PUS7-dependent pseudouridylation of ALKBH3 mRNA inhibits gastric cancer progression

Abstract

Background: RNA pseudouridylation is a critical post-transcriptional modification that influences gene expression and impacts various biological functions. Despite its significance, the role of mRNA pseudouridylation in cancer remains poorly understood. This study investigates the impact of pseudouridine synthase 7 (PUS7)-mediated pseudouridylation of Alpha-ketoglutarate-dependent Dioxygenase alkB Homolog 3 (ALKBH3) mRNA in gastric cancer.

Methods: Immunohistochemistry and Western blotting were used to assess PUS7 protein levels in human gastric cancer tissues. The relationship between PUS7 and gastric cancer progression was examined using 3D colony formation assays and subcutaneous xenograft models. Real-time quantitative PCR (RT-qPCR), Western blotting, and polysome profiling assays were conducted to investigate how PUS7 regulates ALKBH3. A locus-specific pseudouridine (Ψ) detection assay was used to identify Ψ sites on ALKBH3 mRNA.

Results: Our findings indicate a significant reduction of PUS7 in gastric cancer tissues compared to adjacent non-tumour tissues. Functional analyses reveal that PUS7 inhibits gastric cancer cell proliferation and tumour growth via its catalytic activity. Additionally, PUS7 enhances the translation efficiency of ALKBH3 mRNA by modifying the U696 site with pseudouridine, thereby attenuating tumour growth. Importantly, ALKBH3 functions as a tumour suppressor in gastric cancer, with its expression closely correlated with PUS7 levels in tumour tissues.

Conclusions: PUS7-dependent pseudouridylation of ALKBH3 mRNA enhances its translation, thereby suppressing gastric cancer progression. These findings highlight the potential significance of mRNA pseudouridylation in cancer biology and suggest a therapeutic target for gastric cancer.

Highlights: PUS7 enhances the translation efficiency of ALKBH3 through its pseudouridylation activity on ALKBH3 mRNA, thereby inhibiting gastric tumourigenesis. The expression levels of PUS7 and ALKBH3 are significantly correlated in gastric tumours, which may be potential prognostic predictors and therapeutic targets for patients with gastric cancer.

Keywords: alkbh3; gastric cancer; pseudouridylation; pus7.

FIGURE 1

PUS7 is significantly downregulated in gastric tumour tissues and inhibits cancer cell proliferation. (A, B) Immunohistochemistry analysis with anti‐PUS7 antibody of gastric cancer tissue array from cohort 1. Relative PUS7 expression was analyzed. (C, D) Western blots of gastric tumour tissues and their corresponding non‐tumour tissues with anti‐PUS7 and β‐actin antibodies. β‐actin, a loading control. N, non‐tumour; T, tumour. Relative levels of PUS7 are shown. (E, F) Western blotting with anti‐PUS7 and β‐actin antibodies in various gastric cancer cell lines. β‐actin, a loading control. The intensity of PUS7 was measured based on the normalization to β‐actin (F). (G–I) MKN45 cells infected with lentivirus‐carrying shRNAs targeting PUS7 (shPUS7‐1 or shPUS7‐2) were subjected to western blots and 3D colony formation assays. Quantification of colony number per field is presented. Scale bar, 200 µm. shnc, negative control shRNA. (J–L) MKN45 cells treated with the indicated shRNAs were infected with lentivirus expressing PUS7 or PUS7‐D294A plasmid and subjected to western blots and 3D colony formation analyses. The colony number was analyzed. Scale bar, 200 µm. (M‐O) AGS cells were infected with lentivirus expressing PUS7 or PUS7‐D294A plasmid and applied for western blots and 3D colony formation assays. Quantification of colony number per field is shown. Scale bar, 200 µm. (P‐R) AGS cells infected with lentivirus expressing the indicated plasmids were processed to subcutaneous implantation in BALB/C nude mice. The gloss morphology of subcutaneous tumours (Q) and tumour growth curve (R) were shown. Data are expressed as means   ± SD. Student's t‐test (tumour growth data (R) are expressed as means ± SEM. Two‐way ANOVA); *p < .05, **p < .01, ***p < .001, ns, not significant.

FIGURE 2

PUS7 increases the expression of ALKBH3 protein and the translation efficiency of ALKBH3 mRNA. (A, B) Western blotting with anti‐ALKBH3 and PUS7 antibodies in MKN45 cells treated with the indicated shRNAs. β‐actin served as a loading control. The intensity of ALKBH3 was measured based on the normalization to β‐actin (B). (C) The sequence of indels in the PUS7 locus of MKN45 cells generated by the CRISPR/Cas9 system is shown. The sgRNAs target site and the protospacer adjacent motif (PAM) site are indicated in blue and red, respectively. (D, E) Wild‐type (WT) or PUS7 knockout (KO) MKN45 cells were subjected to western blots with the indicated antibodies. β‐actin, a loading control. The intensity of ALKBH3 was measured based on the normalization to β‐actin (E). (F–I) MKN45 or AGS cells treated with the indicated shRNAs or plasmids were applied for western blots with the antibodies as shown. β‐actin, a loading control. The intensity of ALKBH3 was measured based on the normalization to β‐actin (G, I). (J) MKN45 cells infected with lentivirus‐carrying shRNAs targeting PUS7 (shPUS7‐1 or shPUS7‐2) were processed for RT‐qPCR analysis. snRNA U6 was used as an internal control. (K) AGS cells infected with lentivirus expressing PUS7 or PUS7‐D294A were subjected to RT‐qPCR analysis. snRNA U6, an internal control. (L, M) MKN45 cells treated with shPUS7‐1 were lysed and subjected to sucrose gradient centrifugation and polysome profiling. The distribution of ALKBH3 mRNA in different fractions is shown (L). And ALKBH3 mRNA distribution in the fractions of non‐polysome and polysome was analyzed (M). (N, O) Wild‐type or PUS7 knockout MKN45 cells were lysed and subjected to sucrose gradient centrifugation and polysome profiling. The distribution of ALKBH3 mRNA in different fractions is shown (N). And ALKBH3 mRNA distribution in the fractions of non‐polysome and polysome was analyzed (O). LMW, low molecular weight; HMW, high molecular weight. Data are shown as mean ± SD. Student's t‐test; *p < .05, **p < .01, ***p < .001, ****p < .0001, ns, not significant.

