Identification of Itgb1 as a critical gene for radiation-induced injury by bioinformatic analysis combined with experimental verification

doi:10.21203/rs.3.rs-2983643/v1

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Identification of Itgb1 as a critical gene for radiation-induced injury by bioinformatic analysis combined with experimental verification

https://doi.org/10.21203/rs.3.rs-2983643/v1

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Radiation injury is a common side effect of nuclear and radiation accidents as well as clinical oncologic radiotherapy. The organism undergoes a series of pathological responses after irradiation, especially in the short term, accompanied by an intense inflammatory storm [1], and effective targets for intervention have not been identified [2]. In this study, we screened differential genes in gene microarray data from the GEO database and then identified the core gene Itgb1 by enrichment analysis. Subsequently, the expression of Itgb1 was knocked down by siRNA interference and was functionally blocked by RGD, a Itgb1 inhibitor. Next, the proliferation and apoptosis of irradiated cells was detected, and injury of lung tissues and hemopoietic system were also investigated. As a result, knockdown of Itgb1 protected pulmonary epithelial cells and blood cell from irradiation, and RGD remitted the irradiation-induced lung injury and hematopoietic injury. This study suggests that Itgb1 plays a key role in radiation injury and provides new ideas for the prevention and treatment of radiation therapy.

Itgb1

irradiation (IR)

radiation injury

Acute radiation damage occurs when the human body is exposed to high doses of ionizing radiation, and in 50% of cancer treatments, most patients suffer varying degrees of damage from radiation therapy[3]. The most common injuries including hematopoietic failure and radiation-induced lung injury (RILI) usually lead to a large number of deaths and a decline in the quality of life of patients.

In the process of radiation damage generation, abundant free radicals are generated from water molecules in a very short time. At the same time, the ion beam emitted by the radioactive source can disrupt the structure of genes, leading to the breakage of DNA chains linked by phosphodiester bonds, denaturation of base molecules, DNA-protein cross-linking, etc[4]. The cells are affected in subsequent transcription and translation processes if the radiation dose exceeds the body's repair capacity[5].

Radioactive injury caused by ionizing radiation is particularly important in nuclear radiation accidents and in radiotherapy techniques that are widely used today[6]. However, the molecular mechanism of radiation damage is not fully understood.

Currently, bioinformatics gene chip technology and data integration analysis have been used in the research on the pathogenesis of radiation injury. In this study, we used bioinformatics analysis methods to perform integrated analysis on the GEO database high-throughput data set, so as to screened differentially expressed genes between irradiated tissues and normal groups, and further identify the critical genes.

Finally, we verified that Itgb1 mediates radiation injury in body tissues as an involved radiosensitive gene.

Cell culture

The murine-derived alveolar epithelial cell line (MLE-12-12) and the human-derived chronic myeloid leukemia cell line (K562) were obtained from Biobw. MLE-12-12 and K562 cells were cultured in high-sugar DMEM medium and 1640 medium (basic media.China) with 10% fetal bovine serum (BioInd, Israel), at 5% CO₂ at 37°C. MLE-12-12 and K562 cells were exposed to a total dose of 8 Gy irradiation, conducted at a dose rate of 1.2 Gy/min. The radiation source was ⁶⁰Co cobalt (Naval Medical University. China). The mice were C57BL/c males, aged 6 weeks, and given a total body dose of 8 Gy.

Animals and Treatments

Six to eight weeks old male C57BL/c were obtained from Shanghai Jihui Experimental Animal Breeding Co(Shanghai, China). C57 received food and water at room temperature under a circadian rhythm of 12h. Mice irradiated whole body at a rate of 1.2 Gy/min under a ⁶⁰Co cobalt radiation source in a plastic fixation box. The ethics committee of the Naval Medical University approved the animal experiments.

