Reciprocal regulation of microRNA-122 and c-Myc in hepatocellular cancer: role of E2F1 and transcription factor dimerization partner 2

Abstract

c-Myc is a well-known oncogene frequently up-regulated in different malignancies, whereas liver-specific microRNA (miR)-122, a bona fide tumor suppressor, is down-regulated in hepatocellular cancer (HCC). Here we explored the underlying mechanism of reciprocal regulation of these two genes. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and northern blot analysis demonstrated reduced expression of the primary, precursor, and mature miR-122 in c-MYC-induced HCCs compared to the benign livers, indicating transcriptional suppression of miR-122 upon MYC overexpression. Indeed, chromatin immunoprecipitation (ChIP) assay showed significantly reduced association of RNA polymerase II and histone H3K9Ac, markers of active chromatin, with the miR-122 promoter in tumors relative to the c-MYC-uninduced livers, indicating transcriptional repression of miR-122 in c-MYC-overexpressing tumors. The ChIP assay also demonstrated a significant increase in c-Myc association with the miR-122 promoter region that harbors a conserved noncanonical c-Myc binding site in tumors compared to the livers. Ectopic expression and knockdown studies showed that c-Myc indeed suppresses expression of primary and mature miR-122 in hepatic cells. Additionally, Hnf-3β, a liver enriched transcription factor that activates miR-122 gene, was suppressed in c-MYC-induced tumors. Notably, miR-122 also repressed c-Myc transcription by targeting transcriptional activator E2f1 and coactivator Tfdp2, as evident from ectopic expression and knockdown studies and luciferase reporter assays in mouse and human hepatic cells.

Conclusion: c-Myc represses miR-122 gene expression by associating with its promoter and by down-regulating Hnf-3β expression, whereas miR-122 indirectly inhibits c-Myc transcription by targeting Tfdp2 and E2f1. In essence, these results suggest a double-negative feedback loop between a tumor suppressor (miR-122) and an oncogene (c-Myc).

Figure 1. miR-122 is downregulated in c-Myc-induced liver tumors

A. Northern blot analysis of precursor and mature miR-122 levels in the normal liver (L) from FBVN and (Myc-off) mice, as well as benign liver (N) and tumor (T) from the same (Myc-on) mouse. Total RNA (25 µg) from each sample was separated by denaturing PAGE (15% acrylamide), transferred onto Nylon membrane, cross-linked and subjected to Northern blot analysis with ³²P-labeled anti-miR-122 deoxyoligonucleotide. Membrane was exposed to 4hr and 72hr at −80°C to detect mature and precursor miR-122, respectively. The blot was hybridized to anti-sense 5S rRNA probe to demonstrate equal loading of RNA. B. qRT-PCR and Western blot analysis of c-Myc expression in the liver (Myc-off), Myc-on benign liver (N) and tumor (T) tissues in the presence and absence of doxycycline, respectively. Whole tissue extracts (100 µg protein) were immunoblotted with an antibody specific for human c-Myc and reprobed with Gapdh antibody. C. qRT-PCR analysis of primary and mature miR-122 in Myc-off livers and Myc-on tumors using Taqman kit. Data was normalized to RNU6B. D. qRT-PCR analysis of mRNA levels of specified miR-122 targets in livers and tumors. C: control liver, T: tumor. E. Western blot analysis of selected miR-122 targets in liver and tumor whole tissue extracts (100 µg). L: FBVN liver, N: benign liver and T: Tumor. F. Luciferase reporter assay demonstrating Pkm2 is a target of miR-122. Upper panel: miR-122 cognate site in 3’-UTR of Pkm2 gene. Lower panel: relative luciferase activity in Hepa cells transfected with the psiCHECK2 harboring wild type (Pkm2-3’-UTR) or mutant (Pkm2-3’-UTR-mut) site along with miR-122 mimic (miR-122) or negative control RNA (NC).

