| Background and ObjectivesIn different clinical staging of NSCLC patients,radiotherapy is an important part of the treatments,but radiation pneumonia(RP)occured after radiotherapy in some patients.in non small cell lung cancer,the incidence rate of radiation pneumonia is very high,about 10%-37% of non small cell lung cancer patients after radiotherapy occurred radioactive pneumonia and some patients ultimately resulting in death,however,there is no definite biomarker for predicting radiation pneumonitis in patients with lung cancer.In recent years,Micro RNAs(Mi RNAs)have become a hot research topic.It was reported expression of at least 20–30% of human protein-coding genes were modulated by MiRNAs and MiRNA plays a great role during disease development.So the specific MiRNAs and their differentially expressed target genes of lung cancer may play a certain role in the occurrence and development of radiation pneumonitis.Gefitinib,a small molecule epidermal growth factor receptor tyrosine kinase inhibitor,has been widely used in non-small cell lung cancer(NSCLC),in the gefitinib treatment of non small cell lung cancer,part of patients appeared different degree of pulmonary interstitial damage,some patients even are life-threatening.The rate of gefitinib induced pulmonary interstitial disease in Japan is 4-6%,in the United States is 0.3%.The toxicity of this kind of interstitial lung injury has attracted the attention of clinicians.After regression study,the risk factors of pulmonary interstitial injury include: female,smoking,previous history of pulmonary fibrosis,and so on.Study shows that one of the predictive factors of interstitial lung injury in patients is previous chest radiotherapy.Some patients in the whole treatment process need to be treated with Gefitinib and chest radiotherapy separately or at the same time.But a previous clinical study of non-small cell lung cancer,patients with non-small cell lung cancer treated with the combination of gefitinib and chest radiotherapy.But the lung toxicity is too large and it was early closure.The above evidence shows that gefitinib may increase radiation pneumonia severity,but there are few studies reported in this area at present.The objectives of this subject are as follows: 1.Search for the specific MiRNA and its target genes(biomarkers)and relative factors/index in lung cancer patients,which may influence the occurrence and development of radiation pneumonitis;2.The establishment of radiation pneumonia rats model;3.Make clear that whether different gefitinib administration mode would influence the degree of radioactive pneumonia in rat model,to explore the internal mechanism of the gefitinib aggravated radiation pneumonia.4.We collect lung cancer patients who were treated with chest radiotherapy and gefitinib in our hospital,review the relationship between the therapeutic method of radiotherapy and different discontinuation of gefitinib and the incidence rate of radiation pneumonia to further verify the effect of different gefitinib delivery modes on radiation pneumonia.Methods1.The gene expression profile GSE32863 was downloaded from Gene Expression Omnibus database(GEO),which included 15 lung cancer tissue samples and 10 normal lung tissue samples.In addition,the MiRNA microarray profile GSE17681 with 10 lung cancer and 10 normal tissue samples was also downloaded.Then the probe-level data were pro-processed by Significance Analysis of Microacrray(SAM),and the differentially expressed genes(DEGs)and MiRNAs were screened with multi-test package in R language.The selected DEGs were further subjected to functional enrichment analysis using DAVID online tool.Following that,the verified target genes based on screened MiRNAs were selected from mi RTar Base and mi Records databases.Then MiRNA-target gene regulation network was constructed to identify symbolic MiRNAs of lung cancer.Then the specific MiRNA and its target genes and relative factors/index,which might play a role in the development and progression of lung cancer patients with radiation pneumonitis,were screened according to the biological function of target genes.Then the serum ICAM-1 levels of 114 patients with lung cancer who received radiotherapy in our hospital were detected,and the incidence of radiation pneumonitis was evaluated.2.SD rats were used to establish rat model of radiation pneumonia,divided into group of gefitinib treatment before radiotherapy for 8 weeks,group of gefitinib treatment before radiotherapy for 4 weeks,group of gefitinib treatment and radiotherapy at the same time,group of radiotherapy and control group.We will harvest rat blood and lung tissue specimens after radiotherapy for 4 weeks,8 weeks and 12 weeks.To detection of radioactive pneumonia severity,we use enzyme-linked immunosorbent assay to detected interleukin-1 beta and interleukin-6 levels in rat bloods and lung tissue.We use immunohistochemical method for the detection with CD68 expression in lung sections of rats in each group.We use alkaline hydrolysis methods to detection ratio of hydroxyproline contents in lung tissue of rats.The inflammatory score of lung tissue of rats in each group were scored.3.We collect lung cancer patients who were treated with chest radiotherapy and gefitinib in our hospital,divided into group of gefitinib treatment before radiotherapy for 8 weeks,group of gefitinib treatment before radiotherapy for 4 weeks,group of gefitinib treatment and radiotherapy at the same time,group of radiotherapy.We review the relationship between the therapeutic method of radiotherapy and different discontinuation of gefitinib and the incidence rate of radiation pneumonia to further verify the effect of different gefitinib delivery modes on radiation pneumonia.ResultsWe construct a specific MiRNA target gene regulatory network of lung cancer patients,and screen out the mi R-222 targeting ICAM-1 and relative factors/index: interleukin-1 beta,interleukin-6,hydroxyproline contents and activated macrophage,which may play a role in the development of lung cancer patients with radiation pneumonitis.Data analysis showed that the level of serum ICAM-1 in patients with radiation pneumonitis was significantly higher than that of patients without radiation pneumonitis(p<0.05).The inflammatory score result of lung tissue of rats in each group show that the group of gefitinib treatment and radiotherapy at the same time has higher scores then other group at 8 and 12 weeks after radiotherapy(p<0.05).The results of enzyme-linked immunosorbent assay(ELISA)for the detection with interleukin-1 beta,interleukin-6 in rat bloods and lung tissue show that the group of gefitinib treatment and radiotherapy at the same time has more interleukin-1 beta and interleukin-6 expression then other groups at 12 weeks after radiotherapy(p<0.05).The results of immunohistochemical method for the detection of activated macrophage in lung sections of rats in each group show that the group of gefitinib treatment and radiotherapy at the same time has more activated macrophage expression at 4,8 and 12 weeks after radiotherapy(p<0.05).The results of alkaline hydrolysis methods to determine hydroxyproline contents in lung tissue of rats show that there were not significantly differences in all the groups.Retrospective analysis showed that patients in the gorup of gefitinib treatment and radiotherapy at the same time got more incidence rate of radiation pneumonia than other groups(p<0.05).ConclusionsWe construct a specific MiRNA target gene regulatory network of lung cancer patients,and screen out the mi R-222 targeting ICAM-1 and relative factors/index,Serum ICAM-1 level in patients with radiation pneumonitis was higher than patients without radiation pneumonitis after radiotherapy,the serum level of ICAM-1 may become a biomarker for predicting radiation pneumonitis.Mi R-222 targeting ICAM-1 which may play a role in the development of lung cancer patients with radiation pneumonitis.The application of gefitinib and radiotherapy could increase the sensitivity of the radiation pneumonia in rat models,and the application mode of gefitinib treatment and radiotherapy at the same time is markedly.Retrospective analysis showed that the application of gefitinib and radiotherapy could increase the incidence rate of the radiation pneumonia,and the application mode of gefitinib treatment and radiotherapy at the same time is markedly. |
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