| Colorectal Carcinoma(CRC)is a common malignant tumor of the digestive system.The patient usually visits with diarrhea,abdominal pain,bloody stools,and lower abdominal mass.At this time,the disease has progressed to the middle and late stages,and the patient's quality of life is often low,and the prognosis is mostly poor.Early diagnosis and early treatment will greatly improve the patient's prognosis.With the improvement of people's living standards and the growing pursuit of health,it is urgent to find some new methods for diagnosis and treatment of malignant tumor diseases.In recent years,many scholars have been working hard to find molecular markers of colorectal carcinoma.Cyclophilin A(CypA),originally thought to be an intracellular protein with catalytic and chaperone activity,plays a role in protein folding and transport.With further research,it is found that CypA protein is differentially expressed in tumor diseases and participates in many malignant biological behaviors of tumors,but its role in colorectal carcinoma is still unclear.At the same time,its role in the molecular mechanism of colorectal carcinoma has not been confirmed.Based on the backgrounds above,we propose following scientific questions : Is there a differential expression of CypA in colorectal tumor tissues and adjacent tissues? What is the effect of CypA on the biological behavior of colorectal malignancies? What is the role of CypA in the mechanisms of colorectal carcinoma? In order to answer the questions above,this study uses colorectal malignant tumor as a model,CypA as the core,from the clinical tumor tissue level,in vivo,in vitro cell level,gene molecular level research,We conducted the following study:Chapter 1 Expression and clinical significance of CypA in colorectal carcinoma tissuesA large number of literatures have found that CypA is highly expressed in a variety of tumor tissues in order to investigate whether CypA is differentially expressed in colorectal tumor tissues and adjacent tissues.In this study,Western-Blot was used to detect the relative expression of CypA in paired colorectal malignant tumors and adjacent tissues.The results showed that the expression of CypA in colorectal tumor tissues was higher than that in adjacent tissues(p<0.05).Then,the content of CypA in the serum of the healthy subjects(60 cases)and colorectal malignant tumors(70 cases)by enzyme-linked immunosorbent assay(ELISA)were applied.The results showed that the serum CypA level in patients with colorectal carcinoma was higher than that in controls(p<0.05).Finally,immunohistochemistry was used to observe the expression of CypA in pathological sections of 135 patients with colorectal carcinoma.The results showed that most of the CypA were highly expressed in carcinoma tissues.The high expression of CypA was statistically different from the TNM staging of the tumor and whether it invaded the surrounding lymph nodes,blood vessels and nerves(p<0.05).The high expression of CypA was not statistically different from the patient's gender,age,tumor location,tumor differentiation,and tumor diameter(p>0.05).Conclusion: CypA is highly expressed in tumor tissues.CypA is highly expressed in serum of tumor patients.Chapter 2 The effect of CypA on the biological behavior of colorectal carcinoma cells HT29 and SW620 in vitroPrevious experiments have demonstrated the high expression of CypA in colorectal tumor tissues in order to investigate whether CypA has an effect on the biological behavior of colorectal tumor cells.In this study,Western-Blot was used to detect the endogenous expression of CypA in normal colorectal mucosal cells and colorectal carcinoma cells.It was found that CypA is highly expressed in colorectal carcinoma cells.Next,the morphology of the cells was observed using fluorescence microscopy.As a result,it was found that cells transfected with the CypA plasmid showed significant green fluorescence by fluorescence microscopy.Western-Blot showed that the expression of fusion protein GFP-CypA was detected in the cells after transfection of CypA plasmid,and the expression of CypA in the cells was decreased after transfection of siRNA,which could be used in subsequent experiments.Next,the proliferation ability of tumor cells was examined by the CCK8 method.The results showed that after overexpression of CypA,the cell proliferation ability increased significantly(p<0.01);After CypA knockdown,the cell proliferation ability