The Effect Of DCC Gene In Colorectal Cancer And Its Changes After Chemotherapy

Colorectal cancer is a common alimentary tumor threating human health.The disease incidence ascends obviously in these years, specially colon cancer in cities and developed villages. The colorectal cancer incidence will ascend accompaning with the changes of life style, food and drink. So, it is important to research the pathogenesy and strategy about prevention and cure.It is a long and complicated process that normal cells change to tumor. It includes muti-stages, muti-steps and muti-genes. A lot of genes were discovered involved in the process of colorectal cancer genesis and development, including oncogene activation, anti-oncogene inactivation and mismatch repair mutation. Normal mucous membrane to adenoma to cancer is the most important mode. It is important to explore the gene changes in the process for researching the pathogenesy and developing the strategies about prevention and cure.The abnormality of anti-oncogene plays a key role in the colorectal cancer genesis and development. Its inactivation or deletion may resμlt in cells unregμlated proliferation. DCC (Deleted colorectal carcinoma) gene is the longest human anti-oncogene at present. DCC gene located in 18q21.3.Its gene total length is 1400Kb. It includes 29 exons. Its cDNA encodes a transmembrane protein of 1447 amino acids, including transcription genes to start, signal peptide genes and transmembrane protein genes. DCC proteins after the transcription and translation from the gene includes two parts, these are interspongioplastic substance (325 amino acids) and corpuscular substance (1100 amino acids). DCC gene keeps cell nomal growth and differentiation throμgh participating inter-cell and cell-matrix interaction. When DCC gene missed or muted, DCC proteins dyspoiesis, cell growth and differentiation will be chaotic and evolve to malignant tumor.This article discuss the effect of DCC gene in colorectal cancer and its changes after chemotherapy in four directions.Empirical methodPart I The expression of DCC gene in colorectal cancer using immunohistochemistryObjective:To discuss the meaning of DCC gene in colorectal cancer.Method:DCC gene was detected in 120 colorectal cancer tissues using imm unohistochemistry.Resμlts:DCC gene expressed in colon and rectum, and its distribution has no difference (P>0.05). DCC proteins expressed more in well differentiatated tumor than in moderately or low differentiatated tumor (P<0.05). DCC proteins expressed more in stage A and B than in stage C and D, correlated with Dukes stage.They expressed less in metastasis group than in non-metastasis group (P<0.05).Conclusions:DCC gene is correlated with the invasion and metastasis abilities in colorectal cancer, and it can reflect the prognosis of colorectal cancer.lt can be an index to conjecture the biological behaviour of colorectal cancer.Part II Construction of expression vectors of DCC gene and its effects on biological behaviour of colorectal carcinoma cell line SW1116.Objective:To investigate the effects of exogenous wild DCC gene stably transfection on growth of colorectal carcinoma cell line SW1116 in vitro.Method:DCC gene domain was amplified from human normal colon tissue by RT-PCR. At first,a recombinant expression plasmid pcDNA3.1(+)-DCC was constructed. Human colorectal carcinoma cell line SW1116 without DCC gene was transfected with pcDNA3.1-DCC. Cell viability was tested by MTT assay. Western blot and immunofluorescence staining were used to determine effects of pcDNA3.1-DCC and expression of CEA in human colorectal carcinoma cell line SW1116 transfected with pcDNA3.1-DCC.Resμlts:DCC gene domain was amplified from human normal colon tissue by RT-PCR. Its amplification production length is 335bp.DCC gene domain PCR production was connected with pMD18-T vector,and JM109 competent cell was transfected with it. Positive recombinant plasmid colonies were identified by enzymetomy. Destination strap showed at 335bp indicating that pMD18-T-DCC recombinant plasmid was in the colony. The insertion element in this plasmid was tested by DNA sequencing.The resμlt was completely correct after compare the test resμlts with the destination gene. PCDNA3.1(+)-DCC recombinant plasmid construction and amplification: PCDNA3.1(+)-DCC plasmid was double enzymetomy, and its production was electrophoresis in agarose gel, than the vector element was retrieved. DCC gene element was connected with pcDNA3.1(+) vector element, JM109 competent cell was transfected with it. Positive recombinant plasmid colonies were identified by enzymetomy. pcDNA3.1(+)-DCC JM109 strain and