Molecular And Cellular Mechanism(s) By Which Nrf1? Represses Malignant Behavior Of Human Hepatocellular Carcinomas

Hepatocellular carcinoma (HCC) is a serious threat to human healthy survival, inasmuch as it is one of the most frequently occurring tumour in the world, and a high incidence exists in our country of China. More interestingly, spontaneous development of heptoma resembling human HCC was determined to result from severe oxidative stress endogenously occurring in conditional knockout mice with a liver-specific deletion of CNC-bZIP (cap'n'collar basic-region leucine zipper) family member Nrfl (nuclear factor-erythroid 2-related factorl, with its gene being annotated Nfe2ll). In turn, this is supportively consistent with the notion that Nrfl confers cytoprotectionon the host against oxidative stress so that the adaptive antioxidant and detoxification responses insomuch as to intrinsically acclimatize extracellular changes. However, to date, the putative anti-cancer role of Nrfl, particularly its full-length isoform Nrfl a, in the host cytoprotection against hepatocellular carcinogenesis and progression has not been clearly elucidated. In order to determine molecular mechanism(s) underlying the pathogenesis of HCC, we have chosen HepG2 as an objective to investigate cytoprotective effects of Nrfl a on malignant behavior of hepatocellular carcinoma cells. For this, the proposed aim has been focused in this study.RESULTS:? TALEN (transcription activator-like effector nuclease) directed homozygous bi-allelic knockout Nrfla from the HepG2-based monoclonal cellsThe constitutive abundance of Nrfl was expressed at much lower levels in three different HCC cell lines than that obtained from the non-cancerous hepatocellular line HL7702. By using TaqMan assays, the results demonstrate that both Nrfla and TCF11 isoforms appear to be expressed at similar levels in HL7702 cells. By contrast, HCC (particularly HepG2) cells are allowed for a quite lower expression level of TCF11, besides Nrfla Hence, HepG2 cells have been chosen to investigate whether Nrfla has an effect on HCC malignant behavior.Subsequently, TALEN-directed mutagenesis system was employed to site-specific knockout Nrfla from HepG2 cell, and then homozygous bi-allelic Nrfla-/- knockout monoclonal cell lines was established, one of which was called HEA157 (Nrfla-/-). In addition, Nrfl-targeting shRNA were transfected by lentivirus into HepG2 cells so as to establish a stably shNrfl expressing knockdown cell line. Conversely, over-expression of Nrfla occurred in its restored HEA157 (Nrfla-/-) cells; the stably expressing Nrfla cells were designated HEA157Nrf1?.? The behavior of hepatic cells is restricted by knockout of NrflaThe results of both MTS and cloning formation assays revealed that proliferation of HepG2 cells is promoted by knockout of Nrfla. Further data from flow cytometry showed that loss of Nrfladecreased cell apoptosis at the later stage. By contrast, no obvious differences in the early apoptosis and relevant cell-cycle GO/Gl phases between HEA157 (Nrfla-/-) and wild-type HepG2 cell lines were detected. Moreover, both wound healing and transwell experiments revealed that knockout of Nrfla enhances the migration and invasion of hepatoma cell in vitro.Collectively, Nrfla plays a negativeeffect on the biological behavior of hepatocellular carcinoma, but its knockout enhanced invasion and migration abilities of the deficient HEA157 (Nrfla-/-) cells. Loss of Nrfla promotes changes in HepG2 cell shape as EMT (epithelial-mesenchymal transition), which is attributable to hepatic metastasis in the tumour-bearing mice injected with HEA157 (Nrfla-/-) cells.? Knockout of Nrfla promotes the EMT and activates PTEN-PI3K/AKT signaling in the deficient cancer cellsLoss of Nrfla caused remarkable increases in expression of MMP-9, MMP-17 and N-cadherin, with an exception of down-regulated E-cadherin. Both real-time PCR and western blotting revealed that knockout of Nrfl a promotes the EMT, which triggers up-regulation of N-cadherin, SNAI2, Vimentin and MMP-9.The xenograft model showed the host nude mice injected with Nrfla"'" HEA157 were enabled to bear so big tumours under skin. The resultant curve of xenograft in size displays that Nrfla-deficient carcinoma were growing graduallywithin the first two weeks, but thereafter they were expanding exponentially in size until day 35. Subsequently, the growing tumours ruptured insomuch as to becomebleeding ulcers. In addition, it should also be noted that the Nrfla-deficient carcinoma xenograft mice, rather than the control mice, suffered from a severe syndrome that resembles human cancer cachexia, which was much likely to be attributed to hepatic metastasis. This pathology was also accompanied by potential cancer-promoting inflammation in the liversof tumour-bearing mice injected withHEA157 (Nrfla-/-) knockout cells.Furthermore, pathohistological sections combined with immunostaining revealed low expression of Nrfl and E-cadherin in mouse xenogafts, instead of high expression of N-cadherin. Similar results were obtained from FLEA157 (Nrfla-/-) tumour-bearing mouse tissues, real-time PCR and western blotting demonstrating decreased expression of E-cadherin, as accompanied by increased expression of SNAI2.Collectively, the extent of disturbed expression of Nrfl mRNA and Nrfla protein is relevant to the pathological severity of HCC with distinct differentiation and metastatic potentials, together with additional concomitant lesions. The real-time qPCR analysis revealed a markedly low abundance of total Nrfl mRNA in poorly low-differentiated hepatocellular carcinoma and its para-carcinoma tissue, and much lower level of mRNA expressed in the carcinoma compared to para-carcinoma.High-throughput transcriptome sequencing, followed by pathway-clustering of HEA157 (Nrfla-/-), revealed that knockout of Nrfla activates PTEN-PI3K/AKT signaling in deficient cancer cells. Relevant real-time qPCR and western blotting demonstrated that down-regulation of Nrfla promotes Akt activation (p-AKT2T308 and/or p-AKT2S473) and PI3K/Akt downstream target genes expression. Dual luciferase experiments implied that Nrfla have a direct effect on CDH1 and PTEN.Conclusions:? We have established the homozygous bi-allelic Nrfla-/- knockout from the HepG2-based monoclonal cells HEA157 (Nrfla-/-), by using TALEN-directed mutagenesis.? Nrfla plays a negativeeffect on the biological behavior ofhepatocellular carcinoma, but its knockout enhanced invasion and migration abilities of the deficient HEA157 (Nrfla-/-) cells. Loss of Nrfla promotes changes in HepG2 cell shape as EMT, which is attributable to hepatic metastasis in the tumour-bearing mice injected with HEA157 (Nrfla-/-) cells.? The extent of disturbed expression of Nrfl is relevant to the pathological severity of HCC. For instance, low abundance of total Nrfl was found in poorly low-differentiated hepatocellular carcinoma and its para-carcinoma tissue, together with much lower expression level of mRNA in the carcinoma as compared with that detected in the para-carcinoma tissues.? Knockout of Nrfla activates the PTEN-PI3K/AKT signaling in HCC cells.