A novel long noncoding RNA SP100-AS1 induces radioresistance of colorectal cancer via sponging miR-622 and stabilizing ATG3

Abstract

Although radiotherapy is an essential modality in the treatment of colorectal cancer (CRC), the incidence of radioresistance remains high clinically. Long noncoding RNAs (lncRNAs) reportedly play critical roles in CRC radioresistance by regulating genes or proteins at the transcriptional or post-translational levels. This study aimed to identify novel lncRNAs involved in radioresistance. We found that SP100-AS1 (lncRNA targeting antisense sequence of SP100 gene) was upregulated in radioresistant CRC patient tissues using RNA-seq analysis. Importantly, knockdown of SP100-AS1 significantly reduced radioresistance, cell proliferation, and tumor formation in vitro and in vivo. Mechanistically, mass spectrometry and bioinformatics analyses were used to identify the interacting proteins and microRNAs of SP100-AS1, respectively. Moreover, SP100-AS1 was found to interact with and stabilize ATG3 protein through the ubiquitination-dependent proteasome pathway. In addition, it could serve as a sponge for miR-622, which targeted ATG3 mRNA and affected autophagic activity. Thus, lncRNA SP100-AS1 could act as a radioresistance factor in CRC patients via RNA sponging and protein stabilizing mechanisms. In conclusion, the present study indicates that SP100-AS1/miR-622/ATG3 axis contributes to radioresistance and autophagic activity in CRC patients, suggesting it has huge prospects as a therapeutic target for improving CRC response to radiation therapy.

Fig. 1. SP100-AS1 was upregulated in CRC and positively related to radiotherapy.

A The expression profile of lncRNA in radiosensitive and radioresistant colorectal cancer tissues. B SP100-AS1 was upregulated in colorectal cancer tissues, as shown by RNA-seq. C The relative expression of SP100-AS1 in colorectal cancer tissues (CRC, n = 44) and normal tissues (Normal, n  =  44). D The relative expression of SP100-AS1 in radiotherapy-sensitive (Sensitive, n = 22) and resistant patients’ colorectal tissues (Resistant, n = 22). E The relative expression of SP100-AS1 in normal human colon mucosal epithelial cell line NCM460 and a series of human colorectal cancer cell lines (n = 3). F The relationship between SP100-AS1 levels and overall survival of colorectal cancer patients, obtained from the GEPIA database. *P < 0.05, **P < 0.01 compared with the indicated group.

Fig. 2. Downregulation of SP100-AS1 exacerbated radio-induced cell death.

A The cellular survival curves of HCT116 following SP100-AS1 knockdown using siRNA. B The cell viability of HCT116 cells at a radiation dose of 4 Gy. C The cellular survival curves of SW480 following SP100-AS1 knockdown. D The cell viability of SW480 following 4 Gy of IR. E The expression of γ-H2AX in HCT116 and SW480 cell lines following SP100-AS1 knockdown and the gray intensity histogram of γ-H2AX expression. F Cell apoptosis was evaluated by flow cytometry, and the apoptotic cell percentage was statistically analyzed. n = 3, *P < 0.05, **P < 0.01 compared with the indicated group.

Fig. 3. SP100-AS1 downregulation reduced the growth of irradiated-CRC cells in vivo.

A SP100-AS1 was stably knocked down in HCT116, and cells were injected subcutaneously into the axilla of nude mice. 2 Gy IR every other day for 10 days was given until the tumors reached 90 mm³. The image of excised tumors is presented. B The growth curves of tumors from (A). C The tumor weight from (A). D The images following TUNEL staining and representation of relative Ki67 expression. Scale bars = 100 μm. n = 5, *P < 0.05, **P < 0.01 compared with the indicated group.

Fig. 4. SP100-AS1 regulated cell proliferation through the autophagy pathway in HCT116 cells.

