| Colon cancer is one of the most common malignant tumors in digestive tract worldwide. Its morbidity rate ranks the third only after gastric cancer and esophageal cancer in our country; moreover, its morbidity rate is continuously increasing in recent years and its prognosis is poor. Research on the pathogenesis, prevention and treatment of colon cancer is one of the hot points in biomedical field.The colon cancer results from the mutual effects of activation of oncogenes and inactivation of tumor suppressive genes under the different genetic backgrounds. The post-transcriptional regulation is the key component of gene expression, which is mainly regulated by RNA-binding proteins(RBPs). More than 800 kinds of RBPs have been confirmed in human genome,some of which are found to control the gene network associated with carcinogenesis. Further studies have revealed that RBPs, similar to transcriptional factors, have suppression or promotion effect on the cancer development.Musashi-1(Msi1), one of the conserved RNA-binding proteins(RBPs)family, plays an important role to maintain the balance between self-renewal and differentiation. A great deal of studies have indicates that Msi1 acts as one oncogene, and is also an important marker in a variety of cancer stem cells and/or progenitor cells to stimulate the formation and development of those cells. Overexpression of Msi1 in intestinal epithelial progenitor cells can promote tumorigenicity of progenitor cells and xenografted tumor through boosting cell proliferation via activation of Wnt and Notch signaling pathway or regulating p21. It is generally recognized that the resistance of cancer stem cells to chemo- radiotherapy is the bases for tumor occurrence, development,recurrence and metastasis. Msi1 may increase the insensitivity of colon cancerto radiotherapy and facilitate the progression of aggressive tumor through promoting nuclear accumulation of β-catenin. In addition, as the intersection of multiple signaling pathways and the key regulator of gene expression, Msil plays important roles in cell development and homeostasis. Furthermore, Msi1 is involved in the formation and development of cancer because it acts as a key regulator for many kinds of carcinogenesis. So Msi1 is expected to become a potential molecular target for prevention and cure of cancer.Therefore, the determination of the roles and mechanisms of Msi1 in colon cancer has an important theoretical significance and potential application value. The specific roles and underlying mechanisms of Msi1 in colon cancer, however, remain unclear at present. In this study, we aimed to investigate the roles and mechanisms of Msi1 in the formation and development of colon cancer, as well as and the affection of Msi1 on sensitivity of colon cancer cells to radiation. This study was divided into the following four parts: Part one: To verify Msi1 overexpression in cancerous tissues of colon cancer patients and colon cancer cell lines, and to establish stable Msi1 silencing cell lines via RNA interference technique; Part two: To investigate the effects of Msi1 silencing on biological characteristics of HCT116 colon cancer cells; Part three: To explore the molecular mechanisms underlying the effects of Msi1 silencing on cell proliferation; Part four: To study the effects of Msi1 silencing on radiosensitivity in HCT116 cells.Part 1 Protein expression of Musashi-1 in colon cancer tissues and cell lines and establishment of stable Musashi-1 silencing colon cell lineObjective: To detect the expression level of Musashi-1 in colon cancer tissues and cell lines; to construct the stable Msi1 silencing colon cell line.Methods: Western blot was used to determine the protein expression of Msi1 in cancerous and normal tissues from colon cancer patients and in three colon cancer cell lines including HCT116, SW480 and SW620. The colon cancer cell lines with the highest expression level were selected for the following studies. RNA interference technique was used to design the silencing sequence targeting for Msi1 and negative control, tool cells wereinfected after the ligation with plasmid, and the interference plasmid and control plasmid were inserted into lentiviral vector to infect colon cancer cells;stable silencing cells were harvested with the selection of antibiotics.Real-time PCR and Western blot methods were used to examine the silencing effect in Blank, negative control(NC), and knock down(KD) groups after infection at RNA and protein levels.Results: The result of Western blot assay showed that the Msi1 protein was selectively high expressed in colon cancer tissues and three colon cancer cell lines compared with normal intestinal mucosa; among three colon cancer cell lines of HCT116, SW480 and SW620, the protein level of Msi1 was the highest in HCT116 cells which was chosen as the target cells for the following studies. In this part of study, lentiviral vector carrying GV248-Msi1-shRNA targeting Msi1 and negative control GV248-NC-shRNA were successfully constructed. HCT116 colon cancer cell lines were infected with these two lentivirus, and puromycin was applied to select the stable expression cells. The results of Real-time PCR and Western blot confirmed that the expression of Msi1 in HCT116 cells was decreased by 73.85% and 59.01% at RNA and protein levels after the infection of interference lentiviral vector targeting Msi1,respectively.Conclusions: Msi1 protein was highly expressed in colon cancer tissues and cell lines. The colon cancer cell line HCT116 can be used to construct the stable Msi1 silencing cells.Part 2 The effects of Musashi-1 silencing on biological characteristics of HCT116 colon cancer cell lineObjective: To investigate the effects of Musashi-1 silencing on biological