br MODEL br Introduction br PCa
PCa is one of the most common types of cancer expected to occur in all males and accounts for 1 in 5 new diagnoses, excluding lung and bronchus, of the male patients; PCa is the second leading cause of cancer death in men in the United States.1 Cancer metastasis is the primary cause of morbidity and mortality and is responsible for more than 90% cancer-associated deaths. Despite the advancement in treatment strategies over the past 30 years, early diagnosis of PCa to improve the prognosis has not been achieved.2e4 Although the molecular alterations in PCa have been stud-ied extensively, the specific molecular markers responsible for the metastasis and progression of PCa remain to be identified.5
The cell migration cycle is a highly regulated multi-step process that begins with the membrane polarization and extension in the direction of migration.6 The polari-zation of a cell is a key process for migration, which means that the direction of cell movement is determined by different molecular processes. The membrane pro-trusions establish contact with their environment through a diverse array of receptors via controlled adhesive in-teractions with the CORM3 cytoskeleton and the extracel-lular matrix (ECM). Recent researches have focused on the main role of the focal adhesion complexes focal ad-hesions and adherens junctions, in epithelial cell behavior.7e9
The central role of the Rho family proteins is to regu-late membrane protrusions to direct cell migration. Rho family small guanosine triphosphate (GTP) e binding proteins (GTPases) play a key role in cancer malignancy via regulation of multiple biological processes.10 control the formation of cellular pseudopods. Rho GTPases act as pivotal molecular switches by alternating GTP activation and GDP inactivation, where the conversion of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs) in the Dbl family. Some of the Rho GEFs have been found to possess oncogenic functions playing a crucial role in the migratory process.11e13 ARHGEF38 being a member of the Rho GEFs family might be of significant interest to study their cellular roles and mode of regula-tion in cancer.
Materials and methods
The gene expression array data sets were selected from the GEO public database. GSE21034 dataset with 126 PCa and 29 BPH tissues (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?accZGSE21034), GSE54808 dataset with 18 PCa and
12 normal prostate tissues (https://www.ncbi.nlm.nih.gov/ geo/query/acc.cgi?accZGSE54808), and TCGA dataset including 497 PCa tissues and 52 normal samples (http:// gdac.broadinstitute.org/runs/stddata__2016_01_28/data/ PRAD/20160128/) were used in this study. We changed all data to (log)2(ratio) format and then used BRB software to investigate mRNA expression of ARHGEF38 and further survival analyses were carried out.
Identification of DEGs
R limma package (version 3.4.2) and BRB software were used to identify DEGs (p < 0.0001) and calculate the fold change of their expression in PCa (jFCj>2.0). The DEGs were clustered using Consensus Clustering Plus and visual-ized using Java TrueView software. These DEGs were then uploaded to the web-based GEne SeT AnaLysis Toolkit database and KEGG to process for their function enrich-ment and pathway analyses. Finally, the copy number variations (CNVs) of DEGs were analyzed and the co-expression network was obtained.
Patient information and clinical specimens
Prostate tissue samples were obtained from patients with PCa (n Z 100) and BPH (n Z 20) who were treated at Second Hospital, Tianjin Medical University, between July 2013 and July 2014. The patients who had undergone radiotherapy or chemotherapy before surgery were excluded. This study was approved by the Institutional Research Ethics Committee, Second Hospital, Tianjin Med-ical University. Pathological diagnosis and clinical infor-mation (age, clinical stage, GS, lymph node metastasis, and differentiation) were determined by two pathologists. Based on the work of Borley,14 a combination of pre-treatment GS and clinical stage was used to divide the patients into normal, low-grade, intermediate-grade, high-grade, and aggressive groups. A caseecontrol study of Chinese man with BPH and the five PCa groups were strat-ified according to WHO classification: (i) 20 BPH tissues; (ii) 20 low-grade GS 2e6 (LGPCa); (iii) 40 intermediate GS7 (MGPCa) (3 þ 4 and 4 þ 3); (iv) 20 high-grade GS8 (HGPCa); and (v) 20 aggressive GS9 and GS10.
Immunohistochemistry staining and scoring
absence of staining (<10%). IHC image processing was per-formed using Image Pro-plus 6.0 software.15,16