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Radiation-induced pulmonary fibrosis (RIPF) is one of the most serious late complications after nuclear radiation accident, bone marrow transplantation pretreatment and thoracic tumor radiotherapy. The formation process of RIPF is complicated and the pathogenesis has not been fully elucidated. Recent studies have shown that radiation-induced epithelial-mesenchymal transition (EMT) of lung epithelial cells is an indispensable segment of RIPF. This article reviews the role of radiation-induced lung EMT in the occurrence and development of RIPF and related drugs with EMT as a potential therapeutic target, providing ideas for the development of therapeutic drugs for RIPF in the future.
ABSTRACT
Radiation induced lung injury (RILI) is a common complication after radiation therapy of breast tumors and bone marrow transplantation pretreatment, and it is a critical limiting factor of radiotherapy doses in patients. Once RILI progresses to the radiation-induced pulmonary fibrosis stage, it seriously reduces the patient’s quality of life, while causing the patient’s respiratory failure and eventually leading to death. Ionizing radiation (IR) can induce cell injuries, including apoptosis, epithelial-mesenchymal transition, senescence, pyroptosis and ferroptosis, and these injuries can play an important role in the occurrence and development of radioactive lung injury. Starting from discussion of the occurrence of different forms of injury in different cells after IR stimulation, this review summarizes the pathogenesis of RILI and its clinical prevention and treatment.
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Objective To observe the effect of PIF1 knockdown on cell growth and cell cycle arrest induced by ionizing radiation.Methods HeLa cell lines that consistently down-regulated PIF1 were prepared by the lentivirus granules interfering technology and confirmed by real-time PCR and Western blotting.The effect of down-regulation of PIF1 on cell growth and cell cycle arrest induced by ionizing radiation was evaluated by cell counting and flow cytometry.Results HeLa cell lines consistently down-regulating PIF1 were established.The growth of HeLa that down-regulated PIF1 was inhibited greatly after 4 Gy of γ-ray irradiation.There was little cell proliferation until the 5th day post 4 Gy γ-ray.Moreover, the S phase block and G2/M phase block of PIF1 knock-downed cell lines were significantly delayed after 8 Gy γ-ray irradiation.Conclusion Knockdown of PIF1 can significantly enhance the radiation sensitivity and delayes the S phase block and G 2 /M phase block induced by ionizing radiation.
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Objective To construct the recombinant eukaryotic expression plasmids of human adenylyl cyclase-associated protein 1 (CAP1)and to explore its intracellular location and functions.Methods By using Hela cDNA as the template,the cDNAs encoding CAP1 was amplified by PCR and inserted into pCMV-Myc vector to construct the recombinant plasmid.The recombinant plasmid was transfected into 293 cells using lipofectamine 2000.The protein expression and the intracellular location of the inserted gene were confirmed by Western blotting and immunofluorescence,respectively.Scratch-repair experiment was used to detect the cancer cells’ migration ability.Results The recombinant eukaryotic expression plasmid of human CAP1 was successfully constructed and transfected into eukaryote cells.The recombinant plasmid was successfully expressed in eukaryote cells.CAP1 was located in the cytoplasm.The results of scratch-repair experiment showed that the overexpression of CAP1 could significantly inhibit the cells’ migration.Conclusion CAP1 recombinant plasmid was successfully expressed in eukaryotic cells.CAP1 protein was located in the cytoplasm.The overexpression of CAP1 inhibited cell migration. The present study provides important experimental evidence for further study on CAP1.
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Objective To construct recombinant plasmids of SIK2 cDNA and its truncated mutants and induce its expression in E.coil.Methods We designed primers of SIK2 and its truncated mutants.The gene fragments of SIK2,SIK2-Δ1 (280-926),SIK2-Δ2 (400-926),SIK2-Δ3 (1-400),and SIK2-Δ4 (700-926)were amplified by polymerase chain reaction (PCR)and cloned into pGEX-4T-2 vector to construct recombinant plasmids with GST. The plasmids were transformed into E.coil BL2 1 respectively,and induced with IPTG to express fusion protein. The results were confirmed by Coomassie blue staining and Western blot.Results We successfully constructed recombinant plasmid of SIK2 cDNA and its truncated mutants.Coomassie blue staining and Western blot resutls showed that these plasmids were induced to be expressed in E.coil BL21.Conclusion SIK2 cDNA and its truncated mutants were overexpressed in E.coil BL2 1 ,which lays expereimental foundation for further study on the function of each domain of SIK2 .
