The Role And Mechanism Of FKBP51 In The Development Of Colorectal Cancer

Objective:Colorectal cancer(CRC)is one of the malignant tumors of the digestive tract.Its incidence ranks third in the world and is the fourth leading cause of cancer-related deaths.CRC is often insidious,and peripheral invasion and distant metastasis have occurred at the time of discovery.Even after therapeutic surgery,40-50%of patients are still prone to recurrence and metastasis.Finding new CRC development mechanisms and therapeutic targets is of great significance for predicting the onset of CRC,improving the level of treatment,improving prognosis,and reducing patient mortality.FK506 Binding Protein 51(FKBP51)is a very important member of many tumor-related genes.In recent years,it has been widely studied in many tumor-related fields(such as melanoma,glioma,and leukemia).FKBP51 is highly expressed in tumor cells such as oral squamous cell carcinoma,prostate cancer,thyroid cancer,and lymphoma.However,the research on FKBP51 in CRC is very limited,and it is still unclear whether the role of FKBP51 in CRC promotes or suppresses cancer.Epidemiological studies have shown that chronic persistent inflammation not only plays a key role in the occurrence and progression of many cancers,but also contributes to the invasion and metastasis of CRC.The nuclear factor kappa-light-chain-enhancer of activated B cells(NF-?B)pathway is a very important inflammatory pathway in the body.NF-?B is an important nuclear transcription factor in the cell.The normal NF-?B signaling pathway regulates various biological processes.It is involved in the body's inflammatory response,immune response,cell apoptosis and stress response.Some studies have shown that FKBP51 can activate NF-?B by regulating IKK to promote cancer.Therefore,this experiment uses clinical specimens,in vitro experiments and in vivo experiments to study and explore the role of FKBP51 in the development of CRC and related mechanisms of action,and whether FK506 can effectively control the development of CRC,hoping to provide a new theoretical basis for the treatment of CRC And therapeutic targets.Methods:1.From 2014 to 2015,a total of 96 patients with primary colorectal cancer undergoing surgical resection without neoadjuvant treatment were collected from Shengjing Hospital,Affiliated to China Medical University.The clinical tissue specimens consisted of surgical resection of matched primary colorectal cancer and matched adjacent normal colorectal tissue.Use q RT-PCR,Western blot,Immunohistochemical staining,HE staining and other methods to observe the expression of FKBP51,NF-?B and related factors.Use COX analysis and survival curve to analyze the relationship between the expression of FKBP51 and the prognosis of CRC patients;2.Cultivate colorectal cancer cell lines and observe the expression of FKBP51 in colorectal cancer cells.Using silencing and lentivirus overexpression of FKBP51,observe the effect on cell proliferation,migration and invasion and other biological behaviors,and check the expression changes of FKBP51 on tumor-related inflammation factors such as NF-?B,MMP-2 and MMP-9.Use flow cytometry to observe the effect of FKBP51 on the apoptosis of intestinal cancer cells.The cells were treated with FKBP51inhibitor FK506 to observe the expression changes of FKBP51 and its effect on cell proliferation.3.Inject SW620 colon cancer cells into BALB/c nude mice to make a nude mouse tumor model.The mice were treated with different concentrations of FK506 solvent.After the treatment,the tumor tissues of each group of mice were extracted for measurement and weighing,and then the FKBP51,NF-?B and other indicators in the tumor were subjected to detection such as q RT-PCR,Western blot and Immunohistochemical staining to study and analyze the effect of FK506 on the expression of FKBP51 and NF-?B in the body,and whether FK506 has a therapeutic effect on colorectal cancer.Results:1.Among 96 pairs of colorectal cancer tumors and adjacent normal tissues,the m RNA and protein levels of FKBP51 in 77 tumor tissues were significantly higher than normal tissues(p<0.01).In addition,Western blot detection also found that among 77specimens of the High-FKBP51 group,there were 37 tumor tissues in addition to a main band of FKBP51 at about 51 k Da,and a variant band near 65 k Da;and 19 Among the Low-FKBP51 specimens,only 3 cases had isoforms in the tumor tissues;FKBP51 in the High-FKBP51 group was mainly expressed in the malignant glandular epithelial cells of the mucosal layer of colorectal cancer,with nuclear expression mainly.FKBP51 was also strongly expressed in smooth muscle cells in the muscularis mucosa and muscularis propria of colorectal cancer.In the corresponding distal