| Glioma is the most common tumor in central nervous system, which originates from neuroepithelial cell and accounts for 70% in brain primary tumor. Growing glioma could oppress and damage the surrounding tissues. With boundary-less growth pattern of glioma, it’s hard to excise the glioma completely by the surgery, and this leads to its fast recurrence, high recurrence, large lethality and bad prognosis. The most outstanding growth characteristic of glioma is robust expansibility and invasiveness. Thus, to inhibit the invasion potential of glioma becomes an important strategy in glioma treatment.In Traditional Chinese Medicine theory, all the physical activity is promoted by air. The move pattern of air is lift, including air ascending and descending. Once the air runs disorderly, the clear air cannot ascend and foul smell cannot descend, leading to the disease occurrence. So there comes the saying, “Every Disease Was Born in Air”. “Head for ‘the sun’”. Air ascending makes the foul smell descending. The weakness, stun, inverse and drop of air represents the abnormal air-lift, and it would lead to disorderly runof blood, thrombus formation, and even that the water cannot expand to the whole body. Then, the accumulation of water will turn into phlegm in the long run and will lead to tumor recurrence ultimately. Therefore, without the clear Yang ascending and smell Yin descending, the accumulation of smell Yin in brain may be one of the kernel factor in pathogenesis of glioma. The microgravity is special for its air-lift condition, which can affect the air lift in body. However, whether modeled microgravity(MMG) could affect the migration and invasion of glioma and the involved mechanisms have not been elucidated. Therefore, under the guidance of the “Theory of Air Lift” in Traditional Chinese Medicine, our study aims to investigate if MMG could affect the invasion and migration potential of glioma, explore the relative mechanisms, and discover a new therapeutic strategy for the glioma.Part 1. Effect of MMG on the Biological Characteristics of Glioblastoma U87(U87MG)1. Objective To observe the effect of MMG on the biological characteristics of U87 MG.2. Methods U87 MG was divided into two groups, the normal gravity(NG) group and the MMG(MMG) group. U87 MG are cultured under NG and MMG condition for 24 h, 48 h and 72 h respectively. Using inverted phase contrast microscope to observe the changes of morphology; Using Hoechst staining and MTT assay to examine the proliferation of U87MG; Using the wound healing assay to examine the migration potential and using Transwell invasion assay to measure the invasion potential; Using Western Blot to detect the expression changes of MMP-2, MMP-9 and GFAP protein; Using intracranial glioma transplantation to examine the growth of U87 MG subjected to MMG condition in vivo.3. Results(1) Effect of MMG on the morphology of U87MG: Compared with that of the NG group, the morphology of U87 MG in MMG group changed into being irregular and turned to be acuity after cultured for 72 h, and the effect was in time-dependent.(2) Effect of MMG on the proliferation of U87MG: Compared with that of NG group,the growth rate of U87 MG was significantly inhibited under MMG condition for 48 h and 72h(p<0.05). Compared with that of NG group, the growth rate of U87 MG treated with MMG for 48 h or 72 h, was significantly inhibited under normal culture condition(p<0.05).(3) Effect of MMG on the migration potential of U87MG: Compared with that of NG group, the wound closure area of U87 MG in MMG group is reduced, especially after treatment for 48 h and 72h(p<0.05).(4) Effect of MMG on the invasion potential of U87MG: Compared with that of NG group, the invasive number of U87 MG treated with MMG for 24 h, 48 h or 72 h is significantly decreased under normal culture condition(p<0.05).(5) Effect of MMG on the expression of MMP-2, MMP-9 and GFAP protein of U87MG: Compared with that of NG group, MMG could upregulated the expression of GFAP and downregulated the expression of MMP-2 and MMP-9 significantly(p<0.05).