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Objective To investigate the effect of salt-induced kinase 2 (SIK2) in the G2/M checkpoint in response to ionizing radiation and the possible mechanism.Methods HeLa cells were irradiated with 60Co γ-rays.The cell model of knockdown SIK2 expression was constrcuted by transfecting HeLa cells with a pSicoR-based lentivirus vector of expressing SIK2 shRNA by lipofectamin 2000.Western blot and flow cytometry were performed to measure the changes of SIK2 protein level and cell cycle distribution.The phosphorylated histone protein H3 on Ser 10 was used as a molecular marker of mitotic cells for detecting the function of G2/M checkpoint.Results The expression level of SIK2 protein increased in HeLa cells after 60Co γ-ray irradiation.A cell model of knockdown SIK2 expression was successfully generated by transfecting the specific shRNA against SIK2.Depression of SIK2 significantly increased the cellular sensitivity at 1,2,4,6 Gy post-irradiation (t =-3.445,-2.581,-3.251,-2.553,P ＜0.05),and led cells to release earlier from the G2/M boundary arrest compared to control cells at 5,6 h post-irradiation (t =4.341,6.500,P ＜ 0.05).Western blot analysis indicated that the irradiation-induced phosphorylated CHK2/T68 in SIK2 knock-down cells was earlier than that in control cells.Conclusions salt-induced kinase 2 (SIK2) participates in the regulation of G2/M checkpoint induced by ionizing radiation and affects cellular radiosensitivity.
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Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4 degrees C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operates in the cell.
Subject(s)
Humans , DNA Helicases , Genetics , Metabolism , HeLa Cells , RNA, Transfer , Genetics , Recombinant Proteins , Genetics , MetabolismABSTRACT
Objective To investigate the expression profile of human genes in response to acute sodium arsenite treatment by cDNA microarray. Methods The RNA was purified from the L-02 cells without and with arsenite sodium induction for 2 hours, 15 hours and 24 hours, respectively. Results The hybridization patterns were different between every interval of arsenite induction. Expression of hCYR61 increased after 2 hours' induction, but decreased after 15 hours and 24 hours. Expression of metallothionein Ⅳ and Ⅲ elevated at the whole induction phase. HSP86 was up-regulated after 15 hours and 24 hours' induction, but it did not alter at two hours' induction. Conclusion When exposed to arsenite, the cells are under a meet-an-emergency situation to synthesize the most necessary protein and inhibit synthesis of unessential proteins.
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Objective To elucidate physiological functions of human PIF1 helicase at the molecular level,purify N-terminal truncated PIF1 helicase,PIF1△N,and assay its biochemical properties.Methods The N-terminal cDNA sequence of PIF1 helicase was amplified by PCR using the Hela cell cDNA library as template.The cDNA with a histidine tag at the N-terminus was inserted into the pET20b vector to produce recombinant plasmid.The recombinant PIF1△N was successfully expressed by co-transforming a plasmid encoding rare rRNA.At 4 ℃ through a series of affinity column the recombinant PIF1△N protein was purified by fast protein liquid chromatograph.The biochemical activity of PIF1△N was assayed.Results The cDNA fragment of human PIF1 from 540～1 923 was cloned from Hela cDNA library,and the recombinant PIF1△N protein was successfully overexpressed in E.coli.The purification procedure of PIF1△N protein was established and its biochemical activity was identified.Conclusion N-terminal truncated PIF1 helicase,PIF1△N,has ATPase activity,which is DNA and Mg2+ dependent.