| The data of China's malignant tumor report in 2018 shows:bladder cancer ranks first in terms of the incidence or mortality of urinary tumors.With the rapid development of China's economy,high industrial development will inevitably lead to environmental pollution,and the improvement of medical environment and technology will lead to an increase in life expectancy.The combination of the two causes the incidence of bladder to increase year by year,and the trend is increasingly younger,bladder cancer.The diagnosis and treatment have gradually become an urgent problem in the field of public health.The 2014 edition of the guidelines for the diagnosis and treatment of bladder cancer pointed out that surgery is still the most important treatment,and some immunotherapy and neoadjuvant radiotherapy and chemotherapy have also achieved certain effects in the treatment of bladder cancer.However,it is worth noting that about 15%of patients with bladder cancer have developed distal organ metastases at the time of diagnosis.After radical cystectomy,there are still about 40%of patients with invasive bladder cancer who have distant recurrence and metastasis,but still have 30%.Superficial bladder cancer can be transformed into metastatic or invasive cancer,which leads to poor long-term efficacy and poor prognosis in patients with bladder cancer.Part of the reason for recurrence and even death after treatment failure in patients with malignant tumors is metastasis and invasion..Therefore,it is urgent to clarify the molecular mechanism of invasion and metastasis of bladder cancer,which may also lay the foundation for the search for gene therapy targets and evaluation of patient prognosis.A large number of literature reports indicate that many important pathways are constructed by chemokines and corresponding receptors in the human biological axis signaling pathway.These signaling pathways are closely related to tumor biological behavior.CXCL12,also known as stromal cell-derived factor-1,has been shown to play a role in CXCL12 and its specific receptor CXCR4 during tumor invasion,metastasis,and drug resistance.However,it is worth noting that there is a certain degree of difference between CXCL12 and CXCR4 at the expression level.As the research progressed,another specific receptor CXCR7,which is highly similar to the CXCR4 sequence,was gradually discovered.The data show that CXCR7 expression levels in cervical malignant tumors,renal malignant tumors and other tissues are higher than normal tissues,but there are few data to analyze the expression level of CXCR7 protein in bladder cancerIn this paper,we mainly carried out the biological effects and molecular mechanism of CXCR7 in human bladder urothelial carcinoma:1.To investigate the expression of CXCR7 in bladder urothelial carcinoma and normal bladder mucosa,Western blot and immunization were used in this study.The histochemical staining method was used to analyze the expression level of CXCR7 protein in both,aiming to explore whether CXCR7 plays a key role in the proliferation,differentiation,proliferation and metastasis of bladder urothelial carcinoma;2.Reverse transcription-polymerase chain reaction analysis The expression of CXCR7 mRNA in different urothelial carcinoma cell lines was screened,and the cell line with higher CXCR7 expression level was screened.The CXCR7-shRNA vector with higher CXCR7 silencing efficiency was screened and the CXCR7 expression level was transfected.After the higher cell line,the expression of CXCR7 protein and mRNA was analyzed to verify the interference effect after transfection;the proliferation and invasion ability of bladder urothelial cell line were analyzed to verify the biology of bladder cancer cells.Whether the behavior is regulated by CXCR7 lays the foundation for the molecular mechanism of bladder cancer formation and the study of subsequent treatment.The research content of this topic is divided into the following two parts:Part IExpression of chemokine receptor 7 in human bladder urothelial carcinomaResearch purposes:To investigate the expression of CXCR7 in bladder urothelial carcinoma and normal bladder mucosa,and to analyze the expression level of CXCR7 protein in both by Western blot and immunohistochemical staining,in order to explore the proliferation of bladder urothelial carcinoma Whether CXCR7 plays a key role in differentiation,proliferation and metastasis;whether it can be an effective indicator for prognosis of patients with bladder