Figure 2. c-Myc represses miR-122 expression in vitro

A. Relative c-Myc and mature miR-122 expression in Hepa cells transfected with MYC expression vector (pBH-c-Myc) or pBH (vector control). Hepa cells were transfected with specified plasmids and RNAs isolated 36–48 hr post-transfection were subjected to qRT-PCR analysis. The data was normalized to Gapdh or RNU6B. Relative level in vector-transfected cells was assigned a value of 1. B. Relative c-Myc, mature and primary miR-122 expression in Hepa cells transfected with c-Myc siRNA (sic-Myc) or negative control siRNA (siNC). Cellular RNAs isolated after 36–48 hr were analyzed by qRT-PCR. C. Conserved miR-122 promoter region upstream of +1 site harbors a candidate noncanonical c-Myc binding site predicted by rVista program. Sequence conservation of miR-122 gene between mouse and human as analyzed by rVista. Arrows denote the chromatin immunoprecipitation (ChIP) analysis primers spanning c-Myc site. D. ChIP analysis showed increased binding of c-Myc but reduced association of RNA polymerase II (Pol II) and histone H3K9Ac (a marker of active gene) with miR-122 promoter region in c-Myc induced tumors compared to Myc-off livers. Equal amounts of chromatin (DNA) from livers and tumors were subjected to ChIP assay with specific antibodies. Rabbit IgG and protein G beads alone were used as negative controls. 1:100 dilution of input was used for amplification of miR-122 promoter. Upper panel: A representative photograph of ethidium bromide stained agarose gel containing PCR products obtained with specified antibodies in ChIP assay. Lower panel: Quantitative analysis of ChIP data from 3 different livers and tumors. E. Western blot analysis of c-Myc, LETFs and C/ebpα expression in whole cell extracts (WCE) or nuclear extracts (NE) of Myc-off liver, as well as benign liver (N) and tumor (T) from Myc-on.

Figure 3. c-Myc is upregulated in miR-122KO mouse livers

A. Western blot analysis of c-Myc protein levels in the control and miR-122KO liver nuclear extracts using an antibody that detects both mouse and human Myc proteins. Histone H3 was used as a loading control. B. qRT-PCR analysis of endogenous c-Myc mRNA expression in Hepa and KO hepatocytes transfected with miR-122 mimic (miR122) or negative control mimic (NC) as well as in Hepa cells and wild-type (WT) hepatocytes transfected with miR-122 inhibitor (anti-miR122) or negative control inhibitor (anti-NC). C. Western blot analysis of c-Myc in KO and WT hepatocytes transfected with miR-122 mimic and inhibitor, respectively along with respective negative controls as described in 3B.

Figure 4. Tfdp2 and E2f1, targets of miR-122, are involved in the regulation of c-Myc expression by miR-122

A. qRT-PCR analysis of c-Myc mRNA and hnRNA (primary transcript) levels in control (floxed) and miR-122KO (KO) mouse livers demonstrated comparable increase in both in KO livers. B. Potential miR-122 seed match in Tfdp2 and E2f1 3’UTRs predicted by TargetScan and RNA22 programs, respectively. C. qRT-PCR analysis of Tfdp2 and E2f1 mRNA levels in control and KO mouse livers. D. Western blot analysis of Tfdp2, E2f1 and Gapdh in the whole liver extracts. E. qRT-PCR analysis of Tfdp2 and E2f1 in Hepa cells and KO hepatocytes transfected with miR-122 mimic (miR-122), negative control mimic (NC) as well as in Hepa cells and WT hepatocytes transfected with miR-122 inhibitor (anti-miR-122) or negative control inhibitor (anti-NC). F. Western blot analysis of Tfdp2 and E2f1 in KO and WT hepatocytes transfected with miR-122 mimic or inhibitors as described in 4E. G. Tfdp2 and E2f1 3’UTR driven luciferase assays were performed as described in 1F.

Figure 5. Tfdp2 and E2f1 are involved in the regulation of c-Myc expression in miR-122KO mouse livers

A. qRT-PCR analysis of Tfdp2 and E2f1 in respective siRNA or negative control siRNA (siNC) transfected KO hepatocytes. B. qRT-PCR analysis of c-Myc in KO hepatocytes transfected with siRNA specific for Tfdp2, E2f1 or negative control siRNA (siNC). C. Western blot analysis of c-Myc in Tfdp2 and E2f1 siRNA transfected KO hepatocytes. D. Western blot analysis of E2f1 and Tfdp2 in c-Myc-induced tumors (T), benign (N) and control livers. E, F. qRT-PCR analysis of miR-122 (E) and western blot analysis of c-MYC, E2F1 and TFDP2 (F) in Huh7 cells transfected with 50 nM of anti-miR-122 (122-AS) or anti-sense NC-RNA (NC-AS) and Hep3B and PLC/PRF5 cells transfected with 50nM miR-122 mimic (122-S) or sense NC-RNA (NC-S). G. qRT-PCR analysis of c-MYC, E2F1 and TFDP2 in Huh7 and Hep3B cells.

Figure 6. A model depicting reciprocal regulation between c-Myc and miR-122 based on the observations in miR-122KO and c-MYC transgenic mouse models