[miR-15a and miR-16 modulate drug resistance by targeting bcl-2 in human colon cancer cells]
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Published on: September 30, 2016
Min Xu1, Bingjie Zhou1, Guoying Wang1
1Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China.
Objective:
To investigate the reversal effect of targeted modulation of bcl-2 expression by miR-15a and miR-16 on drug resistance of human colon cancer cells.
Methods:
Mimics or inhibitors of miR-15a and miR-16 were transfected into HCT8 or HCT8/VCR cells with the help of Lipofectamine 2000. The expressions of miR-15a and miR-16 mRNA were detected by RT-qPCR. The levels of bcl-2 and P-gp proteins were measured by Western blot. The inhibitory effects of VCR on growth of HCT8 and HCT8/VCR cells were detected by CCK8.
Results:
After transfection of the mimics, the expression of miR-15a in the blank control group, negative control group and miR-15a mimic group was 1.00, 0.87 ± 0.24, and 223.44 ± 59.07, respectively, and miR-15a was increased significantly (P < 0.001). The expression of miR-16 in the blank control group, negative control group and miR-16 mimic group was 1.00, 0.66 ± 0.19, and 107.32 ± 22.58, respectively, and miR-16 expression was increased significantly (P < 0.001). The Western blot assay showed that the relative expressions of bcl-2 protein in the blank control group, negative control group, miR-15a mimic group and miR-16 mimic group were 1.00, 0.97 ± 0.02, 0.51 ± 0.06, and 0.65 ± 0.03, respectively, and the expression of bcl-2 protein was decreased significantly (P < 0.05), however, the expressions of P-gp protein showed no significant difference. The CCK8 test showed that at 1, 5, 25 and 125 µg/ml concentration of VCR, the survival rates of HCT8/VCR cells were basically the same in the blank control group, negative control group, miR-15a mimic group and miR-16 mimic group, but the survival rate of HCT8/VCR cells was significantly decreased after transfection of mimics (P < 0.05). After transfection of the inhibitors, the expressions of both miR-15a and miR-16 were decreased significantly (P < 0.001). The Western blot showed that the expression of bcl-2 protein was increased (P < 0.05), while the expression of P-gp protein showed no significant difference. The CCK8 test showed that the survival rate of HCT8 cells which were transfected with inhibitors was significantly higher than that of the blank control group (P < 0.05).
Conclusions:
miR-15a and miR-16 may reverse the drug resistance in human colon cancer cells. A possible mechanism is regulating the expression of bcl-2.
Insights
MicroRNAs miR-15a and miR-16 can reverse drug resistance in human colon cancer cells by regulating bcl-2 expression. This finding offers a potential new strategy for treating colon cancer.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- Drug resistance is a major challenge in colon cancer treatment.
- MicroRNAs (miRNAs) play crucial roles in cancer development and drug resistance.
- bcl-2 is a key protein involved in apoptosis and drug resistance.
Purpose of the Study:
- To investigate the effect of miR-15a and miR-16 on drug resistance in human colon cancer cells.
- To explore the role of bcl-2 expression modulation by miR-15a and miR-16 in reversing drug resistance.
Main Methods:
- Transfection of miR-15a/miR-16 mimics or inhibitors into HCT8/VCR colon cancer cells.
- Quantitative real-time PCR (RT-qPCR) to measure miRNA expression.
- Western blot to assess bcl-2 and P-gp protein levels.
- Cell Counting Kit-8 (CCK8) assay to evaluate cell viability and drug sensitivity.
Main Results:
- Transfection with miR-15a/miR-16 mimics significantly increased miRNA levels and decreased bcl-2 protein expression.
- miR-15a/miR-16 mimics enhanced the sensitivity of HCT8/VCR cells to vincristine (VCR).
- Inhibitors of miR-15a/miR-16 decreased miRNA levels, increased bcl-2 expression, and reduced sensitivity to VCR.
Conclusions:
- miR-15a and miR-16 play a significant role in reversing drug resistance in human colon cancer cells.
- Modulating bcl-2 expression is a potential mechanism by which miR-15a and miR-16 exert their effects.
- These findings suggest therapeutic potential for targeting miR-15a/miR-16 in overcoming colon cancer drug resistance.
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