| Objective: Worldwide,colorectal cancer(colorectal cancer,CRC)incidence is in the third place of malignant tumours,and CRC is the fourth member in death caused by malignant tumors.There are about one million new cases each year,and the 5-year survival rate is less than 65%.CRC is found frequently in gastrointestinal malignant tumors,which incidence is still on the rise.Though a lot of progress has promoted significantly prognosis of CRC,but metastasis is still a common problem of CRC after surgery and neoadjuvant therapy,more and more research have focused on finding new molecules to regulate progression,development and metastasis of CRC.COL1A2 codes type I collagen,which is the most abundant and the most widely expressed protein in human,and it plays role on the structure skeleton in matrix.Type I collagen constitutes the interstitial part in most solid tumors,this special extracellular matrix has played an important role in tumor growth,function,and has excessive methylation in a wide variety of tumors.Specific methylation PCR measure in the colorectal cancer shows that the COL1A2 happened excessive methylation,when methylation inhibitors added in colorectal cancer cell lines,COL1A2 increases obviously.COL1A2 methylation also appears in the head and neck cancer,bladder cancer,and melanoma,and it induces cell proliferation and invasive in bladder cancer.Proliferation,invasion,metastasis and apoptosis of CRC is a multi-step process caused by polygene,which also involves a variety of signal transduction pathways.COL1A2 was thought to play an important role in progression of tumor,but it is still not clear that if it regulate development of CRC and relative molecular mechanism.NF-κB signaling pathway is essential for cell migration,which plays an important role in conduction extracellular signals to the nuclear transcription factors,also can improve the ability of transcription.There was no report to prove that NF-κB signaling pathway took part in the regulation of COL1A2 in proliferation,migration and invasion of CRC.This study reveals function of COL1A2 in cell proliferation,migration,and invasion mechanism of CRC through finding relationship of COL1A2 and the clinical pathologic correlation,cell function change,and the regulation of the downstream pathway.Methods: 1、We collected 88 pairs of CRC tissues and adjacent normal tissues from the patients who were performed colorectal cancer radical operation in general surgery department in Shengjing Affiliated Hospital of China Medical University between Jan 2015 to July 2016.Normal tissue adjacent to cancer refers to that more than 5 cm from the tumor edge(pathologically proven residual tumor cells is negative for cut edge).Fast for organization,tissues were washed by 1 x PBS fluid flush with precooling,placed in cryopreserved tubes without RNA enzymes,save to-80 ℃ in the fridge waiting for inspection.Western blot and Real Time-PCR detected COL1A2 of CRC tissues and adjacent normal tissues.COL1A2 expression level was analyzed with pathological staging and prognosis.We followed up patients until they died or 48 months after surgery.Fourty cases of colorectal cancer and adjacent pathological section were resected from January 2014 to December 2014 in Shengjing hospital.2、The lentiviral vector containing COL1A2 DNA sequence was constructed in SW480 and SW620 cells,fluorescence photography and PCR detection proved successful transfection.SW480 and SW620 cells were divided into negative control group,COL1A2-over group,and CCK 8,wound healing,Transwell were used to measure the effects of cell proliferation,migration and invasion of each group.On 3rd day,intergroup differences were observed.The values of SW 480 cell NC group and COL1A2 group OD were 1.119±0.090,0.813±0.031,and that of SW 620 cell NC group and COL1A2 group were 0.989±0.034,0.613±0.008,p<0.05.The significant difference was observed on 4th day.The values of SW 480 cell NC group and COL1A2 group OD were 1.637±0.045,0.947±0.061,SW 620 cell NC group and COL1A2 group OD were 1.388±0.063,0.992±0.018,respectively.Both cells indicated that the