Establishment And Identification Of Lewis Lung Cancer Cell Lines With MPcdh18-siRNA Expression Induced By Tetracycline (Tet-on)

Cell adhesion is a form of communication to share information between cells. The cadherin family is a kind of calcium ion-dependent adhesion molecule.Protocadherins constitute the largest subgroup within the cadherin family, and Protocadherinl8(Pcdh18)is a member of it. In zebrafish, two Pcdh18 homologous proteins are found, Pcdhl8a and Pcdhl8b, of which the homology is over 70%. Even though the expression patterns of the two are not the same, they both play important roles in the embryonic neurogenesis. Pcdh18 amino acids of mammals and zebrafish are more than half of the same, which suggests that the expression and positioning of Pcdh18 may have high similarity between mammals and zebrafish.In order to study the biological function of protocadherins in the nervous system, biologists remove genes by knockout technology to observe the nervous system of knockout mice. And it is the main idea to explore the the function of Pcdh18 gene. Combined with RNA interference technology and transgenic mice technology, the expression of target gene is regulated by RNA interference technology, which is mediated by Tet system, to avoid the death of embryos, and obtain the defective phenotype to know the function of Pcdh18 gene.The paper aims to screen out the suitable sequence of siRNA, which has a great inhibition of the expression of mPcdh18, establish and identify Lewis lung cancer cell lines induced by Tet-on system with stable expression of mPcdhl8-siRNA, and lay the foundation for the establishment of RNAi transgenic mice model in the future.The study is performed from the following two aspects.1. The expression of mPCDH18-siRNA mediated by Tet system in 293T cellsAccording to the mPcdh18 gene sequence, three pairs of siRNA were designed, including a pair of unrelated sequence. These three pairs of siRNA and pEGFP-mPcdhl8, which has been already successfully constructed in our laboratory, were co-transfected into 293T cells, with blank control set. We then observed the green fluorescence by fluorescence microscope, and detected at mRNA level with RT-PCR method, to obtain the efficiency of the gene inhibition. The results showed that the siRNA we designed all have strong inhibitory effect in cells.We then cloned the reverse complementary DNA sequence of the three pairs of siRNAs into pSingle-tTS-shRNA vector by enzyme digestion and connection method to construct the siRNAs expression vector pSingle-tTS-shRNA respectively. The vector was then co-transfected with pEGFP-mPcdhl 8 in 293T, and induced by an appropriate concentration of DOX. We observed the green fluorescence by fluorescence microscope, and detected the mPcdhl8 expression at mRNA level with RT-PCR method, to obtain the efficiency of the gene inhibition. The results showed that by the regulation of Tet system, the siRNA we designed still have strong inhibitory effect in cells.2. Establishment and identification of a LLC cell line with stable expression of mPcdh18-siRNAWe screened several mouse and human cells with RT-PCR and Western-Blotting methods, and confirmed that the LLC cell has a high expression of mPcdhl8 gene and protein. The pSingle-tTS-shRNA recombinant plasmid was transfected into LLC cells, and under the selection of G418, a stable cell lines was obtained. The expression level of mPcdh18 of the stable cell line induced by different concentration of DOX was detected with qRT-PCR and Western-Blotting methods. The results showed that with the increase of the concentration of DOX, the expression level of mPcdhl8 descended. The inhibition rate reached 80% by the concentration of DOX around 1000μg/ml. With observation of changes of proliferation activity and cell morphology, and determination of the growth curve and cell migration, it was proved that the proliferation activity went down, the condition of the cells turned worse and the ability of migration became weak.In summary, this study has screened out two pairs of siRNA, which can highly inhibit the expression of mPcdh18 gene, established and identified a Lewis lung cancer cell line regulated by Tet-on system with stable expression of mPcdhl 8-siRNA, and laid the foundation for establishment of RNAi transgenic mice model in the future.