Mechanism Analyse Of Epithelial-mesenchymal Transition Induced By Hepatitis C Virus NS4B Protein

Objective: Try to figure out the function and mechanism of HCV NS4B protein in epithelial to mesenchymal transition（EMT）.Methods: 1.The target gene HCV NS4B was amplified from a plasmid harboring whole length of HCV 1b type by polymerase chain reaction（PCR） using Pfu Taq DNA polymerase. Choosing Eco RI and Bam HI double enzyme digestion method to construct a PEGFPC1-NS4B plasmid. Use q RT-PCR, Western blot assay to verify if HCV NS4B protein could be expressed in cells.2.Then the empty PEGFPC1 plasmid and construction PEGFPC1-NS4B plasmid were transfected into HepG₂ cells by cationic liposome seperartely or empty PEGFPC1, 1μg, 3μg, 5μg PEGFPC1-NS4B plasmid were transfected into HepG₂ cells for 48 h. Ecadhrin and N-cadhrin m RNA level were tested by q RT-PCR and protein level were measured by Westernblot to find out whether HCV NS4B protein could cause EMT changes.3. Use the same method as point 2, the m RNA and protein level of transcription factor Snail were measured. Then utilize Snail sh RNA to knockdown snail expression to understand what role Snail played in HCV NS4B caused EMT changes.4. Use would-healing assay to analyze the migration rate of NS4B transfected or NS4B, Snail si RNA cotransfected HepG₂ cells. 5. The changes of Scribble-Hippo-PI3K/AKT signal pathway was measured by westernblot to figure out the mechanism of HCV NS4B caused EMT changes.Results: 1. The construction plasmid sequencing and double enzyme digestion Results showed that the sequence of inserted target fragment and the insert direction was totally right. The construction plasmid was cleavage into two fragment, 4725 bp and 796 bp length by double enzyme digestion. The length of the two fragment was right, which stated that NS4B gene was successfully inserted into PEGFPC1 plasmid. And HCV NS4B protein could be expressed in eukaryocyte.2. The HepG₂ cells transfected with PEGFPC1-NS4B plasmid showed the down-regulation of E-cadherin and up-regulation of N-cadhrin, no matter in m RNA level or protein level, compared to the empty vector. What’s more, the protein level of E-cadhrin was decreased by the increasing expression of NS4B and the N-cadhrin was increased by the increasing expression of NS4B, which means that NS4B expression was the real reason of EMT changes.3. The HepG₂ cells transfected with PEGFPC1-NS4B plasmid showed the up-regulation of Snail protein level, which increased with the increasing expression of NS4B. However, the m RNA level of Snail had no obvious changes in NS4B transfected or not transfected groups, which means that the regulation of Snail protein level was occurred at post-transcription level. Expression of Snail knocking down could reverse the EMT changes that caused by HCV NS4B protein.4. The HepG₂ cells transfected with PEGFPC1-NS4B plasmid showed increasing migration rate, but the HepG₂ cells co-transfected with PEGFPC1-NS4B plasmid and Snail sh RNA showed no obvious changes of migration rate.5. HCV NS4B protein can interact with Scribble and cause its decrease. The HepG₂ cells transfected with PEGFPC1-NS4B plasmid showed decrease of p-yap/total-yap, which means that Hippo signal pathway was inhibited. And as a result, the downstream pathway PI3K/AKT was activated, which performed in up-regulation of p-AKT/total-AKT. The Scribble-Hippo-PI3K/AKT signal pathway may be a mechanism of HCV NS4B caused EMT changes.Conclusions: Hepatitis C virus NS4B protein induces epithelialmesenchymal transition by upregulation of Snail. The upregulation of Snail may due to Scribble-Hippo-PI3K/AKT signal pathway.