| Lung cancer is the first disease in all cancers caused death. Every year more than one million people die of lung cancer, of which 80% is NSCLC. The effect of radiotherapy is depended on radiosensitivity and its application is restricted by radioresistance. Although a series of blockers or activators targeting molecules related to radioresistance is developed as a radiation sensitizer, compensatory mechanisms or drug resistance due to a single molecule limits its clinical efficacy. Looking for the key molecule of lung cancer cells radioresistance or a effective molecular target is a difficult and important problem in Radiation Oncology. Research background:NRP1 is expressed in many tumor tissues, including liver cancer, prostate cancer, breast cancer, melanoma and pancreatic cancer, etc. but is not expressed in the corresponding normal tissues. The study found that NRP1 is related to the radioresistance of A549 and plays its role by VEGF、PI3K-Akt、MARK-ERK、P38、NF-κB and TGF-β. Inhibite NRP1 can improve the radiosensitivity of A549 cells. Therefore NRP1 may be a molecular target of radiotherapy sensitization drug in lung cancer.HOX genes are master genes of proliferation and differentiation, they play important roles in the development of central nervous system, limbs and the axial skeleton. Recent studies have found that, a variety of cancers such as breast cancer, liver cancer and lung cancer have been detected aberrant expression of HOX genes. It has been reported that HOXB9 can induce EMT associated radioresistance by accelerating DNA damage responses.MLL genes are transcription factors, closely related with 70 different partner genes. Knock out MLL1 can downregulate the expression of HOXA10. Bertani found Lnc RNA Mistral can recruite MLL1 to chromatin to activate Hoxa6 and Hoxa7 expression In mouse embryonic stem cells. Ansari proved MLL2 and MLL3 can regulate the transcription of HOXC6 by interacting with estrogen receptors. Many evidences show that, abnormal MLL genes can lead to disorder transcription, and abnormal expression of HOX genes. Research objectives:1. Select the downstream key genes of NRP1 in lung cancer cells radio-resistance;2. Verified the regulation rules between NRP1 and HOX genes;3. Clarify the role of NRP1-HOXs pathway in lung cancer radioresistance regulation;4. Verify the regulation rules between NRP1 and HOXs and mechanism in tumor radiotherapy through the clinical tissues and nude mouse models. Research methods:1. A549, A549-NRP1 low and A549 cells irradiated 10 Gy were detected by gene microarray to screen out key genes regulated by NRP1 in the cell radioresistance, and verify their expressions in A549 cells;2. Established the A549-NRP1 high, A549-NRP1 low cell model, and used Real-time PCR to detect NRP1, MLL5, HOXA6 and HOXA9 after transient transfection;3. Detected the m RNA and protein expression of NRP1, HOXA6, HOXA9 and MLL5 of various cell models by Real-time PCR and Western Blot at different times or in different doses after radiation, explored the role of NRP1-MLL5-HOXA pathway in lung cancer cells radiation effects;4. MTT assay, colony formation and flow cytometry methods were used to detect the cell phenotype changes such as proliferation, cell cycle and apoptosis of MLL5, HOXA6 and HOXA9 interference models after irradiation, explored the biological role of NRP1- MLL5-HOXA pathway in lung cancer cells radiation resistance;5. Tested the expression of NRP1, HOXA6, HOXA9 and MLL5 in clinical tissue samples and nude mouse models by Real-time PCR and Western Blot, further tested the expression of NRP1 by immunofluorescence, explained the regulation of NRP1-MLL5-HOXA pathway further in non-small cell lung cancer radioresistant;6. Detected the expression of CD31 protein in clinical tissue and nude mouse models to observe the effects of irradiation on angiogenesis in NSCLC. Research results:1. Screen the key regulatory genes of NRP1 in the radiation resistanceA549, A549-NRP1 low and A549 cells irradiated 10 Gy were detected by gene microarray to screen out key genes regulated by NRP1 in the cell radioresistance: MLL5, HOXA6 and HOXA9, and verified their expressions in A549 cells by Real-time PCR.2. The regulatory relation between NRP1 and its downstream genes MLL5、 HOXA6、HOXA9Real-time PCR showed that downregulated NRP1 can reduce MLL5, HOXA6 and HOXA9 expression in A549 cells. Overexpressed NRP1 gene can reduce the HOXA6 and MLL5 but still higher compared the interference NRP1 A549, except MLL5. Downexpressed MLL5 can decrease NRP1 but HOXA6 and HOXA9 can not.3. The role of NRP1-MLL-HOXA path in lung cancer cells radiation resistanceMLL5, HOXA6 and HOXA9 were increased with the change of NRP1 after the 10 Gy X-ray irradiation. HOXA6 increased significantly after irradiation in the up or down expressed cell models, MLL5 increased only in upexpressed model but HOXA9 changed none. They all upregulated in the A549 radiation-resistant lung cancer cell modelCell proliferation, apoptosis and cell cycle were detected on the A549 cell models which is interferenced MLL5, HOXA6 and HOXA9. The results showed that cell viabilities were decreased when HOXA9 and MLL5 interference cells irradiated, si HOXA9 can inhibit radiation-induced apoptosis rate, which may be subject to the regulation of radiation in cell cycle, especially in S phase shorten.4. Evaluate the biological significance of NRP1-HOXA-MLL pathway in lung cancer radiation resistance as a wholeRetrospectively analysed the expression levels of NRP1 and its downstream key genes in 45 pairs of NSCLC tissues and adjacent noncancerous tissues compared to normal tissues. There was no significant difference between normal and peritumor tissues. The expression of NRP1, MLL5, HOXA6 and HOXA9 were all downregulated in tumor tissues, which is uniformly with the in vitro results. Correlation analysis showed that NRP1 is related with HOXA6, HOXA9 and especially MLL5.The number of tissues with radiotherapy can not up to the sample size, we used a nude mouse model of NSCLC xenografts to evaluate the biological significance of NRP1-HOXA-MLL pathway in lung cancer radiation resistance.Results showed that the m RNA and protein expression of NRP1 were all increased, so were HOXA6, HOXA9 and MLL5. Conclusions:1. NRP1 positive regulated HOXA6, HOXA9 and MLL5 in A549;2. MLL5, HOXA6 and HOXA9 interference has no effect on NRP1, but MLL5 interference can reduce HOXA6 and HOXA9 in A549, illustrated MLL5 regulate HOXA6 and HOXA9, they all can’t regulate NRP1;3. The m RNA and protein expression of NRP1, MLL5, HOXA6 and HOXA9 were all induced by ionizing radiation in vivo and vitro;4. Ionizing radiation induced G2/M phase arrest and apoptosis in vitro, but si HOXA9 can inhibit this phenomenon; 5. The expression of MLL5, HOXA6 and HOXA9 were all decreased in tumor tissues and positive correlated with NRP1; 6. NRP1 expression was significantly lower in squamous cell carcinoma than adenocarcinoma, and lymph node metastasis occurred more often in lung cancer patients whose MLL5 and NRP1 expression were higher. |
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