O-GlcNAcylated RALY Contributes to Hepatocellular Carcinoma Cells Proliferation by Regulating USP22 mRNA Nuclear Export

Abstract

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly tumors; however, its pathogenic mechanism remains largely elusive. In-depth researches are needed to reveal the expression regulatory mechanisms and functions of the RNA-binding protein RALY in HCC. Here, we identify RALY as a highly expressed oncogenic factor that affects HCC cells proliferation both in vitro and in vivo. O-GlcNAcylation of RALY at Ser176 enhances its stability by protecting RALY from TRIM27-mediated ubiquitination, thus maintaining hyper-expression of the RALY protein. Mechanistically, RALY interacts with USP22 messenger RNA, as revealed by RNA immunoprecipitation, to increase their cytoplasmic localization and protein expression, thereby promoting the proliferation of HCC cells. Furthermore, we develop a novel RALY protein degrader based on peptide proteolysis-targeting chimeras, named RALY-PROTAC, which we chemically synthesize by linking a RALY-targeting peptide with the E3 ubiquitin ligase recruitment ligand pomalidomide. In conclusion, our findings demonstrate a novel mechanism by which O-GlcNAcylation/RALY/USP22 mRNA axis aggravates HCC cells proliferation. RALY-PROTACs as degraders of the RALY protein exhibit potential as therapeutic drugs for RALY-overexpressing HCC.

Keywords: O-GlcNAcylation; RALY; hepatocellular carcinoma; mRNA nuclear export; peptide proteolysis-targeting chimera.

Figure 1

The expression and function of RALY in hepatocellular carcinoma (HCC). (A, B) Bioinformatics analysis predicted RALY mRNA (A) and protein (B) levels in HCC tissues and normal adjacent tissues by UALCAN. (C) Kaplan-Meier analysis was used to measure the survival rate of HCC patients by UALCAN. (D) Immunohistochemistry images of RALY expression in paracancerous tissues and HCC samples were obtained from the Human Protein Atlas database. (E) Western blotting analysis of RALY expression in paracancerous tissues and HCC samples. (F) Western blotting analysis of RALY expression using four HCC cell lines (Hep3B, Huh7, MHCC-97H and MHCC-97L) and normal liver epithelial THLE-2 cells. (G) The expression of RALY protein was detected after RALY knockdown in HCC cells. (H) CCK-8 assays showed the growth rate of HCC cells upon manipulation of RALY. (I) EdU incorporation assays were carried out in HCC cells (Green fluorescence, EdU-positive cells; blue fluorescence, total cells). (J) Statistical analysis of the EdU incorporation assays. (K) Gross appearances of liver samples from the primary liver cancer mouse mode established with C57BL/6 mice. (L) The levels of RALY protein expression in livers from the indicated groups. (M, N, O) Liver-to-body weight ratios (M), tumor nodules numbers (N) and maximal size of tumors (O), n = 6/group.

Figure 2

OGT mediated O-GlcNAcylation of RALY on Ser176. (A) Venn diagram illustrating the overlapped proteins bound with OGT in Hep3B and BEL7402 cells. (B, C) Co-immunoprecipitation (CO-IP, B) and immunostaining assays (C) validation of the interaction between RALY and OGT in hepatocellular carcinoma (HCC) cells. (D) CO-IP of O-GlcNAcylated proteins in the immunoprecipitates pulled down by anti-RALY antibodies in HCC tissue and cells. (E) Western blotting showed the expression levels of RALY O-GlcNAcylation using two HCC cell lines (Hep3B and Huh7) and normal liver epithelial THLE-2 cells, and paired HCC specimens and their paracancerous tissues. (F) Western blotting showed the expression levels of RALY O-GlcNAcylation by stimulation and block of O-GlcNAcylation in HCC cells. (G) The predicted O-GlcNAcylation sites of RALY using the YinOYang 1.2 server. (H) Western blotting analyzed the O-GlcNAcylated expression levels of RALY in IP-RALY immunoprecipitates from Hep3B cells transfected with plasmids expressing wild-type or O-GlcNAcylated site mutants.

Figure 3

O-GlcNAcylation maintained the protein stability of RALY. (A) RALY was measured by western blotting in hepatocellular carcinoma (HCC) cells treated with TMG, PUG, OE-OGT and siOGT. (B) RALY was measured by qRT-PCR in HCC cells treated with TMG, PUG, OE-OGT and siOGT. (C, D, E). Half-life and quantitative analysis of RALY in HCC cells treated with or without O-GlcNAcylation stimulators (TMG or PUG). HCC cells were transfected with RALYKO-WT (D) or RALYKO-S176A (E) and treated with CHX.