proliferation capacity of COL1A2 group was significantly lower than that of NC group(P < 0.01).Transwell migration and invasion experiments showed that the number of COL1A2 cells was reduced by 24 hours and the number of invasive cells was decreased.Transwell migration showed that the mean number of cells in SW480 cell NC group and COL1A2 group was 183±8,38±6,and the number of cells in SW 620 cell was 218±11,112±8 respectively.Transwell invasion showed that The mean number of cells in SW480 cell NC and COL1A2 group was 166±7,33±7,and the mean number of cells in SW620 cell was 206±13,105±11.3、Total RNA from SW480 cells infected with lentivirus expressing COL1A2 or NC was extracted using Trizol reagent.Then,A microarray-based mRNA expression profile screening was performed.IPA analysis was performed to get different genes,and western blot tested some downstream molecules which may be regulated by COL1A2,CXCL1,EGFR,BIRC3,CDK6,IGF1 R,SNAI2,ETS1,TNFAIP3,IRS1 and CCND1.Relative molecules on NF-κB signaling pathway were also been tested by western blot,such as p65,p-p65,IKKβ,p-IKK.Results:1.The immunohistochemical test indicated that the COL1A2 was low in the carcinoma tissue,mainly expressed in the cytoplasm between the tissues of the cancer,but rarely expressed in the cytoplasm between the tissues of the cancer.The positive staining rate respectively was 77.5 %,27.5 %,P < 0.01.PCR suggested that the relative expression of COL1A2 mRNA in carcinoma and paracentesis was 0.64±0.54 and 1.44±0.73,P < 0.01,and was positively correlated with pathological staging,differentiation and lymph node metastasis.COL1A2 mRNA of high/middle differentiation group compared with low differentiation group,was 0.738±0.595,0.378±0.214,P < 0.01,The COL1A2 mRNA levels T1 + T2 was 1.029±0.575,T3+T4 was 0.398±0.345,(P < 0.01).Compared with the lymphatic metastasis group,the COL1A2 mRNA levels were relatively high(0.851 + 0.627 verus 0.366 + 0.177)(P < 0.01).It has nothing to do with age,sex,tumor size.The risk regression analysis of COX suggested that COL1A2 expression level and lymph node metastasis as independent factors could influence survival of CRC patients.2、The lentiviral vector containing COL1A2 DNA sequence was constructed,fluorescence photography appeared green fluorescent and PCR detection proved COL1A2 expression increased compared with the negative control group in SW480 and SW620 cells(p < 0.05).CCK8 assay showed that COL1A2 over-expression group had lower OD value than negative control group both in SW480 and SW620 cells.Wound healing experiment showed migration ability decreased in 24 hours in COL1A2 over-expression group(p < 0.05),transwell invasion experiment also showed COL1A2 over-expression group had less number of cells in 24 hours(p < 0.05).3、Microarray analysis showed 1592 genes were significantly differentially expressed(p < 0.05 and absolute fold-change [FC Absolute] > 3),in which 927 mRNAs were downregulated,whereas 665 mRNAs were upregulated.Expression of CXCL1,EGFR,BIRC3,CDK6,IGF1 R,SNAI2,ETS1,TNFAIP3,and CCND1 was significantly downregulated,while IRS1 was significantly upregulated in COL1A2 overexpression group compared with NC group in SW480 cells.Change in protein level of EGFR,BIRC3,IGF1 R,SNAI2,ETS1,TNFAIP3 and IRS1 were same to changes of mRNA level,while changes in protein level of CXCL1,CDK6,CCND1 were not coincided with that in mRNA level.The protein levels of p65,IKKβshowed no difference in COL1A2 over-expression group compare to the negative control group,while p-p65,,p-IKK showed decreased expression(p < 0.05).Conclusions: 1.COL1A2 has significantly lower expression in colorectal cancer tissues,and was relevant to the pathological feature,especially to the stage,differentiation degree and lymph node metastasis(p < 0.05).2、Over-expression of COL1A2 can cause decrease in cell proliferation,migration and invasion of SW480 and SW620 cells.3、COL1A2 regulated CXCL1,EGFR,BIRC3,CDK6,IGF1 R,SNAI2,ETS1,TNFAIP3,IRS1 and CCND1,COL1A2 may regulate cell function in CRC through NF-κB pathway. |
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