Experimental Research Of VCAM-1-shRNA Lentiviral Vector Construction And Its Suppression Of The Human Oral Squamous Carcinoma HN12 Cell Line In Vitro

Objective:To construct the VCAM-1-RNAi-lenfivirus and investigate the effects of VCAM-1 on the growth of oral squamous carcinoma cell HN12 cells in vitro.Methods:1. Four pairs of complementary small hairpin RNA (shRNA) oligonucleotides targeting the vcam-1 gene were designed synthesized,annealed and inserted into linearized GFP-lentivirus vector. The recombinant plasmid was identified by double restriction digestion with BamHI and EcoR I and DNA sequencing.The recombinant plasmid and a lentivirus packaging mix were co-transfeeted into 293T cells to obtain packaged lentivirus particles.Viral titer was then determined.2. The vcRNAi-lentivirus construct was transfected into FaDu cells. Real-time PCR and western blot were performed to determine the expressing level of vcam-1.CCK8 was used to analyze cell Proliferation.Results:RT-PCR and sequencing revealed that siRNA plasmids was correctly constructed. Virus with a titer of 1×109 TU/ml was successfully packaged at least. VCAM-1 expression in HN12 cells could be knocked down by virus infection signally, compared with negative control lentivirus.Conclusion:The recombinant lentiviral siRNA expressing vector targeting human VCAM-1 gene has been successfully constructed and packaged. VCAM-1 RNAi could be down-regulated availably in HN12 cells.