A Preliminary Study About Inhibition Action On Growth And Mechanism Of U251Human Glioma Cells By MiRNA-29b

Objective:To observe the effect on U251human gliomacell line growth bytransfected with has-miRNA-29bmimics in vitro, and to clarify werther it canmodulate the proliferation and/or apoptosis of U251cells, to explore theexpression of protein which are interrelated with proliferation and apoptosis inorder to seach its mechanism preliminarily. The ultimate objective of thisexperimentation is to provide theoretical basis for Clinical treatment ofglioma.Methods:1. Lipofectamine2000was used to transfect cel-miRNA-67mimics orhas-miRNA-29b mimics into U251glioma cell line in vitro. The finalconcentration of cel-miRNA-67mimics or miRNA-29b mimics used totransfect U251cells is100nmol/l. The control group was transfected withcel-miRNA-67mimics, While the experimental group was transfect withmiRNA-29b mimics. According to the need of different experiments，theU251cells used to MTT assay、flow cyometry and Western bloting wereharvested at different points （24h、48h or72h）after transfection。2.(1) The observation of cell morphology：Inverted phase contrastmicroscope directly observe the culture plates with the control group and theexperimental group U251cells form and photographed at24h、48h.Comparing the difference between the two groups cells，and taking picturesto record. Each group were repeated three times.(2) MTT assay：The opticaldensity value of the U251cells dyed by four methyl thiazolyl tetrazoliumcolorimetric method were measured at the wavelength of490nm and630nm.Calculated A value (A=OD490nm-OD630nm）which was used to evaluate the cellproliferation ability.(3) Flow cytometry detecte cell cycle phase distribution and apoptosis: PI single staining method was used to detecte cell cycle phasedistribution of the two group cells at48h, recording different cell cycle cellratio. Annexin V/PI double staining method was used to detecte cell apoptosisat48h, calculated the apoptosis index which is represented by AI，AI=Thenumber of apoptosis cells/The number of total cells.(4) Western bloting wasemployed to test the expression of CDK6、cyclinD1、Bcl-2、Mcl-1and Bax inthe two groups of U251cells at both24h and48h. Calculation the proteinexpression ratio of gray in two groups，used to quantitative analysis by IPP6.0. The experiment was repeated5times.3. Statistical analysis: All data are expressed as mean±standarddeviation (x±S). The data were analyzed by two-sample t-test or Wilcoxonrank sum test through SPSS17.0statistical software. P <0.05was statisticallysignificant difference.Results:1. Cell morphology: It is found that the control group cells were growingwell. The adherent phenomenon was obviously.The cell were polygonal, whileits transparent was cytoplasm,and nucleus was clear.The growth ofexperimental group cells were inhibited partly. Specifically, cell growth wasslow， adherent phenomenon was weaken， cell shrinkage slightly, andcytoplasm particles increased.2. MTT results: At24h,48h,72h, the value of A in the control group andthe experimental group were （0.14±0.01）,（0.25±0.03）,（0.36±0.02）and（0.13±0.01）,（0.21±0.02）,（0.30±0.02）, there were statisticallysignificant differences between the two groups (P <0.05)，it is the mostsignificant at72h.3. Flow cytometry results：At48h，the control group cells in the G1, S,G2phase respectively wa（s44.78±1.52）%（、50.86±1.52）%an（d4.56±0.47）%;while experimental group cells were （57.27±2.27）%，（39.25±2.12）%，（3.46±0.44）%, there were statistically significant differences between thetwo groups (P <0.05). Apoptosis rate in the experimental group was（14.96±1.27）%, which was significantly higher than （4.52±1.08）%in the control group (p <0.05).4.By Western bloting results showed that: At24h、48h, the expressionlevels of CDK6, Bcl-2and Mcl-1protein in experimental group wereweakened than in the control group, Bax protein expression level wasenhanced,while there was no significant changes in cyclinD1. The changewas more obvious at48h.Conclusion:1. Overexpression of miRNA-29b can inhibit the growth of U251humanglioma cell.2. Overexpression of miRNA-29b can arrest cell cycle of U251humanglioma cell at G1, its mechanism is related to inhibiting the expression levelsof CDK6protein while unrelated with cyclinD1protein.3. Overexpression of microRNA-29b induces apoptosis of U251gliomacells, its mechanism is related to inhibiting the expression levels of Bcl-2，Mcl-1protein and enhancing the expression level of Bax.4. miRNA-29b may be a target spot in targeted therapy of glioma.