HE staining

The lung of mice were fixed in 4% paraformaldehyde for 24h, then paraffin-embedded [7–9].They were stained with hematoxylin and eosin dyes, then treated with anhydrous ethanol for dehydration, microscopic observation, and photography. The alveolar septum was measured using Image J software.

siRNA transfection

siRNAs were obtained from Jiman Biotechnology Co., Ltd (Shanghai, China), and two siRNAs were targeted to the whole transcriptome of Itgb1 from different species. The transfection reagent lipofectamine 3000 (Thermofisher, USA) was used according to the manufacturer's recommendation, and the final concentration of siRNA was 100 nm[10, 11]. Efficiency of gene knockout were verified by qRT-PCR at 24h and Western blotting at 48h after transfection.

Apoptosis assay and cell proliferation assay

The apoptosis assay kit was obtained from TransGen Biotech (Beijing, China). The medium supernatant was collected together with the cells according to protocol guidelines, centrifuged and resuspended, and Blinding Buffer, Annexin V, and PI dye were added [12–14] .After staining, assays were performed using a flow cytometer CytoFLEX (Biomek, USA). Cell proliferation assay kits were obtained from Beyotime (Shanghai, China), and cells were incubated sequentially in EDU working solution, Click reaction solution, and finally on flow cytometer CytoFLEX (Biomek, USA) for detection.

Western Blotting

MLE-12-12 cells and lung tissue were lysed in 1.5 ml EP tubes by RIPA lysis solution (Beyotime, China), while phosphatase and protease inhibitors were added and protein concentrations were measured using a NanoDrop spectrophotometer (Thermofisher, USA). Proteins were transferred from SDS-PAGE gels onto PDVF membranes (Millipore). Next, they were sequentially incubated in 5% skim milk and primary antibody dilutions, and secondary antibody dilutions and finally exposed to Immobilon Western HRP luminescent substrate Western HRP (Millipore, USA) using a chemical imaging system [15–17]. Tublin (ab179513), NLRP3 (ab214185), Itgb1 (ab179471) were purchased from Abcam, Bax (50599-2-lg) was purchased from proteintech, Bcl-2 (A19693), HRP Goat Anti-Rabbit IgG (H + L) (AS014), Caspase-9 (A0281), AIF (A19536), were purchased from Abclonal. Cleaved-Caspase-3 (Asp175) was purchased from CST.

qRT-PCR

A total RNA extraction kit was obtained from Solarbio (Beijing, China), RNA was reverse transcribed into cDNA by RR036A (Takara) kit, and finally, gene expression was analyzed by RR420A [18–20]. Primer sequences: Itgb1 Forward: 5-GTTTTGTAGGAAGAGGGATAA-3, Reverse: 5-CCAGTGTAGTTGGGGTTG-3, GAPDH Forward: 5-CAGGAGGCATTGCTGATGAT-3, Reverse: 5- GAAGGCTGGGGCTCATTT-3.

Statistical analysis

Differential genes were screened according to adj.P.Value < 0.01, |logF|>1.The data was implemented in GraphPad Prism software, using GraphPad Prism version 8.0.2. All data were expressed as mean ± SEM and statistical analysis was performed using one-way ANOVA for multiple-group comparison. The data were visualized in Hiplot online software.

Itgb1 is screened as critical gene by bioinformatic analysis

To explore radiation damage-related genes, two datasets, GSE52690 and GSE104121, were searched from the GEO database; differential genes were derived using GEO2R analysis. The volcano maps of GSE52690 and GSE104121 differential genes before and after irradiation were compared (Fig. 1A,B). Finally, 390 differential genes of GSE52690 and 504 differential genes of GSE104121 were screened.

The DAVID online database was annotated for functional analysis of two sets of differential genes, GSE52690 and GSE104121, which was used as GO enrichment analysis to obtain the enrichment analysis graph (Fig. 1C,D). Protein binding and metal ion binding were the MF terms with the highest enrichment of differential genes in both datasets. The most enriched MF terms were cytoplasm and membrane. KEGG enrichment analysis identifies 7 pathways associated with differential genes (Fig. 1E,F). The differential genes obtained from the two datasets GSE52690 and GSE104121 were processed in Hiplot online software, and a total of 21 differential genes were obtained by using the Wayne diagram module Adcy9, Gsk3b, Trim8, Tbc1d22a, Coro1c, Itga6, Cd44, Ywhag, Hpse, Itgb1, Hpcal1, Ptgs1, Ecm1, Dapk1, Exoc4, Capns1, Prr5l, Ctsd, Tmbim1, Ltbp1, Phlda3 (Fig. 1G). Protein interaction maps were generated in STITCH (Fig. 1H) and Hubba genes were obtained using Cytoscape software (Fig. 1I). By the analysis of interaction maps, the Itgb1 was identified as Hub genes that occupied the highest score.

