Construction of Specific Hammerhead Ribozyme Targeting Cyclin D1 mRNA and Its Activity in Vitro and Hepatec Stellate Cells

Abstract: The secondary structures of hammerhead ribozyme (Rz) and rat cyclin D1 were analyzed and simulated by mfold software. Hammerhead ribozyme DNA sequence was synthesized. Cyclin D1 DNA sequence was obtained by reverse transcription PCR. Ribozyme and cyclin D1 DNA sequences were separately cloned into pGEM-3Zf(+) vectors. Ribozyme and cyclin D1 mRNA were obtained in vitro transcription， and ribozyme cleavage experiment was made in vitro. pLXSN-Rz vector was constructed by inserting ribozyme gene into retroviral vector and transfected into rat-derived HSC-T6 cell line with lipofectin. The expression of cyclin D1 gene was detected by RT-PCR. The results showed that a ribozyme directing to 832 site of cyclin D1 mRNA coding domain was selected by mfold software， and its DNA sequence was synthesized. The transcription vectors of Rz832 and cyclin D1 mRNA were constructed successfully. Ribozyme and cyclin D1 mRNA were expressed in vitro transcription. Cleavage experiments in vitro showed that Rz832 cleaving cyclin D1 mRNA produced 1 014 nt and 65 nt fragments. The cleavage efficiency was 80%. Restriction analysis showed that Rz832 DNA was correctly inserted into plasmid pLXSN as expected. Sequencing showed that there were no mutation in the ribozyme gene. The expression of cyclin D1 at mRNA level was effectively down-regulated in Rz832-transfected hepatic stellate cells (HSCs) by 42.22% of that in control cells (t=-193.443， P<0.01). The results indicated that Rz832 can specifically cleave cyclin D1 mRNA in vitro and significantly inhibit cyclin D1 gene expression in HSCs.

CSTR: 32200.14.cjcb.2007.01.0024