FIGURE 3

PUS7 decorates ALKBH3 mRNA on U696 with Ψ. (A) Workflow of the method to detect the locus‐specific Ψ sites. (B–D) The PCR products of ALKBH3 were subjected to TA cloning and Sanger sequencing. The results of three putative Ψ‐consensus sequences of ALKBH3 mRNA from TA colonies were analyzed. (E–G) MKN45 cells infected with lentivirus‐carrying shRNAs targeting PUS7 (shPUS7‐1) were subjected to immunoblotting and locus‐specific Ψ sites detecting. The results of U696 site mutation frequency of ALKBH3 were analysed. (H–J) Wild‐type or PUS7 knockout MKN45 cells were subjected to western blotting and locus‐specific Ψ sites detection. The results of U696 site mutation frequency of ALKBH3 were measured. Percentage represents the mutation frequency calculated by taking the peak area of ‘C’ and ‘A’ peaks over the sum of ‘T’, ‘C’ and ‘A’ peaks (G and J). Ψ, pseudouridine; CMC, N‐cyclohexyl‐N′‐(2‐morpholinoethyl) carbodiimide. Data are shown as mean ± SD. Student's t‐test; *p < .05, **p < .01.

FIGURE 4

ALKBH3 suppresses gastric cancer cell proliferation and tumour growth. (A–C) MKN45 cells infected with lentivirus‐carrying shRNAs targeting ALKBH3 (shALKBH3‐1 or shALKBH3‐2) were subjected to western blots and 3D colony formation assays. Quantification of colony number per field is presented. Scale bar, 200 µm. (D‐F) MKN45 cells treated with the indicated shRNAs were infected with the lentivirus expressing ALKBH3, and applied for western blots and 3D colony formation analyses. Analysis of colony number per field is shown. Scale bar, 200 µm. (G–I) AGS cells infected with lentivirus expressing ALKBH3 were processed to western blots and 3D colony formation assays. The colony number per field was calculated. (J–N) MKN45 cells infected with lentivirus targeting ALKBH3 were subcutaneously injected into BALB/c nude mice. BLI (bioluminescence imaging) analysis was performed to measure tumour growth (J, K). The tumour growth curve (M) and weight (N) were analyzed. (O, P) AGS cells infected with lentivirus expressing ALKBH3 were applied for subcutaneous implantation in BALB/C nude mice. Arrows indicate tumours. Tumour growth curve is shown (P). Scale bars, 200 µm. Data are expressed as means   ± SD. Student's t‐test (tumour growth data (M, P) are expressed as means ± SEM. Two‐way ANOVA); *p < .05, **p < .01, ***p < .001, ****p < .0001.

FIGURE 5

ALKBH3 reverses the phenotype induced by PUS7 depletion in gastric cancer model. (A–C) MKN45 cells treated with the indicated shRNAs were infected with lentivirus expressing ALKBH3 and subjected to western blots and 3D colony formation assays. Quantification of colony number per field is presented. Scale bar, 200 µm. (D–I) MKN45 cells treated with the indicated shRNAs were infected with lentivirus expressing PUS7 or ALKBH3 and applied for subcutaneous implantation in BALB/C nude mice (6 mice in each group, and one mouse in the shPUS7+ALKBH3 group died from a non‐tumour‐related bacterial infection before the experiment concluded). Tumour growth was detected by BLI analysis. The tumour growth curve (H) and weight (I) were shown. Data are expressed as means ± SD. Student's t‐test (tumour growth data (H) are expressed as means ± SEM. Two‐way ANOVA); *p < .05, ***p < .001.

FIGURE 6

ALKBH3 expression is significantly decreased in gastric tumour tissues and correlated with PUS7 expression. (A, B) Immunohistochemistry analysis with anti‐ALKBH3 antibody in gastric tumour tissue array from cohort 1. Relative ALKBH3 expression was analyzed. Data are shown as mean ± SD. Student's t‐test; ****p < .0001. (C, D) Western blotting of gastric tumour tissues and their corresponding non‐tumour tissues with anti‐ALKBH3 antibody from cohort 2. β‐actin, a loading control. N, non‐tumour; T, tumour. The relative levels of ALKBH3 were quantified. (E, F) Pearson correlation analysis of PUS7 and ALKBH3 protein levels in gastric tumour tissues from two independent cohorts.

FIGURE 7

Schematic illustration shows that PUS7 modifies ALKBH3 mRNA with pseudouridine (Ψ), enhancing its translation efficiency and suppressing gastric carcinogenesis.