ABALON regulates mitophagy and 5-FU sensitivity in colorectal cancer via PINK1-Parkin pathway

Abstract

Background: Growing evidence demonstrated that long non-coding RNAs (lncRNAs) are closely related with chemoresistance in colorectal cancer (CRC). Mitophagy serves as an essential factor to maintain the quality of tumor cells. However, it is unclear whether lncRNAs are involved in mitophagy regulation in CRC. The aim of this study is to evaluate whether lncRNAs are involved in regulating mitophagy and chemoresistance in CRC.

Methods: In this study, gain/loss of function was used to analyze the biological function influenced by apoptotic BCL2L1-antisense long non-coding RNA (ABALON). Western blot and JC-1 probe were carried out for detecting mitophagy. Chemosensitivity of CRC cells to 5-fluorouracil (5-FU) was determined using cell counting kit-8 (CCK-8), flow cytometry, colony formation and trans well assays.

Results: We found that ABALON expression was increased in CRC, especially in consensus molecular subtype 1 (CMS1) and highly expressed ABALON was related with tumor differentiation, tumor node metastasis (TNM) staging, and lymph node metastasis (P<0.05). ABALON knockdown led to impaired proliferation and enhanced apoptosis in CRC. Mitophagy variations primed by ABALON enhanced mitochondrial homeostasis. The half maximal inhibitory concentration (IC50) of 5-FU in ABALON interference groups declined, while ABALON overexpression elevated IC50. Furthermore, defective mitophagy not only rescued the proliferation, metastasis, and apoptosis induced by ABALON overexpression, but also, enhanced the anti-tumor effect of 5-FU in vivo.

Conclusions: Collectively, our study proposed that ABALON potentiates CRC progression via PINK1/Parkin mediated mitophagy, and ABALON is a promising therapeutic target in reversing 5-FU resistance.

Keywords: 5-fluorouracil (5-FU); Colorectal cancer (CRC); long non-coding RNA (lncRNA); mitophagy; sensitivity.

Figure 1

ABALON was significantly upregulated in CRC tissues, especially in CMS1 subtypes. (A,B) ABALON expression across pan-cancer in TCGA and GTEx database. (C) Relative expression of ABALON in CRC tissues and adjacent normal tissues (n=65). (D) ABALON expression in different CMS subtypes. (E) The expression level of ABALON in CRC cell lines. (F) The copy number variation of ABALON in CRC. (G) Survival curves illustrating disease-free interval among patients with CRC in the high and low-risk groups using Kaplan-Meier analysis. ns, nonsense; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. GBM, glioblastoma multiforme; GBMLGG, glioblastoma multiforme lower grade glioma; LGG, brain lower grade glioma; UCEC, uterine corpus endometrial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; LUAD, lung adenocarcinoma; ESCA, esophageal carcinoma; STES, stomach and esophageal carcinoma; KIRP, kidney renal papillary cell carcinoma; KIPAN, pan-kidney cohort; COAD, colon adenocarcinoma; COADREAD, colon adenocarcinoma/rectum adenocarcinoma esophageal carcinoma; PRAD, prostate adenocarcinoma; STAD, stomach adenocarcinoma; HNSC, head and neck squamous cell carcinoma; KIRC, kidney papillary cell carcinoma; LUSC, lung squamous cell carcinoma; LIHC, liver hepatocellular carcinoma; WT, high-risk Wilms tumor; SKCM, skin cutaneous melanoma; BLCA, bladder urothelial carcinoma; THCA, thyroid carcinoma; READ, rectum adenocarcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; TGCT, testicular germ cell tumors; UCS, uterine carcinosarcoma; ALL, acute lymphoblastic leukemia; LAML, acute myeloid leukemia; PCPG, pheochromocytoma and paraganglioma; ACC, adrenocortical carcinoma; KICH, kidney chromophobe; CHOL, cholangiocarcinoma; ABALON, apoptotic BCL2L1-antisense long non-coding RNA; CMS, consensus molecular subtypes; TPM, transcripts per million; HR, hazard ratio; CI, confidence interval; L, low; H, high; CRC, colorectal cancer; TCGA, The Cancer Genome Atlas; GTEx, Genotype-Tissue Expression.