A HCT116 cells were irradiated at 4 Gy, and relative protein fold change of autophagy-related proteins LC3 and p62 were quantified. B Fluorescence images of HCT116 cells treated in (A) infected with LC3-GFP-RFP overexpressing lentivirus and subjected to 4 Gy radiation. Quantification of average dots per cell of RFP and GFP signals in each cellular condition was presented. Scale bars = 20 μm. n = 3. *P < 0.05, **P < 0.01 compared with the indicated group.

Fig. 5. SP100-AS1 regulation of autophagy was rescued by ATG5 and Beclin1 overexpression.

A HCT116 cells overexpressed ATG5 and Beclin1 following SP100-AS1 knockdown. The relative expression of LC3 was detected. B Histogram representing the apoptosis rates of HCT116 in A measured by flow cytometry. C Fluorescence images of HCT116 cells in A infected with LC3-GFP-RFP lentivirus. The red puncta level per cell was statistically analyzed. D Both HCT116 (left panel) and SW480 (right panel) cells overexpressed ATG5 and Beclin1 following SP100-AS1 knockdown. The cellular survival curve of the indicated cells was presented. Scale bars = 20 μm. n = 3, *P < 0.05, **P < 0.01 compared with the indicated group.

Fig. 6. SP100-AS1 interacted with ATG3 and regulated its protein stability.

A FISH image of SP100-AS1 in HCT116 and SW480 cells. B The localization rate of SP100-AS1 in the cytoplasm (Cyto) and the nucleus (Nucleu) of HCT116. C The localization rate of SP100-AS1 in the cytoplasm (Cyto) and nucleus (Nucleu) of SW480. D Proteins retrieved from the SP100-AS1 pull-down assay were analyzed by mass spectrometric analysis. E RIP assay using anti-ATG3 antibodies showed that ATG3 interacted with SP100-AS1 in HCT116 cells. F The enrichment level of anti-ATG3 antibodies pulling down SP100-AS1. G Western blot analysis of the proteins retrieved from the SP100-AS1 pull-down assay, with its antisense sequence used as a negative control. H The ATG3 expression in HCT116 cells following knockdown or overexpression of SP100-AS1. I The ubiquitination level of ATG3 was analyzed. J HCT116 knockdown SP100-AS1 cells were treated with CHX for the indicated time, and the stability and expression of ATG3 were analyzed. K HCT116 cells used in (H) were treated with MG132, and the expression of ATG3 was analyzed. Scale bars = 50 μm. n = 3, **P < 0.01 compared with the indicated group.

Fig. 7. SP100-AS1 serves as a sponge for miR-622 in CRC.

A RIP assay showed SP100-AS1 interacted with AGO2 in HCT116 cells. B SP100-AS1 interacted with AGO2 in SW480 cells. C RNA pull-down assay was carried out using an SP100-AS1 probe. D The enrichment level of SP100-AS1 and miR-622 using the indicated probes. E Bioinformatic analysis screened for miR-622 interaction with wild-type or mutated SP100-AS1. F RNA pull-down assay for the luciferase activity of SP100-AS1-WT and SP100-AS1-MUT in HCT116 co-transfected with miR-622 mimics. G RNA pull-down assay as in (F) in HCT116 cells co-transfected with anti-miR-622. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 compared with the indicated group.

Fig. 8. SP100-AS1 regulated ATG3 expression by interacting with miR-622.

A The indicated protein expression in HCT116 cells overexpressing miR-622 and SP100-AS1. B The relative protein change in (A). C Bioinformatic analysis screened for miR-622 interaction with wild-type and mutant ATG3 3’UTR. D The luciferase assay of ATG3 3’UTR in HCT116 cells co-transfected with miR-622 and SP100-AS1. E The expression of ATG3 in HCT116 cells co-transfected with miR-622 and SP100-AS1. F The relative expression of ATG3 was represented using a histogram. G The relative mRNA level of ATG3. H Schematic illustration depicting a proposed model of the molecular mechanism of SP100-AS1 in initiating radioresistance in human colorectal cancer. n = 3, *P < 0.05, **P < 0.01 compared with the indicated group.