| Background:Colorectal cancer (CRC) is the third most common cancer in men (746,000 cases, 10.0% of the total) and the second in women (614,000 cases,9.2% of the total) worldwide. Every year, nearly 0.7 million people died of CRC, the high mortality is mainly due to chemoresistance and tumor metastasis. It is reported that approximate 50% of the CRC patients would develop distant metastasis eventually. Chemotherapy is an essential treatment to metastatic cancer patients, however, drug resistance leads to treatments failure and cancer progression. So better understanding of the molecular mechanism underlying CRC chemoresistance and metastasis is critical to the treatments of CRC. CD 147 (also named EMMPRIN, basigin, M6 and tumor cell-derived collagenase stimulatory factor), is a glycosylated cell surface transmembrane protein of the immunoglobulin superfamily (IgSF). The CD 147 protein has 269 amino acids, exists in two forms:glycosylated form (HG,～40-60 kDa) and core-glycosylated form (LG,～32 kDa), the ratio isvariable in different cell types, while HG-CD147 isconsidered to be the functional form. CD 147 has a broad tissue distribution, implicating in many physiological processes. Aberrant CD 147 expression has been observed to correlate with several diseases, especially cancers. During tumorigenesis, CD 147 contributes to cell proliferation, metastasis, drug resistance and angiogenesis, moreover, its level may also predicts tumor relapse and patient outcome. CD 147 has been shown to interact with hyaluronan, multidrug transporters of the ABC family and monocarboxylate transporters to mediate drug resistance. In addition, CD 147 is capable of inducing the expression of several matrix metalloproteinases (MMPs), including MT1-MMP, MMP-1, MMP-2 and MMP-9. MMPs are major proteases in degrading the extracellular matrix (ECM) and basement membrane, which are vital to tumor initiation, progression and invasion. It was reported that CD 147 was upregulated in CRC, nevertheless, whether the aberrant expression of CD 147 correlates with drug resistance and metastasis of CRC are still unclear. In this research, we aimed to evaluate the effects of CD 147 on drug resistance and metastasis of CRC cells. We assessed the expression pattern of CD 147 and analyzed its correlation with clinicopathological factors of CRC patients. Next, the effects of CD 147 on drug resistance, cell invasion and EMT were elucidated. Further we investigated the involvement of MAPK/ERK signaling pathway in CD147-induced cell invasion and migration, which would provide new evidences for the research of CRC drug resistance, and contribute to the research of controlling invasion and metastasis of CRC.Part IExpression of CD 147 in Colorectal cancer tissues and cell linesObjective:To evluate the expression of CD147 in Colorectal cancer (CRC) tissues and cell lines.Methods:40 samples were collected in the gastrointestinal surgery department, Shandong provincial hospital from 2013 to 2014, which were diagnosed and radical cured by the same surgical team. Samples were divided into various groups by the differences of gender, age, tumor differentiation, lymph node metastasis, local infiltration and distant metastasis to count. Colorectal cancer tissues and the corresponding negative margin of normal cells were selected to detect the expressions of CD 147 in two groups of cells by the methods of Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analysis respectively. Meanwhile, SW480, Caco-2, HCT116, HCT15 cell lines were purchased from the Cell Bank of Chinese Academy of Sciences to detect the expressions of CD 147 in two groups of cells by using methods of qRT-PCR and Western blot respectively to explore the standard of CD 147 in above tissues and cells, and to analyze the differences of them by using statistical method. At the same time,we analyzed the expression of CD 147 in CRC tissues and the clinicopathological features by using statistical method to find the relationships between the expression pattern of CD 147 and clinicopathological features of patients with CRC.Results:Results showed that CD 147 mRNA expression was upregulated in 55% (22/40) tissues, and protein levels in 62.5%(25/40) tissues. In CRC cell lines, we found CD147 was expressed in SW480, Caco-2, HCT116 and HCT15 cell lines, and SW480 cells exhibited the highest expression level of CD 147, while Caco-2 cells showed lowest CD 147 level. To further explore the relationship between the expression pattern of CD 147 and clinicopathological features of patients with CRC, 40 patients were categorized into two groups according to the CD 147 expression level difference (≥1.5-fold). Statistical analyses revealed the following results:gender factor:P=0.804, age factor:P= 0.8, degree of tumor differentiation:P= 0.673, partial infiltration:P= 0.055, lymph node metastasis:P= 0.033, distant metastasis:P= 0.327.Conclusion:CD 147 is expressed both in CRC tissues and cell lines. Furthermore, in all the 4 cell lines, SW480 cells exhibited the highest expression level of CD 147, while Caco-2 cells showed lowest CD 147 level. These results suggested that upregulation of CD 147 may play an important role in CRC metastasis.Significance:This experiment detected the expression of CD 147 in CRC tissues and cell lines, and revealed the relationship between upregulation of CD 147 and CRC invasion and metastasis. Meanwhile, results of the experiment showed that two