This study aims to design and generate recombinant lentiviral vector containing the complete coding sequence (CDS) region of human serine protease inhibitor Kazal type 1 gene (SPINK1) and establish a human pancreatic cancer cell line (AsPC-1) stably overexpressing SPINK1. Then, to assess the proliferative and oncogenic effects of upregulated SPINK1 gene on pancreatic cancer AsPC-1 cells using different methods. Initially, the target coding sequence (CDS) of SPINK1 was amplified by polymerase chain reaction (PCR) and the synthesized product was subsequently subcloned into the lentiviral vector. The construction of recombinant SPINK1 gene was verified by the restriction digestion and sequencing analysis. The lentiviral particles carrying SPINK1 gene were produced by co-transfection of the recombination lentiviral vector and the packaging mix plasmids into 293 T cells and filtered and concentrated before AsPC-1 cells were infected by the virus particles. The cells transduced were differentially selected with puromycin, and the clones that highly expressed SPINK1 were chosen by real-time PCR and confirmed by Western blot after 7 weeks. The stably transduced AsPC-1 cell line showed significantly increased metabolic and proliferative capability using CCK-8 and Trypan Blue assays (P < 0.001). Cell cycle and DNA content analysis by flow cytometry showed that upregulated SPINK1 elicited significant increase in the percentage of AsPC-1 cells in the S and G2/M phase (P < 0.001). Clone formation assay demonstrated that the number of the colonies formed in the experimental group was greater than that in the control parental cells (P < 0.001). It was concluded that a stable AsPC-1 cell line capable of overexpressing SPINK1 had been successfully created, and that the proliferative capacity of AsPC-1 pancreatic cancer cells was significantly raised by SPINK1 upregulation as well as the ability of a single AsPC-1 cell to grow into a colony.
Serine protease inhibitor Kazal type 1 (SPINK1) Lentiviral vector Pancreatic cancer Proliferation
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This study was financially supported by the National Natural Science Foundation of China (NSFC), Grant no.: 81270542, and Research Foundation of Dalian Medical University.
The sequencing data for the constructed recombinant lentiviral vector. The whole SPINK1 coding sequence subcloned in pLenti-CMV-2A-GFP vector was confirmed by the sequencing. The inserted EcoRI (site), “A” nucleotide, Kozak sequence, CDS of SPINK1 gene, and BamHI (site) were represented in the sequence graph. (JPG 2511 kb)
The amplification plot and melting curve from q-PCR reaction for the detection of mRNA expression of SPINK1. a An amplification plot showed the variation of log (ΔRn) with PCR cycle number. The results revealed three typical “S-shaped” amplification curves. b An amplicon either from 293 T (positive control), stable clone #10 or parental AsPC-1 cells produced only one peak in the plot. (JPG 1575 kb)
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