PRMT5 imposed a dual repression on DDIT3 transcription to promote the malignancy of hepatocellular carcinoma

Figure 1 PRMT5 predicted poor prognosis of hepatocellular carcinoma patients and inhibition of PRMT5 significantly reduced the viability of hepatocellular carcinoma cells. A and B: Using online database (GEPIA2) to analyze the overall survival and disease-free survival of hepatocellular carcinoma (HCC) patients with PRMT5 low and high expression; C and D: The protein levels of PRMT5 in stable cell lines were assessed by western blot, and the effect of PRMT5 on clone formation of HCC cells was examined by crystal violet assay; E and F: The growth of PRMT5 overexpression and knockdown HCC cells was detected by methylthiazolyldiphenyl-tetrazolium bromide assay; G: The cell viability of HCC cells treated with HLCL-61 at different concentration for 24 hours; H: The inhibition of HLCL-61 on growth of HCC cells with different malignancy at different concentration for 72 hours. Data are presented as the mean ± SD. ^bP < 0.01. ^cP < 0.001. VC: Vector; Scr: Scramble; PVTT: Portal vein tumor thrombus; OD: Optical density; shPRMT5: Short hairpin RNA of PRMT5.

Figure 2 Inhibition of PRMT5 induced cell apoptosis and increased DDIT3 expression in hepatocellular carcinoma cells. A-D: Cell apoptosis was examined in knockdown and PRMT5-inhibited hepatocellular carcinoma (HCC) cells by flow cytometry analysis; E: Heatmaps showed that knockdown PRMT5 upregulated the messenger RNA (mRNA) level of DDIT3 from RNA-sequencing cohort (GSE168745); F and G: The expression of DDIT3 mRNA was evaluated in PRMT5 overexpression and knockdown HCC cells; H and I: DDIT3 mRNA expression was assessed in HCC cells treated with HLCL-61 at different times or different concentrations of HLCL-61; J: Western blot was performed to examined the expression of DDIT3 and PRMT5 protein in stable cell lines; K: DDIT3 protein level was detected in HCC cells treated with HLCL-61 (8 μM) at 0, 12, 24, 36, 48 hours; L: The PRMT5 and DDIT3 expression were examined in paired HCC tissues and their adjacent normal tissues; M: Based on the results of western blot, the correlation between PRMT5 and DDIT3 expression was analyzed by statistical analysis. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; Scr: Scramble; FITC: Fluorescein isothiocyanate; PI: Propidium iodide; mRNA: Messenger RNA; N: Normal; T: Tumor; shPRMT5: Short hairpin RNA of PRMT5.

Figure 3 PRMT5 suppressed DDIT3 transcription by increasing H4R3me2 modification at the DDIT3 promoter. A and B: The effect of PRMT5 overexpression and knockdown on DDIT3 promoter activity in hepatocellular carcinoma (HCC) cells; C: Location diagram of designed chromatin immunoprecipitation (ChIP) primers on DDIT3 promoter; D: ChIP examined the enrichment of PRMT5 on DDIT3 promoter; E and F: The level of H4R3me2 of DDIT3 promoter was evaluated in PRMT5 overexpression and knockdown HCC cells by ChIP assay. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; Scr: Scramble; IgG: Immunoglobulin G; shPRMT5: Short hairpin RNA of PRMT5.

Figure 4 PRMT5 interacted with STAT3 and recruited it to co-suppress DDIT3 transcription. A: Using JASPAR online database to analyze the binding of STAT3 on DDIT3 promoter; B: The activity of DDIT3 promoter was assessed in HERK293T cells respectively transfected with vector, PRMT5 or PRMT5 and STAT3; C and D: Endogenous PRMT5 interacted with exogenous and endogenous STAT3; E: Localization of PRMT5 and STAT3 protein in HepG2 cells was examined by immunofluorescence assay, scale bar = 25 μm; F: Endogenous PRMT5 interacted with exogenous phospho-STAT3; G: Truncations of STAT3 were constructed as shown in graphic; H: The interaction between PRMT5 and DNA-binding domain was examined by immune-coprecipitation in HEK293T cells; I: The binding of STAT3 on DDIT3 promoter was testified in hepatocellular carcinoma (HCC) cells by chromatin immunoprecipitation (ChIP) assay; J: ChIP-Re-ChIP assay was used to verify that the PRMT5-STAT3 interaction occurred at DDIT3 promoter; K and L: The binding of STAT3 on DDIT3 promoter in PRMT5-overexpressing and knockdown HCC cells was examined by ChIP assay. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. IP: Immunoprecipitation; FL: Full length; shPRMT5: Short hairpin RNA of PRMT5; ChIP: Chromatin immunoprecipitation; IgG: Immunoglobulin G; DAPI: 4’,6-diamidino-2-phenylindole; p-STAT3: Phospho-STAT3; NTD: Amino-terminal domain; CCD: Coiled-coil domain; DBD: DNA-binding domain; LD: Linker domain; SH2: SH2 domain; TAD: Transactivation domain; Scr: Scramble.

Figure 5 DDIT3 restored the phenotype mediated by PRMT5 or HLCL-61. A and B: The protein levels of PRMT5 and DDIT3 were examined by western blot; C and D: DDIT3 rescued the growth of hepatocellular carcinoma (HCC) cells mediated by PRMT5; E and F: DDIT3 restored the clone formation of HCC cells mediated by PRMT5; G and H: Down-regulation of DDIT3 reduced the cell apoptosis of HCC cells induced by knocking down PRMT5 expression. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; Scr: Scramble; OD: Optical density; FITC: Fluorescein isothiocyanate; PI: Propidium iodide; shPRMT5: Short hairpin RNA of PRMT5; shDDIT3: Short hairpin RNA of DDIT3.

Figure 6 HLCL-61 was a promising strategy of hepatocellular carcinoma therapy. A-F: The growth curve and weight of tumors generated by huh7 or HepG2 cells, which treated with vehicle and HLCL-61 (50mg/kg); G-I: HLCL-61 significantly reduced the volume and weight of tumors in patient-derived xenografts models; J and K: The Ki67 (scale bar = 50 μm) and DDIT3 (scale bar = 100 μm) staining of tumor tissues. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. PDX: Patient-derived xenografts.

Figure 7 The mechanism of PRMT5 promoting malignant growth of hepatocellular carcinoma via anti-apoptosis. HCC: Hepatocellular carcinoma.