| Colorectal cancer(CRC)is one of the common malignancy of the digestive tract.Among all types of malignant tumors,it has the third-highest incidence and the second-highest death rates,both worldwide and in China.It has the characteristics of high morbidity and high mortality.Colorectal cancer has no obvious positive signs or only slight symptoms in the early stage,which is easy to be ignored.When the clinical symptoms are obvious,the lesions have developed into the advanced stage,and the opportunity of operation was lost.Although with the improvement of diagnostic methods and means,the number of cases found in the early stage are increasing,the overall survival is still not statisfied.With the progress of research,although the advanced patients have the chance to be treated with radiotherapy and chemotherapy,targeted treatment and immunotherapy,the efficiency is still low.It is important to find the exact targets to guide the individualized treatment.Biliverdin reductase A(biliverdin reductase A,BLVRA)is a conserved soluble NADH dependent enzyme,which is widely exsisted in the cytoplasm of animals and plants.It converts biliverdin to bilirubin in the heme metabolism pathway,which is also an antioxidant.It maintains the dynamic balance of redox reaction in cells,regulates cell growth,participates in cell signal transduction,and promotes the proliferation of tumor cells,invasion and migration.It also inhibits the apoptosis of cells.BLVRA is highly expressed in a varietyf of tumors,but whether it acts as "oncogene" in colorectal cancer remains unclear.Wogonin is a kind of flavonoid extracted from Scutellaria baicalensis Georgi.It has wide antitumor activities that can induce cell cycle arrest,promote cell apoptosis,reduce tumor neovascularization,and can also enhance the sensitivity of tumor cells to chemotherapy drugs.Its antitumor effect is related to multiple signal pathways.Whether the effects were regulated by BLVRA has not been studied yet.In this study,first,the expressions of BLVRA in peripheral blood and tumor tissues of patients with colorectal cancer were detected by ELISA,qRT-PCR and immunohis-tochemistry respectively.Second,the effects of BLVRA knockdown and overexpression on proliferation,migration,invasion,epithelial-mesenchymal transition(EMT)and apoptosis of colorectal cancer cells were observed after cell transfection.Third,whether the effects of BLVRA was regulated by Wnt/p-catenin signaling pathway was also been investigated.Fourth,the effects of Wogonin on the proliferation,migration,invasion,epithelial-mesenchymal transition(EMT)and apoptosis of colon cancer cells were also been researched.Part ?:The expression of BLVRA in colorectal cancer patients and its clinical significanceObjective:To investigate the expression of BLVRA in peripheral blood and tumor and adjacent tissues of colorectal cancer patients,evaluated the relationship with clinicopath-ological parameters and survival.Methods:The study was carried out using samples from 110 patients who were histopathologically diagnosed as colorectal adenocarcinoma cases at the Affiliated Hospital of Yangzhou University from 2015 to 2017.As controls,110 healthy volunteers joined in this study.The tissues and the plasma of peripheral blood were acquired before the first treatment.ELISA,Real-time PCR and Immunohistochemistry(IHC)were applied to test the level of BLVRA in patients and volunteers.Kaplan-Meier was used to draw PFS and OS survival curves of different expression levels of BLVRA,and log rank test was used to compare the cumulative progression free survival and cumulative total survival differences.Cox regression analysis was performed according to age,location,stage,differentiation,lymph node metastasis,distant metastasis and BLVRA expression.Results:The expression of BLVRA in the peripheral blood of patients with colorectal cancer was higher than that of healthy people.It was expressed both in tumor tissue and adjacent tissue,which is significantly higher in tumor tissue(P<0.001).The PFS and OS of patients with high expression of BLVRA were shorter than low expression of BLVRA(P<0.001).The expression level of BLVRA has no connection with age(P=0.881),gender(P=01.43)and the location of the disease(P=0.387).COX univariate analysis showed that stage,differentiation,lymph node metastasis,distan t metastasis and the level of BLVRA were all related to poor prognosis(P<0.001),but age(P=0.594),gender(P=0.903)and location(P=0.649)were not connected with prognosis.COX multivariate analysis showed that stage,degree of differentiation,lymph node metastasis,distant metastasis and BLVRA expression were all related to poor prognosis(P<0.001),but age(P=0.220),gender(P=0.057)and location(P=0.202)are without relationship to prognosis.Conclusions:The expression of BLVRA in the peripheral blood and tumor tissues of colorectal cancer patients was significantly higher than that of the healthy people and the adjacent tissues;the PFS and OS of patients with high expression of BLVRA were significantly shortened,indicating poor prognosis;tumor stage,differentiation,metastasis and expression level of BLVRA were independent factors affecting the poor prognosis of colorectal cancer patients.Part ?:The biological effects of BLVRA in colorectal cancer cellsObjective:To investigate the effects of BLVRA on the proliferation,apoptosis,migration,invasion and epithelial stromal transformation of colon cancer HT-29 and SW620 cells after transfected with BLVRA interference and overex-pressionvectors.Methods:First,the expression of BLVRA mRNA and protein in colon cancer cell lines HT-29,SW620,SW480,HCT116,LoVo and normal intestinal epithelial cells FHC were detected