normal colon tissue,FKBP51was expressed in the nucleus of glandular epithelial cells and mesenchymal cells at a moderate to high level;in 19 Low-FKBP51 specimens,the expression of FKBP51 in the tumor was low,and it was only present in a few nuclei.The expression was even lower than that of normal glands next to the tumor.Smooth muscle cells can be seen with low-to-medium FKBP51 signals.The corresponding FKBP51 in normal tissues adjacent to tumors was mainly expressed in glandular cell nuclei and mesenchymal cells,and the signal was weak,which was significantly lower than that in normal tissues adjacent to tumors in the High-FKBP51 group.Comparing the expression of FKBP51 in colorectal cancer,lymph node metastasis and liver metastasis of the same origin,it was found that FKBP51 was expressed more strongly in tissues farther from the original site.NF-?B,MMP-2 and MMP-9 in High-FKBP51 group tumors were significantly higher than normal tissues(p<0.01).COX analysis and survival analysis found that the prognosis of patients in the High-FKBP51 group was worse,and FKBP51 can be used as an independent prognostic factor for CRC.2.Among the three cell lines of NCM460,SW480 and SW620,SW620 had the strongest expression of FKBP51,MMP-2 and MMP-9 had the highest expression,and NF-?B had the largest activation degree.After down-regulating and up-regulating FKBP51,the expression trends of NF-?B,MMP-2,MMP-9 and IKK were consistent with FKBP51,and the expression trends of p-IKK?and I?B?were opposite to FKBP51.Si FKBP51reduced the viability of SW620 cells by 74.5%,71.2%and 70.2%compared with the NC group at 24 h,48 h and 72 h,respectively.The healing of cell scratches at 24 h was only half that of the NC group(p<0.01).In the Transwell invasion experiment,the number of cells in the si FKBP51 group was 20.4±4.6(units),which was significantly lower than that of the NC group,43.3±8.7(units)(p<0.05).The migration experiment showed that the number of cells in the si FKBP51 group was 17.9±6.2(units),which was significantly lower than the NC group 39.5±6.3(pieces)(p<0.05).When the expression of FKBP51 was up-regulated,oe FKBP51 increased the viability of SW620 cells,reaching 1.46 times,1.52 times and 1.61 times that of the PC group at 24 h,48 h and 72h,respectively.The results of the scratch test suggested that up-regulation of FKBP51promoted cell migration,which showed a difference from the PC group starting at 6 h.At24 h,the average migration rate of the PC group was 32.47%,while that of the oe FKBP51 group was 55.32%.Transwell experiment oe FKBP51 group 24 h cell migration and invasion numbers were 79.3±12.9(units)and 76.6±8.9(units),respectively,PC group were 43.3±8.7(units)and 37.8±.9.2(units),oe FKBP51 group was significantly higher In the PC group(p<0.05).The high expression of FKBP51 can inhibit the expression of apoptosis factor BAX and reduce the rate of apoptosis.The expression of FKBP51 was regulated by FK506,but not regulated by NF-?B agonist TNF-?and inhibitor PDTC.3.Injection of different doses of FK506 to tumor-forming nude mice can significantly reduce the tumor volume.The average tumor size in each group was 657.93 mm3 in the control group,441.38 mm~3 in the 1 mg/kg group,and 297.41 mm~3 in the 3 mg/kg group,5 mg/kg group was 257.47 mm~3(n=6,p<0.05).FK506 significantly weakened the expression of Ki67 and reduced the degree of malignancy.After the mice were treated with FK506,the expressions of FKBP51,NF-?B,MMP-2 and MMP-9 in the tumor were significantly reduced(p<0.05).At the same time,Tunel staining showed that FK506 can promote the apoptosis of colon cancer tumor cells,and the degree of apoptosis was positively correlated with the dose of FK506.Conclusion:1.The high expression of FKBP51 in CRC tumors was related to the malignancy of tumors.FKBP51 was expected to be an effective reference for the evaluation of the prognosis and survival rate of CRC patients.2.The expression intensity of FKBP51 was positively correlated with the malignancy of colorectal cancer cells;in colorectal cancer cells,FKBP51 was the upstream of NF-?B,regulating the expression of NF-?B and related factors,and affecting the proliferation,invasion and migration of tumor cells.It also had the effect of inhibiting tumor cell apoptosis;FK506 can regulate the expression of FKBP51 and NF-?B in cell lines,and was expected to be used as a treatment for colorectal cancer.3.In the body,FKBP51 can still regulate the changes of tumor-related factors by regulating the expression of NF-?B;FK506 inhibited the growth and malignancy of colorectal cancer tumors by down-regulating the expression of FKBP51,and promoted apoptosis of colorectal cancer tumors;FKBP51 was expected to be a new target in the treatment of colorectal cancer.