Effects Of SiRNA Inhibit HMGA1on LX-2Cell Biological Functions

Objective：To investigate the effects of siRNA mediate HMGA1silence on proliferationand the gene expression of HMGA1,a-SMA and E-cad in activated hepatic stellatecells LX-2and the mechanisms is involved.Methods:1. LX-2cells are cultured in vitro and divided into six groups:①normalcontrol group;②blank group;③negative control group;④HMGA1-siRNA-1transfection group;⑤HMGA1-siRNA-2transfection group;⑥HMGA1-siRNA-3transfection group. After48hours of transfection, HMGA1mRNA and proteinexpression are detected by RT-PCR and western blot. Filter out the best sequence ofthe siRNA interference.2. LX-2cells are cultured in the medium containing different concentrationsof TGF-β1(Final concentration are1,5and10ng/ml), and a control group. cells arecollected at24h of incubation, the RNA and protein are isolated and HMGA1geneexpression level are measured by RT-PCR and western blot.3. Cultured LX-2cells are transfected with HMGA1siRNA by liposometransient transfection. The cells are divided into four group:①control group;②TGF-β1group;③TGF-β1+NC-siRNA group and④TGF-β1+HMGA1siRNAgroup. The mRNA expression of HMGA1,α-SMA and E-cad is determined byRT-PCR, and their protein expression is detected by Western blotting. Cellproliferation is assessed by MTT assay.Results：1. Compared with that of in normal control group, blank group and negativecontrol group, the expression level of HMGA1in HMGA1siRNA groups are significantly decreased(P<0.05), and the HMGA1-siRNA-1group had the besteffects (P<0.01).2. The HMGA1gene expression levels of HSCs increased gradually with theincrement of TGF-β1concentration, and significant differences in HMGA1geneexpression were found between each group (P<0.05).3. Compared with control group, the cell proliferation and the mRNA andprotein expression of HMGA1and α-SMA in TGF-β1group and TGF-β1+NC-siRNA group were significantly increased (P<0.05), without significant differencesbetween the two groups (P﹥0.05), while the expression of E-cad in TGF-β1groupand TGF-β1+NC-siRNA group is significantly decreased compared with controlgroup. Meanwhile, the cells in TGF-β1+HMGA1siRNA group show significantlydecreased proliferation level,down-regulated mRNA and protein expression ofHMGA1and α-SMA but up-regulated expression of E-cad compared with TGF-β1group and TGF-β1+NC-siRNA group(P<0.05).Conclusions：1. HMGA1expression in hepatic stellate cells LX-2could be inhibitedsignifieantly by three targeting HMGA1siRNAs, and the best inhibited effect isHMGA1-siRNA-1.2. TGF-β1strongly up-regulates HMGA1gene expression in hepatic stellatecells LX-2.3. HMGA1intereference could significantly down-regulate the expression ofα-SMA in hepatic stellate cells LX-2cultured with TGF-β1, and up-regulate theexpression of E-cad, thus inhibiting the proliferation and activation of the cells.