Role of a novel functional variant in the PPP2R1A promoter on the regulation of PP2A-Aalpha and the risk of hepatocellular carcinoma

Abstract

Previously, we identified the genetic variant -241 (-/G) (rs11453459) in the PP2A-Aα gene (PPP2R1A) promoter and demonstrated that this variant influences the DNA-binding affinity of nuclear factor-kappa B (NF-κB). In this study, we further confirmed that the transcriptional activity of PPP2R1A may be regulated by NF-κB through the functional genetic variant -241 (-/G). Moreover, we also demonstrated that the methylation status of CpG islands in the promoter of PPP2R1A influences the activity of this gene promoter. Few studies have examined the role of this -241 (-/G) variant in genetic or epigenetic regulation in hepatocellular carcinoma (HCC). To investigate whether this functional variant in the PPP2R1A promoter is associated with the risk of HCC and confirm the function of the -241 (-/G) variant in the HCC population, we conducted a case-control study involving 251 HCC cases and 252 cancer-free controls from a Han population in southern China. Compared with the -241 (--) homozygote, the heterozygous -241 (-G) genotype (adjusted OR = 0.32, 95% confidence interval (CI) = 0.17-0.58, P<0.001) and the -241 (-G)/(GG) genotypes (adjusted OR = 0.38, 95% CI = 0.22-0.67, P = 0.001) were both significantly associated with a reduced risk of HCC. Stratification analysis indicated that the protective role of -241 (-G) was more pronounced in individuals who were ≤ 40 years of age, female and HBV-negative. Our data suggest that the transcriptional activity of PPP2R1A is regulated by NF-κB through the -241 (-/G) variant and by the methylation of the promoter region. Moreover, the functional -241 (-/G) variant in the PPP2R1A promoter contributes to the decreased risk of HCC. These findings contribute novel information regarding the gene transcription of PPP2R1A regulated by the polymorphism and methylation in the promoter region through genetic and epigenetic mechanisms in hepatocarcinogenesis.

Figure 1. Functional analysis of the−241 (−/G) variant in the PPP2R1A proximal promoter.

A. Transcriptional activity of fragments bearing the PPP2R1A proximal promoter with variant allelotype of −241 (−) or −241 G was measured by Dual Luciferase Assays in immortalized human normal hepatocyte L02 cells. The results are expressed as fold increases in RLU over the empty pGL3-Basic vector (Ctrl). Compared with the basal activity of Ctrl, the promoter activity of the pGL3b-2R1Ap-241(−) or −241G with or without treatment was higher (∗ P<0.05). Compared with pGL3b-2R1Ap-241(−) construct, the construct with −241 G allele had lower activity (^# P<0.05). Compared with the Ctrl of pGL3b-2R1Ap-241(−), the promoter activity of constructs containing −241 (−) was significantly decreased when treated with parthenolide (PN) (^a P<0.05), and was significantly increased after TNF-α induction (^b P<0.05). Compared with the TNF-α treatment alone, the promoter activity was decreased after treatment with PN+TNF-α in constructs carrying the −241 (−) allele (^c P<0.05). However, there was not changed of the promoter activity in the constructs with the −241 G allele among the Ctrl and the different treatment groups (P>0.05). B. Immunoblot characterization of the nuclear NF-κB p65 translocation after treatment with NF-κB activator or inhibitor in L02 cells. The expression of p65 was inhibited 28% after treatment with PN, while the expression of p65 was significantly increased by 21% after treatment with TNF-α. When pretreated with PN for 4 h and stimulated with TNF- for another 24 h (PN+TNF-α), the expression of p65 was still decreased by 35% in L02 cells (P<0.05). The numbers under the graph represent the fold of relative expression level of nuclear NF-κB p65 (Ctrl group level set as 1.0). The results represent the mean ± SD of three independent experiments.

Figure 2. Effect of plasmid DNA methylation on PPP2R1A promoter activity.

A. The PPP2R1A genomic reference sequence (with GenBank accession no. AC 010320) was used with the transcription start site (TSS) set as nucleotide +1. The variant identified in the current study, −241 (−/G) (rs11453459), is located in the PPP2R1A promoter region (−1801 nt to +237 nt). The two fragments, designated as 2R1Ap-Fs, including F1 ((−)⁻²⁴¹) and F2 ((G) ⁻²⁴¹), are located in the PPP2R1A proximal promoter (−448 nt to +237 nt, 685 bp). B. The CpG islands of the 5′-flanking region of PPP2R1A. The region from −400 nt to +105 nt was identified as a CpG island using the CpG finder at EMBL (http://www.ebi.ac.uk/) and the following parameters: CpG length >200 bp, G+C >50% and a “CpG value” of at least 0.6. C. The PPP2R1A promoter activity with various methylation statuses was measured by Dual Luciferase Assays in L02 cells. The promoter activity in the luciferase reporter assays decreased significantly following plasmid DNA methylation. Compared with the basal activity of the pGL3-Basic control (Ctrl, which the relative luciferase activity was given a value of 1 RLU), the promoter activity of all constructs was increased by statistically significant levels (∗ P<0.01). Compared with the luciferase activity of unmethylated pGL3b-2R1Ap-241(−) construct or −241G construct, the promoter activity of the methylated −241(−) construct or −241G construct was decreased by statistically significant levels (^a P<0.01 and ^b P<0.01, respectively). Compared with the luciferase activity of the methylated F1 construct with −241 (−), the promoter activity of the methylated F2 construct with −241 G was decreased by statistically significant levels (^# P<0.01). D. The PPP2R1A promoter activity with methylation was measured in HepG2 cells. The promoter activity in the luciferase reporter assays decreased significantly following plasmid DNA methylation. Compared with the basal activity of Ctrl (given as 1 RLU), the promoter activity of all constructs was in significant higher levels (∗ P<0.01). Compared with the activity of unmethylated pGL3b-2R1Ap-241(−) construct or −241G construct, the promoter activity of the methylated −241(−) construct (^a P<0.01) or −241G construct (^b P<0.01) was decreased significantly respectively. Compared with the luciferase activity of the unmethylated or methylated pGL3b-2R1Ap-241(−) construct, the promoter activity of the −241G construct was decreased by statistically significant levels (^# P<0.01). E. Methylation status was confirmed by comparing the plasmid’s digestion profile with HpaII (methylation sensitive) and MspI (methylation insensitive) restriction enzyme. A series of PPP2R1A luciferase reporter constructs and its empty vectors were treated with or without M. SssI and its substrate SAM. The results represent the mean ± SD of three independent experiments.

Figure 3. Treatment with 5-Aza-dC upregulates the transcription of PPP2R1A in L02 cells.

RNA was extracted from immortalized human normal hepatocyte L02 cells treated with or without 5-Aza-2′-deoxycytidine (5-Aza-dC), a DNA methylation inhibitor, for 48 hours at the indicated concentrations (2.5, 5.0 and 10.0 µM), and the PPP2R1A transcripts in the control group and the 5-Aza-dC treated group were quantified using real-time RT-PCR (normalized against ACTB). The results were normalized against the untreated control (Ctrl), which was given a value of as 1.0. Compared with the Ctrl, the levels of PPP2R1A mRNA in the 5-Aza-dC treated groups were increased by statistically significant levels (∗ P<0.05). The level of PPP2R1A mRNA in the 10.0 µM 5-Aza-dC treatment group was significantly higher than the mRNA level in the 2.5 µM 5-Aza-dC treatment group (^# P<0.05). The results represent the mean ± SD of at least three independent experiments performed in duplicate.