by real-time PCR and Western blot.BLVRA knockdown and overexpression vectors and their negative control vectors were constructed for cell transfection,real-time PCR and Western blot were used to test BLVRA mRNA and protein levels after transfection to confirm that BLVRA knockdown and overexpression cell lines were successfully established.MTT,flow cytometry,Transwell,immunofluorescence and Western blot were performed to gauge cell proliferation,apoptosis rate,expression of apoptotic factors,cell migration and invasion ability,and the changes of epithelial mesenchymal transformation(EMT).Results:The results of Real-time PCR and Western blot showed that the expression level of BLVRA mRNA and protein in HT-29 cells was the highest in the five colon cancer cell lines(P<0.01),and that in SW620 cells was the lowest(P<0.05).HT-29 and SW620 cells were selected for the follow-up experiments.The expression of BLVRA mRNA and protein was decreased(P<0.001),and the expression of BLVRA mRNA and protein was increased in SW620 cells(P<0.001)after transected;MTT results showed that after BLVRA knockdown,the proliferation ability of HT-29 cells was inhibited,and with the time dependent(P<0.001).Flow cytometry showed that the apoptosis rate was increased(P<0.001),western blot revealed that the protein level of Bcl-2 and Survivin were reduced(P<0.05),while Bax and caspase-7 were enhanced(P<0.05).Transwell implied that the number of cell migration and invasion were significantly decreased(P<0.001).The expression of E-cadherin was increased,and vimentin was decreased(P<0.05)with immunofluorescence and western blot(P<0.05).On the other hand,MTT showed that after overexpression of BLVRA,the proliferation ability of SW620 cells was increased,which also with the time dependent(P<0.001).Flow cytometry exhibited that the apoptosis rate was decreased(P<0.001),western blot indicated that the protein levels of Bcl-2 and Survivin were upregulated(P<0.05),while the level of Bax and caspase-7 were downregulated(P<0.05).Transwell displayed that the number of cell migration and invasion were increased significantly(P<0.001).The expression of E-cadherin was reduced,and vimentin was increased(P<0.05)with immunofluorescence,which is consistent with the results of western blot(P<0.05).Conclusions:The expression of BLVRA in colon cancer cell lines was higher than that in normal intestinal epithelial cells;siRNA-BLVRA could inhibit the proliferation,apoptosis,migration and invasion of HT-29 cells,and restrain the process of epithelial mesenchymal transition(EMT),while the overexpression of BLVRA played an opposite role in SW620 cells.Part ?:The potential mechanisms of BLVRA in colon cancer cellsObjective:To discuss whether the effects of BLVRA was regulated by Wnt/?-catenin signal pathway.Methods:Western blot were applied to test the expression of Wnt/?-catenin and its targeted proteins before and after been treated with inhibitor(IWR-1)and activator(CHIR98014).Results:Knockdown of BLVRA downregulated the expression of Wnt,?-catenin and its targeted proteins,such as cyclin D1?C-myc?p-GSK-30?COX-2,while the expression of p-?-catenin and p-PTEN were upregulated.O n the contrary,overexpression of BLVRA promoted the expression of Wnt,?-catenin and its targeted proteins,such as cyclin D1?C-myc?p-GSK-3??COX-2,the expression of p-?-catenin and p-PTEN were restrained.After been treated with activator and inhibitor,the opposite expression of the Wnt/?-catenin targeted protein were observed.Conclusions:siRNA-BLVRA can inhibit Wnt/?-Catenin signal pathway,and it can be stimulated again after treating with activator.Overexpression of BLVRA can activate Wnt/?-Catenin pathway,and it can be inhibited again after treating with inhibitor.All the results indicated that the effects of BLVRA were regulated by Wnt/?-Catenin signal pathway.Part ? The mechanisms of Wogonin inhibiting the colon cancer cells proliferation,migration,invasion and inducing apoptosis via BLVRAObjective:To investigate the effects of Wogonin on the proliferation,migration,invasion,apoptosis and EMT in conlon cancer cells HT-29 and SW620,and to certify whether its effect is regulated by BLVRA.Methods:CRC cell lines HT-29 and SW620 in logarithmic growth phase were used.Wogonin with different concentrations(0,20,40,80,160?g/mL)and 5-FU(12.5?g/mL)were added in the cells.MTT assay was used to test the proliferation of HT-29 and SW620 treated with Wogonin.Flow cytometry was adopted to examine the apoptosis rate of HT-29 and SW620 treated with Wogonin for 24 h.Transwell was applied to detect the migration and invasion of HT-29 and SW620 after Wogonin treatment.The apoptosis related proteins of Bcl-2,BAX and EMT related proteins E-Cadherin,Vimentin,snail were detected with western blot.Results:Wogonin could inhibite the proliferation of HT-29 and SW620 with time and concentration dependence(P<0.01).Wogonin could significantly enhance the apoptosis rate of HT-29 and SW620 cells with concentration dependence(P<0.01).It also decrease the expression of anti-apoptosis protein Bcl-2,increase the expression of BAX(P<0.01).Wogonin restrained the migration and invasion of HT-29 and SW620 in a dose-dependent manner(P<0.01).Western blot showed Wogonin promoted the expression of E-Cadherin,reduced the expressions of Vimentin and snail with concentration dependence(P<0.05).In both HT-29 and SW620 cells,Wogonin inbibited the expression of BLVRA in a dose-dependent manner(P<0.05).Conclusions:Wogonin could obviously inhibit the proliferation,migration and invasion,induce apoptosis in HT-29 and SW620 cells,and the mechanism may be related to the expression of BLVRA. |
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