Specifically Deoxyribozyme of the Protein Kinase C-δ Expression Treated in Human Hepatocellular Carcinoma HepG2 Cells

Abstract:

The tumor suppressor function of protein kinase C isoforms (PKC-δ) was evaluated by design and synthesis the 10-23 deoxyribozyme (DRz), thio-modified DRz (DRz-s) and antisense oligonucleotide (asON) of the PKC-δ genomic mRNA to detect the catalytic cleavage activity for tumor cells. Firstly, the cDNA fragment of PKC-δ gene was amplified from total cellular RNA of the HepG2.2.15 cells by reverse transcription PCR (RT-PCR). Subsequently, the fragments were cloned to pcDNA3.1(+) plasmids and generated a recombinant plasmids, then sifted the positive recombinant plasmids out to amplify. The expression vector of PKC-δ mRNA was obtained in vitro transcription by using T7 RNA polymerase. The results of transfection indicated that when PKC-δ mRNA gamyed with deoxyribozyme which activity disappeared, and DRz-s had more intensive specific catalytic cleavage activity than DRz by cells transfecting, but the asON wasn't detected with this activity.

• Periodical: Advanced Materials Research (Volumes 915-916)
• Pages: 917-921
• DOI: https://doi.org/10.4028/www.scientific.net/AMR.915-916.917
• Online since: April 2014
• Authors: Zhuo Wei*
• Keywords: 10-23 Deoxyribozyme (DRz), Catalytic Cleavage Activity, Protein Kinase C-δ(PK-δ), Reverse Transcription Rt-PCR

References

[1] Pearson, R. B, Kemp,B. E. Methods Enzymol, 200 (1991), pp.62-81.

[2] Newton. A. C, J. Biol. Chem., 270 (1995), pp.28495-28498.

[3] Xia.J. H, Matsuhashi. S, Hamajima. H, Iwane. S, Takahashi. H, Eguchi. Y, Mizuta. T, Kuroda. S, Ozaki. I, Journal of Nutritional Biochemistry, 23 (2012) 1668– 1675.

DOI: 10.1016/j.jnutbio.2011.11.010

[4] Taketoshi. K, J. Biol. Chem., 279(2004), 12668-12676.

[5] Michael. J, Humphries, Kirsten. H. J. Biol. Chem., 281(2006), 9728-9737.

[6] Voss. O. H, Kim. S, Wewers.M. D, J. Biol. Chem., 280(2005), 17371-17379.

[7] Kamada. S, Kikkawa. U, Tsujimoto. Y, J. Biol. Chem., 280(2005), 857-860.

[8] Lin. C. S, Lin. F. Y, Yang. S. P, Lai. J. H, Cardiovascular Research, 95(2012), 346–355.

[9] Kao.H. H, Wu. C. J, Won. S. J, Shin. J. W, Liu. H. S, Su. C. L, Plant Foods Hum Nutr 66 (2011), 136-142.

[10] Fahmy.R. G, Waldman. A, Zhang. G, Mitchell. A, Tedla N, Cai H, Geczy C. R, Chesterman C. N, Perry. M, Khachigian.L. M, J. Nat. Biotechnol. Vol. 24(2006), p.856.

DOI: 10.1038/nbt1225

[11] Yen. L, S.M. Strittmatter, R.G. Kalb, Ann. Neurol. Vol. 46(1999), p.366.

[12] Chakraborti. S, A.C. Banerjea, Molec. Ther. Vol. 7(2003), p.817.

[13] Lu Z. X, Ye.M., Yan, G.R., Li, Q., Tang, M., Lee, L.M., Sun, L.Q., Y. Cao, Cancer Gene Ther. Vol. 12(2005), p.647.

[14] Takahashi. H, Hamazaki, H., Habu, Y., Hayashi, M., Abe, T., Miyano-Kurosaki, N., H. Takaku, FEBS Lett. P. Vol. 560(2004), p.69.

DOI: 10.1016/s0014-5793(04)00073-0

[15] Zhao.C. A, Zhao, X. D, Yu, H.G., Wu, Y. P, X.Q. Yang, 2003. ZhonghuaEr. Ke. Za Zhi. Vol. 41(2003), p.594.

[16] Li. Y, Breaker R. R, Curr Opin Struct Biol, Vol. 9(1999), p.315.

[17] Liu Y, Breaker R. R, Biochemistry, Vol. 39(2000), p.3106.

[18] Carmi N, Breaker R. R, Vol. 9(2001), p.2589.

[19] Breaker R. R, Science, Vol. 290(2000), p. (2095).

[20] Breaker R. R, Natl Biotechnol, Vol. 15(1997): p.427.