Effect Of Down-regulation Human Angiogenin On Proliferation And Apoptosis In Human Bladder Cancer T24Cells

Objective To determine the effects of down-regulation angiogeninby vector-mediated siRNA on proliferation, apoptosis in human bladdercancer T24cells.Methods1.Construct two specific siRNA expression vectors andone no homology control vector，and identify the vectors by SalI digestionand DNA sequencing. Then the vectors were stably transfected into T24cell by Lipofectamine2000; Using G418to screen the positive clone andrespectively named T24-NC group (transfected with pGenesil-1siRNA3plasmid), T24-siANG1(transfected withp pGenesil-1siRNA1plasmid)group and T24-siANG2(transfected withp pGenesil-1siRNA2plasmid)group. The efficiency of ANG siRNA was detected by Q-PCR, westernblot.2.The change of morphology was determined by HE staining; MTTassay and Flow cytometry examination were done to detect the inhibitoryeffect of ANG siRNA on the cell proliferation; Immunofluorescence LaserScanning Confocal was detected whether RI and ANG colocalized; and then using cell/tumor Immunofluorescence way to assess the expression ofANG, RI, p-Akt, p-mTOR and p-GSK3β.3. Flow Cytometry examination, TUNEL kit and Hochest(33342)assay were done to evaluate the effect of ANG siRNA on the cell apoptosis;The expression level of protein RI, AKT, GSK3β, mTOR, p-Akt, p-mTOR,p-GSK3β, Caspase3, Bax and Bcl-2were determined by Western blotting.4. The T24cells, T24-NC cells and T24-siANG1cells wererespectively collected and injected into the backs of the BALB/C nudemice,35days after injection, All the mice were killed and then the tumorswere weighted. Immunofluorescence detection was done to explore thelevel of ANG, RI, Bax, Caspase-3, Bcl-2, p-AKT, p-GSK3β, p-mTOR,AKT, GSK3βand mTOR; The micorvessels of tumor and the metastasis oflung were assessed by HE staining. In the mine time, the influence of ANGsiRNA on micorvessels of tumor was doned to determine viaimmunofluorescence assay of CD31Results1. The ANG siRNA expression vectors were constructedand identified with endonuclease digesting and DNA sequencing.Compared with negative control, the ANG expression of mRNA andprotein levels was significantly decreased inT24-siANG1(p<0.05).2. The HE staining showed that the transfected T24-siANG1cellsbecame lower malignant phenotype including less overlapping growth, asmaller nucleus, and weaker alkalophilic quality of cytoplasm compared with the other two control groups； T24-siANG1cell group showedremarkable lower cell proliferation than those of the T24and the vectorcells groups；The results indicated that the proliferations of T24-siANG1cells were inhibited with G1phase arresting, S phase and G2-M reducing;The results demonstrated that ANG and RI colocalized in cytoplasm；Theexpression of ANG, p-Gsk3β, ANG downstream targets p-Akt and p-mTORdecreased, but the expression of RI was increased.3. Flowcytometry showed that (30.23±15.81)%of counted cellsbecame apoptotic in T24-siANG1cells, however, about only (5.44±3.09)%and (3.96±1.58)%of counted cell became apoptotic in T24cells andT24-NC cells groups respectively. The number of TUNEL-positive cellswas significantly higher in the T24-siANG1cells, compared with T24cellsand T24-NC cells. The results of Hochest demonstrated thatT24-siANG1cells appeared typical apoptotic morphology features such aschromatin condensation, the nuclear fragmentation and brighter bluefluorescent. However, the control cells did not emerge apoptoticcharacteristics. the expression of Bcl-2was significantly decreased inT24-siANG1cells compared with T24and T24-NC cells(P＜0.01),but theexpression of Caspase-3and Bax protein was stably increased (P＜0.05).4. All the cell groups were implanted into the backs of BALB/C nudemice. The results showed that the T24-siANG1cell group significantlyinhibited the growth of bladder cancer compared with the other control groups; Immunofluorescence assay of CD31and HE stain was performed.T24-siANG1group showed low CD31expression and remarkableinhibition of angiogenesis in tumor tissue than two other control groups;Mice injected with the T24-siANG1cells also showed a significantsuppression of the spontaneous lung metastasis; The T24-siANG1groupshowed higher expression of RI, Bax and Caspase3, as well as lowerexpression of ANG, p-Akt, p-GSK3β and p-mTOR compared with thecontrol groups.Conclusion Inhibiting of ANG expression vectors were constructedsuccessfully, and significately inhibited proliferation and induced apoptosisof bladder cancer T24cells via regulating the apoptosis-related proteins andPI3K/AKT/mTOR signaling pathway in vivo and in vitro.