**4. Possible KEGG pathway following activation and suppression of KCNMA1 in glioma cells**

Glioma cell line U-87 MG was obtained from the American Type Culture Collection (Manassas, VA) and cultured in MEM supplemented with 10% FBS and 0.1 mM nonessential amino acids. Cells were maintained at 37°C in 5% CO<sup>2</sup> . In order to study the biological significance of up- and down-regulation of *KCNMA1* on associated genes, we performed microarray using the Affymetrix Human Genome U133 Plus 2.0. Array analyses of U-87 MG cell lines where *KCNMA1* was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation, angiogenesis, cell cycle, and invasion (**Figure 3**). Class comparison tests indicated significant changes in global expression patterns. Twenty genes highly downregulated by suppression but upregulated by overexpression of *KCNMA1* or vice versa are shown in **Figure 3**. This data support our rationale that *KCNMA1* plays a critical role in the above cellular processes.

Array analyses of U-87 MG cell lines where *KCNMA1* was either overexpressed or suppressed showed significant changes in genes involved in cell proliferation, angiogenesis, cell cycle,

and invasion (**Figure 4; see arrows**). Cluster analysis of 476 transcripts that are altered in opposite directions by expressing *KCNMA1* gene up and down in U-87 cells. These transcripts were identified from the significantly altered genes by ANOVA at p < 0.05, and the fold-change thresholds such that one of *KCNMA1* up or down altered the gene expression by

**Figure 4.** Cluster analysis of 62 genes altered by twofold in opposite directions by expressing KCNMA1 gene up and down in U-87 cells. These transcripts were identified from the significantly altered genes by ANOVA at p < 0.05.

Role of an Alternatively Spliced *KCNMA1* Variant in Glioma Growth

http://dx.doi.org/10.5772/intechopen.74509

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The KCNMA1 encodes the pore-forming α-subunits of large-conductance Ca2+-activated K+ (BKCa) channels. More than 20 variants of this gene are associated with alternative splicing at ten or more different sites [12, 13], while majority of the splice sites are located in the large cytoplasmic domain. This domain is called the C-terminal half of the channel that contains multiple Ca2+ binding sites [14–16]. Gating properties and kinetics with regard to the voltage and Ca2+ dependence of gating are altered by alternative splicing in these regions [17–19]. Expressions of different BKCa isoforms have been implicated in auditory processing [20] and alter the sensitivity of BKCa to modulation by phosphorylation [21] and other processes [22]. However, the role of BKCa isoforms in cancer is now being investigated [23]. More specifically, KCNMA1 is altered in a wide variety of cancers, and their overexpression liked to increased malignancy in gliomas [4–7]. The BKCa protein isoform transcribed by its alternatively spliced mRNA in cancer cells is known as likely to respond differently to changes in intracellular

twofold and the other altered it at least by 1.5-fold in the opposite direction.

**5. KCNMA1 splicing in glioma**

**Figure 3.** High-grade glioma cells, U-87 MG were transfected to make stable cell line over-expressing KCNMA1. In addition, we transfected U-87 MG cells with shKCNMA1; this suppressed basal KCNMA1 expression. A microarray was performed on these cells. The heat map shows the differential expression of genes that were directly or indirectly affected by upregulation or down regulation of KCNMA1. We found 8102 and 7259 significant features at p < 0.05, respectively, for overexpression and suppression of *KCNMA1*. From these, features having at least a signal value of 255 were selected to reduce false positives (false discovery rate < 0.0079).

**Figure 4.** Cluster analysis of 62 genes altered by twofold in opposite directions by expressing KCNMA1 gene up and down in U-87 cells. These transcripts were identified from the significantly altered genes by ANOVA at p < 0.05.

and invasion (**Figure 4; see arrows**). Cluster analysis of 476 transcripts that are altered in opposite directions by expressing *KCNMA1* gene up and down in U-87 cells. These transcripts were identified from the significantly altered genes by ANOVA at p < 0.05, and the fold-change thresholds such that one of *KCNMA1* up or down altered the gene expression by twofold and the other altered it at least by 1.5-fold in the opposite direction.
