Analyzing The Effects of ProL1 Upregulation On LNCaP Global Gene Expression

Purpose: To examine the effects of proL1 upregulation on global gene expression in LNCaP cells using RNA sequencing (RNA-Seq) Methods: LNCaP cells were transformed to overexpress proL1 using lentiviral vectors. The proL1 overexpressing variant (LNCaP proL1+) and their parental cell line were plated in 6-well plates in triplicate (3 wells for each cell line). After 3 days, their RNA was isolated using the Qiagen RNeasy Mini kit and their global gene expression patterns were analyzed using RNA-Seq Results: We identified 1110 differentially expressed genes between LNCaP proL1+ and its parental cell line. Of these differentially expressed genes (DEGs), 209 were in common with PC3 when proL1 was overexpressed. To identify biological functions that may be regulated through overexpression of proL1 in LNCaP cells, the list of DEGs was submitted to the DAVID, GOC, and KEGG databases. According to these databases, proL1 overexpression regulated gene expression in ontological groups involved in morphogenesis and signal transduction. DEGs were also overrepresented in ontological groups potentially involved in vascularization and blood flow regulation. However, there were differences in the regulation of specific genes related to the androgen response between LNCaP and PC3. Unlike PC3, where overexpression of ProL1 caused a significant increase in expression of the androgen receptor gene (AR) and a decrease in expression of the estrogen receptor (ESR1), in LNCaP neither of these genes were changed in expression. In contrast, proL1 overexpression in LNCaP reduced expression of the progesterone receptor gene (PGR) but not in PC3 cells. Conclusion: Global analysis of the changes in gene expression caused by proL1 overexpression in PC3 and LNCaP cells supports that it has a role both in the modulation of genetic pathways involved in both overcoming hypoxia and the development of androgen-insensitivity. Opiorphins have previously been shown to be directly involved in regulation of blood-flow to tissues through their modulation of smooth muscle tone, and therefore their upregulation in tumors may directly contribute to overcoming the hypoxic barrier that develops in the growing tumor. In addition, overexpression of ProL1 in both PC3 and LNCaP cells modulated expression of genes involved in angiogenesis and morphogensis; the activation of these pathways would likely be involved in vascularization of the tumor and thereby also contribute to overcoming hypoxia. Overexpression of proL1 also affected genes involved in steroid metabolism and response pathways in both LNCaP and PC3, which could potentially contribute to the modulation of androgen sensitivity. For example, overexpression of ProL1 in LNCaP reduced expression of the progesterone receptor gene (PGR). Activation of PGR is a positive modulator of cell division and the reduced expression of PGR in LNCaP proL1 cells might therefore explain the negative effect on tumor growth in female mice. Overall, our data associates the development of PrCa with overexpression of opiorphin and suggests genetic pathways by which they contribute to tumor growth and development of androgen-insensitivity. Total RNA of LNCaP cells overexpressing proL1 and their parental cell line