Study On The Correlation Between NRP1 And MiR-9 Expression In NSCLC And Their Roles In Radiation Resistance

Lung cancer is the leading cause of cancer related death in the world. In recent years, many factors, such as air pollution and smoking lead to increasing incidence of lung cancer, the number of patients with lung cancer increases sharply. Non-small cell lung cancer approximately accounts for about 80% of lung cancer, and is easy to resistant to radiation therapy than small cell lung cancer. How to overcome the radiation resistance of tumor cells has become a hot issue of radiation biology and radiation oncology research.Research background: Neuropilin 1, also denoted NRP1, CD304 or BDCA-4, is involved in a series of process, such as the immune, cardiovascular development, neuronal orientation, cell migration, angiogenesis and Tumorigenesis. NRP1 expression has been reported in a wide variety of solid cancer including pancreatic cancer, prostate cancer, breast cancer, colorectal cancer and renal cell carcinoma, melanoma, glioma, leukemia, lymphoma. Its interactions with several ligands such as VEGF, PDGF and EGF, or its function promoting EMT, involved in tumor cell proliferation, metastasis and angiogenesis. Generally speaking, NRP1 expression which associated with more aggressive clinical tumor behavior has been linked to a poor prognosis. Taking into account the multiple functions of NRP1 and its interactions with the extracellular ligands, NRP1 may be used as a potentially valuable therapeutic target. Micro RNAs(mi RNAs) are a group of 22-nucleotide single-stranded non-coding RNAs, widespread in eukaryotic cells. They regulate gene expression by hybridizing to complementary sequences in the 3’ untranslated region of target m RNA, which causes transcriptional repression or m RNA degradation,and finally inhibites the synthesis of the target protein to play its functions. More and more researches have revealed that mi RNAs play a significant role in lung cancer development and pathogenesis. mi R-9, initially found that high expression in vertebrate brain tissue, plays an important role in the nervous system, liver cell growth and negative regulation of the innate immune response. Studies have shown that aberrant expression of mi R-9 has often been found in a variety of human tumors, and is involved in tumorigenesis and cancer development. Our previous results indicate not only high expression of NRP1 in NSCLC cells is related to radiation resistance, but also that overexpression mi R-9 can enhance the radiosensitivity of A549 cells by targeting NRP1. This study is aimed at NRP1, analyze its role in the radiation resistance through NSCLC specimens and tumor-bearing nude mice model of NSCLC, and verify whether or not mi R-9 is involved in this process. Studying on the relationship between the expression of NRP1 and mi R-9 and tumor clinical stage, histological type and prognosis of disease,may provide further experimental evidence for NRP1 becoming a potential marker for NSCLC radiotherapy and improving radiotherapeutic effect.Research objectives: 1. Research on the expression of NRP1 and mi R-9 before and after irradiation, to verify the relationship between mi R-9 and NRP1. 2. To analyze of the relationship between the expression of NRP1 and mi R-9 and clinicopathological features in tissue samples. 3. To clarify the molecular mechanisms that NRP1 involved in NSCLC radiation resistanceResearch methods: In this study, q RT-PCR, Western-blot and immunohistochemistry were used to analyze the expression of NRP1 and mi R-9 before and after IR and the molecular mechanism they involved in NSCLC radioresistant downstream signaling pathways at the overall level. First, study analyzed the expression level of NRP1 and mi R-9, the relationship between their expression and clinical characteristics of patients, expression of the relevant signaling pathways and angiogenesis-related factors in clinical specimens. And then analyzed the tumor tissue morphological changes before and after IR, expression levels of NRP1 and mi R-9, expression of the relevant signaling pathways and angiogenesis-related factors in tumor-bearing nude mice model of NSCLC using the same means in the former.Research results: 1. The expression levels of NRP1 and mi R-9 in NSCLC tissue samples Western-blot,q RT-PCRand immunohistochemistry were used to analyze the expression level of NRP1. The expression level of NRP1 m RNA was found to be remarkably lower in NSCLC tissues compared to normal lung tissues and adjacent carcinoma tissues, and so is the NRP1 protien expression level; q RT-PCR was used to analyze the expression level of mi R-9, and the expression level of mi R-9 was found to be remarkably higher in NSCLC tissues compared to normal lung tissues and adjacent carcinoma tissues, their expression show a reverse tendency(r =-0.478 P<0.01). 