| Background and aims:Gastric cancer(GC) is one of the most common malignant tumors in China. The invasion and metastasis are the main factors of poor prognosis and survival in GC patients. Growing evidence shows that the invasion and metastasis of GC are closely related to epithelial-mesenchymal transition(EMT). Caudal-type homeobox transcription factor 2(CDX2) plays a crucial role in the process of differentiation for gastric epithelial stem cells. Moreover, for CDX2-positive expression GC patients, postoperative survival rate is significantly higher than that of CDX2-negative expression. Thus, CDX2 is a key transcription factor for gastric epithelial cell differentiation and inhibition of GC occurrence. Our preliminary study showed the micro RNA-32(mi R-32) was up-regulated higher than other mi RNAs by mi RCURY? LNA Array after CDX2-forcing expression. Recent literature reported that mi R-32 played an anticancer role in lung cancer and oral cancer. Our study also found that enforcing mi R-32 overexpression could obviously inhibit the growth and invasiveness of GC cells SGC-7901. However, CDX2 whether directly regulates mi R-32 expression and inhibits GC cells proliferation, migration and invasion? And are these effects related to EMT? Currently the answers remain unknown. In present study, the aims are to explore the relationship between expression of CDX2, mi R-32 and clinical pathological features, recurrence, and survival in GC patients. Next, we are to explore the possibility that CDX2 regulate the transcription of mi R-32 in GC cells. And, to investigate the effect that CDX2-targeted mi R-32 modulate GC cells proliferation, migration, invasion and EMT. Finally, to probe the effect that CDX2-tageted mi R-32 expression affect the growth and EMT of subcutaneous transplantation tumor in nude mice model.Methods:1. Surgically resected specimens of GC were obtained from the Affiliated Hospital of Nantong University between November 2009 and August 2010. Immunohistochemistry technique was used to detect CDX2 protein expression in GC tissues, analyzing its relationship between clinical pathological features, postoperative recurrence and survival in GC patients. Next, the expressions of CDX2 m RNA and mi R-32 were detected by Real-time quantitative fluorescent PCR(RT-PCR) in 50 surgically dissected GC and matched normal gastric mucosal specimens. The relationship between CDX2 m RNA, mi R-32 expression and clinicopathological characteristics of GC was discussed. Moreover, the correlation between CDX2 m RNA and mi R-32 expression was to explore in GC patients.2. RT-PCR and Western blot analyses were used to detect the expression of CDX2 in different GC cell lines. Then, CDX2 plasmid expression vector, mi R-32 inhibitor vector, sh RNAs against CDX2 vector, mi R-32 mimic vector or empty plasmid vector(EV) control group were respectively transfected into the GC cells by LipofectamineTM 2000. RT-PCR was used to detect the expression of CDX2 m RNA and mi R-32 in each group of GC cells. The effects of mi R-32 regulated by CDX2 in GC cells were evaluated with cell proliferation assay using by Cell Counting Kit 8(CCK8) and colony formation assay. The migration and invasion assay using by wound-healing assay and Matrigel invasion Assay. Levels of CDX2, E-cadherin and Vimentin proteins were examined by Western blot analysis, respectively.3. MKN-45 cells transfected with a CDX2 plasmid expression vector(MKN-45/CDX2 group), CDX2 plasmid expression vector and mi R-32 inhibitor vector(CDX2+mi R-32 inhibitor group), sh RNAs against CDX2 vector(MKN-45/CDX2 sh RNA), and an empty plasmid vector(MKN-45/EV group), respectively. The four groups GC cells were subcutaneously inoculated into nude mice. Then, subcutaneously transplant the histo1ogical1 y intact xenograft tissues to nude mice, observed and compared the condition of nude mice, described the rates of tumor survival and growth velocity. The morphology changes of tumor cells were observed by hematoxylin-eosin(HE) staining. The levels of CDX2 m RNA and mi R-32 were determined by RT-PCR, and the expressions of CDX2, E-cadherin and Vimentin proteins were detected by immunohistochemistry and Western blot, respectively.Results:1. Immunohistochemistry technique showed the positive CDX2 expression was mainly