ADAR1 promotes cisplatin resistance in intrahepatic cholangiocarcinoma by regulating BRCA2 expression through A-to-I editing manner

Abstract

Aberrant A-to-I RNA editing, mediated by ADAR1 has been found to be associated with increased tumourigenesis and the development of chemotherapy resistance in various types of cancer. Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive malignancy with a poor prognosis, and overcoming chemotherapy resistance poses a significant clinical challenge. This study aimed to clarify the roles of ADAR1 in tumour resistance to cisplatin in iCCA. We discovered that ADAR1 expression is elevated in iCCA patients, particularly in those resistant to cisplatin, and associated with poor clinical outcomes. Downregulation of ADAR1 can increase the sensitivity of iCCA cells to cisplatin treatment, whereas its overexpression has the inverse effect. By integrating RNA sequencing and Sanger sequencing, we identified BRCA2, a critical DNA damage repair gene, as a downstream target of ADAR1 in iCCA. ADAR1 mediates the A-to-I editing in BRCA2 3'UTR, inhibiting miR-3157-5p binding, consequently increasing BRCA2 mRNA and protein levels. Furthermore, ADAR1 enhances cellular DNA damage repair ability and facilitates cisplatin resistance in iCCA cells. Combining ADAR1 targeting with cisplatin treatment markedly enhances the anticancer efficacy of cisplatin. In conclusion, ADAR1 promotes tumour progression and cisplatin resistance of iCCA. ADAR1 targeting could inform the development of innovative combination therapies for iCCA.

FIGURE 1

ADAR1 is upregulated in iCCA tissues, particularly in chemoresistant iCCA, and is associated with poor clinical outcomes. (A) Comparison of ADAR1 mRNA levels in tumour versus normal tissues across various cancers in TCGA database. (B) ADAR1 mRNA expression in iCCA tumour and paracancerous tissues in TCGA and GEO databases. (C) Representative immunohistochemistry (IHC) images of ADAR1 protein expression in iCCA tumours, highlighting variations in staining intensity. (D) Analysis of the relationship between ADAR1 expression and tumour size, tumour number, and TNM stage in our in‐house cohort 128 iCCA cases. (E) Kaplan–Meier plots depicting overall survival (OS) and disease‐free survival (DFS) based on ADAR1 IHC scoring. (F) Multivariate COX analysis of OS in our in‐house cohort 128 iCCA cases. (G) Representative IHC staining images and staining scores of ADAR1 expression in the cisplatin chemo‐resistant group (n = 12) and chemo‐sensitive group (n = 12).

FIGURE 2

ADAR1 promotes iCCA resistance to cisplatin via A‐to‐I RNA editing. (A) ADAR1 mRNA and protein levels in shADAR1 and control iCCA cells, assessed by RT‐qPCR and Western blot. (B) Cell viability of shADAR1 and control groups post‐cisplatin treatment (20 or 10 μM, respectively). (C) Colony formation in shADAR1 and control groups post‐cisplatin exposure (20 or 10 μM, respectively). (D) mRNA and protein expression of ADAR1 in wild‐type ADAR1‐overexpressing (OE) and control iCCA cells. (E) Cell viability in wild‐type ADAR1‐OE and control groups post‐cisplatin treatment (10 or 5 μM, respectively). (F) Colony formation assay results of the wild‐type ADAR1‐OE and control groups in iCCA cells after cisplatin treatment (10 or 5 μM, respectively). (G) Flow cytometry and γ‐H2AX fluorescence intensity in shADAR1 and control groups post‐cisplatin treatment (20 or 10 μM, respectively). (H) Schematic and transfection efficiency validation of wild‐type and catalytic mutant (E/A) ADAR1 plasmids via Western blot. (I) Cell viability in ADAR1‐OE, E/A, and control groups post‐cisplatin treatment (10 or 5 μM, respectively). (J) Colony formation assay results of the ADAR1‐OE group, E/A group and control group in iCCA cells after cisplatin treatment (10 or 5 μM, respectively). (K) Representative flow cytometry analysis and γ‐H2AX mean fluorescent intensity of the ADAR1‐OE group, E/A group and control group in iCCA cells after cisplatin treatment (10 or 5 μM, respectively).

FIGURE 3

ADAR1 regulates BRCA2 expression through A‐to‐I RNA editing. (A) Volcano plot illustrating differential gene expression obtained by RNA sequencing of HuCC‐T1 with stable knockdown of ADAR1, down‐regulated genes are highlighted in blue, and up‐regulated genes in red. (B) GO enrichment analysis for differentially expressed genes (DEGs) in shADAR1 and shControl cells, covering biological processes (BP), cellular components (CC), and molecular functions (MF). (C) Venn diagram showing the overlap between genes downregulated after ADAR1 knockdown and genes exhibiting A‐to‐I RNA editing in the 3′UTR. (D) Comparison of BRCA2 mRNA expression between iCCA tumour and paracancerous tissues in TCGA and GSE107943 datasets. (E) Correlation analysis between ADAR1 and BRCA2 mRNA expression in TCGA iCCA tumour and paracancerous tissues. (F) Representative IHC staining images and protein correlation analysis between ADAR1 and BRCA2 expression in the in‐house cohort (n = 30). (G) Assessment of the ADAR1 and BRCA2 mRNA expression of the shADAR1 group and shControl group in iCCA cells via RT‐qPCR. (H) Evaluation of ADAR1 and BRCA2 protein levels of iCCA cells from shADAR1 and shControl groups using western blot. (I) Sanger sequencing chromatograms of BRCA2 A‐to‐I RNA editing sites in iCCA cells from shADAR1 and shControl groups. (J) RT‐qPCR analysis of ADAR1 and BRCA2 mRNA expression in iCCA cells from ADAR1‐OE group, E/A group, and control group. (K) Western blot analysis of the ADAR1 and BRCA2 protein levels in iCCA cells from ADAR1 OE group, E/A group, and control group. (L) Sanger sequencing chromatograms of BRCA2 A‐to‐I RNA editing sites in iCCA cells from ADAR1‐OE group, E/A group and control group.

