Study On The Relationship Between HOXB7 And Hepatocellular Carcinoma Invasion And Metastasis

Hepatocellular carcinoma (HCC) is one of the most prevalent tumor types, and both the incidence and mortality rates of HCC have increased in recent years. Newly diagnosed cases of HCC in China account for 55% of the total cases worldwide per year. Surgical resection remain the best approach for a potential cure, but a high rate of recurrence and metastasis after resection has prevented further improvements in HCC survival. Therefore, to further explore the mechanism of metastasis and recurrence and identify effective ways to inhibit these process become the key point to further improve the survival.HOX genes, a subset of the homeobox gene family, are well conserved at the genomic level during evolution. In humans 39 HOX genes have been identified and they are organized into 4 paralogous clusters (HOXA, HOXB, HOXC and HOXD) and mapped on chromosomes 7,17,12, and 2, respectively. In addition to their roles as master transcriptional factors critical in the regulation of embryonic development, a growing body of literature has emerged in the last decade on the involvement of HOX genes in the pathogenesis of cancer. Many studies have reported aberrant expression of a number of HOX in cancer. HOX is highly expressed in leukemias, melanomas and in breast, oral, colorectal, prostate and ovarian cancers. HOXB7 is a found transcriptional factor of HOX family. It has been reported that HOXB7 plays an important role in proliferation, migration, and invasion in several cancer. And the expression of HOXB7 in human cancer is related with clinical features. HOXB7 can aberrantly transactivate bFGF, confer the biological and molecular characteristics of EMT and induce tumor-associated neoangiogenesis. The role of HOXB7 was studied in melanomas and in breast, ovarian and oral cancer, but not in hepatocellular carcinoma.In the present study, we compared systematically the expression of HOXB 7 in HCC cell lines with different metastatic potential and HCC specimens with or without recurrence, and then explored the relationship between HOXB7 and HCC invasion and metastasis. By transfected the HOXB7-siRNA, we studied the effects of silencing HOXB7 gene on HCCLM3 proliferation, migration, adhesion, invasion and metastasis in vitro. By transfected the HOXB7-pCDNA3 plasmid, we studied the effects of over-expression of HOXB7 gene on MHCC97L tumor growth and metastasis behavior in vivo. And we explored the mechanisms of HOXB7 promoting HCC malignant phenotypes. Last, we assayed the clinical significance of HOXB7 in 394 HCC samples using tissue microarray.These experiments are divided into 3 parts, as following,1. HOXB7 expression in HCC cell lines with different metastatic potential and HCC samples; 2. Effects of expression of HOXB7 on the biological characteristic in the HCC cell lines and related mechanism; 3. Study on the expression of HOXB7 in human HCC samples and analysis with clinical features.Part I. HOXB7 expression in HCC cell lines with different metastatic potential and HCC samplesObjective:to identify the HOXB7 expression in HCC cell lines with different metastatic potential and HCC samples, and the relationship between HOXB7 expression and HCC invasion and metastasis. Methods:Real time PCR, Western blot, immunofluorescence and immunocytochemistry were applied to detect HOXB7 expression in different metastatic potential HCC cell lines (HepG2, MHCC97L, MHCC97H, HCCLM3). Western blot was used to detect the HOXB7 protein expression in 12 HCC tissues with or without recurrence within 5 years. Results:The expression of HOXB7 mRNA and protein gradually increased with the increasing metastatic potential of cell lines (p<0.05), immunofluorescence and immunocytochemistry results suggested that HOXB7 is mainly expressed in the nuclear. The expression of HOXB7 protein in HCC tissues which recurrence in 5 years was higher than that without recurrence ones (p<0.001). Conclusion:The high expression of HOXB7 might play an important role in the process of invasion and metastasis in HCC. Part II. Effects of expression of HOXB7 on the biological characteristic in the HCC cell lines and related mechanismObjective:to study the effects of up or down regulate the HOXB7 expression