| Background and purposesRadiation-induced pulmonary fibrosis（RIPF）is one of the common complications in patients receiving radiation therapy for thoracic tumors and those exposed to high doses of nuclear radiation accidents.The main symptoms are limited physical activity,shortness of breath,decreased blood oxygen saturation and respiratory failure.Currently,there is no effective treatment other than living donor lung transplantation.The pathogenic mechanisms of RIPF are complex,and the dysregulation of tissue damage repair in type II alveolar epithelial cells（AT2）is the main pathogenic mechanism.Radiation exposure leads to damage in AT2 cells,which in turn results in the secretion of a significant quantity of cytokines,inflammatory factors,and pro-fibrotic factors.The production of numerous fibroblasts occurs,which then transform into myofibroblasts,ultimately leading to the deposition of collagen fibers and extracellular matrix（ECM）.This process ultimately culminates in the formation of tissue fibrosis.The latest research confirms that fibroblasts in RIPF lesions are mainly derived from epithelial mesenchymal transition（EMT）of lung epithelial cells,and EMT of AT2 cells is one of the main sources,the damage of AT2 cells and the occurrence of EMT play a key role in the pathogenesis of pulmonary fibrosis.micro RNA-21（miR-21）is one of the first discovered micro RNAs,located at the vacuolar membrane protein 1（VMP1）locus on chromosome 17.Studies have shown that miR-21 is involved in radiation-induced EMT.Meanwhile,miR-21 expression was increased in the serum and alveolar lavage fluid of clinical patients with pulmonary fibrosis,suggesting that miR-21 may be an important regulator affecting the process of pulmonary fibrosis.However,the downstream molecules of miR-21 and the signaling pathways involved in the regulation of EMT have yet to be confirmed.Therefore,this study aims to explore the biological function of miR-21 in the formation of RIPF.And screen the differentially expressed molecules downstream of miR-21 to elucidate its mechanism of radiation-induced EMT in AT2 cells.Methods1.To evaluate the role of miR-21 in RIPF（1）Establishment of RIPF animal model:The chest of 6-week-old male C57BL/6wild-type（WT）mice was irradiated with a single dose of 15 Gy of cobalt sourceγ-rays to establish the RIPF model.Before modeling（F0,n=50）,2 weeks（F2,n=15）,8 weeks（F8,n=30）,and 16 weeks（F16,n=30）after modeling,through visual observation,living lung function test,chest Micro-CT examination and lung histopathological analysis to evaluate the RIPF animal model.（2）RT-q PCR detection of miR-21 expression in lung tissue of RIPF animal model and radiation-treated rat AT2 cell line（RLE-6TN）.（3）The RIPF model of miR-21-/-mice was constructed.RT2Profiler PCR Array（QIAGEN）and RT-q PCR were used to analyze the differentially expressed genes in the lung tissues of miR-21-/-mice and WT mice after 2 weeks irradiation.A dual-luciferase reporter gene was used to verify the direct interaction between miR-21 and the differential expression molecules.（4）RT-q PCR and WB were used to detect the expression of CREBL2 in lung tissue of RIPF animal model and radiation-induced RLE-6TN cells.（5）The effect of knocking out the miR-21gene on pulmonary fibrosis in a mouse RIPF model was assessed by immunohistochemical（IHC）staining for collagen I（Col-1）and the method described in（1）.2.Effect of miR-21/CREBL2 on radiation-induced EMT in AT2 cells（1）Establishment of the RLE-6TN cells irradiation model:RLE-6TN cells were irradiated with cobalt sourceγ-rays when they were in the logarithmic growth phase,with a total dose of 8 Gy and a dose rate of 0.65 Gy/min.The epithelial cell cadherin（E-cadherin）,mesenchymal cell cadherin（N-cadherin）and fibroblast-specific protein-1（FSP-1）were detected by RT-q PCR,WB and cell immunofluorescence staining after 48 h of irradiation.（2）RT-q PCR detected the effects of radiation on the expression of E-cadherin,N-cadherin,and FSP-1 in RLE-6TN cells transfected with miR-21 mimic or inhibitor.（3）RT-q PCR and WB detected the expressions of E-cadherin and N-cadherin in RLE-6TN cells induced by radiation after overexpression or inhibition of CREBL2.3.miR-21/CREBL2-mediated endoplasmic reticulum stress regulates the mechanism of radiation-induced EMT.（1）IHC staining was used to detect endoplasmic reticulum stress（ERS）marker molecule glucose regulated protein 78（GRP78）and DNA-damage inducible transcription factor 3（CHOP）in lung tissue of RIPF animal model.（2）Fluo-4AM,RT-q PCR and WB were used to detect the calcium ion concentration and the expression of CHOP in RLE-6TN cells induced by radiation.（3）RT-q PCR and WB detected the expression of CHOP in RLE-6TN cells induced by radiation after overexpression or inhibition of CREBL2.（4）RT-q PCR and IHC staining were used to detect the expression of CHOP in lung tissue of RIPF animal model.（5）RT-q PCR detected the expression of E-cadherin,N-cadherin andα-smooth muscle actin（α-SMA）in RLE-6TN cells induced by radiation after overexpression or inhibition of CHOP.