The Axl-Regulating Tumor Suppressor miR-34a Is Increased in ccRCC but Does Not Correlate with Axl mRNA or Axl Protein Levels

Background

High expression of the receptor tyrosine kinase Axl is associated with poor prognosis in patients with Renal Cell Carcinoma (RCC), the most common malignancy of the kidney. The miR-34a has been shown to directly regulate Axl in cancer cells. The miR-34a is a mediator of p53-dependent tumor suppression, and low expression of miR-34a has been associated with worse prognosis in several cancers. Our aim was to elucidate whether miR-34a or the other members of the miR-34 family (miR-34b/c) regulate Axl in RCC.

Methodology and Results

Using western blot, flow cytometry, and RT-qPCR, we showed that Axl mRNA and protein are downregulated in 786-O cells by miR-34a and miR-34c but not by miR-34b. A luciferase reporter assay demonstrated direct interaction between the Axl 3’ UTR and miR-34a and miR-34c. The levels of miR-34a/b/c were measured in tumor tissue in a cohort of 198 RCC patients, and the levels of miR-34a were found to be upregulated in clear cell RCC (ccRCC) tumors, but not associated with patient outcome. Neither of the miR-34 family members correlated with Axl mRNA, soluble Axl protein in serum, nor with immunohistochemistry of Axl in tumor tissue. In addition, we measured mRNA levels of a known miR-34a target, HNF4A, and found the HNF4A levels to be decreased in ccRCC tumors, but unexpectedly correlated positively rather than negatively with miR-34a.

Conclusions

Although miR-34a and miR-34c can regulate Axl expression in vitro, our data indicates that the miR-34 family members are not the primary regulators of Axl expression in RCC.     

An HNF4α-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis

Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.     

Germ-Line Mutations in the von Hippel–Lindau Tumor-Suppressor Gene Are Similar to Somatic von Hippel–Lindau Aberrations in Sporadic Renal Cell Carcinoma

von Hippel–Lindau (VHL) disease is a hereditary tumor syndrome predisposing to multifocal bilateral renal cell carcinomas (RCCs), pheochromocytomas, and pancreatic tumors, as well as angiomas and hemangioblastomas of the CNS. A candidate gene for VHL was recently identified, which led to the isolation of a partial cDNA clone with extended open reading frame, without significant homology to known genes or obvious functional motifs, except for an acidic pentamer repeat domain. To further characterize the functional domains of the VHL gene and assess its involvement in hereditary and nonhereditary tumors, we performed mutation analyses and studied its expression in normal and tumor tissue. We identified germ-line mutations in 39% of VHL disease families. Moreover, 33% of sporadic RCCs and all (6/6) sporadic RCC cell lines analyzed showed mutations within the VHL gene. Both germ-line and somatic mutations included deletions, insertions, splice-site mutations, and missense and nonsense mutations, all of which clustered at the 3' end of the corresponding partial VHL cDNA open reading frame, including an alternatively spliced exon 123 nt in length, suggesting functionally important domains encoded by the VHL gene in this region. Over 180 sporadic tumors of other types have shown no detectable base changes within the presumed coding sequence of the VHL gene to date. We conclude that the gene causing VHL has an important and specific role in the etiology of sporadic RCCs, acts as a recessive tumor-suppressor gene, and appears to encode important functional domains within the 3' end of the known open reading frame.     

A premature termination codon interferes with the nuclear function of an exon splicing enhancer in an open reading frame-dependent manner

Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5' element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5' and 3' elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3' enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3' splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.     

Molecular cloning and characterization of a complete Chinese hamster provirus related to intracisternal A particle genomes.

We report here the nucleotide sequence of a full-length Chinese hamster genomic proviral element, CHIAP34. CHIAP34 is 6,403 bp long with long terminal repeats of 311 bp at each end. The genetic organization of CHIAP34 was determined by comparison with intracisternal A particle (IAP) genetic elements from the mouse and Syrian hamster. Extensive homology at the nucleotide and deduced amino acid sequence levels was observed between CHIAP34 and the mouse and Syrian hamster IAP elements. CHIAP34 may represent a defective Chinese hamster IAP genetic element. The gag gene consists of 837 codons, of which 558 codons are in a single long open reading frame followed by several frameshifts. The pol gene begins with a -1 frameshift and consists of a long open reading frame of 753 codons followed by a short open reading frame of 103 codons. The putative env region contains multiple termination codons in all reading frames. CHIAP34 is representative of the predominant retroviral elements in the Chinese hamster ovary cell genome present at around 80 copies per haploid genome.     

Sequence requirements of the bidirectional yeast TRP4 mRNA 3'-end formation signal.

The yeast TRP4 3'-end formation signal functions in both orientations in an in vivo test system. We show here that the TRP4 3'-end formation element consists of two functionally different sequence regions. One region of approximately 70 nucleotides is located in the untranslated region between the translational stop codon and the major poly(A) site. The major poly(A) site is not part of this region and can be deleted without a decrease in TRP4 3'-end formation. 5'and 3'deletions and point mutations within this region affected 3'-end formation similarly in both orientations. In the center of this region the motif TAGT is located on the antisense strand. Point mutations within this motif resulted in a drastic reduce of 3'-end formation activity in both orientations. A second region consists of the 3'-end of the TRP4 open reading frame and is required for 3'-end formation in forward orientation. A single point mutation in a TAGT motif of the TRP4 open reading frame abolished TRP4 mRNA 3'-end formation in forward orientation and had no effect on the reverse orientation.     

Mechanism of translational regulation by miR-2 from sites in the 5' untranslated region or the open reading frame

MicroRNAs (miRs) commonly regulate translation from target mRNA 3' untranslated regions (UTRs). While effective miR-binding sites have also been identified in 5' untranslated regions (UTRs) or open reading frames (ORFs), the mechanism(s) of miR-mediated regulation from these sites has not been defined. Here, we systematically investigate how the position of miR-binding sites influences translational regulation and characterize their mechanistic basis. We show that specific translational regulation is elicited in vitro and in vivo not only from the 3'UTR, but equally effectively from six Drosophila miR-2-binding sites in the 5'UTR or the ORF. In all cases, miR-2 triggers mRNA deadenylation and inhibits translation initiation in a cap-dependent fashion. In contrast, single or dual miR-2-binding sites in the 5'UTR or the ORF yield rather inefficient or no regulation. This work represents the first demonstration that 5'UTR and ORF miR-binding sites can function mechanistically similarly to the intensively investigated 3'UTR sites. Using single or dual binding sites, it also reveals a biological rationale for the high prevalence of miR regulatory sites in the 3'UTR.