| Purpose:According to the strategic plan of China space exploration,Chinese space station will be launched and put into routine operation around 2022 and the Chinese landing on moon surface is also under discussion.With the development of space exploration,Chinese astronauts will stay longer and longer in space,and their safety has become an important issue that cannot be avoided.As the two most important space environmental factors threatening the life activities,space radiation and microgravity induce severe effects of on human body.But there is few reports on the combined effects of space radiation and microgravity.In this study,human lung epithelial BEAS-2B cells were treated by simulated space radiation and microgravity,and the changes in malignant phenotypes were observed during continuous subculture.The epithelial-mesenchymal transition(EMT)was investigated in this process.We also tried to clarify whether space radiation and microgravity have synergistic effects in the process of inducing EMT and malignant transformation,and further elucidate the mechanisms underlying the combined effect of these two factors on the induction of EMT.Methods:(1)We used immortalized human lung epithelial cells(BEAS-2B)for the studies.For simulating space radiation environment,we used ?-particles for exposure because of its 14%abundance in the galaxy cosmic rays and relative high ionizing ability.The a source was 241Am radioactive isotope in the School of Radiation Medicine and Protection of Soochow University.Three dose groups of 0.2 Gy,0.4 Gy,and 0.5 Gy were constructed by single exposure.Three multiple exposure groups were set,and the total doses of 0.2 Gy,0.4 Gy,and 0.5 Gy were achieved at a dose rate of 0.02 Gy/time with 10 times,20 times,and 25 times correspondingly.Each irradiation was conducted every three days,and then cells were subcultured once.For comparison,three single irradiation groups with the same total dose and a control group were prepared parallelly.The seven groups of cells were labeled as Ctrl(control group),R1-02(single irradiation of 0.2 Gy),R1-04(single irradiation of 0.4),R1-05(single irradiation of 0.5 Gy),R10-02(multiple irradiation of 0.2 Gy,10 times),R20-04(multiple irradiation of 0.4 Gy,20 times)and R25-05(multiple irradiation of 0.5 Gy,25 times).After the accomplishment of exposures,all cells of the seven groups were subcultured for different passages before submitted for various analysis.Cell proliferation ability was detected by CCK-8,cell migration was detected by cell scratch test,invasion ability was detected by transwell assay,and the proportion of cancer stem-like cells were detected by flow cytometry with two markers,CD 133 and CD44.Western blot and real time quantitative PCR were used to detect the expression of EMT markers.We then treated the cells with protons and heavy ions to study the effect of different particles on EMT.Proton irradiation was performed at the China Institute of Atomic Energy(Beijing Tandem Accelerator Nuclear Physics National Laboratory),while carbon ions and iron ions irradiations were conducted at the HIMAC,National Institute of Radiological Sciences,Japan.Finally,basing on our previous experimental results,Ctrl,R1-05,and R25-05 cells at 50th passage were subcutaneously injected into NOD/SCID mice to test their tumor-forming ability.Finally,we did transcriptomic sequencing to find differential genes in cells with different irradiation patterms and analyzed the functions of these differential genes.(2)To examine the combined effects of space radiation and microgravity,human lung epithelial BEAS-2B cells were first irradiated with 2 Gy X-rays,and then immediately placed on a random position machine for simulated microgravity.The random rotational speed was 0-10 rpm to achieve a simulated microgravity of about 0.1 g.Forty-eight hours later,the cells were removed for further analysis.The experiment was divided into four groups,Ctrl,IR(2 Gy irradiation group),RPM(simulated microgravity group),and IR+RPM(combined group).The Ctrl and IR groups were cultured in the same incubator.The three-dimensional structure of the cells was observed by atomic force microscopy,and the Young's modulus of the cell surface was analyzed.The expression of EMT biomarkers was detected by Western blot and real time quantitative PCR.The changes of cytoskeleton were observed by immunofluorescence.High-speed centrifugation was used to separate F-actin and G-actin,and Western blot to analyze the changes of the proportion of the two after microgravity treatment.Western blot,real time PCR,and immunofluorescence assay were used to detect the expression of the FN1/YAP/F-actin pathway after treated by radiation and microgravity.After knockdown of the Fibronectinl with siRNA,cell proliferation and cell migration ability were detected,and immunofluorescence assay was used to detect the localization of YAP and cytoskeletal protein.Luciferase reporter assay was used to detect the regulation of Fibronectinl on the transcriptional activity of YAP.Results:(1)After the establishment of the seven group cells,the morphology at the 50th passage was found to be different from the 10th passage cells,the difference between the control group and the single-irradiation group was not obvious.In the 10th.passage cells with multiple exposures,cell become elongate,and the cell junctions became less tight.When subcultured to the 50th passage,cells in multiple irradiation groups basically lost their epithelial shape and the cytoplasm elongated,which were especially conspicuous in the R25-05 group.Cell proliferation results showed that when the cells subcultured to the 10th passage,there was no significant difference between the seven groups and subcultured to the 30th passage,proliferation ability in single irradiation of 0.4 Gy and 0.5 Gys and multiple irradiation of 0.5 Gy were increased compared with the control group(P<0.05).However,the proliferation of all irradiated cells at the 50th passage