. State Key Laboratory of Molecular Oncology, Department of Etiology and Carcinogenesis, Beijing Key Laboratory for Carcinogenesis and Cancer Prevention, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China . Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou 510060, China
Identifying biomarkers for predicting radiotherapy efficacy is crucial for optimizing personalized treatments. We previously reported that rs1553867776 in the miR-4274 seed region can predict survival in patients with rectal cancer receiving postoperative chemoradiation therapy. Hence, to investigate the molecular mechanism of the genetic variation and its impact on the radiosensitivity of colorectal cancer (CRC), in this study, bioinformatics analysis is combined with functional experiments to confirm peroxisomal biogenesis factor 5 (PEX5) as a direct target of miR-4274. The miR-4274 rs1553867776 variant influences the binding of miR-4274 and PEX5 mRNA, which subsequently regulates PEX5 protein expression. The interaction between PEX5 and Ku70 was verified by co-immunoprecipitation and immunofluorescence. A xenograft tumor model was established to validate the effects of miR-4274 and PEX5 on CRC progression and radiosensitivity in vivo. The overexpression of PEX5 enhances radiosensitivity by preventing Ku70 from entering the nucleus and reducing the repair of ionizing radiation (IR)-induced DNA damage by the Ku70/Ku80 complex in the nucleus. In addition, the enhanced expression of PEX5 is associated with increased IR-induced ferroptosis. Thus, targeting this mechanism might effectively increase the radiosensitivity of CRC. These findings offer novel insights into the mechanism of cancer radioresistance and have important implications for clinical radiotherapy.
Just Accepted Date: 27 June 2024Online First Date: 29 August 2024Issue Date: 29 October 2024
Cite this article:
Qixuan Lu,Ningxin Ren,Hongxia Chen, et al. Polymorphism in the Hsa-miR-4274 seed region influences the expression of PEX5 and enhances radiotherapy resistance in colorectal cancer[J]. Front. Med.,
2024, 18(5): 921-937.
Fig.1 miR-4274 functions as a radioresistant oncogene in CRC. (A) miR-4274 expression in blood was upregulated in patients with digestive system tumor than healthy control group. Colorectal, colorectal cancer; colon, colon cancer; stomach, stomach cancer; esophageal, esophageal cancer; liver, liver cancer. (B) Transfection miR-4274 mimics in HCT116 (left) and HCT8 (right) cells significantly increased the cell proliferation and induced radioresistance. (C) Transfection miR-4274 inhibitor in HCT116 (left) and HCT8 (right) cells significantly inhibited cell proliferation and increase the radiosensitivity. Growth curves data are mean ± SEM from 3 independent experiments and most error bars are with the symbols. (D) Western blot analysis demonstrated that the upregulation of miR-4274 resulted in a decrease in γ-H2AX protein levels, while the downregulation of miR-4274 led to an increase in γ-H2AX protein levels treated with radiation. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant of Student’s t-test. NC, NC mimics; miR [del], deletion-type miR-4274 mimics; miR [ins], insertion-type miR-4274 mimics; NC inh, NC inhibitor; miR inh [del], deletion-type miR-4274 inhibitor; miR inh [ins], insertion-type miR-4274 inhibitor. IR, irradiation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Fig.2 PEX5 is a target of miR-4274 in CRC. (A) Venn diagram of the predicted results in 6 miRNA data sets and cancer databases followed by flowchart of miR-4274 target selection. (B) Schematic diagrams showing the putative binding sequence of miR-4274 in the wild-type PEX5 and the mutant PEX5 3′UTR. The miR-4274 was divided into miR-4274 mimics [del] and [ins] by simulating transcription products of different genotypes in rs1553867776. (C, D) The relative luciferase activity of psiCHECK-2 vector bearing PEX5 3′UTR was reduced by the miR-4274 mimics, especially the miR-4274 mimics [del], and increased by the miR-4274 inhibitor; however, mutation of the PEX5 3′UTR reversed these trends. Data are presented as mean ± SEM; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 and ns, not significant of Student’s t-test. (E) Protein level of PEX5 was downregulated by the miR-4274 mimics, especially the deletion type, and upregulated by the miR-4274 inhibitor. NC, NC mimics; miR [del], deletion-type miR-4274 mimics; miR [ins], insertion-type miR-4274 mimics; NC inh, NC inhibitor; miR inh [del], deletion-type miR-4274 inhibitor; miR inh [ins], insertion-type miR-4274 inhibitor; WT, wild-type PEX5 3′UTR; MUT, mutant PEX5 3′UTR.