characteristics of HCT116 colon cancer cell lineMethods: MTS assay was utilized to test the proliferation of stable Msi1 silencing HCT116 cells, Flow cytometry analysis was used to examine the change of cell cycle and apoptosis, Transwell chamber assay in vitro was used to check the invasive abilities, and tumor sphere experiment was conducted to test the stemness of tumor cells. Besides, nude-mouse xenograft model wasestablished to observe the effects of Msi1 silencing on tumorigenicity in nude mice.Results: The results of MTS and Transwell assays showed that the proliferation and invasiveness of HCT116 cells were significantly decreased in vitro after silencing Msi1 gene. Flow cytometry results indicated that the apoptosis of cancer cells and the number of G0/G1 stage cancer cells were significantly increased after silencing Msi1. Tumor sphere experiment revealed that Msi1 silencing decreased the number of tumor spheres significantly. The results of nude-mouse subcutaneous xenograft model of HCT116 cells showed that the growth of transplanted tumor was significantly inhibited when Msi1 expression was reduced.Conclusions: Interference of Msi1 expression can inhibit the proliferation and invasiveness of HCT116 cells in vitro, resulting in the arrest of G0/G1 cell cycle and the induction of cell apoptosis. The stemness and xenograft growth of HCT116 colon cancer cells can be significantly reduced by silencing Msi1.Part 3 The molecular mechanisms underlying the effects of Msil silen-cing on cell proliferationObjective: To study the molecular mechanisms underlying the effects of Msil silencing on cell proliferation.Methods: Real-time PCR and Western blot were used to examine the mRNA and protein levels of p21 after silencing Msi1, and p21 3’-UTR luciferase reporter gene vector(p21WT) and mutant(p21MU) were constructed. These reporter gene vectors were transfected into Msi1 silencing cells and control cells, and the fluorescence ratio in these cells was calculated and compared between the two groups to evaluate the activity of p21 3’-UTR region.Results: Western blot results found that the protein level of p21 in Msi1 silencing HCT116 colon cancer cells was significantly upregulated compared to blank and negative control groups; the results of Real-time PCR showed that the mRNA level of p21 was not significantly altered among three groups.The further luciferase reporter assay showed that the activity of p21 WT luciferase in KD group was significantly increased compared to blank and negative control groups and no significant changes were found in the activity of mutant p21 MU.Conclusions: In HCT116 colon cancer cells, Msi1 can specifically bind with the mRNA 3’-UTR region of target gene p21, which inhibits its translation and downregulates p21 protein expression, induces change of cell cycle and suppresses cell apoptosis,and thus promotes tumor cell growth.Silencing Msi1 can inhibit tumor cell growth via upregulating p21 protein expression.Part 4 The effects of Msil silencing on radiosensitivity in HCT116 cells.Objective: To explore the effects of Msi1 silencing on radiosensitivity in HCT116 cells.Methods: Lentivirus-mediatd Msi1 silencing expression plasmid was used to infect HCT116 cells; the infected HCT116 cells were treated with X-ray irradiation; clone formation assay was conducted to detect the effects of Msi1 silencing on radiosensitivity in HCT116 cells. MTS assay was used to check the proliferation of colon cancer cells after irradiation and the inhibitory rate was calculated. Flow cytometry analysis was utilized to examine the cell cycle and apoptosis after irradiation.Results: The clone formation assay showed that after 0 ~ 8Gy X-ray irradiation the survival curve was decreased in cells of KD group, the values of D0, Dq, N and SF2 in cells of KD group were significantly lower than those in blank and negative control groups, and the sensitization enhancement ratio(SER) in cells of KD group were 1.56 and 1.47 respectively compared to blank and negative control groups. The MTS experiment indicated that the proliferation activity in each period was significantly lower than those in blank and negative control groups, and the inhibitory rate gradually increased with the increment of time and dosage. Flow cytometry analysis revealed that the rate of cell apoptosis was apparently increased in cells of each group in time-dependent and dosage-dependent manners after irradiation, but theincrease of apoptotic rate in KD group was more obvious in each period. Also the ratio of G2/M stage cells in KD group cells was significantly reduced after irradiation compared to the blank and negative control groups.Conclusions: The inhibition of Msi1 expression in HCT116 colon cancer cells could improve the radiosensitivity, which may be related to the promotion of cell apoptosis and the relief of cell G2/M stage arrest.Conclusions:1 Msi1 protein was highly expressed in both colon cancer tissues and cell lines, which implies the important roles of Msi1 in formation and development of colon cancer;2 The colon cancer cell line HCT116 can be used to construct the stable Msi1 silencing cell line successfully;3 Interference of Msi1 expression can inhibit the proliferation and invasiveness of HCT116 cells, resulting in the arrest of G0/G1 cell cycle, the induction of cell apoptosis, the reduction of cell stemness, and the suppression of xenograft tumor growth.4 In HCT116 colon cancer cells, Msi1 silencing can inhibit tumor cell growth via upregulating its target gene p21 protein expression.5 Msi1 silencing in HCT116 colon cancer cells could enhance the radiosensitivity by promoting cell apoptosis and releasing G2/M stage arrest after radiation. |
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