Effect Of SiRNA Targeting Neuropilin-1on Platelet-derived Growth Factor-BB-induced Activation,Proliferation And Migration Of Hepatic Stellate Cells

Background and objective:Through100years of research, the role of hepatic stellate cells in the liver has been basically confirmed the pathological and physiological. HSC activation is the central link in the liver fibrosis development. The formation of liver fibrosis is a more complicated process factors, and the different causes of liver fibrosis in early have the HSC activation, which is the cellular basis of fibrosis.Despite the increasing number of functions for HSC they are best known for their role as wound-healing cells in the injured liver. The so-called "activated" HSC (aHSC) adopts a smooth muscle myofibroblast-like phenotype, secretes a variety of extracellular matrix (ECM), causes the imbalance of MMPs (Matrix Metalloproteinase)and TIMPs (inhibitors of metalloproteinase) and inhibits HSC apoptosis. Activaed HSC can migrate to damaged areas and cause the damage zone fibrosis.So the hepatic stellate cell activation is the direct cause and the core aspects of liver fibrosis.Cirrhosis is a major risk factor for the occurrence of hepatocellular carcinoma (HCC), almost of the HCC patients with chronic liver disease and cirrhosis background. Activaed HSC can be located within the tumor stroma, and the close correlation exists between tumor cells. Recent studies show that the activated HSC are widely present in mesenchyme of the liver cancer, as part of the tumor microenvironment, playing a role the occurrence development and invasion of liver cancer.Platelet derived growth factor (PDGF), which is generated and secreted by a variety of cells,is a class of wide range of biological activated cytokines,and is closely related to the growth, development and cancer, involving a variety of physiological and pathological process.Neuropilin-1(NRP-1) which was found in1987,is a multifunctional type Ⅰ transmembrane glycoprotein in Cell membrane frequently expressed by cancer cells, endothelial cells and several other normal cell types. It was first identified for their key role in mediating axonal guidance in the developing nervous system through their interactions with class3semaphorins.Later,Growing evidence supports that NRP-1is widely expressed on endothelial cells and tumor cells,and can be used as the receptor of VEGF165and several other angiogenesis-related VEGF family members. Now the function of NRP-1has already from the development of nervous system extended to angiogenesis, tumor occurrence and development of the hematopoietic system diseases, digestive diseases, immune system function, especially the role in tumor angiogenesis become a hot spot.Recent studies revealed NRP-1can bind several other growth factors, and/or their receptors, such as hepatocyte growth factor, the hepatocyte growth factor receptor, some fibroblast growth factors and platelet-derived growth factor(PDGF). Many researchs show that neuropilin-1co-immunoprecipitated and co-localized with phosphorylated PDGFRs in many cells, thereby regulating cell signalling,migration, proliferation and network assembly. Neuropilin-1knockdown reduced PDGF-BB-induced PDGFRβ activation and migration. Some researchers found that neuropilin-1was upregulated in activated HSCs in rat models of liver fibrosis and human livercirrhosis,and had Correlation with the expression of PDGF-receptor ββ (PDGFR-ββ). Other researchs show that NRP-1expressed in the liver endothelial cells, hepatocellular carcinoma cells, but not in the normal livercells. These data indicate that NRP-1plays an important role in liver physiology, pathology.RNA interference is the phenomenon of silence in a post-transcriptional gene expression (PTGS).When a certain sequence homology with the endogenous mRNA coding region of the double-stranded RNA (dsRNA) into cells, it can lead to the endogenous mRNA-specific degradation, thereby lead to silence gene expression, resulting in a corresponding loss of function The technique has been widely used in biological experiments.NRP-1is widely expressed inhuman tissues, playing an important role in the occurrence of certain diseases, especially the occurrence and development of tumor. Currently the research of NRP-1on the liver pathhology, physiology research is still very rare abroad, the country has not been reported in the literature.In this study, the RNA interference method was used to observe the role of NRP-1in PDGF-BB in hepatic stellate cells, in order to pave the way to further explore the NRP-1on the liver pathology, physiology research.MothedImmunofluorescence double-staining was used to detect the expression of NRP-1and PDGF-receptor B in the cytomembrane of HSCs;one pair of chemically synthesized small interfering RNA(siRNA) targeting neuopilin-1and one pair of negative control siRNA were transfected into the cell line of HSCs-LX2. HSCs were devided into three groups:positive interfering group(A group)、negative interfering group(B group) and nomal control group (C group).Real time-PCR and West-blot was carried out to eaxmined the mRNA and protein of NRP-1after transfection;Used PDGF-BB (20ng/ml) to sitmulate the HSCs. mRNA expression of α-SMA was analyzed by RT-PCR. Cell proliferation was asseccde by CCK8assay.The migration was eaxmined by wound-healing-assaySatistical analys:The analysis was conduced by SPSS13.0. The data are expressed as mean±standard deviation (SD). Comparisons between groups were tested by one-way analysis of variance (ANOVA), While comparison among groups was test by Bonferroni. Dunnetts T3method was exployed for heterogeneity of ariance.Differences were considered to be statistically significant at P<0.05.Result1.From the Imaging,both NRP-1and PDGFR-ββ expressed in the cytomembrane widely, signifying NRP-1and PDGFR-ββ colocatized on HSCs.2. Quantitative real-time PCR showed that total mRNA in A group decreased after transfected for24h and48h compared with B group and C group(P=0.000).There was not difference between B group and C group (P=0.120, P=1.000).However, mRNA levels were not significantly among three groups after72h (F=0.800, P=0.492)。3. Western blotting analysis show that protein levels in A group decreased after transfected for24h and48h compared with B group and C group(P=0.000). There was not difference between B group and C group (P=1.000, P=1.000).But the protein levels were not significantly among three groups after72h (F=0.303, P=0.750)4.Following incubation with PDGF-BB for24h, significant differences in the mRNA expression of α-SMA were found among A group、B group and C group (F=33.492, P=0.001). Compared with B and C group,A group is deceased significantly (P=0.001, P=0.001).Howerer,there no significant difference between B group or C group(P=1.000)=RT-PCR in this study showing that the siRNA of NRP-1decreased α-SMA expression in LX-2cells, implicatde that NRP-1silencing suppressed activation of hepatic stellate cells induced by PDGF-BB5. We examined the effect of reducing NRP-1levels on PDGF-BB-induced proliferation in HSCs using CCK8assay.We found that NRP-1A group cells showed decrease in PDGF-BB-induced proliferation at24h after addition of PDGF-BB in comparison to B group and C group(P=0.019, P=0.019), while proliferation in B group was similar to that in C group(P=0.982). These results demonstrated that knockdown NRP-1slightly reduced PDGF-BB-induced mitogenic response of HSCs.6.After incubation with PDGF-BB for48h, we can see The differences in mobility of three group were statistically significant(F=132.732, P=0.000).The mobility of A group(67.83±2.44)%is significantly slower than the mobility of B group(89.26±0.30)%and C group(88.46±1.98)%(P=0.000, P=0.000)ConclusionsIn summary, our study demonstrates that the novel multifunctional receptor NRP-1is expressed in HSCs with PDGFR-ββ. Silencing NRP-1gene results in a significant decrease of PDGF-BB-induced cell activation,proliferation and migration in HSC cells.NRP-1may be taken as a novel target for clinical therapeutics of liver cirrhosis and hepatocellular carcinoma in the future.