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Let-7b expression determines response to chemotherapy through the regulation of cyclin D1 in glioblastoma.

Guo Y, Yan K, Fang J, Qu Q, Zhou M, Chen F - J. Exp. Clin. Cancer Res. (2013)

Bottom Line: Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells.Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis.Collectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurosurgery, Xiangya Hospital, Central South University, 410008, Changsha, China.

ABSTRACT

Background: Glioblastoma is the most common type of primary brain tumors. Cisplatin is a commonly used chemotherapeutic agent for Glioblastoma patients. Despite a consistent rate of initial responses, cisplatin treatment often develops chemoresistance, leading to therapeutic failure. Cellular resistance to cisplatin is of great concern and understanding the molecular mechanisms is an utter need.

Methods: Glioblastoma cell line U251 cells were exposed to increasing doses of cisplatin for 6 months to establish cisplatin-resistant cell line U251R. The differential miRNA expression profiles in U251 and U251R cell lines were identified by microarray analysis and confirmed by Q-PCR. MiRNA mimics were transfected into U251R cells, and cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis.

Results: U251R cells showed 3.1-fold increase in cisplatin resistance compared to its parental U251 cells. Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells. Transfection of Let-7b mimics greatly re-sensitized U251R cells to cisplatin, while transfection of other miRNAs has no effect or slightly effect. Cyclin D1 is predicted as a target of Let-7b through bioinformatics analysis. Over-expression of Let-7b mimics suppressed cyclin D1 protein expression and inhibited cyclin D1-3'-UTR luciferase activity. Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis.

Conclusions: Collectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression. Let-7b may serve as a marker of cisplatin resistance, and can enhance the therapeutic benefit of cisplatin in glioblastoma cells.

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Transfection of Let-7b increased cisplatin-induced apoptosis in U251R cells. U251 cells (A), U251R cells (B) or U251R cells transfected with Let-7b (C) were treated with cisplatin at 0.625 μg/mL for 48 hours. Cisplatin-induced apoptosis was assessed by Annexin V staining followed by flow cytometry. Right-hand quadrants indicate Annexin V positive cells, indicative of apoptosis. (D) The percentage of apoptotic cells was calculated from at least three separate experiments. (E) U251, U251R and U251R transfected with Let-7b mimics were treated with cisplatin for 48 hours, and caspase-3 activity was measured. The results were presented as mean±SD (n = 3) (*p < 0.05).
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Figure 5: Transfection of Let-7b increased cisplatin-induced apoptosis in U251R cells. U251 cells (A), U251R cells (B) or U251R cells transfected with Let-7b (C) were treated with cisplatin at 0.625 μg/mL for 48 hours. Cisplatin-induced apoptosis was assessed by Annexin V staining followed by flow cytometry. Right-hand quadrants indicate Annexin V positive cells, indicative of apoptosis. (D) The percentage of apoptotic cells was calculated from at least three separate experiments. (E) U251, U251R and U251R transfected with Let-7b mimics were treated with cisplatin for 48 hours, and caspase-3 activity was measured. The results were presented as mean±SD (n = 3) (*p < 0.05).

Mentions: The cisplatin-induced apoptosis was examined by Annexin V/PI staining (Figure 5A-C). Consistently, Let-7b mimics increased cisplatin-induced apoptosis in U251R cells compared with scramble transfection (16.66±1.57% Vs. 8.32±0.85%, p < 0.05). Notably, the apoptosis in U251R transfected with Let-7b is comparable to that in U251 parental cells (16.66±1.57% vs. 17.82±1.47%, p > 0.05) (Figure 5D).


Let-7b expression determines response to chemotherapy through the regulation of cyclin D1 in glioblastoma.

Guo Y, Yan K, Fang J, Qu Q, Zhou M, Chen F - J. Exp. Clin. Cancer Res. (2013)

Transfection of Let-7b increased cisplatin-induced apoptosis in U251R cells. U251 cells (A), U251R cells (B) or U251R cells transfected with Let-7b (C) were treated with cisplatin at 0.625 μg/mL for 48 hours. Cisplatin-induced apoptosis was assessed by Annexin V staining followed by flow cytometry. Right-hand quadrants indicate Annexin V positive cells, indicative of apoptosis. (D) The percentage of apoptotic cells was calculated from at least three separate experiments. (E) U251, U251R and U251R transfected with Let-7b mimics were treated with cisplatin for 48 hours, and caspase-3 activity was measured. The results were presented as mean±SD (n = 3) (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716992&req=5

Figure 5: Transfection of Let-7b increased cisplatin-induced apoptosis in U251R cells. U251 cells (A), U251R cells (B) or U251R cells transfected with Let-7b (C) were treated with cisplatin at 0.625 μg/mL for 48 hours. Cisplatin-induced apoptosis was assessed by Annexin V staining followed by flow cytometry. Right-hand quadrants indicate Annexin V positive cells, indicative of apoptosis. (D) The percentage of apoptotic cells was calculated from at least three separate experiments. (E) U251, U251R and U251R transfected with Let-7b mimics were treated with cisplatin for 48 hours, and caspase-3 activity was measured. The results were presented as mean±SD (n = 3) (*p < 0.05).
Mentions: The cisplatin-induced apoptosis was examined by Annexin V/PI staining (Figure 5A-C). Consistently, Let-7b mimics increased cisplatin-induced apoptosis in U251R cells compared with scramble transfection (16.66±1.57% Vs. 8.32±0.85%, p < 0.05). Notably, the apoptosis in U251R transfected with Let-7b is comparable to that in U251 parental cells (16.66±1.57% vs. 17.82±1.47%, p > 0.05) (Figure 5D).

Bottom Line: Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells.Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis.Collectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurosurgery, Xiangya Hospital, Central South University, 410008, Changsha, China.

ABSTRACT

Background: Glioblastoma is the most common type of primary brain tumors. Cisplatin is a commonly used chemotherapeutic agent for Glioblastoma patients. Despite a consistent rate of initial responses, cisplatin treatment often develops chemoresistance, leading to therapeutic failure. Cellular resistance to cisplatin is of great concern and understanding the molecular mechanisms is an utter need.

Methods: Glioblastoma cell line U251 cells were exposed to increasing doses of cisplatin for 6 months to establish cisplatin-resistant cell line U251R. The differential miRNA expression profiles in U251 and U251R cell lines were identified by microarray analysis and confirmed by Q-PCR. MiRNA mimics were transfected into U251R cells, and cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis.

Results: U251R cells showed 3.1-fold increase in cisplatin resistance compared to its parental U251 cells. Microarray analysis identified Let-7b and other miRNAs significantly down-regulated in U251R cells compared to U251 cells. Transfection of Let-7b mimics greatly re-sensitized U251R cells to cisplatin, while transfection of other miRNAs has no effect or slightly effect. Cyclin D1 is predicted as a target of Let-7b through bioinformatics analysis. Over-expression of Let-7b mimics suppressed cyclin D1 protein expression and inhibited cyclin D1-3'-UTR luciferase activity. Knockdown of cyclin D1 expression significantly increased cisplatin-induced G1 arrest and apoptosis.

Conclusions: Collectively, our results indicated that cisplatin treatment leads to Let-7b suppression, which in turn up-regulates cyclin D1 expression. Let-7b may serve as a marker of cisplatin resistance, and can enhance the therapeutic benefit of cisplatin in glioblastoma cells.

Show MeSH
Related in: MedlinePlus