was significantly higher than the control group,and the multi-exposed groups were significantly higher than the single exposure in the same dose.Similar results were obtained with cell migration assay.There was no significant change in the migration ability of the cells at the 10th passage,but the migration ability at the 30th passage cells significantly increased,especially the 0.5 Gy group.And in p50 passage cells,all the six irradiation groups' migration ability was increased compared to the control group(P<0.05),and the migration ability of the cells irradiated to multiple exposures with a total dose of 0.2 Gy and 0.4 Gy was significantly stronger than single irradiation group(p<0.05).The results of cell invasion showed that when the cells were subcultured to 50th passage,the invasive ability in the irradiated groups were significantly increased compared with the control(p<0.05).The proportion of CD133+/CD44-cells were increased in both 10th passage and 50th passage group(p<0.05).We also found that there was no significant change in the protein level of EMT biomarkers at the 10th passage cells after irradiation.However,the protein expression of epithelial cell marker EpCAM was suppressed at the 30th and 50th passage.While the expression of mesenchymal cell markers,Fibronectinl and Vimentin increased in 50th passage cells but not 30th passage cells.And the changes were more significantly in multiple-irradiation groups.Longitudinal comparisons in different passages revealed that EMT began to occur in passage 30 cells.Proton beam,carbon ion beam and iron ion beam differed in the induction of the expression of EMT markers.The mesenchymal cell markers,Vimentin and Fibronectinl,were not sensitive to proton irradiation but increased significantly after heavy ion irradiation.The results of xenografts formation in NOD/SCID mice showed that R25-05 group cells at the 50th passage had stronger tumorigenic ability than R1-05 cells,demonstrated by the larger tumor volume and the more malignant pathology whereas the control group did not have tumorigenic ability.Transcriptomic sequencing showed that there were 2410 genes with differential expression in the R25-05 group compared with Ctrl in p50,of which 1457 were up-regulated and 953 were down-regulated,while compared with Rl-05,458 genes were changed in R25-05 group,221 were up-regulated and 231 down-regulated.The GO enrichment analysis found that among the 458 differentially expressed genes,the plasma membrane,extracellular matrix,and NADP enzyme activity relative genes were significantly changed.The KEGG analysis found that these differentially expressed genes accounted for a high proportion of signal transduction,cancer,and immune diseases.(2)The observation with atomic force microscopy showed that the surface morphology of the cells in the irradiation group did not change significantly;however,the microgravity group cells and the combined treatment group cells lost their smoothness and became rough,the cellular pseudopod number and length increased,suggesting that the cytoskeleton may have changed.Young's modulus analysis showed that the mechanical elasticity of the cells decreased after microgravity treatment,while the simple irradiation group did not change significantly.The Young's modulus of the combined treatment group was even lower than that of the microgravity group.Western-blot and qPCR results showed that there is a tendency to undergo EMT transformation after microgravity and radiation treatment,besides,radiation and microgravity had a synergistic effect on inducing EMT by regulating Fibronectinl.The immunofluorescence result showed that F-actin in the microgravity group cells and the combined treatment group cells was depolymerized and collapsed.The ratio of F-actin to G-actin decreased in the microgravity group.The expression of Fibronectinl was significantly up-regulated after irradiation and microgravity treatment,and there were synergistic effects between the two factors(p<0.05).By analyzing the transcription factors that can promote FN1 transcription,we found two transcription factors initiating FN1 transcription,E2F1 and ?-arrestin 1.However,their mRNA levels were not changed after irradiation and microgravity,but immunofluorescence assay showed that?-arrestin1 had a very significant migration from cytoplasm to nucleus after microgravity treatment.western blot results showed that YAP was also sensitive to radiation and microgravity,irradiation and microgravity had a synergistic effect on the up-regulation of YAP expression at protein levels.qPCR results revealed that the expression of CRY61 and PTX3 was significantly up-regulated after microgravity and irradiation which are downstream target genes of YAP(p<0.05),while CTGF did not change significantly.After FN1 was knocked down,the cell proliferation slowed down,and the migration activity was also significantly reduced(p<0.05).The expression of YAP also decreased and the cytoskeleton depolymerized.The results of luciferase report assay showed that FN1 had a certain regulatory effect on the transcription of YAP,but the specific mechanism remains to be further explored.Conclusion:(1)Low doses of alpha particles can induce malignant transformation of human lung epithelial cells in a dose-dependent manner and the effect of multiple exposure was stronger than a single exposure.(2)EMT occurs in cells before malignant transformation,implying that EMT may be a cause of radiation-induced malignant transformation.(3)The proportion of cancer stem cells increases during long-term subculture after irradiation with low-dose a particles,which may be another important reason of malignant transformation.(4)Radiation and microgravity may have synergistic effects in increasing the potential of EMT by depolymerizing the cytoskeleton.(5)The FN1/YAP/F-actin pathway probably plays a key role in EMT process induced by radiation and microgravity. |
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