The Role And Mechanism Of Neurotensin Along With NTSR1in Glioma Proliferation And Migration

Glioma is the most common and high fatal intracranial malignant tumor, which is characterized by extensive invasion at the cellular levels in the whole brain, damage on normal brain tissue, resistance to alternating chemoradiation therapy, and high recurrent rate after surgery and chemoradiation therapy, resulting in high mortality. So it is difficult to eradicate tumor tissue by a surgical operation. At the same time, surgical stimulation could induce cancer cells divorcing from tumor tissues, further increasing the glioma invasion degree deepen in normal brain tissue. Adopts measures such as radiotherapy and chemotherapy are taken to kill the postoperative residual tumor cells. However, the postoperative residual lesions tissues usually showed a greater resistance for radiotherapy, chemotherapy and other measures, leading to tumor recurrence and poor prognosis in glioma patients. Considering that glioma therapy effect is still not satisfactory at the present stage, we investigates the mechanism of glioma cell proliferation and migration. Our purpose is to inhibit the cellular proliferation and migration activities at the early stages of the glioma formation, which could control the glioma tissues within a certain range, avoiding the invasion into important functional areas in brain, in order to eliminate the tumors completely through the surgical resection without causing extra njury to normal tissues. Our study is the first time confirmed that NTS and its receptor NTSR1could play a role in glioma cell proliferation and migration process.By in vitro experiment we studied the NTS and NTSRl affect GL261glioma cell proliferation and migration. We use NTSR1specific antagonist SR48692GL261, analysis NTSR1expression were inhibited after GL261affected the proliferation and migration process. Also discussed the process of the ERK signaling pathway involved, namely NTSR1activation of downstream signaling pathways of ERK1/2level of phosphorylation and the relationship between cell proliferation and migration. We use after intracerebral transplantation tumor model in mice to explore in vivo and in vitro experimental phenomena are consistent. The main results are as follows:1. NTSR1expressed higher in human glioma tissue and GL261mice glioma cell linesTake the other people at all levels for immunohistochemical study glioma samples. Cultivation of logarithmic phase GL261cells by conventional method, and the cells collected and transferred to the package how much is poly lysine growth onto a glass slide, then fluorescence detection by immune cells NTSR1expression in GL261cells. Experimental results show that the NTSR1expressed in all levels of human brain glioma tissues, in which low grade glioma tissues expressed in low and high grade glioma tissues expression was higher, and its positive expression was also found in GL261cells with higher expression rate.2. NTSR1by SR48692antagonism and siRNA silence effects on GL261cell proliferationFirst of all, we through the CCK8GL261cell growth curve drawing method. In the case of joining NTS, respectively using5um and10um GL261SR48692cells, antagonism to NTSR1. SR48692, relative to the control group after treatment of GL261proliferation was significantly inhibited and this inhibition is dose dependent effect. And then we build NTSR1-siRNA and sc-siRNA interference on GL261cells respectively, found that cell proliferation significantly suppressed after NTSR1was silent. And sc-siRNA group basic cell proliferation is not affected, it contains NTS of sc-cell proliferation best siRNA group. After we using BrdU labeled method to detect GL261cell proliferation. GL261cells after NTS and5um SR48692processing after48hours, BrdU positive rate decreased obviously. And after NTS and10um SR48692processing after48hours, GL261cells BrdU positive signals without visible to the naked eye, SR48692also showed a dose dependent inhibition effect. In the presence of NTS, relative to sc-siRNA group, group NTSR1-siRNA GL261cell proliferation significantly suppressed. It suggests that NTSR1by SR48692antagonism and siRNA silence, GL261cell proliferation can be obviously suppressed, prove NTSR1can promote glioma cell proliferation. 