Whole genome oligo microarray

To know the targets of miR-192/215, we performed gene microarrays, which were carried out by the Agilent Whole Genome Oligo Microarrays (4×44K, Agilent, Santa Clara, CA, USA). Different expression genes were identified between two groups of cells, BGC823 cells with inhibition of miR-192/215, and HFE145 cells with mimics of miR-192/215. Upregulated or downregulated at least two-fold genes were counted as significance. Briefly, TRIzol Reagent (Invitrogen, Carlsbad, California, USA) lyses cells, and RNA Integrity was tested by Agarose Gel Electrophoresis. Agilent Quick Amp Labeling Kit and Agilent’s SureHyb Hybridization Chambers (Agilent, Santa Clara, CA, USA) were applied for the sample labeling and hybridization as per the manufacture protocols. Data were extracted and further analysis was performed by Agilent GeneSpring GX 11.5.1 software.

Microarray experiments were performed by KangCheng Bio-tech, Shanghai, China. The Agilent Whole Genome Oligo Microarray was used to identify mRNA transcripts with differential expression between LUAD and adjacent normal tissues. Tissue samples were used for the array analysis according to the manufacturer’s protocol. Related data was uploaded in GEO datasets (GSE 140797).
Genome-wide transcriptional sequencing were performed by Baimaike, Beijing, China. Transcriptome sequencing (NEB, USA) was used to identify mRNA transcripts with differential expression between Mex3a-silenced H1299 cells and control H1299 cells. The RNA preparation and Library preparation for Transcriptome sequencing were performed according to the manufacturer’s instructions.
The gene expression and clinical data of LUAD patients from the Cancer Genome Atlas (TCGA) were downloaded from UCSC Xena Browser (https://xenabrowser.net/). The gene expression and clinical data of LUAD patients from the GEO datasets were obtained using GSE 116959 and GSE 19804 (https://www.ncbi.nlm.nih.gov/gds).

Total RNAs were harvested using TRIzol (Invitrogen) and the RNeasy kit (Qiagen) according to manufacturer's instructions, including a DNase digestion step. After having passed RNA measurement on the Nanodrop ND-1000 and denaturing gel electrophoresis, the samples were amplified and labeled using the Agilent Quick Amp labeling kit and hybridized with Agilent whole genome oligo microarray in Agilent's SureHyb Hybridization Chambers. After hybridization and washing, the processed slides were scanned with the Agilent DNA microarray scanner (G2505B) using settings recommended by Agilent Technologies. The resulting text files extracted from Agilent Feature Extraction Software (version 10.5.1.1) were imported into the Agilent GeneSpring GX software (version 11.0) for further analysis.

Fifty-seven GK male rats were used, of which 48 (300–330 g) were randomly separated into 3 groups: uninjured group (n = 18), Day 3 post-injury group (n = 12) and Day 7 post-injury group (n = 18). Three rats from each group were used for morphological and immunohistochemical analysis. Six rats from each group, with the exception Day 3 post-injury group GK rats, were used for expression analysis with an Agilent Whole Genome Oligo Microarray. Six rats were used for Western blotting experiments, and the remaining 3 rats were used for quantitative real-time PCR (qRT-PCR). Another 9 GK rats (150–180 g) were used for cell culture. Weight-matched non-diabetic Wistar rats, separated exactly like GK, were used as controls. All rats were fed a standard diet ad libitum. Room temperature was maintained at 23–25°C, with 50–60% humidity and 8 h period light daily. Animals and forage were purchased from SLAC Laboratory Animal Limited Company (Shanghai, China). The experimental procedures were carried out in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 85-23, National Academy Press, Washington, DC, revised 1996). The experiments were approved by the Animal Research Committee of Xuzhou Medical College (permit number, XMCACUC2010-08-114).

Total RNA was isolated with TRIzol reagent (Invitrogen) from HASMCs infected with or without pAd-GFP or pAd-GFP-KLF4 (n = 3 per group). The RNA quantity was assessed using a NanoDrop 2000, and RNA integrity was assessed by standard denaturing agarose gel electrophoresis. Microarray analysis was performed by Shanghai KangChen Biotech on an Agilent Array platform. The subsequent steps were performed according to the Agilent Whole Genome Oligo Microarray (one-color) protocol. Array data preprocessing, normalization, and quality control were conducted using GeneSpring software V12.1 (Agilent Technologies). To select differentially expressed genes, ratio change threshold values of g2.0 or e0.05 were used. Hierarchical clustering was performed using log2-transformed data in Cluster 3.0, and heat maps were generated with the Ggplot2 R environment. Gene Ontology (GO) term analysis was performed using the topGO R software. KEGG pathway analysis was applied to determine key signaling pathways and relationships between differentially expressed genes.