（6）Evaluation of the effects of 4-Phenylbutyric acid sodium（4-PBA）intraperitoneally injected continuously for 4 weeks on the pulmonary fibrosis of RIPF animal model through in lung function test,hydroxyproline detection,and lung histopathological analysis.Results1.miR-21 promotes the formation of RIPF（1）After 16 weeks of irradiation,the chest hair of WT mice became white and less,and the lung tissue was reddish brown with scattered white nodules on the surface;pulmonary function,such as respiratory system resistance,tissue energy attenuation,small airway resistance and other indicators reflecting tissue damage increased significantly;Micro-CT scan showing lung inhomogeneity,patchy shadows,and cellular changes;pathological staining showed a large number of alveolar atresia and alveolar septal collagen fiber deposition in lung tissue.These results indicate that the RIPF animal model was successfully established.（2）The expression of miR-21 was upregulated in the RIPF animal model,and the expression of miR-21 was also upregulated in RLE-6TN cells after irradiation.（3）Gene chip screening and RT-q PCR showed that CREBL2 was negatively correlated with miR-21,and dual luciferase assay confirmed that miR-21 directly acts on the CREBL2-3’UTR region.（4）CREBL2 is downregulated in RLE-6TN cells and WT mouse irradiation models,while upregulated in lung tissue of miR-21 knockout mice.（5）After 16 weeks of irradiation,miR-21-/-mice had more chest hair and pink lung tissue than WT mice;Micro-CT examination showed increased lung tissue density,and CT 3D reconstruction showed no tissue defect;the degree of lung function damage was significantly smaller than that of WT mice;the lung coefficient was not significantly increased,and a small amount of hydroxyproline and Col-1 were deposited in the lung tissue.The above results indicate that knockout of miR-21 gene significantly alleviated RIPF in mice.2.miR-21/CREBL2 activates radiation-induced EMT in AT2 cells（1）Observation by the microscope revealed that the number of RLE-6TN cells decreased after irradiation,and the morphology changed from cobblestone irregular epithelial cells to elongated or spindle cells;WB detection and immunofluorescence staining showed that E-cadherin was downregulated and upregulated in N-cadherin and FSP-1 at 48h after irradiation of RLE-6TN cells.（2）Transfection of miR-21 inhibitor,RT-q PCR detection found that the expression of E-cadherin was upregulated,and the N-cadherin and FSP-1 were downregulated after irradiation in RLE-6TN cells.Conversely,transfection with miR-21 mimic resulted in downregulation of E-cadherin expression and upregulation of N-cadherin and FSP-1 expression in RLE-6TN cells after irradiation.（3）After transfection of the CREBL2 overexpressing plasmid,the expression of E-cadherin was upregulated in RLE-6TN cells after irradiation,and the expression of N-cadherin and FSP-1 were downregulated.Inhibition of the CREBL2 expression downregulated E-cadherin and upregulation of N-cadherin and FSP-1 after irradiation of RLE-6TN cells.In summary,miR-21/CREBL2 promotes radiation-induced EMT in AT2 cells.3.The miR-21/CREBL2 promotes radiation-induced AT2 cell transformation to mesenchymal cells by activating ERS（1）IHC staining showed that the expression of GRP78 and CHOP in the lung tissue of RIPF animal model increased with the increase of irradiation time.（2）The intracellular calcium ion concentration in RLE-6TN cells significantly increased after irradiation,and the expression of CHOP was upregulated.（3）After transfection with CREBL2 overexpression plasmid,CHOP expression in RLE-6TN cells was downregulated after irradiation;conversely,inhibition of CREBL2 upregulated CHOP expression in RLE-6TN cells after irradiation.（4）Through RT-q PCR and IHC staining,it was found that the expression of CHOP in the lung tissue of WT mice in the RIPF animal model was significantly higher than that of miR-21-/-mice.（5）After transfection with CHOP overexpression plasmid,the expression of E-cadherin was downregulated and the expression of N-cadherin andα-SMA were upregulated in RLE-6TN cells after irradiation.Inhibition of CHOP expression resulted in an upregulation of E-cadherin expression and downregulation of N-cadherin andα-SMA expression in RLE-6TN cells after irradiation.（6）The 4-PBA group showed significantly better lung function indexes such as morphological constant,deep inspiratory volume,and static compliance in the RIPF animal model after 16 weeks of irradiation compared to the saline group;and the content of hydroxyproline and collagen fibers were lower than the saline group.These results suggest that miR-21/CREBL2 promotes radiation induced EMT in AT2 cells through ERS and mediates the formation of RIPF.ConclusionThis study revealed that miR-21 is involved in the pathological process of RIPF.A novel target gene of miR-21,CREBL2,was discovered.To reveal the regulatory mechanism of miR-21/CREBL2 through enhancing the ERS signaling pathway-induced EMT,and provide a potential target for the prevention and treatment of RIPF. |
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