Construction Of SiRNA Plasmid Targeted Human Ribonuclease Inhibitor And The Silencing Effects To B16 Cancer Cells

Cancer is one of serious diseases threatening the human health and life, thus the world medical arena is explaining the cause of cancer and to seek the substitution of tradition surgical operation, radiotherapeutic, chemotherapy and so on. In recent years, medical scientists believe the cause of tumorigenesis is the activation of oncogene and tumor suppressor abnormity .They believe that human organism has anti-tumor system, and there are a few of regulator against tumorgenesis, propagation and cell division among the system. When these regulator factors have the flaw or the deactivation, they can cause the cell canceration .Human ribonuclease inhibiting factor (hRI) is an acidic cytoplsmic glycoprotein with 50KDa estimated molecular weight and locates at the 15pl5.5. hRI has various function and is widespread existence in mammal cytoplasm. Its structure richly contains leucine and the massive cysteines, the spatial structure by richly contains the leucine remnant base repetition area (Leucin-rich reapeat, LRR)alpha/beta spiral fold the curving U-shaped structure which the structure repetition forms many times. The domestic and foreign research discovery that, hRI acts as RNase inhibitor (Ki=4.0xl0^-14 M) and can regulate the RNA level in the cells to control the protein synthesis; hRI also may act as the inhibitor of Angiogenin(Ang) (Ki=7.1×10¹⁶ M)and suppresses the tumor vascularization, infiltrates and migration and so on; hRI has the anti-organism oxidation damage function. Analysing the domestic and foreign research, our research group thought that hRI has the anti-tumor function and hri gene might act as one of candidates of tumor suppression gene.In order to prove whether hri might be the tumor suppression gene, weconstructed siRNA vector pKD-dsRI targeting hri gene and pLNCX-pKD-dsRI, then co-transfected into B16 cells with report vector expressing green fluorescenc protein fused hri gene. After 48h, we found that 80% hRI was silenced through the decrease of green fluorescence among cytoplasm by cell fluorescence locating experiment and RT-PCR antiquantity analysis.RT-PCR semiquantity analysis demonstration that the inhibit effect may reach above 80%.Westen-blotting analysis indicated that the RI expression obviously dropped. After inhibiting hri gene with carrier pLNCX-pKD-dsRI, we detected the cell growth ability with MTT. The results showed that the cell growth ability was increased remarkably and the growth ratio is the 120-145%, compary with B16 cells (P < 0.05); FCM examine the cell cycles was changed obviously, while S phase increased G2-M phase reduced. Those initially proved the hri gene may belong to the tumor suppression system and might be one kind of tumor inhibiting factors.In this research the hri gene firstly was silented by siRNA vector in the B16 cells. The siRNA of hri influenced on the multiplication and cell cycle of B16. Our reseach lays the foundation of hri applied in the tumor gene treatment.