Investigation On Mechanism Of Bmi-1shRNA Inhibiting Cell Proliferation, Invasion And Migration Of Bladder Cancer5637Cells

Objective: To investigate the effect of Bmi-1shRNA on cellproliferation, invasion and metastasis of bladder cancer5637cells, byconstructing the vector pGeneClip^TM-shRNA for human Bmi-1gene, thenexplore the molecular marker for early diagnosis, tumor grade and prognosisof bladder cancer, and provide a viable solution of gene therapy for bladdercancer.Methods:1. Construct the vector pGeneClip^TM-shRNA: According to the siRNAdesign program from Promega Corporation and Bmi-1mRNA (NM₀05180)from GenBank inquiry system, we synthesized two target nucleotidefragments for the ring finger structure (shRNA1) and H-T-H-T structure(shRNA2) from506-1486bp core coding domain of Bmi-1, and a missensenucleotide fragment sequence. Synthesized short harpin DNA was connectedwith linearization pGeneClip^TMplasmid vector after being annealled andpurified. We transformed the products into competent DH5α E.coli. After therecombinant being screened and identified by PstⅠrestriction endonuclease,we extracted the plasmid to analysize DNA sequence and confirm thecorrectness of the short harpin DNA sequence. We got the correct expressionvector of shRNA: pGeneClip^TM-B1, pGeneClip^TM-B2, pGeneClip^TM-B-neg,and extracted the recombinant plasmids.2. Transfect5637cells with pGeneClip^TM-shRNA: We let the frozenbladder cancer5637cells recovery, be cultured and passaged. It was dividedinto four groups:(1) untransfected group (blank control group);(2)pGeneClip^TM-B-neg group (negative plasmid control group);(3)pGeneClip^TM-B1group (interfering ring finger structure of Bmi-1);(4) pGeneClip^TM-B2group (interfering H-T-H-T structure of Bmi-1). Accordingto CodeBreakerTMsiRNA Transfection Reagent protocol, the5637cells weretransfected with recombinant plasmid.3. Test the parameters of5637cells after being transfected: After testingthe expression levels of Bmi-1mRNA by RT-PCR and Bmi-1protein byWestern-Blot, we detected the cell proliferation activity by MTT, cellapoptosis by Flow Cytometry, cell invasion by Transwell invasion assay, andthe cell migration velocity by Cell scratch test.4. Statistical methods: Measurement data was figured by (χ±SD), alloutcomes were analysized by software SPSS13.0.Results:1. The constructions of pGeneClip^TM-shRNA met the design requirement:The three recombinant plasmids, which coding two target sequences (plasmidpGeneClip^TM-B1and pGeneClip^TM-B2) and a negative plasmid control(pGeneClip^TM-B-neg), met the design requirement by Pst Ⅰ restrictionendonuclease analysis.2. Bmi-1shRNA treatment significantly inhibited the expression levels ofboth Bmi-1mRNA and protein in5637cells: After5637cells beingtransfected with recombinant plasmids for48hours, we extracted the totalRNA and protein for the test of RT-PCR and Western-Blot. Bmi-1mRNA andprotein expression levels of B1and B2group were significantly lower than theB-neg group and untransfected group (P<0.05). It was no significantdifference between the B-neg group and untransfected group (P>0.05).3. Cell proliferation activity was tested by MTT: Cell growth of B1andB2was significantly inhibited (P<0.05), while the B-neg group anduntransfected group had no significant effect on the growth (P>0.05).4. Cell apoptosis was tested by Flow Cytometry: The rate of B1and B2cell apoptosis were (19.83±0.17)%and (27.33±0.15)%, comparing with theuntransfected group (7.13±0.11)%and B-neg group (6.70±0.13)%. Apoptosisrate of B1and B2group was importantly increased (P <0.05).5. Transwell chamber was used to detect cell invasion: By200times magnification, the transmembrane cell count of group B1, B2were78.0±5.8,75.0±7.2; untransfected group, B-neg were153.0±4.6,158.0±6.7. Comparingwith the other two groups, B1and B2groups had statistically significantdifference (P <0.05).6. Cell migration was detected by Cell scratch assay: The convergencetrend of B1, B2groups after48hours was still not obvrious, while most of thecells were close to fusion in untransfected group and the B-neg control group.Conclusions:1. pGeneClip^TM-shRNA for ring finger and H-T-H-T structure of Bmi-1gene can effectively inhibit the expression levels of Bmi-1mRNA and protein.2. Bmi-1shRNA can inhibit the cell proliferation, cell invasion andmigration of bladder cancer5637cells.