(6) Effect of MMG on the growth of U87 MG in vivo: After cultured in NG and MMG condition for 72 h, the U87 MG was injected into the brain stereotaxically. MRI examination showed that the volume of tumor of the MMG group is significantly smaller than that of the NG group after injection for 4w.4. Conclusion After stimulation of MMG, the biological characteristics changes of U87 include: time-dependent morphology changing, proliferation rate reducing, invasion and migration potential inhibiting, expression of MMP-2 and MMP-9 protein upregulating, and expression of GFAP downregulating.Part 2. Effect of Store-Operated Calcium Entry(SOCE) Inhibition on the Migration and Invasion Potential of U87MG1. Objective To observe the effect of SOCE inhibition on the migration and invasion potential of U87 MG.2. Methods Using Western blot to examine the transfection efficiency after transfection; Using calcium imaging to measure the cell cytosolic Ca2+ concentration of U87 MG after 2-APB,SKF-96365 or STIM1 RNAi treatments; Using wound healing assay to examine the effect of SOCE inhibition on the migration potential of U87MG; Using Transwell invasion assay to examine the effect of SOCE inhibition on the invasion potential of U87 MG.3. Results(1) Efficiency of STIM1-sh RNA transfection: compared with that of Scr-sh RNA group, the expression of STIM1 was significantly down-regulated in STIM1-sh RNA group,(P<0.05).(2) Changes of SOCE of U87 MG after 2-APB, SKF-96365 treatments or STIM1 RNAi: SOCE was inhibited significantly(P<0.05), but there was no differences in calcium release.(3) Effect of SOCE inhibition on the migration potential of U87MG: Compared with that of the normal group, the migration potential was significantly inhibited in 2-APB, SKF-96365 and STIM1-RNAi groups(P<0.05).(4) Effect of SOCE inhibition on the invasion potential of U87MG: Compared with that of normal group, the invasion potential was significantly inhibited in 2-APB, SKF-96365 and STIM1-RNAi groups(P>0.05).4. Conclusion STIM1 RNAi could down-regulated the expression of STIM1 of U87MG; SOCE inhibitors and STIM1 RNAi could inhibit the TG-induced SOCE, but had no effects on the TG-induced calcium release; SOCE inhibition of U87 MG could attenuate the migration and invasion potential of U87 MG.Part 3. Effect of MMG on SOCE of U87MG1. Objective To observe the effect of MMG on SOCE of U87 MG.2. Methods Using calcium imaging to measure the changes of SOCE of U87 MG after cultured in NG and MMG condition for 72h; Using q RT-PCR to examine the m RNA expression of STIM1, STIM2, Orai1, Orai2; Using Western Blot to detect the expression of Orai1 protein.3. Results(1) Effect of MMG on SOCE of U87MG: Compared with that of NG group, MMG stimulation for 72 h significantly decreased the TG-induced SOCE to 41% of that of NG group(P<0.05), but there was no significantly difference in TG-induced release of calcium store.(2) Effect of MMG on the m RNA expression of STIM1, STIM2, Orai1 and Orai2 of U87MG: Compared with that of NG group, the m RNA expression of Orai1 in MMG group was significantly downregulated to 48.7% of that of NG group(P<0.05), but there was no significantly difference in expression of STIM1, STIM2 and Orai2 protein(P>0.05).(3) Effect of MMG on the expression of Orai1 protein of U87MG: Compared with that of NG group, the protein level of Orai1 in MMG group was downregulated significantly(P<0.05).4. Conclusion TG-induced SOCE of U87 MG was inhibited significantly after MMG stimulation for 72h; the m RNA and protein expression of Orai1 of U87 MG was down-regulated after MMG stimulation for 72 h.Part 4. Research of Relative Mechanism of MMG Suppressed Invasion and Migration of U87 MG through Downregulating SOCE1. Objective To explore the relative mechanism of MMG suppressing invasion and migration of U87MG: involvement of downregulating SOCE2. Methods After U87 MG was transfected with pc DNA3.1-Orai1 plasmid for 48 h, and then cultured in MMG for 72h: Using Western Blot to detect the expression of Orai1 of U87MG; Using calcium imaging to examine the changes of SOCE, and using wound healing assay and Transwell invasion assay to measure the migration and invasion potential.3. Results(1) Effect of overexpression of Orai1 on the MMG-induced downregulation of Orai1 of U87MG: Compared with that of MMG group, the expression of Orai1 was up-regulated(1.87 fold) significantly after MMG stimulation(P<0.05).(2) Effect of overexpression of Orai1 on the MMG-induced decreasing SOCE of U87MG: Compared with that of MMG group, overexpression of Orai1 successfully rescued the decreasing SOCE caused by MMG and slightly exceeded the SOCE of that of normal group(P<0.05).(3) Effect of overexpression of Orai1 on the MMG-induced inhibition of the migration and invasion potential of U87MG: Compared with that of MMG group, overexpression of Orai1 increased the migration and invasion potential of U87 MG to normal level(P<0.05).4. Conclusion Overexpression of Orai1 could successfully eliminated MMG-induced downregulation of Orai1 and the decreasing SOCE of U87MG; Overexpression of Orai1 could attenuate the MMG-induced decrease of the migration and invasion potential. Above all, MMG might decrease the migration and invasion potential in U87 MG by downregulating the expression of Orai1, and sequentially inhibiting the SOCE. |
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