3. NTSR1by SR48692antagonism and siRNA silence effects on GL261cell migrationWe first through the scratch experiment test GL261migration ability. The exponential phase GL261cells, respectively treated with SR48692and siRNA interference, and scratches for the cell. Under inverted microscope, respectively at0hours and36hours of the two time points observed scratch on healing of cells. Nick healing situation can intuitively reflect the GL261cell migration. Nick healed well, cell migration, scratches the healing is poorer, then decreased cell migration. SR48692antagonism experiment results show that:after5um SR48692processing after36hours, Nick basically no change. And without SR48692control and NTS scratch wound healing in good condition, scratch space compared with the former shrank by47%. SiRNA silences the results display that scratches after36hours, after NTSR1-siRNA transfection GL261cell scratch healing degree is low. And relative to the NTSR1siRNA transfection group, sc-siRNA transfection group of scratches spacing is narrowed by58%. After we test by transwell perforated experiments GL261migration ability. Logarithmic phase cell also digest to collect back all to join the NTS, then respectively using SR48692processing and siRNA transfection, and transferred to the Transwell Chambers. After3h with crystal violet staining and observed under inverted microscope staining cells, or on behalf of the migration of cells and staining, the more that cell migration ability. The results of SR48692antagonism shows that5um SR48692GL261cells after treatment by crystal violet staining cells quantity is20%more than the control group and the NTS group. Via the inverted microscope observation of siRNA silence shows that NTSR1-siRNA transfection GL261cell dyeing quantity is less, and relative to the NTSR1-siRNA transfection group, sc siRNA transfection group staining cells was increased by67%. It suggests that NTSR1by SR48692antagonism and siRNA silence, GL261cell migration activity has also been significantly suppressed, NTSR1promote glioma cell migration.4. After animal experiments NTSR1by SR48692antagonism effects on GL261cell migrationWe established the C57mouse within GL261glioma model, and then were given different doses of SR48692and DMSO respectively. By observing the survival groups of mice found that minimum survival time in mice of control group, only injection DMSO is16-22d, and the average lifetime is19d. Injection SR48692dose of2mg/kg of experimental mice survival period for13d-21d, the average survival period of17d. Injection SR48692dose of5mg/kg of experimental mice survival for21d-33d, the average survival for27d. Injection SR48692dose of10mg/kg of experimental mice survival period, the longest of25d-41d, the average survival period of33d. Four groups of C57mice out brain take photos after death, found that in the control group of mice brain edema, and10mg/kg SR48692experimental mice brain boundaries clear. After this we do the brain slices and H.E. stain, the experiment found that in the control group and larger nucleus dyeing is quite deep, cell arrangement, distribution is more dense, and invasive growth, invade the surrounding normal tissue.5mg/kg group the number of cells less relative to the control group, and there is no infection occurred. And10mg/kg group did not see obvious tumor cells, and suggests that suppressed tumor cell proliferation. This indicates SR48692NTSR1inhibition in mice can also through the antagonism GL261cell migration, further proof NTSR1glioma cells can promote the migration process.5. ERK1/2phosphcrylation effects on tumor cell proliferation and migrationFirst use of NTS, SR48692and NTS GL261neutralizing antibody after same time cells, analysis of protein phosphorylation of ERK1/2. Western blot-experiment results show that compared with the control group, with NTS GL261ERK1/2protein phosphorylation were significantly increased in cells, and with the increase of NTS concentration ERK1/2protein phosphorylation level is higher. In the presence of NTS to join SR48692, results showed that ERK1/2phosphorylation level individual treatment of the experimental group had significantly lower than the NTS. In the presence of NTS to NTS of neutralizing antibodies, the results show that ERK1/2phosphorylation level compared to NTS separate processing of experimental group have significantly lower. And the same is the presence of NTS, at the same time add SR48692and NTS neutralizing antibodies, the results found that compared with all other experimental group and the control group, the least ERK1/2protein phosphorylation, almost undetectable its phosphorylation stripe. After is the use of NTS, Sc-siRNA and NTSR1-siRNA treatment GL261cells respectively, ERK1/2protein phosphorylation is characterized by:with NTS GL261ERK1/2protein phosphorylation in cells were significantly higher than that of control group, further illustrate NTS promote GL261cell proliferation and migration. In the presence of NTS using NTSR1NTSR1expression-siRNA interference, according to the results of ERK1/2phosphorylation level has decreased significantly. The experimental results show that NTSR1by activating ERK1/2protein phosphorylation cause tumor cell proliferation and migration. Anyhow, illustrate the proliferation and migration mechanism of glioma, can design more effective comprehensive treatment strategies to provide new ideas and direction, this will have a positive effect for the treatment of malignant glioma, and to lay a solid foundation for further study.