All animal study protocols were approved by the Animal Ethical Commission of the Shanghai General Hospital (No. 2020AW067). Female C57BL/6 mice (5-week-old) were purchased from Shanghai SIPPR/BK Laboratory Animal Co. Ltd. According to our previous studies, a single 25 Gy of X-ray elicited from a medical linear accelerator (Varian Clinac IX) was administered to the 1 cm-width of anal area of mice after general anesthesia. The rectal tissues of mice were collected on the 14th day after radiation following cervical dislocation for further research.

The rectal tissue sections were deparaffinized and incubated in a sodium citrate buffer. Following blocking with 10% bovine serum albumin (BSA), the sections were incubated with primary antibodies against cleaved caspase-3 (casp-3) (Cell Signaling Technology, 9661, 1:100) or HIF-1α (Affinity, BF8002, 1:400) overnight at 4°C. The sections were then rinsed with phosphate buffered saline (PBS) and incubated with secondary antibodies (Jackson ImmunoResearch Inc., 111-035-003, 1:100) for 1 h at room temperature. Images of the slides were captured using a microscope (Leica, Germany). The level of hypoxia was evaluated using the immunoreactive score of HIF-1α in nuclei by two pathologists, and an average score was used for the final scores. The intensity scores were as follows, 0, negative; 1, weak; 2, moderate; and 3, strong. The frequencies of positive cells were clarified as follows, 0, negative; 1, 10% positive cells; 2, 11–50% positive cells; 3, 51–80% positive cells; and 4, more than 80% positive cells. Immunoreactive score = intensity score × frequency of positive cells (Xue et al., 2016). An immunoreactive score >8 was considered as high HIF-1α, and ≤8 was considered as low HIF-1α. The degree of cleaved casp-3 was estimated by the number of cleaved casp-3 positive cells per field.

After rinsed with PBS, the cells were treated with 4% paraformaldehyde for 15 min followed by permeabilization with 0.1% Triton X-100 for another 15 min. Then, 1% BSA was used for block before incubation with HIF-1α primary antibody (1:500, 179483, Abcam) overnight at 4°C. The next day, the cells were incubated with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody (1:1000, 150077, Abcam) for 1 h in the dark, after which, the nuclei were stained with DAPI (1:1000, D9542, Sigma).

For the detection of apoptosis, an Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen TM, United States) was used according to the manufacturer’s instructions. Briefly, the cells were digested and resuspended in binding buffer, followed by staining with Annexin V-FITC and PI in turn protected from light. They were then analyzed using an Accuri C6 Flow cytometer (BD Biosciences, United States). The total apoptosis rate was considered to be the sum of the early (lower right area of the scatter diagram) and late (upper right area of the scatter diagram) apoptosis rates.

To isolate EVs, 120 ml of HIEC cell culture supernatant was collected and subjected to sequential centrifugation at 300 × g for 10 min to remove cells, 2000 × g for 10 min to remove dead cells, 10000 × g for 30 min to remove cell debris, and ultracentrifugation at 100000 × g for 70 min at 4°C. The sediments were washed, resuspended in PBS, and subjected to ultracentrifugation at 100000 × g for 70 min at 4°C for final extraction (Beckman Coulter, United States).

To identify the characteristics of EVs, transmission electron microscopy (TEM) was used to observe the morphology of the extract. DiO (Beyotime, C1038, China)-labeled EVs along with DAPI staining were captured under a confocal microscope to define the cellular uptake of EVs by HIEC cells. Specific EV surface markers were examined by Western blotting.

The cells were washed with pre-cooled PBS and lysed with radioimmunoprecipitation assay buffer (Beyotime, China) containing protease and phosphatase inhibitors (Bimake, United States) when the total protein was collected. The nuclear and cytosolic proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0028, China) according the manufactures’ introduction. The protein concentration was determined using a BCA Protein Quantification kit (Thermo, United States). 20 μg of total protein was electrophoresed on a sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel, followed by transfer to polyvinylidene difluoride membranes (Millipore, United States). After blocking with 5% (w/v) BSA in TBST for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The next day, the primary antibodies were eliminated, and the membranes were rinsed in TBST before they were further incubated with secondary antibodies (Cell Signaling Technology, 7074, 7076, 1:3000) at room temperature for 1 h. A chemiluminescent detection system (Tanon, China) was used to visualize the blots. The primary antibodies used were as follows, HIF-1α (Abcam, 179483, 1:1000), Lamin B1 (Proteintech, 66095-1-Ig, 1:10000), a-Tubulin (Sigma, T8203, 1:5000), CD9 (CST, 13403, 1:1000), Alix (Santa Cruz, sc-53540, 1:500), Calnexin (Proteintech, 10427-2-AP, 1:10000), ß-actin (Cell Signaling Technology, 4970, 1:1000), phospho-AKT (p-AKT) (Ser473, Cell Signaling Technology, 4060, 1:1000), and γH2AX (CST, 9718, 1:1000).

MiR-122-5p inhibitor and negative control were synthesized by RiboBio (Guangzhou, China) and were diluted to a final concentration of 200 nM during transfection. Lipofectamine 2000 (Invitrogen, United States) was used to perform the transfection process according to the manufacturer’s instructions.

cDNA was synthetized using a miRcute Plus miRNA First-Strand cDNA Kit (Tiangen, KR201, China). qRT-PCR was conducted using a miRcute Plus miRNA qPCR Kit (Tiangen, FP411, China) on a QuantStudio 6 Flex system (Life Technologies, United States) with U6 chosen to be the reference gene. The 2^−△△Ct method was applied for the calculation of relative expressions of genes. The primers were synthesized by RiboBio (Guangzhou, China) and were listed in Table 1.