located in nucleus, and its overall positive rate was 35/60(58.3%) in GC tissues. The expression of CDX2 protein was significantly correlated with TNM clinical stage and lymph metastasis(all P<0.05). The patients of CDX2-positive 1-year, 2-year, 3-year, 4-year, and 5-year after surgery with the cumulative recurrence rates were 20.0%ã€45.7%ã€71.4%ã€82.8% and 91.4%, respectively. In CDX2-negative patients 1-year, 2-year, 3-year, 4-year, and 5-year after surgery,the cumulative recurrence rates were 40.0%, 80.0%, 96.0%, 100.0%, and 100.0%. The 2-4 years of cumulative recurrence rates, 1-5 years median recurrence time and survival period in the two groups after surgery were significantly different(all P<0.05). RT-PCR showed the expressions of CDX2 m RNA and mi R-32 were correlated significantly with the infiltrating depth, TNM stage and lymph node metastasis(all P<0.05). Moreover, the positive correlation was found between CDX2 m RNA and mi R-32 in GC tissues(r=0.861,P<0.05).2. CDX2 was highly expression in AGS and medium expression in MKN-45, while it was expression rarely in SGC-7901(P<0.05). mi RNA-32 showed a high expression after CDX2 was overexpressed by CDX2 plasmid expression vector transfected in GC cells(P<0.05). Nevertheless, the expression of mi RNA-32 was downregulated by CDX2 sh RNA(P<0.05). Chromatin immunoprecipitation assay(Ch IP) showed the CDX2 antibody can combine with mi RNA-32 gene promoter of primers corresponding area in MNK-45 and AGS cells. mi RNA-32 was upregulated by CDX2 plasmid expression vector transfected, which inhibited GC cells growth, opposed migration and invasion in vitro(all P<0.05). However, both enforcing expression of CDX2 and interfering expression of mi RNA-32, the inhibitory effect was weakened(P>0.05). Inversely, mi RNA-32 was downregulated by CDX2 sh RNA, the GC cells showed a significantly increased proliferation rate, wound-healing rate and invade cell numbers(all P<0.05). Moreover, both downregulating expression of CDX2 and upregulating expression of mi RNA-32, the promotion effect was also weakened(P>0.05). Interestingly, MKN-45/CDX2 cells exhibited a significant upregulation of E-cadherin protein and a significant downregulation of Vimentin protein expression(P<0.05). However, AGS/CDX2 sh RNA cells exhibited a significant downregulation of E-cadherin protein and a significant upregulation of Vimentin protein expression(P<0.05). But the mi RNA-32 inhibitor and mi RNA-32 mimic were used to intervene respectively, E-cadherin and Vimentin protein dynamics have reversed. Collectively, mi RNA-32 inhibitor abrogated CDX2-mediated inhibition of cell proliferation, migration, and invasion in vitro.3. Subcutaneous transplantation tumors were successfully established in nude mice using the medium CDX2 expression level of MKN-45 cell. The success rate of transplanted tumor was 80% for overexpression of CDX2 group. Knockdown CDX2 group was 60%. Both enforcing CDX2/interfering mi RNA-32 groups and control group were all 100%. We found that compared with the other groups, enforcing CDX2 expression group had a slower tumor growth rate and smaller tumor volume, however, knock-down CDX2 expression group had the opposite effect. Accompanied by up-regulating CDX2 expression, the level of mi R-32 was also up-regulated. While the CDX2 expression was down-regulated, the level of mi R-32 was down-regulated, too. The results were in agreement with in vitro(all P<0.05). Furthermore, mi RNA-32 inhibitor also abrogated CDX2-mediated inhibition of cell growth in vivo.Conclusions:Our findings suggested that CDX2 and mi R-32 were related with tumor invasion and metastasis. CDX2 expression was positively correlated with mi R-32. The absent of CDX2 and mi R-32 in postoperative GC tissue were related to worse prognosis and more prone to short-term relapse after surgery. CDX2 was able to directly regulate the transcription and expression of mi RNA-32, inhibit the proliferation, migration and invasion of GC cells. CDX2 participated in the process of EMT of GC by regulating the expression of EMT related proteins such as E-cadherin and Vimentin in vitro. These results were further supported by in vivo tumorigenicity assays, which showed that CDX2 suppressed gastric tumor xenograft growth and inhibited EMT in nude mice. |