urothelial carcinoma,whether it can become a new target for targeted therapy in the treatment of patients with bladder urothelial carcinoma.Research contents:1,Immunohistochemical method was used to detect 97 cases of bladder urothelial carcinoma tissue and corresponding normal bladder in the urology department of the First Affiliated Hospital of Jinzhou Medical University from May 2016 to May 2018.CXCR7 expression in mucosal control group,semi-quantitative analysis of its expression results;2,Western blot was used to determine the expression efficiency of CXCR7 protein in tissue samples;3,According to the staining intensity and positive cell number of CXCR7 expressed in bladder urothelial carcinoma,the expression of CXCR7 protein in bladder urothelial carcinoma and normal mucosa was analyzed,and the relationship between pathological grade and clinical stage was analyzed.Research results:1,CXCR7 is strongly stained in the cytoplasm and/or cell membrane of bladder urothelial carcinoma,respectively.There was no staining in normal bladder mucosa,and the difference in staining intensity between the two groups was statistically significant(p<0.05)2,CXCR7 protein was only weakly expressed in 3 cases of normal bladder mucosa(3/97).Compared with normal tissues,the positive expression rate of CXCR7 in bladder urothelial carcinoma was higher.Statistical analysis x2=45.263,p<0.01,the difference was statistically significant;3,The CXCR7 protein was highly expressed,underexpressed or unexpressed in the high-grade group,34 cases and 1 case,respectively,accounting for 2.86%and 97.14%.CXCR7 protein was high,low or non-expressed in 47 cases,15 cases,accounting for 75.81%and 24.19%,the difference was statistically significant(?2=7.126,r=0.694,p<0.05),it indicates that the expression of CXCR7 protein is positively correlated with the pathological grade of bladder urothelial carcinoma;4,CXCR7 protein was highly expressed,underexpressed or not expressed in the non-invasive group,21 cases and 30 cases,respectively,accounting for 41.18%and 58.82%.CXCR7 protein was highly expressed,underexpressed or not expressed in the invasive group,41 cases,5 cases,accounting for 89.13%and 10.87%,the difference was statistically significant(?2=17.596,r=0.715,p<0.05),it indicates that the expression of CXCR7 protein is positively correlated with the clinical stage of bladder urothelial carcinoma;5,Correlation analysis between CXCR7 protein expression in bladder urothelial carcinoma and other clinical indicators revealed gender(p=0.516),age(p=0.307),tumor size(p=0.884)in patients with bladder urothelial carcinoma and Whether smoking(p=0.917)and other aspects were not related to the expression level of CXCR7 protein:6,Western blot analysis showed that the average relative gray value of CXCR7 protein expression in normal mucosal tissues was 0.315±0.104.In bladder urothelial carcinoma,the average relative gray value of CXCR7 protein expression was 0.964±0.276.CXCR7 between the two groups.There was a statistically significant difference in protein expression(t=6.552,p<0.05);7,The mean value of the relative gray value of the non-invasive group was 0.851±0.319,and the mean value of the relative gray value of the infiltrative group was 1.1481±0.254.The F value was 4.008 by one-way analysis of variance,and the relative gray value of CXCR7 between the two groups.There was a statistical difference in the mean(p<0.05);8,The relative gray scale of the CXCR7 protein in the high-level group was 1.253±0.406,and the relative gray-scale mean of the CXCR7 protein in the low-level group was 0.941±0.305.One-way ANOVA showed that the CXCR7 protein was in the high-level group and the low-level group.The difference was statistically significant(F=3.947,p<0.05),indicating that in bladder urothelial carcinoma,the pathological grade increased with the increase of CXCR7 protein expression level.Analysis conclusions:This section uses immunoblotting hybridization and immunohistochemistry to detect the expression of CXCR7 protein in bladder urothelial carcinoma,and concludes that:1,The expression of CXCR7 protein in bladder urothelial carcinoma and normal tissues was significantly higher than the latter.This indicates that CXCR7 protein may be an important factor in promoting the development of bladder cancer.2,The expression of CXCR7 protein is positively correlated with pathological grade and clinical stage of bladder cancer,and CXCR7 may be an important auxiliary judgment indicator.3,When