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Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

Munoz JL, Rodriguez-Cruz V, Ramkissoon SH, Ligon KL, Greco SJ, Rameshwar P - Oncotarget (2015)

Bottom Line: We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance.TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein.Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells.

View Article: PubMed Central - PubMed

Affiliation: New Jersey Medical School, Rutgers, Newark, NJ, USA.

ABSTRACT
Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

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Increased miR-9 in GBM(A) Computational analyses were performed for potential miRNA interacting site with the PTCH1 3′ UTR. The alignment between the sequence for miR-9 and the 3′ UTR of PTCH1 is shown below. (B) U87 and T98G cells were treated with 200 μM TMZ. At various times, real time PCR (Taqman) was performed for miR-9. (C) The studies in ‘C’ were repeated, except for studies at the 72-h time point, and with primers for miR-9-2, using Sybr-Green. (D) U87 and T98G were transfected with non-targeting oligo or anti-miR-9 and then treated with 200 μM TMZ. After 72 h, cell viability was assessed and presented as % viability, n = 4 ±SD. (E) The heat map shows the analyses from 505 miRNA arrays with GBM tissues from The Cancer Genome Atlas (TCGA). The map represents the miRNAs with +/– 0.5-fold changes from the internal control. Arrow highlights the output for miR-9. *p < 0.05 vs. other time points, **p < 0.05 vs. vehicle. ***p < 0.05 vs. non-targeting oligo.
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Figure 2: Increased miR-9 in GBM(A) Computational analyses were performed for potential miRNA interacting site with the PTCH1 3′ UTR. The alignment between the sequence for miR-9 and the 3′ UTR of PTCH1 is shown below. (B) U87 and T98G cells were treated with 200 μM TMZ. At various times, real time PCR (Taqman) was performed for miR-9. (C) The studies in ‘C’ were repeated, except for studies at the 72-h time point, and with primers for miR-9-2, using Sybr-Green. (D) U87 and T98G were transfected with non-targeting oligo or anti-miR-9 and then treated with 200 μM TMZ. After 72 h, cell viability was assessed and presented as % viability, n = 4 ±SD. (E) The heat map shows the analyses from 505 miRNA arrays with GBM tissues from The Cancer Genome Atlas (TCGA). The map represents the miRNAs with +/– 0.5-fold changes from the internal control. Arrow highlights the output for miR-9. *p < 0.05 vs. other time points, **p < 0.05 vs. vehicle. ***p < 0.05 vs. non-targeting oligo.

Mentions: This set of studied sought the identity of the candidate miRNA(s) in TMZ resistance. We focused on the miRNA(s) that could target PTCH1. An analysis of the 3′ UTR of PTCH1 identified potential interacting sites for mir-9, mir-16, mir-101, mir-141 and mir-200a (Figure 2A, Table S1). Time-course real time PCR with viable TMZ-treated GBM cells indicated a significant (p < 0.05) increase in miR-9 at 72 h (Figure 2B).


Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

Munoz JL, Rodriguez-Cruz V, Ramkissoon SH, Ligon KL, Greco SJ, Rameshwar P - Oncotarget (2015)

Increased miR-9 in GBM(A) Computational analyses were performed for potential miRNA interacting site with the PTCH1 3′ UTR. The alignment between the sequence for miR-9 and the 3′ UTR of PTCH1 is shown below. (B) U87 and T98G cells were treated with 200 μM TMZ. At various times, real time PCR (Taqman) was performed for miR-9. (C) The studies in ‘C’ were repeated, except for studies at the 72-h time point, and with primers for miR-9-2, using Sybr-Green. (D) U87 and T98G were transfected with non-targeting oligo or anti-miR-9 and then treated with 200 μM TMZ. After 72 h, cell viability was assessed and presented as % viability, n = 4 ±SD. (E) The heat map shows the analyses from 505 miRNA arrays with GBM tissues from The Cancer Genome Atlas (TCGA). The map represents the miRNAs with +/– 0.5-fold changes from the internal control. Arrow highlights the output for miR-9. *p < 0.05 vs. other time points, **p < 0.05 vs. vehicle. ***p < 0.05 vs. non-targeting oligo.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359226&req=5

Figure 2: Increased miR-9 in GBM(A) Computational analyses were performed for potential miRNA interacting site with the PTCH1 3′ UTR. The alignment between the sequence for miR-9 and the 3′ UTR of PTCH1 is shown below. (B) U87 and T98G cells were treated with 200 μM TMZ. At various times, real time PCR (Taqman) was performed for miR-9. (C) The studies in ‘C’ were repeated, except for studies at the 72-h time point, and with primers for miR-9-2, using Sybr-Green. (D) U87 and T98G were transfected with non-targeting oligo or anti-miR-9 and then treated with 200 μM TMZ. After 72 h, cell viability was assessed and presented as % viability, n = 4 ±SD. (E) The heat map shows the analyses from 505 miRNA arrays with GBM tissues from The Cancer Genome Atlas (TCGA). The map represents the miRNAs with +/– 0.5-fold changes from the internal control. Arrow highlights the output for miR-9. *p < 0.05 vs. other time points, **p < 0.05 vs. vehicle. ***p < 0.05 vs. non-targeting oligo.
Mentions: This set of studied sought the identity of the candidate miRNA(s) in TMZ resistance. We focused on the miRNA(s) that could target PTCH1. An analysis of the 3′ UTR of PTCH1 identified potential interacting sites for mir-9, mir-16, mir-101, mir-141 and mir-200a (Figure 2A, Table S1). Time-course real time PCR with viable TMZ-treated GBM cells indicated a significant (p < 0.05) increase in miR-9 at 72 h (Figure 2B).

Bottom Line: We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance.TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein.Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells.

View Article: PubMed Central - PubMed

Affiliation: New Jersey Medical School, Rutgers, Newark, NJ, USA.

ABSTRACT
Glioblastoma Multiforme (GBM), the most common and lethal adult primary tumor of the brain, showed a link between Sonic Hedgehog (SHH) pathway in the resistance to temozolomide (TMZ). PTCH1, the SHH receptor, can tonically represses signaling by endocytosis. We asked how the decrease in PTCH1 in GBM cells could lead to TMZ-resistance. TMZ resistant GBM cells have increased PTCH1 mRNA and reduced protein. Knockdown of Dicer, a Type III RNAase, indicated that miRNAs can explain the decreased PTCH1 in TMZ resistant cells. Computational studies, real-time PCR, reporter gene studies, western blots, target protector oligos and ectopic expression identified miR-9 as the target of PTCH1 in resistant GBM cells with concomitant activation of SHH signaling. MiR-9 mediated increases in the drug efflux transporters, MDR1 and ABCG2. MiR-9 was increased in the tissues from GBM patients and in an early passage GBM cell line from a patient with recurrent GBM but not from a naïve patient. Pharmacological inhibition of SHH signaling sensitized the GBM cells to TMZ. Taken together, miR-9 targets PTCH1 in GBM cells by a SHH-independent method in GBM cells for TMZ resistance. The identified pathways could lead to new strategies to target GBM with combinations of drugs.

Show MeSH
Related in: MedlinePlus