on biological characteristic of HCC cell. Methods:In the present study, the alterations of cell proliferation, migration, adhesion, movement, invasion, and apoptosis, cell cycle was detected by CCK8, Scratch test, adhesion experiments, transwell assay and flow cytometry technology in HOXB7-siRNA interference HCCLM3 cells. MHCC97L cells were stable transfected with pcDNA3-HOXB7, the expression of bFGF, cell proliferation was detected, growth and metastatic capacity of the MHCC97L-pcDNA3-HOXB7 cell in vivo was also detected. The changes of the expression of E-cadherin, N-cadherin, Vimentin, MMP2, Cyclin D1 and Cyclin E were detected by Real time PCR, Western blot or immunofluorescence. Results:HOXB7 expression was effectively inhibited up to 80% under the optimized condition in HCCLM3 cells by Real time PCR and Western blot. The HCCLM3 cell proliferation (p<0.01), migration (p<0.001), adhesion (p<0.01), movement (p<0.05) and invasion (p<0.05) were obviously depressed but apoptosis with the comparison to untreated group. Cell cycle arrests at GO-G1 phase, S phase proportion was significantly reduced (p<0.05). The cell shape from irregular shape to fusiform shape in MHCC97L cells with upregulated HOXB7, the expression of bFGF is significantly increased (p<0.001), cell proliferate more quickly (p<0.01), tumor formation (p<0.01) and metastatic potential increased compared with untreated group, more metastatic lesions were found in lung compare with untreated group (p<0,001). Real time PCR, Western blot and/or immunofluorescence result show that the expression of E-cadherin was correlated negatively with HOXB7 expression in HCCLM3/MHCC97L cells, significantly positive correlations were found between the expressions of N-cadherin, Vimentin, MMP2, Cyclin D1 and Cyclin E and HOXB7. Conclusion:These results suggested that the expression of HOXB7 might play an import role in cell proliferation, migration, adhesion, movement and invasion in HCC cells by regulate the expression of MMP2 and bFGF, cell cycle progression and EMT progression. Part III. Study on the expression of HOXB7 in human HCC and analysis with clinical featuresObjective:To investigate the clinical significance of the expression of HOXB7 in hepatocellular carcinoma. Methods:394 cases of HCC tissue were accumulated and made into tissue microarrays. Expression of HOXB7 proteins was detected by immunohistochemical staining, and then the clinical significance of HOXB7 expression was analyzed by SPSS 16.0. Results:The positive rates of HOXB7 in cancerous tissue were significantly higher than paracancerous tissue. The over expression of HOXB7 were significantly associated with liver cirrhosis, tumor number, blood vessel invasion, BCLC staging and TNM staging (p<0.05). Over-expression of HOXB7 group with decreased the 8-year overall survival (OS) and relapse-free survival (RFS) compared with low-expression of HOXB7 in HCC, results of Kaplan-Meier analysis showed that high-expression of HOXB7 in non-blood vessel invasion group, no satellite lesion group, low TNM staging, low AFP level, poor differentiation group with a short OS and RFS compared with minimal HOXB7 group. Multivariate analysis by Cox regression showed that with liver cirrhosis, multiple tumor numbers, with blood vessel invasion and high-expression of HOXB7 were independent predictors for OS and RFS. High AFP and the diameters of tumor>5cm were independent predictors for OS. Conclusion: The over-expression of HOXB7 was closely related the invasion, metastasis and prognosis of HCC. Novelty1. We firstly demonstrated that there was differential gene expression profile of HOXB7 from HCC cell lines with stepwise metastatic potential. Preliminary study on the HOXB7 play an important role in invasion and metastasis of HCC might by activate the EMT, increasing the production of bFGF and MMP2 expression.2. We firstly evaluate the HOXB7 expression in cancerous and paracancerous tissue by tissue microarray, and clinical significance, and may be utilized as a molecular marker of HCC prognosis.Potential merits for clinical application1. HOXB7 may be a potential and valuable biomarker and therapeutic target in HCC.2. Our study provides experimental data to research further for molecular mechanism of HOXB7 in HCC invasion and metastasis.