was significantly reduced(p<0.01).Then,Transwell migration assay was used to detect the migration ability of tumor cells.The results showed that the cell migration ability was significantly increased after CypA overexpression(p<0.01);After CypA knockdown,the cell migration ability was significantly reduced(p<0.01).Finally,Transwell invasion assay was used to detect the invasive ability of tumor cells.The results showed that the cell invasion ability was significantly increased after CypA overexpression(p<0.01);After CypA knockdown,the cell invasion ability was significantly reduced(p<0.01).Conclusion: CypA can promote tumor cell proliferation,migration and invasion.Chapter 3 The effect of CypA on the proliferation of colorectal carcinoma cell HT29 in vivoIn vitro studies have demonstrated that CypA overexpression can promote tumor cell proliferation in order to study the effect of CypA on colorectal tumor cell proliferation in vivo.In this study,pEGFP-N1 and pEGFP-CypA were injected subcutaneously into nude mice(5 in each group),and the size of the subcutaneous tumor was measured with a vernier caliper every 5 days to estimate the tumor volume.After 25 days,the nude mice were sacrificed,and the tumors of the nude mice were taken out,measured and weighed.The results showed that compared with the pEGFP-N1 group,the volume of xenografts in the pEGFP-CypA group was increased and the weight was increased(p<0.05).At the same time,the growth curve of nude mice xenografts was observed.Compared with pEGFP-N1 group,the growth rate of pEGFP-CypA group increased significantly after 15 days(p<0.01).Then,Western-blot was performed to verify the expression of GFP-CypA in the nude mouse tumor.As a result,it was found that the GFP-CypA protein was continuously expressed in the pEGFP-CypA group.Finally,histochemical method was used to determine the expression of CypA and Ki67 in nude mice.The results showed that CypA was more strongly expressed in the pEGFP-CypA group than the pEGFP-N1 group,and Ki67 had a higher proliferation index in the pEGFP-CypA group.Conclusion: Overexpressed CypA promotes proliferation of colorectal tumor cells.X Chapter 4 CypA regulates the proliferation of colorectal carcinoma cells HT29 and SW620In vitro and in vivo studies have confirmed that CypA can promote the proliferation of colorectal tumors in order to study the mechanism of CypA on colorectal tumor proliferation.In this study,10 pairs of TNM III stage colorectal malignant tumors and adjacent tissues were selected,total protein was extracted,protein concentration was analyzed,and proteolytic peptide mixture was prepared and separated by two-dimensional chromatography-mass spectrometry.The results showed that there were 28 proteins differentially expressed in colorectal malignant tumor tissues and adjacent tissues,of which 20 were significantly up-regulated in colorectal malignant tumor tissues,and 8 were down-regulated.Functional analysis of differential proteins in colorectal tumors was then performed by IPA(Ingenuity Pathway Analysis).The results showed that the differential proteins were related to the function of tumor cell movement,proliferation,invasion and migration;the ERK/MAPK pathway in colorectal tumors may be activated.Finally,we used Western-blot analysis of the differential protein CypA,which was screened by two-dimensional chromatography-mass spectrometry,and the related protein in the ERK/MAPK pathway associated with the proliferative function found in IPA analysis.After CypA overexpression,the expression of CD147 protein increased(p<0.05),the expression of p-ERK1/2 protein increased(p<0.05),and the ratio of expression of p-ERK1/2 to ERK1/2 increased(p<0.05).After CypA knockdown,CD147 protein expression decreased(p<0.01),p-ERK1/2 protein expression decreased(p<0.01),and the ratio of p-ERK1/2 to ERK1/2 expression decreased(p<0.01).After overexpression and knockdown of CypA,the expression of ERK1/2 protein,p-p38 and p38 were not significantly changed(p>0.05).Conclusion: CypA promotes the proliferation of colorectal malignant tumors through ERK/MAPK signaling pathway.Through the above experiments,we have reached the following conclusions:1.CypA is highly expressed in tumor tissues and serum of CRC patients.2.Overexpressed CypA can promote tumor cell proliferation,invasion and migration.3.CypA affects the proliferation of colorectal malignancies through the ERK/MAPK signaling pathway. |
PDF Full Text Request