| Colorectalcancer (CRC) is one of the popular malignant tumor in Digestive System, which seriously hazards the human health and lives. The coordination effects of body internal causes (polymorphism and susceptibility) and external causes (diet and environment) result in CRC. The close relationship between CRC and gene mutation has been confirmed by a great deal of experiments. Activation of oncogenes and inactivation of anti-oncogenes could appeare in any steps of growth and differentiation in normal mucosa cells. Mutation or lost of many genes might be concerned with colorectal carcinogenesis. Researches of genes in colorectal carcinogenesis could further clarify the carcinogenesis mechanism and supply new targets for biotherapy of CRC.Recently, it has been found that many signaling pathways which regulate processes of embryo development and tissue differentiation largely affect processes of tumourigenesis, such as EGFR signaling pathway, TGF-βsignaling pathway, Sonic hedgehog signaling pathway, Wnt signaling pathway, Notch signaling pathway. The viewpoint that the singnaling pathway controlling embryo development of vertebrates may be concerned with human tumourigenesis supplies new research directions of tumourigenesis mechanism.Sonic hedgehog is one of major signaling pathways in human embryo development, which regulate cell proliferation and tissue differentiation. Sonic hedgehog signaling pathway is comprised by secretory Sonic hedgehog -glycoprotein (Shh) ligand, transmembrane protein receptor patched (Ptc), transmembrane protein smoothened (Smo) and downstream transcription Gli protein (Gli1,Gli2,Gli3). Recently, a large amount of researches have proved that multiple cancers are closely associated to Sonic hedgehog signaling pathway abnormalities. It has been found the mutation or abnormal expression of key members of Sonic hedgehog signaling pathway in human basal cell carcinoma, small cell lung cancer, pancreatic carcinoma, prostate cancer, medulloblastomas, gastric adenocarcinoma, esophageal carcinoma, bladder cancer, breast cancer and hepatocellular carcinoma. The high levels of Shh and Ptc expression are also found in CRC. Exogenous Shh can promote the proliferation of colorectal mucosa cells and cancer cells, while Shh antibody can restrain the proliferation of colorectal mucosa cells and cancer cells.Previous study of abnormal activation of Sonic hedgehog signaling pathway in CRC emphasized the effects of Shh ligand, ignoring the effects of other members of Sonic hedgehog signaling pathway, particularly Smo protein with activation function. There is no study of the effects of Smo gene in CRC.Expression of Smo protein and Smo mRNA is detected by Western blot and Semi-quantitative RT-PCR to study the relationship between Smo gene and CRC in our research. We explore the proliferation and apoptosis of Lovo cells by MTT method and Flow Cytometry when Smo mRNA is degraded by RNAi with the transfection of specific Smo siRNA in vitro to study the effects of Smo gene in CRC.[Objective]To study the relationship between Smo gene and CRC, and the effects of Smo gene in CRC. To provide experimental evidence of blocking Smo gene site on Sonic hedgehog signaling pathway for the treatment of colorectal adenocarcinoma.[Materials and methods]1,CellsThe colon adenocarcinoma Lovo cells were from experimental animal center of Zhong Shan University.2,Methods(1) Cultivating Lovo cells in RPMI1640 media (10% fetal cattle serum, 1% Streptomycin and Penicillin). (2) To detect the expression of Smo mRNA of Lovo cells by Semi-quantitative RT-PCR. (3) To detect the expression of Smo protein of Lovo cells by Western blot. (4) To design and synthesis of 3 pairs of Smo siRNA and 1 pair of non-specific siRNA in vitro. (5) Five groups of Lovo cells in 6 times: group A (transfection of specific siRNA-1), group B (transfection of specific siRNA-2), group C (transfection of specific siRNA-3), group D (positive control group, transfection of non-specific siRNA), and group E (negative control group, only Lipofectamine). (6) Tansfection of Smo siRNA into Lovo cells by Lipofectamine. (7) To observe the expression of Smo mRNA in Lovo cells of all groups after 48 hours of transfection by Semi-quantitative RT-PCR and to select the group with highest inhibition effect for the experiment group in later experiments. (8) Three groups of Lovo cells in 6 times: experiment group, negative control group, positive control group. (9)To detect proliferation levels of Lovo cells of all groups by MTT after 12h,24h,36h,48h,72h of transfection, respectively. (10) To test apoptosis rates of Lovo cells of all groups by flow cytometry after 48h of transfection.3,StatisticsThe data were analyzed with SPSS11.5 software. Expression levels of Smo mRNA in different groups were compared with One-way ANOVA, and proliferation levels of Lovo cells of differen groups in different time points were compared with Repeated Measures ANOVA, then performed LSD Multiple Comparisons while P<0.05. Apoptosis rates of Lovo cells were compared withx~2 test.[Results]1,High expression of Smo protein and Smo mRNA were found in Lovo cells, detected by Western blot and Semi-quantitative RT-PCR.2,The expression of Smo mRNA were significantly different in Lovo cells among siRNA-1 group, siRNA-2 group, siRNA-3 group, positive control group and negative control group, after 48 hours of transfection (F = 891.496, P <0.001). The level of Smo mRNA expression of siRNA-1 group Lovo cells is lowest, and siRNA-2 group is lower, with significant difference when compared with negative control group (P<0.001) . There were no differences between the levels of Smo mRNA expression in Lovo cells of siRNA-3 group, positive control group and negative control group (P>0.05) . Besides, the expression of Smo mRNA were significantly different in Lovo cells between siRNA-1 group and siRNA-2 group (P<0.001) . The inhibition rates of siRNA-1 group, siRNA-2 group were 63.56%,46.54%, respectively, while siRNA-3 group with no inhibition effect. Therefore, siRNA-1 group was selected for experiment group in later experiments.3,There were different proliferation levels of Lovo cells of siRNA-1 group, positive control group and negative control group after 12,24,36,48,72 hours of transfection, statistically (F =359.299, P<0.001). The proliferation levels of Lovo cells of siRNA-1 group were notably lower than that of negative control group after different time of transfection (P <0.05).4,The apoptosis rates of Lovo cells in Smo siRNA-1 group was significantly higher than positive and negative control groups after 48 hours of transfection, with apoptosis rates of 21.7%,10.1%,4.4%, respectively. (x~2=15.909, P<0.001)[Conclusions]1,The oncogenesis of colon adenocarcinoma is related to the high expression of Smo gene.2,The high expression of Smo gene is related to the proliferation and opoptosis of colon adenocarcinoma Lovo cells. Smo gene may be one of oncogenes, which affect the oncogenesis of colon adenocarcinoma.3,It is feasible to degrade the levels of Smo mRNA expression in Lovo cells mediated by siRNA.4,It may be a new target for colon adenocarcinoma treatment that blocking the Sonic hedgehog signaling pathway in Smo gene sites.
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