Figure 2

ABALON promoted CRC cell proliferation. (A) The knockdown efficiency of ABALON in DLD1 and SW480 cells transfected with shRNA. (B) The overexpression efficiency of ABALON in CACO2; the proliferation of CRC transfected with shRNA and pc-DNA, (C,D) CCK8 assay. (E,F) Colony formation assay. The staining method of colony formation assay: crystal violet staining. (G,H) EDU assay (scale bar =75 µm). *, P<0.05; **, P<0.01; ***, P<0.001. shRNA, short hairpin RNA; pcDNA, pcDNA^TM3.2/V5-DEST; shNC, short hair negative control; OD, optical density; ABALON, apoptotic BCL2L1-antisense long non-coding RNA; CRC, colorectal cancer; CCK8, cell counting kit-8; EDU, 5-ethynyl-2’-deoxyuridine.

Figure 3

ABALON influenced migration, invasion and cell apoptosis. (A,B) Boyden chamber trans-well assay was used to validate the invasion and migration via ABALON knockdown and overexpression (scale bar =100 µm, magnification: ×40). (C,D) Flow cytometry was performed to determine the cell apoptosis after transfected with shRNA and pc-DNA and on DLD1 and SW480 cells. The staining method of trans-well assay: crystal violet staining. *, P<0.05; **, P<0.01; ***, P<0.001. shNC, short hair negative control; pcDNA, pcDNA^TM3.2/V5-DEST; FITC, fluorescein isothiocyanate; PI, propidium iodide; ABALON, apoptotic BCL2L1-antisense long non-coding RNA; shRNA, short hairpin RNA.

Figure 4

ABALON was related with oxidative phosphorylation. (A-C) KEGG and GO analysis showed ABALON was mainly related to mitochondrial related pathways, IFN-γ response and autophagy. (D) GSEA demonstrated ABALON was implicated in autophagy pathway. (E,F) FISH and nuclear cytoplasmic fractionation showed ABALON was mainly expressed in the cytoplasm (scale bar =10 µm, magnification: ×1,000). (G) The cellular ROS generation was detected by DCFH-DA staining (scale bar =200 µm, magnification: ×400). BP, biological process; CC, cellular component; MF, molecular function; KEGG, Kyoto Encyclopedia of Genes and Genomes; ABALON, apoptotic BCL2L1-antisense long non-coding RNA; GO, Gene Ontology; IFN-γ, interferon-gamma; GSEA, gene set enrichment analysis; FISH, fluorescence in situ hybridization; ROS, reactive oxygen species; DCFH-DA, 2’,7’-dichlorodihydrofluorescein diacetate.

Figure 5

ABALON regulated mitochondrial function in CRC cells. (A-C) Western blot was used to detect the expression of mitophagy associated proteins. (D,E) JC-1 staining showed knocking down ABALON increased the green fluorescence intensity (scale bar =200 µm, magnification: ×400). **, P<0.01; ***, P<0.001. shNC, short hair negative control; ABALON, apoptotic BCL2L1-antisense long non-coding RNA; CRC, colorectal cancer.

Figure 6

ABALON knockdown strengthened the sensitivity of CRC cells to 5-FU. (A,B) The IC50 in DLD1 and SW480 cells was reduced following ABALON knockdown. (C) The IC50 in CACO2 cells was upregulated by overexpression of ABALON. (D-G) The effect of 5-FU with different concentration and time on the inhibition and viability of DLD1 and SW480 cells. *, P<0.05; **, P<0.01. IC50, the half maximal inhibitory concentration; 5-FU, 5-fluorouracil; shNC, short hair negative control; ABALON, apoptotic BCL2L1-antisense long non-coding RNA; CRC, colorectal cancer.

Figure 7

Inhibition of mitophagy enhanced the sensitivity to 5-FU treatment. DLD1 and CACO2 were co-treated with 5-FU and CQ (the autophagy inhibitor, 5 µM) or transfected with siPINK1. (A) With the treatment of 5-FU, ABALON knockdown increased the apoptosis ability of DLD1 cells, and mitophagy inhibition weaken the apoptosis. (B) CACO2 transfected with siPINK1 or treated with CQ reversed the apoptotic cells induced by ABALON overexpression. (C-F) Transfection with siPINK1 or CQ treatment inhibited the colony formation and migration. (G,H) The effect of 5-FU (10 or 20 mg/kg) and CQ (50 mg/kg) on nude mice growth. (I) Graphic description shown the regulatory mechanism. ns, nonsense; *, P<0.05; **, P<0.01; ***, P<0.001. 5-FU, 5-fluorouracil; WT, high-risk Wilms tumor; shNC, short hair negative control; CQ, chloroquine; PI, propidium iodide; pcDNA, pcDNA^TM3.2/V5-DEST; ABALON, apoptotic BCL2L1-antisense long non-coding RNA; CNV, copy number variation.