Figure 4

Crosstalk between O-GlcNAcylation and ubiquitination of RALY. (A) The RALY protein level after treated with NH4Cl, 3-MA, Z-VAD-FMK and MG132 in hepatocellular carcinoma (HCC) cells, were analyzed by western blot with indicated antibodies. (B) RALY was examined in total ubiquitinated proteins in HCC cells. (C) Scheme displaying the mass spectrometry procedure used for identifying the specific target TRIM27 of RALY. (D, E) Co-immunoprecipitation (D) and immunostaining assays (E) validation of RALY interaction with TRIM27 in vitro. (F) Western blotting indicated the increased level of RALY ubiquitination in HCC cells with TRIM27 overexpression. (G, H) Western blotting indicated the RALY ubiquitination in HCC cells. HCC cells were treated with or without TMG (G); HCC cells were transfected with RALYKO-WT or RALYKO-S176A and treated with TMG (H). (I, J) The level of TRIM27 protein was measured in purified RALY immunoprecipitation samples from HCC cells. HCC cells were treated with or without TMG (I); HCC cells were transfected with RALYKO-WT or RALYKO-S176A and treated with TMG (J). (K) Western blotting showed the RALY protein expression in TRIM27-overexpression HCC cells with TMG treatment.

Figure 5

Knockdown of OGT weakened the proliferative effect of RALY overexpression. (A, B) CCK-8 (A) and EdU incorporation assays (B) were performed in hepatocellular carcinoma (HCC) cells for rescue experiments. (C) Statistical analysis of the EdU incorporation assays. (D) The photograph of planted subcutaneous tumors of Huh7 cells infected with the indicated lentivirus. (E) The weight of the tumors was measured at the experimental endpoint. (F) The volumes of tumors were measured every week.

Figure 6

RALY increased the cytoplasmic localization of USP22 mRNAs to enhance their protein expressions. (A) Western blotting showed the efficiency of immunoprecipitation. (B) Venn diagram of transcripts bound by RALY and IgG. (C) Venn diagram of RALY-regulated transcripts and RALY RNA immunoprecipitation (RIP) sequencing targets. (D) The qRT-PCR assays showed the abundance of RALY target RNAs (HNRNPC, CHD4, HNRNPA2B1, PCBP2, STIP1 and USP22) from hepatocellular carcinoma (HCC) cells transfected with siRALY or OE-RALY. (E) RIP-qPCR showed the RNA-binding capacity of RALY in HCC cells. (F) Western blotting showed the protein abundance of RALY target RNAs (HNRNPC, CHD4, HNRNPA2B1, PCBP2, STIP1 and USP22) from HCC cells transfected with siRALY or OE-RALY. (G) Cytoplasmic and nuclear mRNA expression of USP22 in RALY silenced HCC cells. (H) Immunostaining assays validation of RALY interaction with ALYREF in vitro. (I) RIP-qPCR verified that the USP22 mRNA-binding capacity of ALYREF was decreased in siRALY HCC cells compared to control.

Figure 7

RALY exerted its biological functions by promoting USP22 expression in hepatocellular carcinoma (HCC) cells. (A) Western blotting showed the expression of USP22 in two HCC cell lines by stimulation and block of O-GlcNAcylation. (B, C) Cell proliferative capacity was determined after cell transfection by CCK-8 (B) and EdU incorporation assays (C). (D) Statistical analysis of the EdU incorporation assays.

Figure 8

Peptide‑based proteolysis-targeting chimeras (p-PROTAC) degrader of RALY. (A) Name and sequence of peptides targeting RALY protein. (B) Name and sequence of peptides conjugated with FITC and TAT sequence at the N-terminus and C-terminus, respectively. (C) CCK-8 showed the growth rate of hepatocellular carcinoma (HCC) cells treated with five screened peptides. (D) The optimized binding modes with lowest binding energy generated by ClusPro web server and interaction between RALY and F-2, Red: protein; Yellow: F-2. (E) Cellular localization of FITC labeled FIP-2 (green) by immunostaining assays. (F) Structure of RALY-PROTAC. (G) Western blotting showed the expression of RALY protein in HCC cells treated with FIP-2 and RALY-PROTAC. (H, I) CCK-8 (H) and EdU incorporation assays (I) of HCC cells treated with FIP-2 and RALY-PROTAC. (J) Statistical analysis of the EdU incorporation assays.