Feed-forward miR-181d degradation modulates population variance of methyl-guanine methyl transferase (MGMT) and temozolomide resistance
Description
To characterize the changes in miRNA expression in response to TMZ therapy, two patient-derived glioblastoma cell lines BT-83 and CMK3 were treated with 500 μM TMZ or vehicle for 6 h. These cell lines were selected because their gene expression profile suggests they capture the two key glioblastoma cell states: BT-83 exhibits an expression pattern suggestive of the mesenchymal-like state, while CMK3 exhibits an expression pattern suggestive of the neural progenitor-like state45. RNA was extracted and profiled by RNA sequencing. In both cell lines, the TMZ treatment did not significantly alter the expression of >95% of the miRNAs.miRNAs exhibiting a >2-fold change in expression following TMZ treatment in both lines were individually assessed to determine whether their expression differed in matched pre- and post-TMZ-treated clinical glioblastoma specimens.Among the miRNAs evaluated, miR-181d was the only one consistently showing lowered levels in the post-TMZ specimens. This finding recapitulated our prior profiling experiment, where miR-181d was downregulated in post-TMZ-treated clinical glioblastoma specimens.
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Steps to reproduce
BT-83 or CMK3 cells were treated with 500 μM TMZ or DMSO (1%) for 6 h. The cells were harvested, and RNA was isolated (miRNeasy Kit, Qiagen). The RNA integrity was determined by Bioanalyzer RNA 6000 Nano kit (Agilent). The RNA samples were submitted to the University of Minnesota Genomic Center (UMGC) for library preparation using TruSeq Small RNA Library protocol and short-read sequencing using HiSeq 2500 High Throughput Sequencer (Illumina). All datasets were analyzed using a uniform processing workflow. Raw sequencing data (FASTQ files) underwent preprocessing and quality control with miRTrace. Reads were discarded if fewer than 50% of nucleotides had a Phred score above 20 nucleotides. Adapter sequences at the 3′end was trimmed, and sequences shorter than 18 nucleotides or consisting of repetitive elements were removed. Samples were excluded if fewer than 25% of reads were 20-25 nucleotides in length, more than 75% of reads were removed during QC, or fewer than 10% of reads were classified as miRNA.
Institutions
- University of Minnesota