The Experimental Study Of MicroRNA-184 Inhibits Cell Proliferation And Invasion, And Specifically Targets TNFAIP2 In Human Glioma

Objective: To detect the expression level of mi R-184 and TNFAIP2 in human brain glioma specimens and glioma cell line and normal human brain tissues; To investigate the effect of miR-184 on the proliferation,invasion and apoptosis of glioma cell;To reveal the relationship between miR-184 and TNFAIP2 in gliomas 。 To lay the foundation for the discovery of new target genes for the diagnosis of gliomas,judging the prognosis and treatment of glioma。Method:1.The expression profile of miRNAs in glioma cell line SHG139 was detected by gene chip technology, and the expression of miR-184 was down- regulated, which was selected as the research object.Real-time quantitative RT-PCR method was used to verify the expression of miR-184 in glioma cell lines and non- tumor brain tissues.2.The expression of miR-184 in 49 cases of glioma tissues was detected by qRT-PCR method after increasing the quantity of the glioma tissues.3.MiR-184 mimics, inhibitor and negative control oligonucleotide were transfected into glioma cel-line U87 and U251 by Lipofectamine2000 liposome. The proliferation of U87 and U251 cells that were effected by miR-184 was detected by CCK-8 cell proliferation assay; The early apoptosis rate of U87 and U251 cells was detected by Annexin V PE Apoptosis Detection Kit PE and flow cytometry. The effect of miR-184 on the invasion ability of U87 and U251 was detected by scratch test and Transwell test.4.By referring to the literature and retrieval of biological information software(miRwalk, Targetscan, miRnada), we believe that miRNA-184 can be paired with the TNFAIP2 3,-UTR region. TNFAIP2 was detected by qRT-PCR technique in 81 cases of glioma, 5 glioma cells and 5 cases of non-tumor brain tissue. The expression of TNFAIP2 protein was detected by Western blot method. The expression of TNFAIP2 protein of U87 and U251 cells after transfected by miR-184 mimics、inhibitors and negative control miRNA was detected by Western blot.5. The use of lentiviral transfection method to construct stable expression of miR-184 and miR-NC in U87 cells, and were inoculated into nude mice subcutaneously and intracranial regularly; The maximum length and width of the subcutaneous mass of nude mice were measured; The changes of subcutaneous tumors in nude mice were observed under magnetic resonance T2 images, and then the mice were sacrificed at thirty-fifth days after inoculation; Then the transplanted tumor was weighed and then embedded in paraffin; The expression of TNFAIP2 and Ki-67 was detected by immunohistochemistry.Result:1.The expression of miR-184 was down-regulated in 49 cases of glioma tissues and 5 human glioma cellsMiRNA microarray data analysis showed that miR-184 expression was downregulated in human glioma cells.Using qRT-PCR method to verify that the expression of miR-184 in 5 glioma cells(U87, U373, U251, SHG44, A172) was significantly lower than that in non-tumor brain tissues(P<0.01) and the expression of miR-184 in 49 cases of glioma tissues was significantly lower than that in 8 cases(P< 0.01),with the increasing of the grade of glioma, the expression level of miR-184 in gliomas were gradually decreased.2.Over-expression of miR-184 can inhibit the proliferation,cell cycle progression and induce apoptosis of U87 and U251 cellsWe transfected U87 and U251 cell lines with miR-184 mimics and miRNA(mi R-NC). and then measured the absorbance at 450 nm wavelength by CCK-8 colorimetric massay for 3 days. Compared with control group mi R-NC, mi R-184 could inhibit cell proliferation(P<0.01); At the same time, the cell cycle was detected by flow cytometry. The over-expression of miR-184 could significantly increase the proportion of G0/G1 phase,decrease the proportion of S phase in U251 and U87 cells(P<0.05). Compared with the control group,overexpression of miR-184 could induce apoptosis of U87 and U251 cells, and down-regulated expression of miR-184 could decrease the number of apoptotic cells.3.Overexpression of miR-184 can inhibit the invasion and migration of U87 and U251 cells.The scratch assay results showed that the expression of miR-184 could inhibit the invasion of U87 and miR-184 cells compared with the control group. Transwell assay results indicated that the number of U251 and U87 cells in miR-184 mimics transfected group was less than that in the control groupm, the difference was statistically significant. This result shows that miR-184 can significantly inhibit the invasion and migration of U87 and U251.4.The expression of TNFAIP2 in glioma tissues and glioma cells was up-regulatedqRT-PCR results showed that the mRNA expression level of TNFAIP2 in 5 glioma cells was up-regulated compared with 5 cases of non-tumor brain tissues;The expression of TNFAIP2 in 81 cases of glioma tissues was up-regulated compared with that in non-tumor brain tissues. Western blot results further illustrated that the expression of TNFAIP2 protein in 5 human glioma cells was higher than that in normal human astrocytes 1800. Thus, it can be speculated that the expression of TNFAIP2 and miR-184 in gliomas were negatively correlated.5.The expression of TNFAIP2 in U87 and U251 can be negatively regulated by miR-184U87 and U251 cells were stably transfected with mi R-184 mimics,inhibitor and miRNA,then the RNA and protein of cells were extracted. Both qRT-PCR and Western blot results showed that overexpression of mi R-184 could significantly down regulate the expression of TNFAIP2; Downregulation of miR-184 can promote the expression of TNFAIP2.6.MiR-184 can inhibit the growth of U87 cells and inhibit the expression of Ki-67 and TNFAIP2 in nude miceThe growth of Subcutaneous tumor in nude mice of U87-miR-NC group was significantly lower than that of U87-miR-184 group, and the tumor of U87-miR-184 group was smaller than U87-mi R-NC group. At the thirty-fifth day, the mice were sacrificed and the tumor was measured. In U87-miR-184 group(0.27±0.10g),the tumor volume was significantly smaller than that in U87-miR-NC group(0.64±0.08g)the difference was statistically significant(P<0.01). The HE staining of intracranial tumor specimen in nude mice showed that the tumor volume in U87-miR-184 group was significantly smaller than that of the control group, and the expression of Ki-67 and target gene TNFAIP2 was significantly inhibited by overexpression of miR-184. In vivo experiments showed that miR-184 can significantly inhibit the growth and proliferation of glioma.Conclusions:(1) The expression of mi R-184 in glioma tissues and glioma cells was downregulated,however, the expression of TNFAIP2 was up-regulated in human glioma tissues and cells;(2) In vitro, the overexpression of miR-184 could inhibit the invasion and migration of U87 and U251 cells; Overexpression of miR-184 inhibited proliferation, cell cycle progression and induced apoptosis of U87 and U251 cells;(3) The expression of TNFAIP2 in U87 and U251 cells could be negatively regulated by miR-184;(4) In vivo, overexpression of miR-184 can inhibit the growth of U87 transplanted tumor, and miR-184 can specifically target the expression of TNFAIP2 protein in nude mice.