expressions of the genes related with hypoxia-induced autophagy is unexplored in salivary adenoid cystic carcinoma. Also the relationship of HIF1A and the pathological features in the adenoid cystic carcinoma has not been well investigated. Further investigations are required for the mechanism of hypoxia-induced autophagy in salivary adenoid cystic carcinoma.The aim of the present study was to examine whether the autophagy pathway was enriched in the salivary adenoid cystic carcinoma microarray dataset and the relationship between hypoxia and autophagy, and to analyze the correlation between HIF1A expression and MAP1LC3B expression in salivary adenoid cystic carcinoma in vitro and in vivo. This study also aimed to facilitate the development of the new strategies for cancer prevention and therapeutic intervention.Methods:1. The microarray data set of salivary adenoid cystic carcinoma was obtained from the GEO website. The expression data was normalized and organized.2. The data set was normalized and the differentially expressed gene analysis on the dataset was performed using Wilcoxon test in R program.3. The genes involved in the autophagy pathway was collected and the enrichment analysis was performed on the dataset.4. The correlation between HIF1A and the autophagy related genes was analyzed in R program.5. The expression of HIF1A and MAP1LC3B was analyzed in the paraffin-embedded specimens using the immunohistochemical method.6. ACC-M cells with exponential growth were exposed to Cobalt Chloride to mimic hypoxic conditions.7. MTT assay was utilized to quantify the viability of ACC-M cells to detect the inhibition effect of CoCl2. Autophagosome was observed by a transmission electron microscopy. HIF1A and MAP1LC3 were detected using immunofluorescence staining, RT-PCR and Western blot. Data were shown as the mean ± SEM of at least three independent experiments. Statistical comparisons between groups were performed using two-tailed Student’s t-test (SPSS 15.0). Values of p<0.05 were considered as significance. Results:1. There were 20201 genes investigated in the dataset GSE59701,764 (3.78%) genes were up-regulated, whereas,545 (2.70%) genes were down-regulated.2.26 genes were defined as the autophagy pathway related genes.3. The autophagy pathway was up-regulated in the dataset GSE59701, and the P value was 0.008223.4. Five genes, ATG3, ATG4A, ATG5, PIK3R4 and MAP1LC3B were found to have the significant correlation with HIF1A.5. MTT assay was utilized to quantify the viability of ACC-M cells treated with CoCl2, which reduced the cell viability of ACC-M cells and the inhibition effect, to be assumed as an obvious dose-response relationship. Numerous autophagosome-like structures consisting of double membranes were observed using a transmission electron microscopy.6. The expression of HIF1A and MAP1LC3 were significantly increased induced by CoCl2 and they had the same expression pattern with time-response relationship.7. Immunohistochemistry results showed that HIF1A was expressed in 53 samples of 71 in all, MAP1LC3B was expressed in 40 samples of 71 in all. Besides, the two genes were both expressed in 25 samples of 71 in all.Conclusions:1. Autophagy was up-regulated in salivary adenoid cystic carcinoma which was proved by bioinformatics analysis and transmission electron microscopy. This pathway may play an important role in the development of salivary adenoid cystic carcinoma.2. The expression of HIF1A and MAP1LC3B were correlated both in the bioinformatics analysis result and the RT-PCR and Western blot results. These findings indicated that there might be important connections between HIF1A and MAP1LC3B during the development of salivary adenoid cystic carcinoma.