groups of CRC cell lines have significant differences, which make a contribution to cell transfection experiments and in vitro experiments in the following parts.Part IIThe association of upregulation of CD147on chemoresistance and metastasis in CRC cellsObjective:To further explore the relationship between upregulation of CD147, cheroresistance of CRC cells and CRC metastasis.Methods:According to results in the first part, Caco-2 and SW480 cell lines were chosen to further study the function of CD 147 by cell transfection experiments. Caco-2 cells transfected with CD 147 expression vector and negative control were named Caco-OvCD147 and Caco-cCD147. SW480 cells transfected with CD 147 siRNA vector and negative control were labeled as SW480-SiCD147 and SW480-cCD147. After cell lines were harvested, qRT-PCR and western blotting were used to evaluate the expression of CD 147 in new cell lines. Furthermore, CCK-8 kit was employed to detect the effects of CD 147 on 5-FU resistance. Cell invasion was measured by using transwell assay. Cell migration was tested by using Wound healing detection.Results:By using cell transfection experiments, two groups of cell lines were created, which are Caco-OvCD147 and Caco-cCD147, and SW480-SiCD147 and SW480-cCD147. Moreover, by using qRT-PCR and western blotting, the expressions of CD 147 were evaluated in new cell lines in each group. Results of CCK-8 kit revealed that increased expression of CD 147 enhanced the 5-FU resistance of Caco-2 cells, while CD 147 reduced its expression in SW480-SiCD147 cells compared to SW480-cCD147 cells. The transwell assay indicated CD 147 overexpression increased cell invasion of Caco-2 cells. In wound healing assay, the migratory activity of Caco-OvCD147 cells was induced compared to the Caco-cCD147 cells. Adverse results were found in CD 147 knockdown cells. By contrast, silencing of CD 147 inhibited SW480-SiCD147 cells migration as compared with SW480-cCD147 cells.Conclusions:The results of CCK-8 kit indicated that CD147 regulates the 5-FU resistance of CRC cells, and the results of Transwell assay revealed that CD147 could promote the invasion and migration of CRC cells. Those results indicated that upregulation of CD 147 expression, and CRC invasion and metastasis are significantly correlated.Significance:Results proved that CD 147 regulates the drug resistance of CRC cells, which provide the basis for the research of drug-induced mechanism. Moreover, results indicated that the relationship between upregulation of CD 147 expression, and CRC invasion and metastasis are significantly correlated, which provide the evidences for further exploration.Part ⅢThe involvement of EMT and MAPK/ERK signaling pathway in CD147-induced cells invasion and metastasis in CRC cellsObjective:To identify the function of the expression of EMT and MAPK/ERK signaling pathway in CD147-induced cells invasion and metastasis in CRC cells.Methods:By using cell transfection experiment, two groups of cell lines, which have dramatic differences, Caco-OvCD147 and Caco-cCD147, and SW480-SiCD147 and SW480-cCD147 were harvested. Moreover, by using qRT-PCR and Western blot, the expressions of CD 147 were evaluated in new cell lines in each group. And then, the expression level of E-cadherin, vimentin, MMP-2, MMP-9, pERK in ERK makers, and the level of total-ERK were detected by using qRT-PCR and Western blot, of cell lines were detected by using those experiments. To validate whether the CD147-mediated cell invasion and migration in CRC cells were regulated by MAPK signaling pathway, CD 147 overexpressed Caco-2 cell lines were treated with ERK inhibitor (U0126; 10μM). Transwell assay and Wound healing detection showed the ability of cells to invasion and migration after U-0126 treatment, and Western blot showed the expression of MMP-2 and MMP-9 in Caco-OvCD147 and Caco-cCD147 respectively after U-0126 treatment.Results:Results demonstrated that overexpression of CD 147 in Caco-2 cells led to decreased expression of E-cadherin and increased level of vimentin, and the level of MMP-2, MMP-9 were increased. Knockdown of CD 147 in SW480 cells induced the expression of E-cadherin and inhibited the level of vimentin, while the expression of MMP-2 and MMP-9 weresuppressed. Moreover, the data indicated that upregulation of CD 147 induced the phosphorylation of ERK (pERK), whereas downregulation of CD 147 reduced the expression of pERK, but there wereno obvious changesof the total-ERK (tERK) in the 2 cell lines. Results showed that U-0126 treatment decreased the expression of MMP-2 and MMP-9 in Caco-OvCD147, as compared with the Caco-cCD147. Transwell assay indicated that U-0126 treatment suppressed the invasion ability of Caco-OvCD147, as compared with Caco-cCD147 after the U-0126 treatment. Furthermore, Wound healing detection demonstrated that U-0126 treatment decreased the migratory ability of Caco-OvCD147, as compared with Caco-cCD147 after the U-0126 treatment.Conclusion:Results suggested that CD 147 promoted cell EMT, stimulated the expression of MMP-2,MMP-9 and activated the MAPK/ERK pathway, and MAPK/ERK signaling pathway may involve in CD147-stimulated cell invasion.Significance:Results in the third part demonstrated the molecular biology process of CD147-induced cells invasion and migratory in CRC cells, which provide a new direction for the research of CRC migratory in the future. |
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