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The purpose of exploring the Na / K-ATPase in Bufalin (Bufalin) inhibit the role of liver cancer cell growth process and mechanisms. Method 1.WST-8 (water-soluble terazoliun salt) assay hepatoma cell lines PLC/PRF/5 Bufalin people, HepG2, SMMC7721, tumor suppression, and draw the cell growth inhibition curve. By RT-PCR and Western Blot technology, the detection of human hepatoma cell line PLC/PRF/5, HepG2, the line SMMC7721. In Na / K -ATPaseα subunit expression differences. 3 Stealth RNAi method, the the of Na / K -ATPaseα3 isoform of high expression strain PLC/PRF/5 interfering. The PLC/PRF/5 cells strain the 4.WST-8 Determination of hepatoma cell lines PLC/PRF/5 and Na / K -ATPaseα3 isoformRNAi on Bufalin sensitivity changes. The results 1.HepG2 PLC/PRF/5 /, line SMMC7721. In application Bufalin, growth suppression, the three larger difference in the sensitivity of cells to Bufalin, HepG2, PLC/PRF/5 /, SMMC7721 application Bufalin The 24-hour IC 50 were 182.3nM, 52.2nM, 97.74nM. Vitro inhibitory curve shows that more than three liver cancer cells PLC/PRF/5 the most sensitive Bufalin, and HepG2 most insensitive. 2.Na / K-ATPaseα1, Na / K -ATPaseα2, Na / K -ATPaseα3, Na / K -ATPaseα4 human hepatoma cell line HepG2, PLC/PRF/5 SMMC7721 were expressed, but the content is different. GAPDH as reference, HepG2, PLC/PRF/5, SMMC7721, in Na / K -ATPaseα1 the mRNA relative expression respectively 2.78,1.76,1.72; Na < sup> / K the -ATPaseα2 in mRNA relative expression levels were 1.28,0.92,0.84; Na / K -ATPaseα3 in mRNA expression were 0.45,1.79,0.88; Na / K the -ATPaseα4 in mRNA expression were 0.79,0.64,0.47 through Na / K -ATPaseα isomers of hepatoma cells relative expression level of the X-axis, three hepatoma cells IC 50 Y-axis mapping can be found that the Bufalin the inhibition of liver cancer capacity and Na , / K the -ATPaseα3 expression was positively correlated. Applications Western Blot detection the hepatoma cell Na / K the high-ATPaseαmRNA expression of two subtypes of Na / K < sup> -ATPaseα1 and Na / K -ATPaseα3 results show that GAPDH as a reference, HepG2, PLC/PRF/5, SMMC7721, in Na / K -ATPaseα1 of protein relative expression amount were: 1.86,1.46,1.64; Na / K-ATPase a3 protein expression respectively: 1.73,2.94,1.914 Application the Stealth RNAi technologies the of Na / K -ATPaseα3 interference, detected by RT-PCR and found siRNA1, siRNA2, siRNA3 of RNA expression levels were blank control group 41.8 %, 10.5% and 74.1%; we choose siRNA2 as positive interference serial follow-up study. By WST-8 was detected Na / the K-ATPaseα3 interference hepatoma cells sensitivity changes of Bufalin, display the of Na / K -ATPaseα3 hepatoma cells after interference Bufalin sensitivity decreased, application Bufalin 48 hours after, PLC/PRF/5/RNAi group IC 50 for 42.10nM negative control group (lipofect) the the IC 50 for 29.29nM; HepG2/RNAi group IC 50 200.10 nM, negative control group (lipofect) IC 50 84.53 nM. The conclusion 1.Bufalin can effectively inhibit the growth of liver cancer cells. 2.Bufalin inhibit the ability of liver cancer cell growth and hepatoma cell Na / K -ATPaseα3 expression levels positively correlated, Na / K < sup> -ATPaseα3 interference, Bufalin inhibiting the growth of liver cancer cells decreased Na / K the -ATPaseα3 may be an important the Bufalin inhibit the growth of liver cancer cells targets. 3.Na / K -ATPaseα3 hepatoma cells on Bufalin reactive differences can lead to differences in expression in hepatoma cells through the liver cancer in Na in / K -ATPaseα3 detected to some extent predict liver cancer as the main active ingredient of anticancer drugs to Bufalin response to treatment, but also conducive to the screened Bufalin advantage of the main active ingredient of anticancer drugs crowd.
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