Fig 1: LL22NC03-N14H11.1 inhibited H-RAS (G12V) ubiquitination by inhibiting LZTR1.a Western blot results of PDI1, CDK1, p-ERK1/2, and total ERK1/2 in HCC cells under LL22NC03-N14H11.1 silence. b Western blot and RT-qPCR detected the expression of H-RAS (G12V) in Huh7 and SK-HEP-1 cells under LL22NC03-N14H11.1 silence. c Cycloheximide (CHX) was added to inhibit protein generation. Protein level of H-RAS (G12V) in SK-HEP-1 and Huh7 cells under LL22NC03-N14H11.1 silence was detected by western blot and quantified at 0, 3, 6, and 9 h after CHX treatment. d CoIP followed by western blot analyzed the levels of H-RAS (G12V) and LZTR1 in the binding complex of LZTR1 and control IgG, as well as their levels in input. e CoIP assay determined the ubiquitination level of H-RAS (G12V) in SK-HEP-1 cells under LL22NC03-N14H11.1 silence with or without MG132 treatment, and the level of input H-RAS (G12V) and LZTR1 was also detected via western blot. f Western blots of H-RAS (G12V) and LZTR1 in HCC cells under LL22NC03-N14H11.1 silence. g RT-qPCR analysis of LZTR1 level in HCC cells under LL22NC03-N14H11.1 silence. h RT-qPCR analysis of LL22NC03-N14H11.1 level in HCC cells under LZTR1 silence. i RT-qPCR analysis of LZTR1 level in 62 pairs of HCC tissues and matched non-tumorous tissues. j Negative correlation between LZTR1 and LL22NC03-N14H11.1 in HCC tissues was confirmed by Pearson’s correlation curve. k RT-qPCR analysis of LZTR1 in HCC cell lines vs. normal cell line. **P < 0.01; n.s. no significance.
Fig 2: LL22NC03-N14H11.1 transcriptionally inhibited ZLTR1 through recruiting c-Myb.a Luciferase activity of LZTR1 promoter reporter in HCC cells under LL22NC03-N14H11.1 silence. b Ten transcription factors potentially targeting LZTR1 promoter was predicted by human TFDB (P < 0.001) and PROMO (P < 0.05). c The enrichment of ten predicted TFs in the pulldown of LL22NC03-N14H11.1 biotin vs. LL22NC03-N14H11.1 no biotin in HCC cells was quantified. d RT-qPCR assessed the expression of LZTR1 at mRNA and protein levels in HCC cells under the silence of ELF1, AR, and c-Myb, e RT-qPCR analysis of LL22NC03-N14H11.1 enrichment in precipitates of c-Myb after RIP assay. Interaction between U1 and SNRNP70 served as the positive control of this assay. f FISH staining of LL22NC03-N14H11.1 and IF analysis of c-Myb showed their co-localization in the nucleus of SK-HEP-1 cells. Scale bar: 25 μm. g Three predicted c-Myb sites on LZTR1 promoter. h Luciferase activity of wild-type (WT) LZTR1 promoter reporter or with site 1/2/3 mutated, respectively, in 293T cells under c-Myb overexpression. i RT-qPCR analysis of LZTR1 promoter with predicted c-Myb site 1, 2, or 3 in the RIP of c-Myb in HCC cells. j Enrichment of LZTR1 promoter in the ChIP complexes of c-Myb under LL22NC03-N14H11.1 silence vs. control. k HCC cells were transfected with sh-NC, sh-LL22NC03-N14H11.1#1, sh-LL22NC03-N14H11.1#1 + pcDNA3.1, or sh-LL22NC03-N14H11.1#1 + pcDNA3.1/c-Myb, respectively. RT-qPCR data of LZTR1 and c-Myb levels in HCC cells of each group. l Western blot results of LZTR1, c-Myb. H-RAS (G12V), p-ERK1/2, total ERK1/2, p-DRP1 (S616) and total DRP1 in HCC cells of each group. **P < 0.01; n.s. no significance.
Fig 3: Graphical presentation of LL22NC03-N14H11.1/c-Myb/LTZR1/H-RAS/MAPK/DRP1 axis in HCC.LL22NC03-N14H11.1 interacted with c-Myb to transcriptionally repress LZTR1, so as to decrease H-RAS (G12V) ubiquitination, activate MAPK pathway, and induce p-DRP1 (S616), resulting in induced mitochondrial fission and accelerated HCC progression.
Fig 4: LL22NC03-N14H11.1 regulated HCC progression and mitochondrial fission through LZTR1/H-RAS (G12V).a–e HCC cells were transfected with sh-NC (a), sh-LL22NC03-N14H11.1#1 (b), sh-LL22NC03-N14H11.1#1 + sh-LZTR1 (c), or sh-LL22NC03-N14H11.1#1 + H-RAS (G12V) (d), respectively. Proliferation, apoptosis, invasion, and migration of HCC cells were evaluated by CCK-8, colony formation, transwell invasion, and wound healing assays, respectively. f Western blot analysis of E-cadherin, N-cadherin, MMP2, and MMP7 in HCC cells of each group. g MitoTracker Red staining and quantification of mitochondrial fission in HCC cells of each group. Scale bar: 3 μm. **P < 0.01.
Fig 5: LL22NC03-N14H11.1 drove tumorigenesis and metastasis in HCC through LZTR1 in vivo.SK-HEP-1 cells were, respectively, transfected with sh-NC, sh-LL22NC03-N14H11.1#1, or sh-LL22NC03-N14H11.1#1 + sh-LZTR1 and injected subcutaneously or from tail vain to monitor tumorigenesis and metastasis of HCC. a Volumes of xenografts in mice of each group was evaluated every 4 days after subcutaneous injection and the growth curve was outlined. b Twenty-eight days after injection, tumors from each group were resected and weighed. c TUNEL staining was used to evaluate the apoptosis in tumors of each group. d RT-qPCR analysis of LL22NC03-N14H11.1 and LZTR1 in tumors of each group. e Western blot was implemented to detect the levels of LZTR1, c-Myb, H-RAS (G12V), p-ERK1/2, total ERK1/2, p-DRP1 (S616), and total DRP1 in tumors of each group. f, g IHC staining and quantification of Ki67, PCNA, N-cadherin, and E-cadherin in tumors of each group. h Metastatic nodules in tumors of each group was stained by HE and quantified. Scale bar: 100 μm. **P < 0.01; n.s. no significance.
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