Identification of gene expression signatures and related microRNAs for chronic glomerulonephritis based on microarray and bioinformatic analysis

To further reveal the key genes and related microRNAs involved in the CGN and to determine the potential molecular mechanisms of CGN pathogenesis. We have employed microarray expression profiling as a discovery platform to identify genes and related microRNAs. To identify the key ceRNA in CGN, we generated a ceRNA network using data from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO). 2334 differentially regulated genes and a total of 673 related miRNAs were identified. We found that the ceRNA network was composed of 31 lncRNAs, 344 mRNAs, and 57 miRNAs. Expression of five genes (FOS, SYK, CYP1A1, HSD3B6, UGT2B15), five microRNAs (miR-34a-5p, miR-214-3p, miR-155-5p, miR-182, miR-592), three lncRNAs (LOC102547703, NONRATT006779, FQ220365) were quantified in the same RNA samples by real-time PCR, which could be potential diagnostic biomarkers and therapeutic targets for CGN. Glomerular tissues from normal, experimental and TCM-treated rats were analyzed based on Agilent Rat 4 × 44 K whole genome microarray, the accumulation of high-throughput gene expression profile data has greatly promoted the application of genome microarray in functional gene studies. RNA quantity and quality were measured by NanoDrop ND-1000. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Briefly, total RNA from each sample was linearly amplified and labeled with Cy3-UTP. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 11 μl 10 × Blocking Agent and 2.2 μl of 25 × Fragmentation Buffer, then heated the mixture at 60°C for 30 min, finally 55 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 100 μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).