pcDNA3.1(+) JM109 were amplified to obtain hypsi-concentration pcDNA3.1(+)-DCC plasmid (1.55μg/μl) and pcDNA3.1(+) plasmid (1.12μg/μl). pcDNA3.1(+)-DCC plasmid suppressed colorectal carcinoma cell line SW1116 amplification:cell line SW1116 was transfected with pcDNA3.1(+)-DCC plasmid and pcDNA3.1(+) plasmid, cellμla digestivum at 1,2,3,4,5,6 days after transfection, and count cells to draw cell growth curve. The popμlation of cells transfected with pcDNA3.1(+)-DCC plasmid was lower than those with pcDNA3.1(+)-DCC plasmid and normal cells (P<0.05) at 3～6 days after transfection, and the proliferation rate of cells transfected with pcDNA3.1(+)-DCC plasmid was lower than those with pcDNA3.1(+) plasmid and normal cells. Cell viability was tested by MTT assay at 1,2,3,4,5,6 days. Cell line SW1116 transfected with pcDNA3.1(+)-DCC plasmid total viability was lower than normal cells (P<0.05) at 2～6 days after transfection. Cell line SW1116 transfected with pcDNA3.1(+)-DCC plasmid total viability was lower than those with pcDNA3.1(+) plasmid (P<0.05) at 2,4,5,6 days after transfection. This resμlts showed that transfected DCC gene coμld suppress colorectal carcinoma cell line SW1116 amplification. Transfected pcDNA3.1(+)-DCC plasmid suppressed colorectal carcinoma cell line SW1116 to express CEA:cell line SW1116 was transfected with pcDNA3.1(+)-DCC plasmid and pcDNA3.1(+) plasmid, than the cells was fixed after 96 hours after transfection, and CEA expression was detected by immunofluorescence staining. CEA was flavo-green colour after immunofluorescence staining under uviol lamp, the population of flavo-green colour cells transfected with pcDNA3.1(+)-DCC plasmid and the fluorescent intensity of these cells were lower than those with pcDNA3.1(+) plasmid and normal control cells. This resμlt showed that transfected DCC gene coμld make CEA expression of cell line SW1116 down regμlation.Conclusions:DCC gene domain was cloned and amplified from human normal colon tissue. At first, a recombinant expression plasmid pcDNA3.1(+)-DCC was constructed. MTT assay results show that pcDNA3.1(+)-DCC plasmid transfected in cell line SW1116 can suppress colorectal carcinoma cell line SW1116 amplification. Western blot and immunofluorescence staining show that pcDNA3.1-DCC plasmid suppress the cell proliferation rate and expression of CEA in human colorectal carcinoma cell line SW1116 transfected with pcDNA3.1-DCC. Transfected DCC gene can suppress the cell proliferation and make CEA expression of cell line SW1116 down regulation to weaken its infiltration and metastasis abilities.Part III The clinical meaning of DCC gene promoter methylation in colorectal cancerObjective:To discuss the clinical meaning of DCC gene promoter methylation in colorectal cancer genesis and development.Method:DCC gene was detected by RT-PCR, and DCC gene promoter methylation was detected by MSP.Resμlts:DCC gene promoter methylation frequency had no relation with depth of infiltration (P>0.05) in colorectal cancer tissues; it correlated with tumor differentiation, DCC gene promoter methylation frequency was higher in low-differentiation or non-differentiation groups than high-differentiation or middle-differentiation groups (P<0.05); it correlated with tumor clinical stages, DCC gene promoter methylation frequency was higher in stage C and D than stage A and B(P<0.05).Conclusions:DCC gene promoter methylation and DCC proteins are different in normal tissues,tissues nearby and primary cancer.They may have relationship with colorectal cancer genesis and development, and can be an index of biological behaviour of colorectal cancer.Part IV The changes of DCC gene in colorectal carcinoma cell line SW1116 after chemotherapy.Objective:To discuss the effects of DCC gene in colorectal carcinoma cell line SW1116 after chemotherapy.Method:DCC gene transcription and protein expression were detected by RT-PCR and Western blot after chemotherapy.Resμlts:The observation from gene transcription level and protein expression showed that DCC gene transcription and protein expression were advanced after used cisplatin and 5-fluorouracil in colorectal carcinoma cell line SW1116. DCC protein expression was up-regμiation after 6 hours, obvious after 12 hours, reaching the highest,and the cell growth was lower, reaching the lowest after 24 hours. Conclusions:Missing or low expression of DCC gene may play a key role in the colorectal cancer genesis and development, especially in its infiltration and metastasis course. commonly used chemotherapeutics has anticancer effect throμgh increacing DCC proteins.It can be a valid target gene in colorectal cancer gene therapy.