MYBL2 Gene Expression And Study Of Mechanisms In Colorectal Cancer

Colorectal cancer(CRC) is a common malignancy and the third cause of cancer death in western countries. Daily diet with a low fiber content may increase the incidence of CRC. Cancer screening and treatment program in China is less standard available than that in western countries. The development of CRC is a long and complicated process, including the activation of multiple oncogenes and the inactivation of tumor suppressor genes. These genes may provide new targets for the future treatment of cancer.MYBL2 gene, also known as B-MYB, is widely expressed in proliferating cells. MYBL2 gene has multiple effects such as maintaining the normal cell cycle progression. It is also supposed to suppress cell apoptosis through multiple pathways and be associated with cellular aging.Some studies have shown that MYBL2 gene is associated with stem cell-like phenotype,for MYBL2 gene may have functions to maintain the pluripotent stem cell characteristics, such as maintaining self-renewal and differentiation.MYBL2 gene is overexpressed in a variety of tumors and associated with the prognosis of cancer patients, which indicated that MYBL2 gene plays an important role in the genisis and progression of tumors. So far, there are few studies to illustrate the role of MYBL2 gene in CRC. The mRNA and protein levels of MYBL2 in colorectal cancer CRC tissues and adjacent noncancerous tissues (ANCT) are still unknown. It is also necessary to establish its relationship with the prognosis of patients with CRC. In addition, current studies of gene function mainly focus on cell cycle. Whether MYBL2 gene have other functions in CRC will also be explored in this research. This study will be divided into three sections:Part I Expression of MYBL2 mRNA in colorectal cancer and its relationship with prognosisObjective:This study is to detect the expression of MYBL2 mRNA in CRC tissues and ANCT, and to explore its relationship with clinicopathological characteristics and prognosis in CRC patients.Methods:RNAlater preserved specimens were collected at Fudan University Shanghai Cancer Center from 2007 to 2009. A total of 70 pairs of cases were used to detect MYBL2 mRNA expression in CRC and ANCT, and another 180 cases of CRC tissues were used to explore the relationship between MYBL2 mRNA level and clinicopathological characteristics. The differences of mRNA level between CRC and ANCT was analyzed by paired T-test. The relationship between mRNA level and clinicalpathologyical characteristics was analyzed using T test. Survival curve was estimated with the Kaplan-Meier method and compared using the log-rank test. Multivariate Cox regression model were used to explore the associations of patient outcome and characteristics. Statistical significance was defined as a P-value less than 0.05.Results:MYBL2 mRNA level was significantly higher in CRC than that in ANCT (P=0.016), suggesting that MYBL2 may be related to the carcinogenesis of CRC. In addition, MYBL2 mRNA level was related to tumor size, tumor stage, and lymph node metastasis. MYBL2 mRNA expression was higher in larger tumors (P=0.048) and in stage III patients (P=0.014). Patients with lymph node metastasis had higher MYBL2 level than those without metastasis (P=0.014). MYBL2 mRNA expression was positively correlated to N stage (P=0.049) and negatively correlated with disease-free survival (DFS) (P=0.017). MYBL2 mRNA level was higher in patients with worse DFS. MYBL2 mRNA expression was also positively related to metastasis and recurrence (P=0.037). MYBL2 mRNA expression is an independent prognostic factor for CRC patients.Part II Expression of MYBL2 protein in CRC and its relationship with prognosisObjective:This part is to detect the expression of MYBL2 protein in CRC and ANCT, and to explore its relationship with clinicopathological characteristics and prognosis in CRC patients.Methods:The formalin-fixed paraffin-embedded (FFPE) samples were collected from CRC patients receiving surgery from 2005 to 2007 at Fudan University Shanghai Cancer. The samples were made into tissue microarrays.97 CRC tissues and 104 ANCT tissues were included in this study. The relationship between protein expression and clinicopathological features of CRC patients was analyzed using χ2 test. Survival curve was estimated with the Kaplan-Meier method and compared using the log-rank test. Multivariate Cox regression model were used to explore the associations of patient outcome and characteristics. Statistical significance was defined as a P-value less than 0.05.Results:MYBL2 protein was expressed mainly in the cytoplasm and nucleus. MYBL2 protein expression in CRC tissues was much higher than in ANCT. MYBL2 protein expression was not significantly related to patient’s gender, age, degree of differentiation, gross type, tumor size, location, and TNM stage. However, the protein expression in recurrence group (93.75%) was higher than the non-recurrence group (70.77%) (P=0.010). MYBL2 protein expression was negatively associated with the prognosis of CRC patients. Patients with higher protein expression of MYBL2 had worse DFS compared to those with lower expression (P<0.05). The COX multivariate regression analysis shows that, MYBL2 protein expression and tumor stage were independent prognostic factors for CRC patients.Part III MYBL2 gene preliminary study of mechanisms in colon cancerBackground and objective:MYBL2 gene may be involved in the development of CRC tumors by affecting tumor proliferation, invasion and metastasis. Current reports indicated that, MYBL2 gene plays an important role in cell proliferation, and is also involved in cell cycle regulation. Therefore, this section will further study the effect of MYBL2 gene on the proliferation and migration in colon cancer cells to explore its possible mechanisms.Methods:We detected mRNA and protein expression of MYBL2 in several CRC cell lines by RT-PCR and western blot. siRNA was used to decrease MYBL2 expression in SW480, which had high expression of MYBL2. Proliferation differences between interference group and the control group were detected by CCK8 test. Cell cycle and apoptosis were investigated by flow cytometry; wound healing assay, transwell assay, and Western blotting were used to detect migration and invasion changes.Results:After successfully transfering siRNA to decrease MYBL2 expression in SW480, CCK8 test showed that MYBL2 could promote the proliferation of CRC cells. Downregulating MYBL2 promoted cell cycles arrest in G2 phase and prevent cell cycle progression. Furthermore, interfering MYBL2 gene expression would facilitate cell apoptosis. This suggested that the MYBL2 might promote colon cancer proliferate by regulating cell cycle and apoptosis mechanisms. Cell scratches and transwell assays showed that MYBL2 could promote cell migration. Finally, MYBL2 could affect the expression of invasion-related proteins such as MMP9, Vimentin and E-cadherin in colon cancer cells, suggesting MYBL2 may promote cell invasion. Further studies should be conducted to explore the possible mechanism of MYBL2 in CRC cells.