FIGURE 4

ADAR1 promotes cisplatin resistance in iCCA cells via BRCA2. (A) Venn diagram illustrating the overlap in differentially expressed genes (DEGs) between shADAR1 and siBRCA2 RNA‐seq. (B) Gene Ontology (GO) enrichment analysis of overlapping DEGs from shADAR1 and siBRCA2 RNA‐seq, including biological processes (BP), cellular components (CC), and molecular functions (MF). (C) Gene Set Enrichment Analysis (GSEA) of overlapping DEGs between shADAR1 and siBRCA2 RNA‐seq. (D) Representative immunohistochemistry (IHC) staining images and scoring of BRCA2 in cisplatin‐resistant (n = 12) and sensitive groups (n = 12). (E) Validation of BRCA2 mRNA expression post‐BRCA2 silencing in iCCA cells using RT‐qPCR. (F) Validation of BRCA2 protein expression post‐BRCA2 silencing in iCCA cells using Western blot. (G) Measurement of BRCA2 mRNA levels in vector, ADAR1 + siControl, and ADAR1 + siBRCA2 groups in iCCA cells by RT‐qPCR. (H) Measurement of BRCA2 protein levels in vector, ADAR1 + siControl, and ADAR1 + siBRCA2 groups in iCCA cells by Western blot. (I) Cell viability curves for vector, ADAR1 + siControl, and ADAR1 + siBRCA2 groups in iCCA cells post‐cisplatin treatment (10 or 5 μM). (J) Colony formation assay outcomes for vector, ADAR1 + siControl, and ADAR1 + siBRCA2 groups in iCCA cells treated with cisplatin (10 or 5 μM). (K) Representative flow cytometry analysis and γ‐H2AX mean fluorescent intensity in vector, ADAR1 + siControl, and ADAR1 + siBRCA2 groups in HuCC‐T1 cells post‐cisplatin treatment (10 μM).

FIGURE 5

A‐to‐I RNA editing on BRCA2 mediated by ADAR1 inhibits miR‐3157‐5p binding to its 3′UTR and regulates the level of BRCA2 expression. (A) TargetScan predicts miRNAs that may bind to the A‐to‐I edited site in the BRCA2 3′UTR. (B) Assessed BRCA2 protein expression in iCCA cells transfected with the miR‐3157‐5p mimic or control using Western blot. (C) Evaluated ADAR1 and BRCA2 protein expressions in vector, ADAR1‐OE, and ADAR1‐EA groups in iCCA cells transfected with miR‐3157‐5p mimics using Western blot. (D) Schematic representation of the BRCA2 3′UTR luciferase reporter plasmid, including both wildtype (WT) and mutant (MUT) versions. (E) Dual luciferase reporter assay results for the BRCA2‐WT and BRCA2‐MUT groups in iCCA cells co‐transfected with miR‐3157‐5p mimics or miR‐Control. (F) Dual luciferase reporter assay results for the BRCA2‐WT and BRCA2‐MUT groups in iCCA cells co‐transfected with miR‐3157‐5p inhibitor or control. (G) Assessed BRCA2 protein expression in iCCA cells transfected with the miR‐3157‐5p inhibitor or control.

FIGURE 6

Enhancing anti‐tumour efficacy of cisplatin in iCCA PDX models via ADAR1 targeting. (A) Graphic depiction of constructing a cisplatin‐resistant iCCA PDX model. (B) PDX tissues were randomly assigned into four groups and treated as follows: siControl (3 nmol per mouse, twice a week), siADAR1 (3 nmol per mouse, twice a week), siControl plus cisplatin (siControl, 3 nmol per mouse, twice a week, and cisplatin, 4 mg/kg, once a week), and siADAR1 plus cisplatin (siADAR1, 3 nmol per mouse, twice a week, and cisplatin, 4 mg/kg, once a week) over 21 days. Tumour growth curves for each group are displayed. (C) Tumour weights for the siControl, siADAR1, siControl + cisplatin, and siADAR1 + cisplatin groups were measured post‐euthanasia. (D) Serum ALT and AST levels, indicators of liver function, were measured in the siControl, siADAR1, siControl + cisplatin, and siADAR1 + cisplatin groups. (E) Serum creatinine (CREA) and blood urea nitrogen (BUN), indicators of kidney function, were measured in the siControl, siADAR1, siControl + cisplatin, and siADAR1 + cisplatin groups. (F) Representative immunohistochemistry (IHC) staining and IHC scores for ADAR1, BRCA2, and Ki67 in tumours from the siControl, siADAR1, siControl + cisplatin, and siADAR1 + cisplatin groups.

FIGURE 7

Schematic diagram illustrating the function and mechanism of ADAR1 in iCC.