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The Impact of Up-regμlating miR-21on the Biological Behaviors of HT-29Cancer Cells and Sensitivity to5-RU and Radiotherapy

Author: DengJun
Tutor: XiongJianPing
School: Nanchang University Medical College
Course: Oncology
Keywords: lentiviral-vector miR-21 HT-29 drμg-resistant
CLC: R735.34
Type: Master's thesis
Year: 2013
Downloads: 22
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Abstract


Objective: To investigate the impact of up-regμlating miR-21on the biologicalbehaviors of HT-29cancer cells and observe the changes of sensitivity to5-FU andradiotherapy.Method: we first transfected with Lentiviral vector-pre-miR-21as theexperimental group,then we transfected the nonsense sequence lentiviral vector-NCand lentiviral vector-U6as the negative control groups,and utilized the HT-29cell asthe mock group.we developed a lentiviral vector for the over expression ofpre-miR-21and evaluated the transfection effeciency under a fluorescencemicroscope. The miR-21expression was measured by TaqMan real-time PCR,thecell proliferation capacity was detected by CCK-8;the colony formation capacity wasdetected by the colony formation assay;cell apoptosis and cell cycle distribution wasanalyzed with flow cytometry;Transwell chamber assay was used to observe the cellmigration ability. chemotherapy sensitivity to5-FU was detected by CCK-8andcalcμlated the half of the inhibition (IC50)to5-FU; the radiation sensitivity wasdetected by MTT.Each test was repeated three times.Resμlts: Real-time PCR resμlt showed that the miR-21expression in thelentiviral vector-pre-miR-21group increased4.228times (p <0.01). compared withthe control group and two negative control groups, the up-regμlating in the expressionof miR-21significantly promoted the proliferation of HT-29cells (p <0.01), andsignificantly increased colony-forming ability(559±19vs375.67±21.01p<0.01),inhibited apoptosis (p <0.05),early apoptotic (1.1±0.26vs6.67±0.81)%,lateapoptotic and dead cells(1.33±0.85vs5.53±1.1)%; furthermore, up-regμlating ofmiR-21re-distributed the cell cycle and promoted G1/S cell cycle transition,leadingto the proportion of G0/G1phase cells reduced(36.53±0.92vs60.18±0.93)%(p<0.01) and the proportion of S phase cells increased(56.8±1.02vs33.0±1.01)%(p<0.01), G2/M phase cells was no significant difference; the invasion ability in vitrowas enhanced(invasion cells104.8±6.72vs34.2±2.59,p <0.01), and reduced chemo-sensitivity to5-FU, the IC50of the experimental group (232.73±21.69μg/ml)is2.07times compared to the control group (112.46±15.26μg/ml)(p<0.01); the inhibition rate to X radiation is lower(16.76±1.87vs36.14±1.54)%(p<0.05).Conclusion:we demonstrated that up-regμlating of miR-21inhibited cellsapoptosis in HT-29cancer cells, and promoted proliferation, colony formation abilityand invasion capacity,and increased the tumor cells to5-FU and X radiationresistance.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Intestinal neoplasms > Colorectal tumors
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