Expression of Itgb1 after irradiation

To verify the changes in Itgb1 protein levels after irradiation, two cells line, the murine-derived alveolar epithelial cell line (MLE-12-12) and the human-derived chronic myeloid leukemia cell line (K562) for their high radiation sensitivity.

In the irradiated group compared with the control group, the protein expression level of Itgb1 was reduced after irradiation, as evidenced by the protein expression at two time points of IR 12h and 24h, with no significant difference at 12h, but reduced expression at 24h (Fig. 2A). Interestingly, in the MLE-12 cell line, we found that expression was increased at 24h (Fig. 2B).

To further understand the involvement of Itgb1 in the regulation of radiation damage, we used small interfering RNA to generate RNAi effects to knock down Itgb1 expression in K562 and MLE-12, and administration of small interfering RNA treatment produced a significant knockdown effect at the transcriptional level (Fig. 2C, D). It was further demonstrated that the knockdown of Itgb1 resulted in RNAi effects at both the transcriptional and protein levels in MLE-12 cell lines (Fig. 2E, F).

Knockdown of Itgb1 aggravates injury induced by irradiation

Under 8Gy irradiation, the IR group showed a significant decrease in proliferation rate compared to the IR + si group (Fig. 3B,F).The effect of Itgb1 on irradiation damage was analyzed using cell proliferation assays. Under 8Gy irradiation, for K562, the IR group showed a significant decrease in proliferation rate compared with the Blank group, and the proliferation rate in the IR + si group was significantly different from the IR group, suggesting that Itgb1 is involved in the proliferation damage effect induced by irradiation, and then Itgb1 may mediate the repair of cellular DNA damage after irradiation (Fig. 3A,B). We reached the same conclusion in the K562 cell line (Fig. 3E,F).

For K562, there were no differences between the blank and NC groups; the rate of apoptosis was significantly increased in the IR + si group compared to the NC and IR + NC groups (Fig. 3C,D). In MLE-12 cells, the apoptosis rate was similar between the Blank and NC groups; the rate of apoptosis was significantly increased in the IR + Si group compared with the IR + NC group (Fig. 3E,F). Above results suggested that Knockdown of Itgb1 aggravates injury induced by irradiation.

Itgb1-mediated radiation injury in the lung and hematopoietic system

Next, the role of Itgb1 in radiation injury in vivo was investigated. RGD, the Itgb1 inhibitor was used. The result of H&E staining showed that without irradiation, no significant alveolar septal thickening between PBS group and RGD group (Fig. 4A, B). 3 days after of irradiation, inflammatory cell infiltration and increased alveolar septal thickness were clearly seen in H&E staining of lungs in the IR + PBS group (Fig. 4A). However, we found that RGD promoted inflammatory cell infiltration and was accompanied by alveolar septal thickening (Fig. 4B).

Therefore, we wanted to further investigate the effect of Itgb1 on the hematological system, including erythrocytes and leukocytes as well as platelets, after irradiation damage. By blood test, we found that irradiation could significantly reduce WBC, RBC and PLT.

Interestingly, mature erythrocytes without nuclei were insensitive to the effects of radiation damage, but the total number of erythrocytes was reduced in the IR + RGD group compared to IR + PBS (Fig. 4D). The leukocyte assay showed that irradiation had a strong leukocidal effect on leukocytes in addition to RGD, which further deepened the damage (Fig. 4C). The platelet assay results showed that all three groups were in the normal range except for the IR + RGD group (Fig. 4E). This tentatively suggests that Itgb1 is involved in the effects on the hematopoietic system under short-term high-dose irradiation.

