Abstract
The full-length Nrf1α is processed into distinct isoforms, which together regulate genes essential for maintaining cellular homeostasis and organ integrity, and liver-specific loss of Nrf1 in mice results in spontaneous hepatoma. Herein, we report that the human constitutive Nrf1α, rather than smaller Nrf1β/γ, expression is attenuated or abolished in the case of low-differentiated high-metastatic hepatocellular carcinomas. Therefore, Nrf1α is of importance in the physio-pathological origin and development, but its specific pathobiological function(s) remains elusive. To address this, TALENs-directed knockout of Nrf1α, but not Nrf1β/γ, is created in the human hepatocellular carcinoma (HepG2) cells. The resulting Nrf1α−/− cells are elongated, with slender spindle-shapes and enlarged gaps between cells observed under scanning electron microscope. When compared with wild-type controls, the invasive and migratory abilities of Nrf1α−/− cells are increased significantly, along with the cell-cycle G2-M arrest and S-phase reduction, as accompanied by suppressed apoptosis. Despite a modest increase in the soft-agar colony formation of Nrf1α−/− cells, its loss-of-function markedly promotes malgrowth of the subcutaneous carcinoma xenograft in nude mice with hepatic metastasis. Together with molecular expression results, we thus suppose requirement of Nrf1α (and major derivates) for gene regulatory mechanisms repressing cancer cell process (e.g. EMT) and malignant behaviour (e.g. migration).
Highlights
To maintain cellular homeostasis and physiological integrity of life systems, all organisms living in oxygenated environments have evolutionally developed efficient cytoprotective strategies against a vast variety of stresses, pathophysilogical stimuli and other biological cues[1,2,3,4]
The cells were selected by treatment with puromycin (2 μg/ml) for 48 h before being subjected to the cloning of single cells grown in 96-well plates, in order to determine the activity of transcription activator-like effector nucleases (TALENs)-mediated gene editing. (f) The genomic DNA from the individual cell clones served as a template of PCR to amplify the TALENs-target region of Nrf[1], followed by sequencing of PCR products
The high-efficiency of TALENs is dictated by targetable nucleases composed of a customizable sequence-specific DNA-binding domain (DBD) fused C-terminally to an effector nuclease domain of Fok[1] (Fig. 2a)
Summary
To maintain cellular homeostasis and physiological integrity of life systems, all organisms living in oxygenated environments have evolutionally developed efficient cytoprotective strategies against a vast variety of stresses (e.g. oxidants, xenobiotics, nutrients), pathophysilogical stimuli (e.g. inflammation and aging) and other biological cues (e.g. metabolites, inducers and inhibitors)[1,2,3,4]. Albeit these N-terminally truncated variants are neither targeted to the ER nor recovered in membrane fractions[58,59], Nrf1β /LCR-F1 only functions a weak activator because it lacks its acidic domain 1 (AD1, which is a major TAD element), whilst Nrf1γ and Nrf1δ act as dominant-negative inhibitors competing against wild-type Nrf[1] (and/or Nrf2)[43,44,46,49,50] These short isoforms are generated primarily by the prototypic Nrf1α processing at both post-transcriptional and post-translational levels, expect that an additional fraction of such short proteins (e.g. Nrf1β /LCR-F1 and Nrf1γ ) are produced though internal translation pathway on the base of the fact that the putative products are significantly diminished by mutation of relevant in-frame translation start codons[42,44,55]. The putative cDNA codons were placed in the black backgrounds
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