| Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. Its morbidity and mortality rank the 3rd and 2nd respectively among all types of malignant tumors. Moreover, it tends to be increasing in recent years. Characteristics of hepatocellular carcinoma (HCC) are rapid development, early metastasis, poor prognosis and short survival times. Traditional surgical therapies, radiotherapy, chemotherapy and a combination of traditional Chinese medicine (TCM) and chemical or biological drugs can not improve the prognosis significantly. Following the development of biological tumor therapy, such as gene therapy for HCC has been suggested based on promising preliminary results.Recently, HCC gene therapy has attached more attention, IGF-â…¡is an important pro-angiogenic factor which can directly promote angiogenesis and a number of liver cancer cell lines secrete it through an autocrine or paracrine mechanism. The human IGF-â…¡gene is complex transcription unit containing four different promoters (P1-P4). The expression of human IGF-â…¡gene is controlled by an intricate mechanism. In adult liver, promoter P1 is activated and promoter P2, P3 and P4 are completely shut off. In cell lines and hepatocellular carcinoma (HCC) expressing IGF-â…¡, P3 is the most active promoter. P3 is also the major promoter in many tumour tissues and IGF-â…¡expressing cell lines. P3 directly promotes angiogenesis and indirectly promotes the formation of liver cancer blood vessels, thereby contributing to the occurrence of liver cancer's development and transfersion.Deoxyribozymes (DNAzymes or DRzs) are synthetic, single-stranded catalytic DNAs which can be engineered to bind by Watson-Crick base pairing to complementary sequences in a target messenger RNA (mRNA) and cleave it at predetermined phosphodiester linkages. The 10-23 DRzs has been proposed as a general model for DNAzymes. In this study,10-23 DNAzyme has a catalytic domain of 15 deoxyribonucleotides, flanked by two arms, each with a 7-9 deoxyribonucleotide substrate-recognition domain. In vitro analysis,10-23 DNAzyme could cleave substrate mRNA effectively at purine and pyrimidine junctions. They have been widely shown either in vitro or in vivo to inhibit the expression of their targeting gene.10-23 DRzs can also block the expression of targeting gene and have potential utility as therapeutic tools.Principle of design:10-23 DNAzymes were designed by man-made, which could block and cut IGF-IIP3 to degress the expression of targeting gene. The application of molecular enzymase was implemented in liver cancer gene therapy.Innovation:We first used 10-23 DNAzyme targeting IGF-IIP3 mRNA. Our results showed that DRzl could significantly inhibit proliferation and induce apoptosis and inhibit the invasion and migration in SMMC-7721 cells.Meaning:IGF-IIP3 may become a new gene therapy's target to HCC and DRz1 may become a new gene drug to treat liver cancer, which may provide new theory to liver cancer's gene therapy.The main contents and results:Contents1. Design and selection of IGF-IIP310-23 deoxyribozymes:Briefly, a region of 10-23 model DRz (DRz1, DRz2, DRz3) was designed according to IGF-IIP3 mRNA and computer prediction. MTT assays were used to select the activity of IGF-IIP3 DRz.2. IGF-IIP3 DRz inhibited IGF-IIP3 protein expression in SMMC-7721 cells:Real time-PCR, western blotting and immunohistochemical analyses were used to detect the effects of DRzs on the expression of IGF-IIP3 (mRNA and protein) in different concentration and different time.3. IGF-IIP3 DRz inhibited cell proliferation and induced apoptosis in SMMC-7721 cells:MTT and flow cytometry were used to detect the effects of DRzs on inhibition proliferation, induse apoptosis and specificity in SMMC-7721 cells; DAPI staining was used to detect the cellular morphology; western blotting analysis was used to study the signal of conduction.4. IGF-IIP3 DRz inhibited the invasion and migration in SMMC-7721 cells:flow cytometry was used to select the minimal effect concentration; wound healing assay was used to detect the effect of DRzl on inhibition invasion, motility and migration of SMMC-7721 cells; the FN, LN and Fb cell were used to detect the anti-adhesion of SMMC-7721 cells; western blotting analysis was used to study the signal of conduction.Results:1. IGF-IIP310-23 deoxyribozymes had the catalytic activity.2. IGF-IIP3 DRzl degressed the expression of IGF-â…¡P3, inhibited the proliferation and induced the apoptosis.3. IGF-IIP3 DRz1 inhibited the invasion and migration of SMMC-7721 cells.4. IGF-IIP3 DRz1 may be a new enzyme and "drug" in liver cacer gene therapy.
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