Study On Effects And Mechanism Of Imp2 In Glioma

This experiment use glioma tissue specimens, glioma cell line as the research object to study Imp2 expression and its impact on the biological functions of glioma and potential molecular mechanisms. This experiment is divided into two parts: 1. Imp2 expression in human GBM and its effects on cell growthInsulin-like growth factor 2 m RNA-binding protein-2(IGF2BP2,Imp2) is protein, which plays an important role in the development of the human brain. The study found that its expression increased obviously in a variety of malignant tumor. A recent study found that Imp2 as an oncogene plays an important role in the process of development in glioma. The expression level of Imp2 in gliomas was positively correlated with malignant degree, which has been confirmed by DNA microarray and tissue microarray technology [3].In order to study the expression differences of Imp2 between GBM and normal brain tissue, we compared 49 cases of clinical GBM glioma tissue samples, and 6 cases of normal brain tissue. Compared to the espression of Imp2 in normal brain tissue(median log2 ratio=-0.13), it was up-regulated in 43 of 49 gliomas specimens(median log2 ratio=1.424). The expression level of Imp2 m RNA in GMB tissue samples was significantly higher than that in normal brain tissue.Immunohistochemical staining showed that compared with normal brain tissue, 32 in 49 cases(65.3%) of GBM tissue specimen high expressed Imp2. Thus, it validated the difference expression of Imp2 between GBM and normal brain tissue at the protein level. Thirty cases of GBM tissue specimens and 4 cases of normal brain tissue in NCBI database was used to compared and calculated the difference expression of Imp2, and Imp2 expression increased significantly in GBM tissue specimens(P=0.001).The next, in order to study the impact on proliferation and invasion of Imp2 in GBM cell, we set up the human glioma cell line U87 and U251 cell model with Imp2 stable overexpression or silence cell line. Western blot results validated the success of Imp2 overexpression or knockdown cells model.Cell proliferation experiment results showed that compared with the U87 and U251 cells transfected Vector, the cells transfected Imp2 plasmid has a higher peak at 570 nm after joining MTT. It proved that Imp2 over overexpression in U87 and U251 cells can increase the cell proliferation ability while Imp2 silence can reduce cell proliferation ability. The results of cell wound healing experiment showed: when U87 and U251 cells transfected with Imp2 or Vector, the migration velocity of Imp2 over expression group is obviouslybigger than Vector group(P<0.05); Imp2 gene silencing group is obviously less than Scramble control group(P<0.05). Cell invasion experimental results found that:Imp2 over expression can markedly increase transwell invasion cell number(P<0.01); Similarly, silent Imp2 can reduce invasion of glioma cells(P<0.05).These results suggest that Imp2 over expression can increase the U87 and U251 GBM cell proliferation, migration and invasion ability, while silence Imp2 can significantly reduce the ability of proliferation, migration and invasion.To sum up,we validated Imp2 m RNA and protein expression in the gliomas tissue samples.In Imp2 abnormal expression glioma cell lines, Imp2 have influence on cell proliferation, migration and invasion ability. We explore its further possible mechanismby experiment. 2. Imp2 regulates cell growth, Epithelial-mesenchymal transition and drug resistancethrough IGF2 / PI3K/Akt signaling pathways.In order to detect the signal transduction pathways which Imp2 invoved in, we examined its downstream m RNA and protein expression both in Imp2 over expressionand silence model.The results suggest that IGF2 m RNA expression has no obvious change between IGF2 over expression and silence cells. We continue to detect IGF2 protein expression and found that Imp2 over expression cells expressed more while Imp2 silence cells decreased. It indicated that Imp2 did not affect the transcription of IGF2, but influenced the translation process of IGF2, which make more IGF2 across the cell membrane and bound IGF1 R receptor.In other cells, we found that IGF2 bound IGF1 R receptor to play a role of IX promoting cell proliferation by activating PI3K/Akt signal pathway.And in GBM cells, we tested the expression of P-Akt and Akt protein. Results indicate that the U87 and U251 cells, Imp2 over expression or silence can make p-Akt expression level rise or fall, without affecting the Akt protein expression levels. It proved that Imp2 regulate cell proliferation perhaps by IGF2/PI3K/Akt signaling pathways.Then we apply key protein inhibitorsofthe pathways blocking the signal pathway to verify our hypothesis through the observation of cell proliferation, migration and invasion abilityfrom the opposition.We added neutralizing antibody of IGF2 IGF2-Ab or PI3 K inhibitors LY294002 in U87 and U251 Imp2 over expression and the control group to inhibit IGF2 or PI3 K protein. The results showed that they offset part or all of the enhancement effect of glioma cell proliferation that the Imp2 over expression brought about. It proved that Imp2 regulate glioma cell proliferation by IGF2/PI3K/Akt signaling pathways.Adding IGF2 neutralization antibody in Vector control cells, we found that blocking IGF2 can significantly reduce migration and invasion ability ofthe GBM cell(P<0.05). Compared with the control group join PBS, add same concentration of IGF2 neutralizing antibodiesin Imp2 over expression cells, cell migration and invasion ability were decreased significantly(P < 0.05). Incells scratche and Transwell experiment, Adding PI3 K blocker LY294002, compared with the control group in DMSO, cell migration and invasion ability has dropped significantly in the Vector transfection cells and Imp2 plasmid transfection cells. It is verified in both forward and reverse ways that Imp2 regulate the GBM cell proliferation, migration and invasion through the IGF2/PI3K/Akt pathway.Epithelial-mesenchymal transition(EMT) refers to the process that at special physiological or pathological conditions, epithelial cells lose their polarity and translated into mesenchymal cells that with activity ability can freely move in cell matrix. EMT is an important mechanism in tumor development and progression, and PI3K/Akt pathway play a core role in EMT. Rt-PCR results showed that Imp2 over expression can increase the expression of E-cadherin and lead to the decrease of the Vimentin, N-cadherin expression while Imp2 silence decrease E-cadherin and increase Vimentin, N-cadherin expression. Western blot results of E-cadherin and Vimentin, N-cadherin expression in U87 GBM were consistent with Rt-PCR. And this indicated the important role of EMT in Imp2 regulating cell proliferation, migration and invasion ability.In drug resistance experiment, compared with Imp2 silence U87 resistance cell and ordinary U87 resistance cell, Imp2 overexpression showed increased cell proliferationin different TMZ concentrations. It suggests that Imp2 regulate sensitivity of GBM cells to TMZ, and the inhibition of Imp2 expression can increase the sensitivity of GBM cells to TMZ.Cell formation cloning experiments suggest: after cultured 14 daysunder the condition of no TMZ, Imp2 over expression and silence U87 resistant cells showed no difference in the number of Cell formation clones between groups.In 50 μM and 100 μM TMZ concentrations,compared with U87-RE cells, Imp2 over expression U87-RE cells cansignificantly form more clone numbers(P<0.05)while Imp2 silence U87-RE cells form less clone numbers(P<0.05). It further proved that Imp2 regulate the sensitivity of GBM cells to TMZ...