Fig.3 PEX5 mediates radiosensitivity of CRC. (A) Expression level of PEX5 in the CPTAC across cancers. Breast, breast cancer; colon, colon cancer; liver, liver cancer; ccRCC, clear cell renal cell carcinoma; UCEC, uterine corpus endometrial carcinoma; lung, lung cancer; PAAD, pancreatic adenocarcinoma, N, normal tissues ; T, tumor tissues. The numbers in brackets represent the sample size. (B) Expression level of PEX5 in the different stages in colon cancer from CPTAC. (C) Kaplan–Meier estimates of survival time by expression of PEX5 from TCGA-READ, n = 166. (D, E, H, I) Silencing PEX5 expression by CRISPR/Cas9 in HCT116 and HCT8 cells significantly decreased sensitivity of cells to IR treatment. (D, E) shows proliferation curves of cells. (H, I) shows fractions of cell survival by limiting dilution assays of HCT116 (H) and HCT8 (I) cells. (F, G) PEX5 overexpression significantly increased sensitivity of cells to IR treatment. Data are mean ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001 of Student’s t-test. KO#1, PEX5-KO#1; KO#2, PEX5-KO#2; OE, PEX5-OE.
Fig.4 PEX5 is associated with response to radiation in CRC cells. (A) Volcano plot displays the 429 upregulated genes (fold change > 1; P < 0.05) and 298 downregulated genes (0 < fold change < 1; P < 0.05) in PEX5-KO cells. (B) Metascape gene enrichment analysis of the 429 upregulated genes (up) and 298 downregulated genes (down). (C–E) The bubble plots shows the specific genes response to radiation pathways (C), positive regulation of cell migration (D), and in wound healing (E). (F) The heatmap shows the q-RT PCR results of HCT116 cells treated with IR. (G) The images of comet assays showing knockdown of PEX5 significantly diminished DNA damage in HCT116 cells, which could be reversed by overexpression of PEX5. Scale bar, 100 µm. (H) Bar charts show the statistics of tail moments in comet assays. Data are mean ± SEM from 3 replicate experiments and 10 fields were randomly selected for each experiment. ****, P < 0.0001 of Student’s t-test. KO#1, PEX5-KO#1; KO#2, PEX5-KO#2; OE, PEX5-OE.
Fig.5 PEX5 participates in Ku-mediated DNA NHEJ repair. (A) Immunofluorescence analysis of PEX5 and Ku70 co-staining in HCT116 and HCT8 cells. Scale bar, 100 µm. (B) Western blot analysis of PEX5 and Ku70 in HCT116. Cell lysates were immunoprecipitated with antibody against PEX5 or IgG. (C) Immunofluorescence analysis of Ku70 co-staining in HCT116 cells with PEX5-OE or PEX5-KO treated with or without IR. Scale bar, 100 µm. (D) Western blot analysis of Ku70, Ku80 and γ-H2AX levels in HCT116 and HCT8 cells with PEX5 OE or PEX5-KO. (E) Western blot analysis of PEX5, Ku70 and Ku80 in HCT116 cells with PEX5 OE or PEX5-KO. Cell lysates were separated from cytoplasm and nucleus with PEX5-KO and PEX5-OE. The blue and red bands are protein markers. KO#1, PEX5-KO#1; KO#2, PEX5-KO#2; OE, PEX5-OE.
Fig.6 PEX5 participates in the IR-reduced ferroptosis. (A) The bubble plot shows the different regulation genes related to ferroptosis. (B) The viability ratio of 3 different inhibitors cell death program inhibitors, Ferrostatin-1 (Fer-1, 4 µM), Necrostatin-1 (Nec-1, 0.5 µM) and Z-VAD(OMe)-FMK (Z-vad, 10 µM) in HCT116 PEX5-OE cells. (C) Western blotting analysis of PEX5, ACSL4, SLC7A11 and GPX4 in HCT116 and HCT8 cells with PEX5 KO or OE. (D, E) HCT116 (D) and HCT8 (E) cells were treated with or without IR, followed by detecting the concentration of MDA. MDA levels were expressed as µM. Data are mean ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001 of Student’s t-test. KO#1, PEX5-KO#1; KO#2, PEX5-KO#2; OE, PEX5-OE.
Fig.7 The effects of miR-4274 and PEX5 on CRC progression and radiosensitivity in vivo. (A, B) The effects of miR-4274 on the volumes of CRC tumors, n = 3. NC, NC ago-mimics; miR [del], deletion-type miR-4274 ago-mimics; miR [ins], insertion-type miR-4274 ago-mimics. (C, D) The effects of PEX5-KO on the volumes of CRC tumors, n = 3. *, P < 0.05; **, P < 0.01 of Student’s t-test at the last measurement. (E) The effects of miR-4274 on the weights of CRC tumors, n = 3. NC, NC ago-mimics; miR [del], deletion-type miR-4274 ago-mimics; miR [ins], insertion-type miR-4274 ago-mimics. (F) The effects of PEX5-KO on the weights of CRC tumors, n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001 and ****, P < 0.0001 of Student’s t-test. (G) The schematic illustration for the possible mechanisms of miR-4274 rs1553867776 regulates PEX5 expression and PEX5-mediated radioresistance in CRC.
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