2. Correlation between clinicopathological features and NRP1 or mi R-9 expression levels in clinical NSCLC tissues By analyzing the expression of NRP1 and mi R-9 in clinical tissue samples, it has been found that the expression of NRP1 was significantly correlated to lymph node metastasis(P <0.05), In contrast, no association was found in the expression level of NRP1 with gender,age, differentiation, TNM stage, histological subtype or clinical stage(P >0.05). The expression of mi R-9 was significantly correlated to gender in the adjacent carcinoma tissue(P<0.05),but no association was found in the expression level of mi R-9 with age, histological subtype, lymph node metastasis,differentiation, TNM stage, or clinical stage(P >0.05). 3. The expression levels of molecules in NRP1 related downstream signaling pathways in clinical NSCLC tissue Compared with normal tissues and adjacent carcinoma tissues, NRP1 protein expression in NSCLC tissues was significantly reduced, which was consistent with its m RNA expression level, there were no significant differences of PI3 K, AKT and ERK1/2 protein expression among normal tissues, adjacent carcinoma tissues and NSCLC tissues. However, p-AKT and p-ERK1/2 expression in NSCLC tissues was significantly lower than that in normal tissues and adjacent carcinoma tissues. NF κB expression in adjacent carcinoma tissues was significantly increased than that in matched normal tissues and NSCLC tissues, P38 and p-P38 expression in adjacent carcinoma tissues was significantly increased compared to NSCLC tissues, but their expression has no significant changes between normal tissues and NSCLC tissues. VEGFR2 expression in NSCLC tissue was significantly lower than that in normal lung tissue, but no significant difference was found compared with adjacent carcinoma tissues, CD31 expression in NSCLC tissues was significantly increased compared with normal lung tissues and adjacent carcinoma tissues. 4. Clarify the roles of NRP1 and mi R-9 and the molecular mechanisms that NRP1 involved in tumor radiation resistance In tumor-bearing nude mice model of NSCLC(containing radiation resistant A549 cells), depending on the situation whether irradiation(irradiation, IR) or transfection with mi R-9, nude mice were divided into four groups, including tumor group, IR group, the group transfected with mi R-9 and mi R-9 + IR group, 6 mice were contained in each group. When compared with the corresponding control groups, volume were reduced in both IR group and mi R-9 + IR group, and the latter was more lower than the former. Compared with Tumor group, tumor weight was decreased in IR group, and compared with mi R-9 group, tumor weight was decreased in mi R-9 + IR group, there was no significant difference of tumor weight in IR group and mi R-9 + IR group, even the latter was a bit lower than the former. In conclusion, over-expression of mi R-9 can promote radiosensitivity of A549 cells. Compared with the tumor group, NRP1 protein was significantly increased in IR group, which was consistent with its m RNA expression level,and mi R-9 was significantly decreased, their expression show a reverse tendency. Our previous results showed that the tumor cells can produce radiation resistance in order to avoid cytotoxic effects caused by IR, the expression of NRP1 was increased, and mi R-9 was decreased, the results were consistent with the earlier results,that NRP1 expression was associated with radiation resistance, and mi R-9 can adjust radiosensitivity of lung cancer cells through targeting NRP1. About the molecular mechanisms, there were no significant differences of PI3 K, NF k B, ERK1/2 and P38 expression level in both groups. AKT, p-AKT and p-ERK1/2 expression were increased in IR group than that in tumor group, and their expression were consistent with NRP1 expression changes. Compared with the tumor group, p-P38 expression were decreased in IR group,and inverse relationship existed in its expression and NRP1 expression. VEGFR2 was significantly higher in IR group than that in tumor group.Conclusions: 1. In NSCLC tumor tissues, there was significant negative correlation between NRP1 expression and mi R-9 expression. 2. In clinical NSCLC patients, NRP1 expression at the transcription level was significantly correlated to pathological type of lung cancer, and NRP1 expression levels at both transcription and translation level were significantly correlated to lymph node metastasis of lung cancer. mi R-9 expression was significantly correlated to gender in the adjacent carcinoma tissues. 3. In tumor-bearing nude mice model of NSCLC, over-expression of mi R-9 can promote radiosensitivity of A549 cells. NRP1 and mi R-9 expression show a reverse tendency. After IR, the formation of microvascular was decreased. 4. In tumor-bearing nude mice model of NSCLC, mi R-9 could enhance radiosensitivity of NSCLC cells by regulating expression of NRP1,and angiogenesis and NRP1 / VEGFR2 signaling pathway may be involved in this process. 5. NRP1 and mi R-9 may be involved in radiation resistance of NSCLC tumor tissues by activating VEGF, PI3 K / AKT and MAPK / ERK pathway.