The miRNAs were extracted using a mirVana RNA Isolation Kit (Life Technologies, United States). The miRNA microarrays were performed using an Agilent (Santa Clara, United States) human miRNA (8 × 60 K) chip to identify differentially expressed miRNAs between hypoxic and normoxic EVs. Briefly, after quantification and integrity assessment, the total RNA sample was subjected to labeling, microarray hybridization, and washing according to the manufacturer’s protocols. Raw data were obtained using Feature Extraction software (version 10.7.1.1, Agilent Technologies). GeneSpring software (version 13.1, Agilent Technologies) was then applied to complete the standardization and subsequent management of the raw data. The differentially expressed miRNAs were screened under the criterion of a Fold change>2.0 or Fold change < −2.0, and a p value <0.05, and their target genes were predicted using Targetscan, microRNAorg, and PITA databases. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to conduct the functional analysis of these target genes.

All statistical analysis were performed using GraphPad Prism 7 software (La Jolla, United States). Student’s t-test was applied for statistical analysis between two groups. A minimum of three biological replicates were performed, and a value of p < 0.05 was considered statistically significant. The data were showed as the mean ± standard deviation.

In the mouse models of radiation-induced rectal injury with a 25 Gy radiation, adjacent sections of the rectum were separately stained for HIF-1α, TUNEL, and cleaved casp-3, and the degree of each staining was measured as mentioned above. The sections were then divided into high-HIF-1α and low-HIF-1α groups based on their immunoreactive scores for HIF-1α. As shown in Figure 1A, both the extent of TUNEL staining (Figure 1B) and the counts of cleaved casp-3 positive cells per field (Figure 1C) were positively correlated with the expression of HIF-1α in their adjacent sections, indicating that hypoxia may promote injury to surrounding tissues after radiation.

To determine the remote effects of hypoxia on normoxic tissues, HIEC cells were used in vitro. We first cultivated HIEC cells in either normoxic or hypoxic conditions for 24 h. Western blotting showed that the hypoxic sensor HIF-1α had a higher expression in the nuclear fraction under hypoxic conditions compared to the normoxic group (Figure 2A). IF detection also exhibited the translocation of HIF-1α in the nucleus under hypoxia (Figure 2B), indicating that the hypoxic culture conditions were successfully established. The Annexin V-FITC staining assay demonstrated that the apoptosis rate of HIEC cells increased under hypoxic conditions (Figures 2C,D). To further clarify the biological influence of hypoxia, the supernatant of hypoxic HIEC cells was collected and utilized to cultivate normoxic cells. After 48 h of incubation, the pro-apoptotic effect of the hypoxic cell supernatant was confirmed by flow cytometry (Figures 2E,F). Collectively, these data indicate that both hypoxia and hypoxic cell supernatants promote apoptosis in normoxic HIEC cells.

To investigate whether EVs were involved in hypoxia-induced apoptosis, EVs from either normoxic or hypoxic HIEC cells were extracted by ultracentrifugation. As shown by TEM, typical saucer-like structures with a diameter of 50–200 nm were captured (Figure 3A). The levels of EV markers CD9 and Alix along with the endoplasmic reticulum marker Calnexin were detected by Western blotting (Figure 3B). The fluorescent confocal images also showed the uptake of DiO-labeled hypoxic HIEC cell-derived EVs by normoxic HIEC cells (Figure 3C). After confirmation of successful EV extraction, we added normoxic or hypoxic EVs into the medium of normoxic HIEC cells. After 48 h, Annexin V-FITC staining revealed that hypoxic EVs promoted apoptosis in HIEC cells compared to normoxic EVs (Figures 3D,E). In addition, we further evaluated the expression of apoptosis-related DNA damage marker γH2AX and the apoptosis-related AKT pathway. Western blotting showed that the expression of γH2AX increased while that of p-AKT decreased owing to the addition of hypoxic EVs (Figure 3F).

It can be concluded from above that hypoxic HIEC cell-derived EVs promoted injury of normoxic HIEC cells. To further elucidate the molecular mechanism responsible for this, a miRNA microarray assay was conducted. The differential miRNA clustering heat map (Figure 4A) and a volcano plot (Figure 4B) demonstrated that there were nine miRNAs upregulated in hypoxic EVs compared to normoxic EVs (Fold change>2.0 or Fold change < −2.0, and p < 0.05). Then, GO and KEGG analyses were performed to analyze the functions of 136 predicted target genes of these differentially expressed miRNAs. The top-ranked terms are displayed in the bubble charts. Apoptosis related pathways, for example, NF-kappaB and MAPK signaling, were among them (Figures 4C,D).

Based on the microarray assay, miR-122-5p was selected for further research because of its highest fold change among all the upregulated miRNAs (Table 2). To determine the role of miR-122-5p in hypoxic EVs-aggravated apoptosis, HIEC cells were transfected with either miR-122-5p inhibitor or negative control and cultured under hypoxic conditions. qRT-PCR showed that miR-122-5p decreased at 24 h after transfection (Figure 5A). EVs from hypoxic HIEC cells at 24 h after transfection were extracted and used to culture normoxic HIEC cells as previously described. Annexin V-FITC staining showed that the apoptosis rate in HIEC cells treated with hypoxic miR-122-5p inhibiting EVs was significantly lower than that in the control group (Figures 5B,C). In addition, Western blotting also showed an increased expression of p-AKT along with a decreased expression of γH2AX upon treatment with hypoxic miR-122-5p inhibiting EVs compared to the control groups (Figure 5D). These results indicated that EV-derived miR-122-5p secreted by hypoxic HIEC cells promoted apoptosis in normoxic HIEC cells, and perhaps the AKT pathway was involved in this process (Figure 6).