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Evidence of calcium-activated potassium channel subunit alpha-1 as a key promoter of glioma growth and tumorigenicity

jglioma. 2019;

Divya Khaitan, Nagendra Ningaraj

Products/Services Used	Details	Operation
PCR Cloning and Subcloning	Cloning of calcium-activated potassium channel subunit alpha-1 in short hairpin RNA expression vector: pcDNA4/TO/HisA (Invitrogen) was modified for the expression of KCNMA1 short hairpin RNA (shRNA) by cloning the H1 promoter from PRNATin-H1.2/Neo vector (GenScript, Piscataway, NJ, USA) using Bgl II and Hin dIII as reported,[11] replacing the cytomegalovirus (CMV) promoter. This yielded the vector pcDNA4/H1.2. The KCNMA1 shRNA sequence was derived from Silencer® small-interfering RNA (siRNA) (Ambion, siRNA ID: 112882) and constructed as long complementary oligos (KCNMA1 si FW-cgt act tca atg aca ata ttt caa gag aat att gtc att gaa gta cgt ctt ttt t and KCNMA1 si RV-aaa aaa gac gta ctt caa tga caa tat tct ctt gaa ata ttg tca ttg aag tac g, containing Hin dIII and Bam HI sites on their respective 5' and 3' ends). The oligos were mixed at 100 μM, heated and amplified through one round of PCR (Amplitaq Gold, Applied Biosystems, Carlsbad, CA, USA), and then TOPO-cloned into the pCR4-TOPO vector (Invitrogen), sequenced and then subcloned via Hin dIII and Bam HI into pcDNA4/H1.2 to create pcDNA4/H1.2/shKCNMA1.	Get A Quote

摘要

Background and Aim: Mechanisms of glioma progression are poorly understood. Upregulation of calcium-activated potassium channel subunit alpha-1 (KCNMA1), which encodes the α-subunit of maxi-calcium-activated potassium (BKCa) channels, is shown to be a novel mechanism for the malignant phenotype of brain tumor cells. The aim of this study was to establish the functional role of KCNMA1 in glioma biology. Materials and Methods: U-87-MG (U-87) cells were transfected to increase BKCa channel expression and activity. Glioma cell proliferation, invasiveness, and transendothelial migration were then measured. BKCa channels were blocked with iberiotoxin or short hairpin RNA (shRNA), which significantly inhibited K+ currents and growth of U-87 cells. It was tested whether KCNMA1 overexpression enhanced tumorogenecity in glioma xenograft mouse models by injecting wild-type and KCNMA1- overexpressing U87-MG cells. In parallel experiment, it was studied whether shRNA KCNMA1-expressing U-87 cells show attenuated glioma growth in mice. The study protocol was approved by the Institutional Animal Care and Use Committee, Mercer University (A0706007_01), Atlanta, GA, USA on July 20, 2007. Results: The effect of KCNMA1 overexpression in glioma growth as well as on associated cell biology functions such as proliferation, invasion, and migration was presented in this study. Messenger RNA and protein analyses revealed that KCNMA1 was amplified in 90% of high-grade gliomas and in high-grade glioma cell line U-87. In contrast, KCNMA1 amplification was not found in normal brain tissues. These data indicate that KCNMA1 plays critical role in glioma biology by interacting with several cellular processes. The data demonstrate that KCNMA1 amplification drives glioma cell proliferation and growth, which can be attenuated by its downregulation. Conclusion: KCNMA1 is a regulator of glioma cell proliferation and growth and thus qualifies as a promising diagnostic and therapeutic target in the treatment of glioma.