In previous studies, Itgb1 was highly expressed in glioma, osteosarcoma, and bone marrow mesenchymal stem cells.Itgb1 is a member of the ITG family [21, 22], which is closely associated with apoptosis. Itgb1 enhances the radiation resistance of non-small cell lung cancer cells by regulating the DNA damage response [23]and YAP1-induced epithelial-mesenchymal transition [24, 25]. Itgb1 mediates the Notch signaling pathway [26, 27], which affects the proliferation of glioma cells, and molecular DNA mutations in the Notch signaling pathway are highly correlated with malignant tumorigenesis [28, 29]. Overexpression of Itgb1 promotes the proliferation of glioma cells. Furthermore, Itgb1 expression was downregulated in in vitro and in vivo experiments, and inhibited collagen, fibronectin, and laminin, thereby mediating metastasis and infiltration of colorectal cancer [30].These previous studies reflect the important role of Itgb1 in tumor radiotherapy and radiation injury. Therefore, we wanted to investigate the role of Itgb1 as a gene associated with radiation damage in radiation injury. Radiation causes acute radiation injury disease, and the survival of mice exposed to 7.5 to 9.5 Gy is only 14 days. The results of the present study showed that inhibition of Itgb1 exacerbated radiation-induced apoptosis in in vivo experiments, but interestingly, the deleterious effect of Itgb1 knockdown was not as high as that of MLE-12 in the K562 cell line, which is not known, perhaps due to the radiation resistance of cancer cells [31]. In vitro experiments verified the effect of Itgb1 on radiation damage to the lung and blood system. Here, we used a pharmacological pathway to enhance the effect of Itgb1 on radiation damage, resulting in a deepening of the inflammatory infiltration in C57 lung tissue. Perhaps the differential expression of Itgb1 in different tissues resulted in the most prominent expression of Itgb1 in radiation-injured lung tissue.

In tissues undergoing radiation damage, Itgb1 acts as an important regulatory gene influencing the biological processes that produce radiation damage in the organism. Unfortunately, this study does not describe the detailed biological pathways involved in Itgb1-mediated radiation injury, and the validation of Itgb1 in many other tumor cell lines was not addressed. In future tumor radiosensitization studies, we are combining biomolecules associated with cancer proliferation and metastasis with radiation damage to provide new targets for subsequent tumor radiotherapy and to provide clues for the prevention and treatment of radiation damage.

To date, efforts to develop targeted drugs for sites of ionizing radiation damage, such as NLRP3 inflammasome [32, 33], have yielded little benefit and therapeutic options for clinical treatment have not been studied in detail [34]. However, none of these treatment options has been studied in detail. Therefore, new biomarkers or targets need to be explored to help prevent and treat it.The present study focuses on the biological effects of Itgb1 on the lung tissue and hematological systems of mice after irradiation. We combine bioinformatics databases and in vivo and in vitro experiments to demonstrate the effects of radiation damage and to highlight the potential research value of Itgb1 as a biomolecule involved in radiation damage in the development of disease.

Ethics approval and consent to participate

All animal experiments conformed to the National Institute of Health Guide for the Care and Use of Laboratory Animals' (NIH Publication No. 85-23, National Academy Press, Washington, DC, revised 1996), with the approval of the Laboratory Animal Center of the Second Military Medical University, Shanghai. The approval ID for this study was 20191512.

Consent for publication

Written informed consent for publication was obtained from all participants

Availability of data and material

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests

The authors declare that they have no competing interests

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No competing interests reported.

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Identification of Itgb1 as a critical gene for radiation-induced injury by bioinformatic analysis combined with experimental verification

Status:

Version 1

Abstract

Figures

Introduction

Materials and Methods

Cell culture

Animals and Treatments

HE staining

siRNA transfection

Apoptosis assay and cell proliferation assay

Western Blotting

qRT-PCR

Statistical analysis

Result

Itgb1 is screened as critical gene by bioinformatic analysis

Expression of Itgb1 after irradiation

Knockdown of Itgb1 aggravates injury induced by irradiation

Itgb1-mediated radiation injury in the lung and hematopoietic system

Discussion

Declarations

References

Additional Declarations

Status:

Version 1