The SOX2 response program in glioblastoma multiforme: an integrated ChIP-seq, expression microarray, and microRNA analysis

Abstract

Background: SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined.

Results: We show that knockdown of the SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells.

Conclusions: We present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.

Figure 1

Functional analysis of SOX2 in GBM cells. (A) Western blot analysis showing the known down efficiency of SOX2 siRNAs (s13295 and s13296). (B) Colony formation assays for SOX2 knockdown and mock control. (C). Cell proliferation assays for SOX2 knockdown and mock control.

Figure 2

ChIP-seq analysis of SOX2 in GBM cells. Quantitative real time PCR for the confirmation of ChIP-seq peaks. Relative amount of PCR products from SOX2-ChIP and IgG-ChIP were shown as bar graph with the amount of IgG-ChIP normalized to 1. Standard deviations were also shown for SOX2-IP. (B) Histograms of SOX2 binding sites around annotated TSS (Transcription start sites) Frequencies of SOX2 island binding were calculated every 10 kilobases (Y-axis). Relative distance to TSS is shown in X-axis, Negative and positive values indicate localization 5' or 3' to TSS respectively. (C). A VENN diagram showing the overlaps of SOX2 targets among human GBM cells, human ES cells and mouse ES cells. (D). The consensus sequence wwTGnwTw with log-likelihood score of 13920.71 identified by the MotifSampler program. The over represented sequences were used as input for the Weblog program (http://weblogo.berkeley.edu/) [53] to display the consensus sequence graphically.

Figure 3

Confirmation of SOX2 regulated genes and microRNAs. (A) Bar graph showing PCR confirmation of the array data of SOX2 gene expression. SOX2-KO: SOX2 knockdown; MOCK, mock known down with negative control siRNAs. Y-axis, relative expression after normalizing the MOCK control to value of 1; X-axis, gene symbols. (B). Bar graph showing PCR confirmation of the microRNA sequencing data. Y-axis, relative expression after normalizing the MOCK control to value of 1; X-axis, microRNA names. (C). Western blot analysis showing the SOX2 expression comparing microRNA 145 precursor mimics and a scrambled negative control. (D). A VENN diagram showing the overlaps of SOX2 targets in GBM and ES cells, and the genes that changed expression (2 fold cutoff) after SOX2 knockdown in GBM cells.

Figure 4

The double negative feedback loop of SOX2 and miR-145. (A) An illustrative drawing showing the double negative feedback loop of SOX2 and microRNA 145. Negative signs and upside down T line drawing indicate inhibitive action. Both SOX2 and miR-145 can receive separate input signals (signal A or signal B). (B) Theoretical bistable output of this double negative feedback loop. Depending on the strength of the input signal A or B, the system could toggle between two stable states: the signal A-SOX2 or the signal B-miR-145 states.