| Low density lipoprotein receptor related protein 5(LRP-5) is one of the members of the family of low density lipoprotein receptor, which belongs to a single transmembrane receptor protein, consisting of 1615 amino acid residues including intracellular area,across the membrane area, extracellular region of single transmembrane receptor protein.LRP-5 is highly homologous with another member LRP-6 in the family. A large number of studies have shown that LRP-5/LRP-6 and seven transmembrane receptor protein FZD constitute the receptor system of WNT/beta-catenin signal transduction pathways,LRP-5/LRP-6 is the complementary receptor binding with WNT protein. The signal path can only be activated, when WNTs and FZD as well as LRP-5/LRP-6 bind together at the same time. At present, the association between abnormalities of WNT/beta-catenin and cancer biology is a hot issue.This study was designed to silence LRP-5 by RNAi technology,and then to detect the changes of WNT/beta-catenin signaling pathway on proliferation, apoptosis, invasion,migration and other biological behaviour and differences in proteomics of gastric cancer cell.First of all, q RT-PCR and immunohistochemical method were used to detect the expression of LRP-5 gene in gastric cancer tissues with different differentiation degrees.As revealed by real-time PCR results, LRP-5 m RNA expression in well and moderately differentiated gastric carcinoma tissues were significantly higher than control group(P<0.05), and gastric cancer tissue accompanied by lymphocyte transfer also presented the LRP-5 high expression. It suggested LRP-5 may involve in the occurrence,development, metastasis and malignant degree of gastric cancer.Second, four interference plasmids with different genetic loci p GPU6/GFP/Neo-LRP-5 were successfully built. The cell proliferation, adhesion, apoptosis, invasion and migration were observed with silence of LRP-5 in MGC-803 cells 48 h post transfection. Results showed that: 1, CCK8 method, post-transfection 48 h in MGC-803 cells within interference group, relative growth rate significantly lower than that of blank and NC group(P < 0.05); 2, Adherent cell number of the LRP-5 interference group was155.00±18.33, It was increased compared with blank control group and NC group. The adhesion level of MGC-803 cell was significantly increased(P < 0.05); 3, Doublestaining methods was used to detect cell apoptosis.The result showed that apoptosis cells were significantly increased in interference group compared with NC group(P < 0.05); 4,Post-transfection 48 h, cells in interference group with high proportion of S phase detected and G2 ratio decreased; 5, Transwell Chambers membrane experiments showed that cell count was 38.67±3.51 when the invasion of LRP-5 interference group migration to the lower of Transwell basement, which was significantly declined compared with that of blank group(52.67±4.51), the Mock(50.67±3.79) and NC group(47.00±4.36; P﹤0.05); 6, Scratch experiments showed that MGC-803 cell migration healing rate of L68 group was significantly(P < 0.05) decreased(0.7509±0.0108) compared with that of blank, Mock, NC group.At last, total proteins were extracted from MGC-803 cells from both interference group(post-transfection 48h) and negative control group. 2D electrophoresis was used to separate and purify proteins. The results showed that a total of 29 differential protein spots were found between two groups. 15 protein spots were selected to do mass spectrum identification, among which 12 was succeed. These proteins include: the cytoskeleton, cell cycle and cell proliferation, the molecular chaperone, sugar metabolism related proteins.This study first used RNAi technology to silence LRP-5 gene,and the effect of silencing on the biological behavior and differential proteomics of MGC-803 gastric cancer cells were determined.Data reported in this study may provide experimental basis for development of drugs for gastric cancer therapy. |
PDF Full Text Request