Identification of genes potentially regulated by human polynucleotide phosphorylase (hPNPase old-35) using melanoma as a model

Abstract

Human Polynucleotide Phosphorylase (hPNPase(old-35) or PNPT1) is an evolutionarily conserved 3'→ 5' exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPase(old-35) in cellular physiology, we knocked-down and overexpressed hPNPase(old-35) in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPase(old-35) resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPase(old-35) caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPase(old-35) knockdown and overexpression datasets allowed us to identify 77 potential "direct" and 61 potential "indirect" targets of hPNPase(old-35) which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPase(old-35) expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes.

Figure 1. Generation of a melanoma cell culture model for hPNPase^old-35 expression.

(A) Phase contrast LM (top) and GFP fluorescent micrographs (bottom) of HO-1 melanoma cell lines following transduction with GFP expressing scrambled shRNA (HO-1 Csh) and hPNPase^old-35 shRNA1 (shown in clone 4; cl4) and 2 (shown is clone 9; cl9) expressing lentiviruses and selection with puromycin. qRT-PCR expression of hPNPase^old-35 (hPNPase^old-35 knockdown) normalized to control (shScramble). Mean values normalized to a GAPDH internal reference; error bars represent mean ± S.E. of three replicate experiments. Anti-hPNPase^old-35 and EF1α loading control immunoblots. (B) qRT-PCR expression of hPNPase^old-35 in HO-1 cells infected with Ad.hPNPase^old-35 normalized to cells infected with Ad.Vec for 36 h. Immunoblot showing hPNPase^old-35 overexpression compared to Ad.Vec post 36 hour of infection. Error bars represent mean ± S.E of three replicate experiments. * P<0.02, *** P<0.001.

Figure 2. Venn diagrams representing number of genes significantly altered when hPNPase^old-35 is knocked down or overexpressed in human melanoma cells.

Shown are total number of dysregulated genes (A), genes “directly” (B) and “indirectly” (C) regulated by hPNPase^old-35.

Figure 3. Functional analysis of genes dysregulated as a result of hPNPase^old-35 depletion.

(A) The biological functions and states associated with genes differentially expressed when hPNPase^old-35 is knocked down in human melanoma cells. (B) Toxicologically related functionalities and pathways associated with genes dysregulated (proportions shown in graphs) after hPNPase^old-35 knockdown in melanoma cells, as identified by IPA Toxicogenomic Analysis.

Figure 4. Functional analysis of genes dysregulated as a result of hPNPase^old-35 overexpression.

(A) The biological functions and states associated with genes differentially expressed when hPNPase^old-35 is overexpressed in human melanoma cells. (B) Toxicologically related functionalities and pathways associated with genes dysregulated (proportions shown in graphs) after hPNPase^old-35 overexpression in melanoma cells, as identified by IPA Toxicogenomic Analysis.

Figure 5. Network visualization of genes potentially “directly” regulated by hPNPase^old-35.

Figure 6. Functional analysis hPNPase^old-35-putative “directly” regulated genes.

(A) The biological functions and states associated with hPNPase^old-35-putative “directly” regulated genes in human melanoma cells. (B) Toxicologically related functionalities and pathways associated with hPNPase^old-35-putative “directly” regulated genes, as identified by IPA Toxicogenomic Analysis.

Figure 7. Network visualization of genes potentially “indirectly” regulated by hPNPase^old-35.

Figure 8. Functional analysis of hPNPase^old-35-putative “indirectly” regulated genes.

(A) The biological functions and states associated with hPNPase^old-35-putative “indirectly” regulated genes in human melanoma cells. (B) Toxicologically related functionalities and pathways associated with hPNPase^old-35-putative “indirectly” regulated genes, as identified by IPA Toxicogenomic Analysis.

Figure 9. Real time qRT-PCR validation of microarray findings.

(A) qRT-PCR verification of hPNPase^old-35-putative “directly” regulated genes identified by microarray analyses in response to hPNPase^old-35 (i) knockdown or (ii) overexpression in HO-1 melanoma cells. (B) qRT-PCR verification of hPNPase^old-35-putative “indirectly” regulated genes identified by microarray analyses in response to hPNPase^old-35 (i) knockdown or (ii) overexpression in HO-1 melanoma cells. Error bars represent mean ± S.E. error of three replicate experiments.