| Hepatocellular carcinoma(HCC) is the third major diseases that cause human death around the world. NFE2L1(nuclear factor-erythroid 2 p45 submit-related factor 1) was also annoted as Nrf1, which belongs to the Cnc-bZIP(cap ‘n’ collar basic-region leucine zipper) family of transcription factors. Nrf1 confers cytoprotection on the host cells against oxidative stress so that the adaptive response enables the cells to acclimatize extracellular stimulations.It is important to note the spontaneous development of HCC in the liver-specific Nrf1 knockout mice, but it is less convincing to be made for the anti-oxidation model., For this reason we aimed to determine the mechanisms underlying the phenotypes of Nrf1 deficiency mice.Furthermore, O-linked N-acetylglucosamine(O-GlcNAc) is known as one of the substrates of O-GlcNAc transferase(OGT), with other O-GlcNAc modifiable proteins. Of note, defective O-GlcNAc modification may correlate with tumorigenesis, invasion and migration. And a recent investigation by liquid chromatography-Spectrometry(LC-SM) indicates interaction between OGT and Nrf1. Hence, it is postulated whether glycoprotein of Nrf1 is GlcNAc modifiable, and its GlcNAcylation state contributes to aggression of HCC by certain signaling pathways.Results:①The behavior of hepatic cell lines is affected by knockdown of hNrf1/TCF11Expression of hNrf1/TCF11 was at much lower levels examined in 3 liver cancer cell lines than that obtained in normal liver cell line HL-7702. The most efficient sequence of shRNA targeting to hNrf1/TCF11 was screened to be confirmed as 5’-ACAACCTGAGGAATACCTT-3’. The shRNA mediated by lentivirus against hNrf1/TCF11 was utilized for transfection of Hep G2, MHCC97 H and MHCC97 L cell lines, in order to make cell lines stably-expressing shNrf1, or shNC(as a negative control).Both MTT and cloning formation assay illustrated that HepG2 cell proliferation is promoted by shNrf1. Further flowcytometry test showed that shNrf1-expressing HepG2 cell cycle was arrested at the G2/M phases, as accompanied by increased apoptosis at the early stage, but equivalents of either MHCC97 H or MHCC97 L were unaffected. Moreover, wound healing and transwell migration experiments revealed that silencing of Nrf1 hastens all 3 cancer cell lines moving.②Knockdown of hNrf1/TCF11 activates ?-Catenin signaling in cancer cell linesSilencing of hNrf1/TCF11 caused remarkable increases in expression of MMP-2, MMP-9 and Vimentin, with an exception of decreased expression of E-cadherin. Both transcriptome sequencing and following pathway clustering of shNrf1-HepG2 revealed that knockdown of hNrf1/TCF11 activates ?-Catenin signaling, which triggers increases in the expression of Cyclin D1, c-Myc and MMP-7. Silencing of Nrf1 enabled the half life of ?-Catenin to be elongated as shown in CHX-chase experiments. The cytoplasmic faction of ?-Catenin was diminished sharply; while the nuclear and total unphosphorylated protein of ?-Catenin was sprung out in shNrf1-Hep G2 cells. Addition experiments indicate that over-expression of Nrf1 promotes ubiquitination of ?-Catenin.Metastasis model showed shNrf1-HepG2 enables nude mice to bear more and bigger tumours in the lungs and livers. Then, pathological sections with immunestaining revealed more expression of ?-Catenin in mouse livers, with more expression of Cyclin D1 in mouse lungs. Similar results were obtained from shNrf1-HepG2 tumour-bearing mice, real-time PCR and western blotting demonstrating decreased expression of E-cadherin, as accompanied by increased expression of ?-Catenin and its targets Cyclin D1, c-Myc and MMP7 in the shNrf1-HepG2-derived tumours.③The half-life of Nrf1 is shortened by O-GlcNAc modification.The input of higher glucose-containing medium enables proteins to be modified at a higher O-GlcNAc status, leading lower expression of hNrf1/TCF11 in HEK293 T cell as detected by western blotting. The OGT enzymatic inhibitor Alloxan(100?M) caused a reduction in total O-GlcNAcylation but an increase in hNrf1/TCF11 expressed in cells that had been cultured in normal glucose(5mM, G5) medium. The contrary effects were exerted by OGA inhibitor PUGNAc(50?M) in high glucose(25mM, G25)-containing medium.Interaction of OGT with Nrf1 was manifested by GST pulldown and coimmunoprecipitation(co IP) experiments; and the interaction is susceptible to the concentration of GlcNH2.siRNA-targeting against OGT caused an enhancement in Nrf1 transactivation as detected in ARE-driven luciferase assay, and siOGT also increase Nrf1 and its downstream proteins Lipin1 and GCLM, whilst over-expression of OGT displayed the opposite effects.OGT increases ubiquitination of Nrf1, and G25 also enhances its ubiquitination but diminishes the protein stability, when compared with G5. Deletion of the TPR motif from OGT(called ΔTPR 1-6) did not promote ubiquitination of Nrf1. However, its stabilization by siOGT was demonstrated in CHX chase assay. By mapping of a series of Nrf1 truncated mutants, the putative O-GlcNAc modification is identified through its serine-rich region of PEST2 degron.Conclusions:①We have established that human liver cancer cell lines HepG2, MHCC97 H and MHCC97 L that were stably transfected with lentivirus-mediated hNrf1/TCF11 such that the latter is knocked down.②Enhanced invasion and migration abilities of hNrf1/TCF11 imply a negative effect on the biological behavior of human hepatocellular carcinoma.③?-Catenin is activated by stable knockdown of hNrf1 /TCF11.④OGT interacts with Nrf1 and enables the CNC-bZIP protein to be O-GlcNAc modified in the serine-rich portion of PEST2 degron, promoting its O-Glc NAcylation-dependent ubiquitination. |
