The Role Of NRP1in Radioresistance Of NSCLC And Its Related Molecular Mechanism

Non small cell lung cancer (NSCLC) accounts for approximately80~85%in alllung cancers，while70%NSCLC patients had developed to advanced disease atdiagnosis, accompanied by distant metastasis, which caused them to lose the chanceof surgical resection, so mainly radiotherapy alone or combined with chemotherapy.However, compared with small cell lung cancer (SCLC), the NSCLC was moretolerance of radiotherapy. Therefore, how to improve the radio-sensitivity of NSCLCis a serious problem.Research background:Neuropilin1(NRP1) is a transembrane glycoprotein which is essential forembryonic development, tumor development, cell migration, angiogenesis andimmune response in vertebrates. NRP1is ubiquitously expressed on tumor vesselsand tumor cells. Furthermore, and the level of its expression was positively correlatedwith the poor prognosis of tumors. It has been shown that inhibition of NRP1couldblock proliferation and angiogenesis of tumor. However, the correlation betweenNRP1and radiation-resistant tumor was rarely studied, especially in the NSCLC.Based on the above studies, this study investigated the role of NRP1inradio-resistance of NSCLC and its related molecular mechanism, and provided newexperimental and theoretical basis for NRP1as a target to enhance theradio-sensitivity of NSCLC, meanwhile provide clinical biomarkers for thedevelopment of individualized treatment programs for NSCLC patients.Research objectives:1. To study the effect of NRP1on the radio-resistant of A549cells;2. To clarify the cellular and molecular mechanisms of NRP1enhancing theresistance to radiation in A549cells;3. To study whether the radio-sensitivity of A549cells could enhance, if the NRP1was inhibite;4. To investigate the suppression effect and its molecular mechanism of shNRP1vianude mouse model of A549. Research methods:Irradiation Conditions: A deep X-ray machine (X.S.S.205(FZ) of fixation type,made in China) was used, the dose was2~20Gy. Build PLNX2-NRP1andpSIREN-RetroQ-shRNA-NRP1recombinant plasmid, and constructNRP1high/NRP1lowcell model by retroviral transfection. To establish A549RR cell line,parental A549cells were exposed to repeated irradiation by repeated highdose X-ray irradiation, each dose of X-ray irradiation was6Gy (total dose of30Gy).MTT assay, colony formation assay, Annexin V-FITC (flow cytometry), scarification,Transwell chamber invasion and comet assay were used to detect the radio-sensitivityof tumor cells. Immunohistochemistry was used to detect the microvascular of tissuetumors. Microarray, Real-time PCR, immune-precipitation, Western-blot method andELISA were used to study the molecular mechanisms of tumor resistance to radiation.Research results:1The effect of NRP1on the Radio-resistant of A549cellsThe results showed that the protein level of NRP1was different in various tumorcells and was highest on A549cells. The inhibitory effect of10Gy X-rayson the proliferation of A549tumor cells were significantly lower than that of othertumor cells, and that of H460tumor cells were higher. We further investigated thepossible relationship between NRP1and NSCLC by evaluating radio-sensitivity ofA549and H460cells. The results showed that the abilities of colony formation andmigration of A549cells were higher than that of H460cells after10Gy X-ray.Meanwhile, the apoptosis of H460cells was significantly higher than that of A549cells. It is noteworthy that NRP1expression was up-regulated in A549cells afterradiation and decreased on H460cells.2The cellular mechanisms of NRP1enhancing theresistance to radiation in A549cellsColony formation assay is the gold standard to evaluate cell radio-sensitivity. Thesurvival fraction (SF) of all cells showed a dose-dependent decrease, while the SF ofNRP1highA549was higher than parent cell at the same dose, while that ofNRP1lowA549cells decreased significantly. Compared with the parental cell line, theapoptosis of NRP1lowA549was significant higher, while the apoptosis ofNRP1highA549cells was lower after radiation. In addition, although G2/M phasearrest occurred in the three cell lines, the extent of arrest in NRP1highA549cell was lower than parental cell line. Interestingly, the extent of arrest in NRP1lowA549cellswas higher than that of NRP1highA549cell and parental cell line, suggesting thatNRP1may alter cell cycle progression induced by ionizing radiation to inhibitapoptosis.3The molecular mechanisms of NRP1enhancing theresistance to radiation in A549cellsThe microarray was employed to explore the molecular mechanisms of NRP1onradiation resistance of A549cells. The results showed that there were784genesregulated by NRP1and ionizing radiation, some of which were mainly involved incell proliferation, differentiation, apoptosis, cycle progression, invasion andmetastasis, and DNA damage repair. We also found that some key genes associatedwith ionizing radiation signaling pathway including PI3K-Akt, MAPK-ERK, NF-κBand TGFβ was affected by NRP1. Western blot was used to further confirm the keygenes associated with ionizing radiation signaling pathway.4Inhibition of NRP1by RNA interference could enhance the radio-sensitivity ofA549Transwell assay was employed to investigate the impact of inhibition of NRP1by RNA interference on cell invasion and migration of A549cells. Compared with theirradiation alone group, shNRP1combined with ionizing radiation could significantlyinhibited cell migration and invasion of A549tumor cells。Comet assay was employedto detect the DNA damage which induced by irradiation, the results showed inhibitionof NRP1could enhance the degree of DNA damage of A549.5Establishment of a radioresistant A549cell line and study the correlationbetween A549RR and NRP1The results showed that SF of A549RR cells were higher than A549cells after10Gy X ray（p<0.01）, and the SF2of A549RR cells was0.995, while the parentalA549was0.887. Then we investigated the A549RR cells cycle, apoptosis, invasion,migration and DNA damage after10Gy X rays. The results showed that A549RR cellline had stronger anti-apoptotic ability than A549cells and irradiation could induceA549RR cells S phase arrest. Meanwhile, the ability to inhibit migration and invasioncapacity and DNA damage were significantly lower than in A549cells. Furthermore,NRP1expression on A549RR cell line was higher than in A549cells.6To investigate the suppression effect and its molecular mechanism of shNRP1 via nude mouse model of A549We further explored that shNRP1combined with radiotherapy has significantanti-tumor effects via nude mouse model of A549cells, showing that the proliferationof tumor was markedly inhibited, the volume and weight of tumor decreased.Meanwhile, the molecular mechanism of shNRP1on tumor was clarified, the resultsshowed the secretion of TGFβ1, the expression of VEGFR2and CD31in tumordecreased significantly, after shNRP1combined with ionizing radiation, in additionthe molecular of signal pathway which were related to the radiation consistent with invitro.Conclusions:This study confirmed that NRP1played a crucial role in the radiation resistanceof NSCLC cells. Specifically in the cell proliferation, colony forming, anti-apoptosis,metastasis and DNA damage repair. Meanwhile, the proliferation of tumor, thesecretion of TGFβ1and the density vessel in tumor decreased significantly, aftershNRP1combined with ionizing radiation. We explored molecular mechanism anddemonstrated that NRP1could regulated radiation resistance of NSCLC throughmultiple signaling pathways (eg: VEGF, PI3K-Akt, MAPK-ERK, NF-κB and TGFβ).This study was the first time confirmed that inhibition of NRP1could enhance theradio-sensitivity of NSCLC cells, which offered a new experimental and theoreticalfor radio-sensitization NSCLC drug.