Study On The Effect Of Knockdown MiR-221by RNAi On Glioma Cell

PART ONE Expression and significance of miR-221and p27in humangliomas【Objective】To analyze the expression and clinical significance of miR-221and p27in human glioma samples.【Methods】The expressions of miR-221and p27were detected by fluorescencequantitative-PCR and Western blot in29glioma samples of different malignancygrades. The relationship of the expressions with the malignancy grades of the tumorwas analyzed.【Results】The expression level of miR-221was positively correlated with gliomamalignancy and negatively correlated with the expression of p27.【Conclusion】 The overexpression of miR-221and downexpression is closelycorrelated to development and progress of human glioma. PART TWO Construction and Screening an EffectiveAnti-miR-221RNAi Vector in vitro【Objective】 To construct and screen an effective anti-miR-221vector of siRNA.【Methods】 Four hairpin structure of siRNA transcript templates targeting miR-221and a negative control were synthesized, then ligated with p GCSIL-GFP vector and a p EGFP-miR-221which express pre-miR-221was also constructed. All therecombinants were sequenced. The confirmed p GCSIL-GFP recombinants bycombining with p EGFP-miR-221were transfected into293T cells seperately. Theexpressed Flag protein was detected by Western blot to evaluate the inhibition effectof targeting sequences.【Results】 The resulting recombinants were confirmed by sequencing whichdemonstrated that the recombinant plasmids contained the correct sequences ofdesigned transcript templates. The results of Western blot indicated the expression ofFlag of No1recombinant plasmid group was inhibited most obviously by comparedwith control group.【Conclusion】 The anti-miR-221expression siRNA espression recombinants wereconstructed successfully, and one sequence with the highest inhibition efficiency wasscreened out, and could be used to suppress target gene for further study in tumorbiology. PART THREE Study on the effect of knockdown of miR-221on U87glioma cell【Abstreact】Objective To construct an anti-miR-221lentiviral vector of siRNA,and study on the effect of knockdown miR-221on U87cells in vitro.【Methods】 A hairpin structure of siRNA transcript templates targetingpre-miR-221and a negative control were synthesized and ligated with lentiviralvector and all the vectors were sequenced. The recombinant lentiviral vectors werethen used to infect U87cells. Then Q-PCR, western blot, MTT, FCM and transwellwere used to detect the effects of knockdown miR-221.【Results】The data of sequencing showed that the recombinant lentiviral vectorscontained the correct sequences of designed transcript templates. The results indicated that the expression of miR-221was inhibited, and at the same time theexpression of p27was upgraded, which is a target of miR-221. Knockdown ofmiR-221also inhibited the growth and invasiveness of U87and induced apoptosis inU87cell.【Conclusion】An anti-miR-221expression siRNAlentiviral vector was constructedsuccessfully, which could inhibit U87cell proliferation and induce cell apoptosis,and could be used to suppress target gene for further study in tumor biology.