| BackgroundBladder cancer is any types of cancer arising from the epithelial lining of the bladder. The bladder cancer we called in common is transitional cell carcinoma which occurs in 90% of all bladder cancer patients. The bladder cancer is the most in common cancer of urinary system in our country. The bladder cancer is the second common cancer which occurred in urinary system after prostatic cancer in the US. The bladder cancer causes 74,000 new cases which are 4.5% of all new cancer cases and 16,000 deaths which are 2.7% of all cancer deaths in the United States for 2015. Five-year survival rates in the United States are 77.4%, based on 2005-2011 data. The bladder cancer incidence is increasing with the age. The most bladder cancer happened at the age of 50-70. The incidence of bladder cancer in men is 3～4 times more than in women. The age-adjusted incidence of bladder cancer is 9/100,000 in men which is ranked 7th of all cancers. The age-adjusted incidence of bladder cancer is 2.2/100,000 in women which is ranked18th of all cancers.The incidence of bladder cancer in developed country (16.6/100,000 in men and 3.6/100,000 in women) is higher than that in developing country 5.4/100,000 in man and 1.4/100,000 in women). European and North American are considered as the highest incidence of bladder cancer which age-adjusted incidence of bladder cancer in men is 20/100,000,5/100,000 in women. However, incidence and death rate of bladder cancer in women are decreasing. The incidence of bladder cancer in men is decreasing too. In our country, the age-adjusted incidence of bladder cancer is 7.37/100,000 in men,1.98/100,000 in women. However, the situation has changed in this decade. With the development of society, industrialization and the aging of population, the incidence of bladder cancer is increasing both in men and women, cities and rural areas.The cause of bladder cancer is complicated which is both effected by gene and environment factors. Smoking is the main known contribution to bladder cancer. Research show that at least half of bladder cancer happened in men and one third happened in women is associated with smoking. Whereas with the development of society, the number of smoking women is increasing, the incidence of bladder cancer in women is decreasing in western country. Moreover the one who has frequent contact with 2-Naphthylamine has more chance to get bladder cancer, such as drivers, hairdressers, leather workers.Bladder cancer is also associated with genetic factors. The people who have relatives with bladder cancer have much more chance to get bladder cancer than others. On one hand they may have similar chemistry contact history. On the other hand, the genes which have related with toxin break down mutation lead to bladder cancer. The risk of bladder cancer among all relatives was slightly elevated, the observed-to-expected ratio being greater among second-and third-degree relatives than among first-degree relatives.It has been confirmed that oncogenes H-ras, FGFR3, erbB2, CCND1, mdm2, cancer suppressor genes INK4A/ARF, Rb, TP53, PTEN, TSC1, PTCH, DBCCR1, are associated with bladder cancer.Bladder cancer is a urinary system cancer which can be occurred both in men and women. The incidence of bladder cancer in men is 2～3 times higher than in women. Usually smoking is considered to be the cause of bladder cancer. Some research shows that smoking cannot explain this completely. It is necessary to looking for the reason why the incidence of bladder cancer is different between female and male in genetics. The research shows that AR (Androgen Receptor) gene which is on x chromosome q11-12 lead to the different incidence between male and female. The Androgen plays an important role in the development and maintenance of the normal urinary bladder. Mutation in Androgen Receptor alters the ligand binding ability that may cause the progression and development of bladder cancer.With the development of high throughput sequencing technology, we can study about the cancer in different levels and data types. Researchers can find the specific gene expression of in certain issue in specific time. All these can support us to investigate the mechanisms of cancer. At present, the mechanisms of bladder cancer and incidence difference in gender cannot be explained completely. So it is necessary to explore further in this issue.ObjectIn this study, we aim at finding the female specific genes related to bladder using the RNA-Seq data of bladder cancer samples which has been downloaded from TCGA database by identifying the differentially expressed bladder cancer related gene which can be found in female but not in male. We further explore the functions and biological process of the indentified genes with regarding to bladder cancer through constructing the co-expression network of female bladder cancer.MethodIn this study the differentially expressed genes are found by two R packages DESeq2 and edgeR. Both of them are used to differential expression analysis of RNA-Seq, and based on the hypothesis of negative binomial distribution. They may have different outputs since they chose different methods to estimate the discrete degree. So we choose both of them to find the differentially expressed gene in order to achieve more reliable results.We use 238 bladder cancer samples to find the female specific bladder cancer related genes. Firstly, we find the differentially expressed genes between tumor and normal samples in female and male respectively. Secondly, we find the differentially expressed genes between female and male in tumor samples. Lastly, we choose the genes which are differentially expressed in female (T/N) but not in male (T/N) and they also can be found as differentially expressed genes between female and male in tumor (F/M),as the female specific bladder cancer related gene. We also conducted functional annotation of these genes using DAVID.In order to explore the relations between the selected genes and female bladder cancer, we constructed the co-expression network using RNA-Seq 52 female bladder cancer samples. We did the GO analysis and KEGG pathway analysis with the module genes which including the female specific bladder cancer related gene, and we used Cytoscape to plot the co-expression network map.In this study, the differential expression analysis and co-expression network analysis were performed using DESeq2, edgeR and WGCNA packages in the R software. Result8,249 and 9,043 differentially expressed genes between male tumor and normal sample were founded by DESeq2 and edgeR, respectively, including 8076 genes which can be found using both methods. In female sample, we found 3,558 and 4,823 genes with DESeq2 and edgeR, respectively, including 3432 genes found using both methods. The differentially expressed genes between female and male in tumor samples are 516 and 3988 with DESeq2 and edgeR, respectively. At last we found 16 female specific bladder cancer related genes consisting of 10 up-regulated genes and 6 down-regulated genes. Among them, CD36 is associated with TSP-1 induced apoptosis in Microvascular Endothelial Cell and PPAR signaling pathway. NRG1 gene is a cancer suppressor gene. NRG1 can combine with ERBB3/ERBB4 promote the proliferation and differentiation of cell. The mutation of COL4A3 leads to the Alport syndrome. TGFBI has the function of antitumor. KLKB1 is associated with Intrinsic Prothrombin Activation Pathway, regard as the biomarker of lung cancer. PAQR8 is associated with female gamete generation and oogenesis.In this study we got 11 modules which are associated with female specific bladder cancer related genes, through constructing the co-expression network of female bladder cancer. Module grey60 contains CD36 and CHIN3 which are hub genes of this module which are associated with PPAR signaling pathway. Module black contains COL4A3 and KLKB1, which are associated with VEGF signaling pathway. Module brown contains SHOX2, which are associated with cell cycle lung cancer and non-small cell lung cancer. Module green contains PEX11A, which is associated with endometrial cancer and P53 signaling pathway. Module pink contains LRRC4, which is associated with pancreatic cancer, non-small cell lung cancer and P53 signaling pathway. Module steel blue contains TGFBI, which is associated with NF-kb signaling pathway.ConclusionIn this paper, we identified the female specific bladder cancer related genes by differential expression analysis and we explored the relations between female specific bladder cancer-related genes found in this study and female bladder cancer by co-expression network analysis.As a paradigm of tumor research, the analysis strategy used in this study could be applied research of other tumors. The future study based on more levels of genomic data should be able to provide more significant knowledge in the bladder cancer. |
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