A novel lncRNA RP11-386G11.10 reprograms lipid metabolism to promote hepatocellular carcinoma progression

Figure 1. RP11-386G11.10 expression is significantly increased in HCC. A HCC and non-tumor tissue RP11-386G11.10 transcription levels based on the analysis of TCGA-LIHC data set. B Overall survival curves of HCC patients based on RP11-386G11.10 expression. C The expression of RP11-386G11.10 in 80 pairs of HCC tissues and its paired non-tumor tissues. D 80 cases of HCC patients based on the overall survival curve of RP11-386G11.10 expression. E The hazard ratio analysis of clinicopathological factors in 80 HCC patients. F RP11-386G11.10 expression in hepatocytes and HCC cell lines. G, H The main position of RP11-386G11.10 analyzed by the nuclear-cytoplasmic RNA fractionation and FISH experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 2. RP11-386G11.10 promotes HCC proliferation and metastasis. A Expression of RP11-386G11.10 in Huh-7 and HCC-LM3 cells transfected with shNC and RP11-386G11.10 targeting shRNA (shHRLL1). B, C CCK-8 and colony formation assay showed the proliferation ability of Huh-7 and HCC-LM3 cells. D Scratch wound-healing assay showed the migration ability of cells. E Transwell Matrigel invasion assay showed the cell invasion ability. F A xenograft model established from control or stable RP11-386G11.10 knockdown HCC-LM3 cells, and evaluate the weight of the anatomical tumor. G The expression of Ki-67 was determined by IHC analysis. H, I The metastasis in the lungs and livers of nude mice injected with shNC or RP11-386G11.10-sh2 HCC cells. Magnification, 100 × . ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 3. RP11-386G11.10 promotes lipid synthesis in HCC. A Volcano plot showed the sum of expression levels of genes affected by RP11-386G11.10 knockdown. B Heatmap showed differential expression of genes across all samples. C Bubble plot showing enriched pathways for mRNA expression affected by RP11-386G11.10 knockdown. D Positive enrichment for the gene signature associated fatty acid metabolism between the low and high RP11-386G11.10 expression groups of TCGA dataset. E Measurement of intracellular triglyceride and cholesterol content in Huh7 and HCC-LM3 cells transfected with shHRLL1. F The intracellular fatty acids of Huh-7 cells transfected with shHRLL1 analyzed by GC–MS. G Intracellular lipids in Huh7 and HCC-LM3 cells transfected with shHRLL1 measured by double staining with Nile Red and Hoechst. Magnification, 400 × . H Pearson correlation analysis of RP11-386G11.10 and FASN, ACLY, ASCD, ACC in 80 HCC tissue samples. I, J In the HCC cells with RP11-386G11.10 knockdown, the mRNA and protein levels of related lipogenic enzymes were significantly reduced. K In the HCC samples with RP11-386G11.10 knockdown, the expression of lipid synthase-related proteins was determined by IF…

Figure 4. RP11-386G11.10 directly binds to miR-345–3p and negatively regulates miR-345–3p expression. A Bioinformatics analysis of potential target genes is performed by prediction software (mirdb, lncbase). B Target miRNA expression in HCC tumors and non-tumor tissues in the TCGA database. C qRT-PCR showed target miRNAs expression in RP11-386G11.10 overexpressing HCC cells. D Pearson correlation analysis of RP11-386G11.10 and miR-345–3p in 80 HCC tissue samples. E Schematic diagram of the potential binding site between RP11-386G11.10 and miR-345–3p. F, G Dual luciferase reporter gene detection and RIP experiment confirmed the interaction between RP11-386G11.10 and miR-345–3p. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5. MiR-345–3p inhibits proliferation and metastasis and lipid synthesis in HCC. A, B RP11-386G11.10 inhibited HCC cell proliferation. C, D RP11-386G11.10 inhibited the migration and invasion ability of HCC cells. E A xenograft model established from control or miR-345–3p overexpressed HCC-LM3 cells, and assess the weight of the anatomical tumor. F, G HE staining showed miR-345–3p overexpression reduced the lung and liver metastasis in the nude mice. Magnification, 100 × . H The intracellular lipids were measured in Huh7 and HCC-LM3 cells overexpressing miR-345–3p by double staining with Nile Red and Hoechst. Magnification, 400 × . I, J Lipogenic enzymes mRNA and protein levels in miR-345–3p overexpressing or knocking down HCC cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6. MiR-345–3p directly binds to HNRNPU and negatively regulates HNRNPU expression. A Prediction software (mirdb, encori and targetscan) screened possible downstream targets of miR-345–3p. B HNRNPU and VPS4B mRNA expression in miR-345–3p overexpressing or knocking down Huh-7 and HCC-LM3 cells. C The expression of HNRNPU protein levels miR-345–3p overexpressing or knocking down Huh-7 and HCC-LM3 cells. D Pearson correlation analysis of miR-345–3p and HNRNPU in 80 HCC tissue samples. E Schematic diagram of potential binding sites between miR-345–3p and HNRNPU. F, G Dual luciferase reporter gene and RIP test confirmed the interaction between miR-345–3p and HNRNPU. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 7. RP11-386G11.10 promotes cell growth and metastasis and lipid synthesis in HCC through the miR-345–3p/HNRNPU axis. A, B The proliferation ability of HCC cells was assessed by CCK-8 and colony formation assays. C The wound healing assay showed the migration ability of HCC cells. D The Transwell invasion assay with Matrigel showed the cell invasion ability. E Tumour growth curves from the xenograft model established with cotransfected HCC-LM3 cells and evaluation of the weights of the excised tumours. F The intracellular lipid content was evaluated in the tumour tissues described in F by double staining with Nile Red and Hoechst. Magnification, 200×. G The triglyceride and cholesterol contents were measured in the tumour tissues described in F. H, I Lipogenic enzyme mRNA and protein levels were measured in the tumour tissues described in F. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 8. RP11-386G11.10 is directly regulated by the transcription factor ZBTB7A. A Transcription factors that may regulate the transcription of RP11-386G11.10 were identified by prediction databases (tfTarget, GeneCards and JASPAR). B The expression levels of ZBTB7A and RP11-386G11.10 in HCC cells with ZBTB7A knockdown were verified by qRT-PCR. C The binding sequence of ZBTB7A and RP11-386G11.10 predicted from the JASPAR database. D The results of the dual luciferase reporter assay confirmed the interaction between ZBTB7A and RP11-386G11.10. E The CHIP assay results confirmed the direct binding between ZBTB7A and the RP11-386G11.10 promoter region. F The expression levels of ZBTB7A and RP11-386G11.10 in HCC cells with HNRNPU knockdown were verified by qRT-PCR. G The protein expression levels of ZBTB7A in HCC cells with HNRNPU knockdown. H Changes in ZBTB7A mRNA stability after actinomycin D treatment of Huh-7 and HCC-LM3 cells. I The effects of different transfectants on the interaction between ZBTB7A and the RP11-386G11.10 promoter were verified by a luciferase reporter assay in HCC cells. J. A model of the regulatory mechanisms of RP11-386G11.10 in HCC. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <…