| Colorectal cancer (CRC) is one of the most common malignant tumors in the world, because of its higher recurrence metastasis incidence and mortality rate, CRC becomes a serious threat to human life and health. With the progress of imageology, surgery and adjuvant chemotherapy, the prognosis of colorectal cancer has improved. However, the postoperative recurrence and metastasis are still the leading causes of death in patients with CRC, and have become the bottleneck of improving survival and prognosis of the patients. Therefore, further study in tumorigenesis, invasion and metastasis molecular mechanism of CRC and trying to explore the effective treatment against its recurrence and metastasis, are important to improve the long-term survival rate and prognosis of CRC patients.Rho GTPase activating protein family (GTPase activating Proteins, GAPs) regulated of Rho GTPase, which act by switching between an inactive GDP-bound and an active GTP-bound conformation, with the latter form could negatively regulate Rho GTPase activity, which involved in the formation of the focal adhesion and the cytoskeletal reorganization.DLC (Deleted in liver cancer) protein family, as a member of Rho GAPs family, has three members DLC1DLC2and DLC3, which have the highly relevant amino acid sequence. They all contain SAM (sterile alpha motif), RhoGAP (Rho GTPase activating protein) and START (StAR related lipid transfer) domains. DLC2(Deleted in liver cancer2), also called STARD13, was cloned in2003as a tumor suppressor gene, located in chromosome13q12.3. First, researches about DLC2were still in infancy, and there were no researches about DLC2expression in colorectal cancer; Second, as a protein closely related to DLC1, whether DLC2plays an important role in CRC invasion and metastasis is still need further research.Rho family proteins are small G proteins of the Ras superfamily members. Rho proteins are guanylic acid binding protein. Their molecular weights are about20kd-30kd and have GTPase activity. In1985, Rho, as a Ras homologue, was first cloned and called Ras related monomer GTPase. Rho has RhoA, RhoB and RhoC three isomers, Rho family proteins were involved in regulating a variety of cell life processes, including actin cytoskeleton reorganization, cell adhesion, cell movement, cell cycle progression, cell division, and gene transcription. In recent years, studies had found that Rho family proteins were associated with many aspects of both tumorigenesis and tumor development, including tumor growth, proliferation, invasion, metastasis, cell apoptosis and tumor new blood vessels formation, etc. Rho family, especially RhoA, highly expressed in a wide variety of tumor tissues and associated with tumor malignant degree, this implied that Rho plays an important role in the tumorigenesis and metastasis. At present, RhoA and its relationship with tumor invasion and metastasis has become one of the hot spot.Because of the RhoGAP domain of DLC2can activate the GTPase hydrolysis activity of Rho protein, which transform the Rho protein by GTP combinated active state into GDP combinated with inactive state. Thus, DLC2plays a negative regulatory factor of the Rho protein family. Recent studies have confirmed that the role of DLC2can inhibit the activity of RhoA kinase and adjust the generation of MMP (matrix metal proteases), this may interfere the cell shape changes and migration. It also implied that DLC2might through the regulation of RhoA expression level to participate the tumorigenesis, invasion and metastasis process of CRC, but the specific mechanism is still unclear.In this study, we first detected DLC2mRNA and protein expression in CRC and PCIT (pericarcinomatous intestine tissues) specimens and investigated the relationships between DLC2expressions and clinicopathological parameters. Second, we investigated the role and molecular mechanism of DLC2in regulating of tumorigenesis, invasion and metastasis of CRC, comprehensive utilization of a series of molecular biology and cell biology methods after inhibiting the expression level of DLC2in HT-29by RNA interference technology. Last, we detected RhoA expression level in CRC and PCIT tissue specimens and investigated the relationships between RhoA expressions and clinicopathological parameters. Even more, we also detected the RhoA expression levels in HT-29cell lines, which DLC2expression were inhibit by RNA interference technology. Classification R656.9 The expression of DLC2in human colorectal cancer and the relationship between DLC2expression and clinic pathological parametersObjective To investigate the expressions of DLC2in CRC tissues and PCIT, even more, to evaluated the expression levels of DLC2in subgroups by clinic pathological parameters.Methods(1) Real-time PCR was employed to detect DLC2mRNA expression in102CRC and PCIT specimens.(2) Western blot was employed to detect DLC2protein expression in102CRC and PCIT specimens.(3) Immunohistochemistry was employed to detect DLC2expressions in CRC and PCIT specimens.Results(1) CRC tissues revealed significantly lower level of DLC2mRNA than PCIT (p<0.05). The expression of DLC2mRNA was correlated with lymph node metastasis, tumor TNM stage and tumor histopathological degree significantly (p<0.05).(2) The DLC2protein expression level had no significant difference between CRC and PCIT. The expression of DLC2protein was correlated with lymph node metastasis and tumor TNM stage significantly (p<0.05).(3) According to the results of immunohistochemistry, the distribution of DLC2protein was mainly in the cytoplasm, and the positive expression rate and scores mean of DLC2in CRC were both higher than PCIT, however, there were no significant difference between them (p>0.05).ConclusionOur data strongly suggested that decreased DLC2expression in CRC correlates with lymph node metastasis and clinicopathologic stage of CRC, underexpression of DLC2is probably associated with poor prognosis in CRC patients. Target to DLC2expression effect on the biological behaviour of colorectal cancer cells by small interfering RNAiObjective To investigate the DLC2expression effect on the biological behaviours of the CRC cells.Methods(1) Western blot was used to detect the expression of DLC2in CRC cells:LOVO, SW-620, HT-29, CaCo-2, HCT-8and HCT-116, chosing the highest one HT-29cell in further RNAi.(2) Using LipofectamineTM2000transfected the small interference RNA into HT-29cell, and acquired three groups of cells:HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.(3) Real-time PCR was employed to detect the expression of DLC2mRNA in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.(4) Western blot was employed to detect the expression of DLC2protein in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.(5) Wound-healing assay, transwell invasion assay, adhesion test were employed to detect migration, invasion and adhesion in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines(6) Immunofluorescence assay was employed to detect the expression of DLC2and cell morphology in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.(7) MTT, colony formation and flow ctyometry assay were employed to detect proliferation and apoptosis in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.Results(1) We chosed the HT-29cell line because its expression level of DLC2was the highest in LOVO, SW-620, HT-29, CaCo-2, HCT-8and HCT-116. In addition, we got satisfactory transfection efficiency and successfully acquired HT-29DLC2RNAi+with downregulated expression of DLC2and HT-29DLC2RNAi-as invalid control, HT-29as blank control.(2) Real-time PCR results showed that the expression of DLC2mRNA in HT-29DLC2RNAi+cell line was much lower than HT-29and HT-29DLC2RNAi- cell lines (p<0.01), and suggested inhibition efficiency of DLC2in HT-29DLC2RNAi+(3) Western blot results indicated that the expression of DLC2protein in HT-29DLC2RNAi+cell line was much lower than HT-29and HT-29DLC2RNAi-cell lines (p<0.01), also confirmed that the inhibition efficiency of DLC2in HT-29DLC2RNAi+was apparently higher than HT-29DLC2RNAi-cell line.(4) The adhesion test demonstrated HT-29DLC2RNAi+cells had significantly higher adhesion ability than HT-29and HT-29DLC2RNAi-cells (p<0.05). Wound-healing assay showed that the closure of HT-29DLC2RNAi+was significantly more quickly than that of HT-29and HT-29DLC2RNAi-(p<0.05). Transwell invasion assay showed that the number of HT-29DLC2RNAi+cells passed through matrigel was much more as compared with HT-29and HT-29DLC2RNAi-cells (p<0.01).(5) Cell immunofluorescence assay showed that DLC2positive expression was in cytoplasma and there was some protuberance likes pseudopodium in HT-29DLC2RNAi+cell membrane.(6) There was no significant difference in cell proliferation between HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cells through MTT and colony formation experiments. However, the result of flow cytometry assay showed that the HT-29DLC2RNAi+cells apoptosis ratio and the ratio of the apoptosis cells in cell cycle was much lower than HT-29and HT-29DLC2RNAi-cells (p<0.01).ConclusionThe down-regulate expression of DLC2may obviously stimulate invasion, metastasis and apoptosis ability of colorectal cancer cell lines in vitro, but may not affect the proliferation of colorectal cancer cell lines. Association between DLC2and RhoA in tumorigenesis, invasion and metastasis of CRCObjective To investigate the expression of RhoA in CRC tissues and PCIT and to evaluate the expression levels of DLC2in subgroups by clinic pathological parameters.Meanwhile, investigated the expression levels of RhoA in DLC2down-regulated HT-29cells by using RNAi.Methods(1) Real-time PCR was employed to detect RhoA mRNA expression in102CRC and PCIT specimens, as well as HT-29, HT-29DLC2RNAi" and HT-29DLC2RNAi+cell lines.(2) Western blot was employed to detect DLC2protein expression in CRC and PCIT specimens, as well as HT-29, HT-29DLC2RNAi" and HT-29DLC2RNAi+cell lines.(3) Immunohistochemistry was employed to detect DLC2expressions in CRC and PCIT specimens.Results(1) CRC tissues revealed significantly higher level of RhoA mRNA than PCIT (p<0.01). The expression of RhoA mRNA was correlated with lymph node metastasis (p<0.01), tumor TNM stage (p<0.05) and tumor histopathological degree significantly (p<0.01).Even more, RhoA mRNA expression level in HT-29DLC2RNAi+was significantly higher than HT-29and HT-29DLC2RNAi-cell lines (p<0.01).(2) RhoA protein expression in CRC tissues also revealed significantly higher level than PCIT (p<0.01). The expression of RhoA protein was correlated with lymph node metastasis (p<0.05), tumor TNM stage (p<0.01) and tumor histopathological degree significantly (p<0.01). Even more, RhoA protein expression level in HT-29DLC2RNAi+was significantly higher than HT-29and HT-29DLC2RNAi-cell lines (p<0.05).(3) According to the results of immunohistochemistry, the distribution of RhoA protein was mainly in cytoplasm, cytomembrane was not common. The positive expression rate and scores mean of RhoA protein in CRC were both higher than PCIT, and there are significant differences between them (p<0.05,p<0.01).ConclusionOur data strongly suggested that RhoA expression was increased in CRC. Its expressions correlated with lymph node metastasis, TNM stage and clinicopathologic stage of CRC. Down-regulate expression of DLC2probably stimulate the up-regulate expression of RhoA in HT-29cell line. |
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