Large Conductance Ca²⁺ Activated K⁺ Channels Regulates The Proliferation,Apoptosis,Migration,and Invasion Of Pancreatic Cancer Cells Through The PI3K/AKT/ERK Signaling Pathway

Objective:To explore the function and mechanism of BK channel(also named Slo1,KCa1.1,Maxi)in the proliferation,apoptosis,migration,and invasion of pancreatic cancer(PC).Methods:1)Cell culture of pancreatic cancer cell lines Mia Pa Ca-2 and PANC-1;2)q PCR detection of BK channel gene expression;3)Recording the potassium currents in pancreatic cancer cell lines Mia Pa Ca-2 and PANC-1 using the patch-clamp technique.4)Small interfering RNA(si RNA)was used to knock down the major subunit gene KCNMA1(BK?)of the BK channel on pancreatic cancer cell lines Mia Pa Ca-2 and PANC-1 and phenotypic changes were observed.5)CCK-8(Cell Counting Kit-8)was used to detect the viability of Mia Pa Ca-2 and PANC-1 in pancreatic cancer cells after knocking down KCNMA1.6)Cell cycle of Mia Pa Ca-2 and PANC-1 after knocking down KCNMA1 was measured by flow cytometry(FCM).7)The apoptotic changes of Mia Pa Ca-2 and PANC-1 cells after knocking down KCNMA1 were detected by Annexin V-FITC/PI method8)Cell migration and invasion changes of Mia Pa Ca-2 and PANC-1 detection by Transwell method.9)RNA sequencing(RNA-seq)was used to find the differentially expressed genes followed by KEGG_PATHWAY enrichment analysis for the involved signaling pathways.10)Western blot was used to verify the key signaling cascades in pathways identified in 9.Results:1)PCR assay showed that KCNMA1 was highly expressed in Mia Pa Ca-2 and PANC-1 cells.The patch-clamp recording showed that Paxilline(10 ? M),a specific BK channel blocker,could significantly reduce the outward potassium currents.2)Compared with the control group,after KCNMA1 knocked down,the absorbance of CCK-8 decreased at 450 nm.The percentage of G0/G1 phase cells was increased.The number of early apoptotic cells and late apoptotic cells was more than that of the control group.3)Contrast to the control group,the cells with decreased KCNMA1 showed less migration tested by Transwell assay..4)m RNA sequencing followed by KEGG_PATHWAY analysis showed that activity of PI3K/AKT/ERK decreased in the tumor cells with decreased BK channel expression.5)Western blot confirmed that phosphorylated AKT and ERK were reduced after silencing the KCNMA1 gene.Conclusion:1)The BK channel was expressed on pancreatic cancer cells.The opening of the BK channel can be detected under physiological conditions.2)In vitro experiments show that silencing KCNMA1 gene can effectively reverse tumor-associated phenotypes including reduced the proliferation,promoted G0/G1 phase arrest,promoted apoptosis,and reduced migration of invasion.3)KCNMA1 regulates the above phenotypic changes via the PI3K/AKT/ERK signaling pathway.