targeted for bladder cancer,CXCR7 may become a new target with far-reaching clinical significance.Part ?Effect of lentivirus-mediated RNAi silencing CXCR7 on biological behavior of human bladder urothelial carcinoma cellsResearch purposes:Reverse transcription-polymerase chain reaction(PCR-PCR)was used to analyze the expression of CXCR7 mRNA in different urothelial carcinoma cell lines.The cell line with higher CXCR7 expression level was screened,and the CXCR7-shRNA vector with higher CXCR7 silencing efficiency was screened and transferred.After staining the cell line with higher CXCR7 expression level,the expression of CXCR7 protein and mRNA was analyzed to verify the interference effect after transfection.The proliferation and invasion ability of bladder urothelial cell line were analyzed.To verify whether the biological behavior of bladder cancer cells is regulated by CXCR7,which lays a foundation for the molecular mechanism of bladder cancer formation and the follow-up treatment.Research contents:1,The growth of human bladder urothelial carcinoma cell lines BIU-87,5637,J82 and EJ-1 was cultured and observed.Reverse transcription-polymerase chain reaction quantitative analysis of CXCR7 mRNA in BIU-87,5637.J82 and EJ-1 cell expression,select cells with higher expression levels as cells for experimental purposes;2,The end-dilution assay was used to determine the virus titer.The three groups of lentiviral vectors CXCR7-shRNA and the negative control group were constructed by fluorescence inverted microscope.3.The optimal infection index of the three groups of lentiviral vector CXCR7-shRNA and the negative control group was determined,and the lentiviral vector CXCR7-shRNA with the best infection index was screened;4.The screened CXCR7-shRNA was transfected into bladder urothelial carcinoma cell line BIU-87,and the silencing efficiency of different lentiviral vectors was determined,and the lentiviral vector CXCR7-shRNA with the highest silencing efficiency was screened.5,The proliferation of BIU-87 cells was analyzed by tetrazolium salt(MTT)colorimetry;6.The amount of BIU-87 cell migration was calculated by Transwell invasion assay,and the inhibition degree of BIU-87 cell invasion after CXCR7 gene silencing was analyzed.7,Cell scratch assay was used to detect the effect of silencing CXCR7 on the migration rate of bladder urothelial carcinoma cell line BIU-87.Research results:1,In bladder urothelial carcinoma cells BIU-87 and J82,CXCR7 mRNA was highly expressed,with higher expression of BIU-87,but lower expression in 5637 and EJ-1 cells,so we BIU-87 cells were selected as the subsequent experimental cell lines;2,The vector CXCR7-shRNA constructed by sequencing confirmed that the vector was inserted correctly,the DNA was recombined successfully,and there was no base mutation.After transfecting the CXCR7-shRNA lentiviral vector into BIU-87,it was found that the cell swelling and rounding or the antennae recovery,the cell detachment suspended in the field of view of the typical CPE phenomenon,accompanied by a large number of green fluorescent expression.The titers of the three groups of lentiviral vector and negative control were:negative control:8.29×108 pTU/ml,CXCR7-shRNA-3:5.1 7×108 pTU/ml;CXCR7-shRNA-2:5.04×108 pTU/ml;CXCR7-shRNA-1:6.57×108 pTU/ml,the virus titer is within the normal range,and subsequent transfection experiments can be performed;3,The expression levels of CXCR7 mRNA in group 1,2,and 3 were significantly lower than those in the control group(group 4)(p<0.05).The transfection group(group 1)significantly inhibited BIU-87 bladder.The expression level of CXCR7 mRNA in urothelial carcinoma cell lines was significantly different fiom that in the transfection group(p<0.05).Therefore,after screening,we selected CXCR7 with the highest lentivirus titer.shRNA-1(6.57×108 pTU/ml)vector was used as a viral vector for subsequent experiments;4,The cells in the experimental group(group A)and the untransfected virus group(group B)were routinely cultured,and the light absorption values were measured at 0,24,48,72,96,and 120 hours,and the growth curves were used to analyze the two groups of BIU-87 cells.Proliferation.The proliferation of cells in group B increased geometrically,and the proliferation rate of group A decreased significantly.The two groups were statistically significant(p<0.05),indicating that lentiviral CXCR7-shRNA-1 can silence CXCR7 gene expression in BIU-87 cells,slowing the proliferation rate of BIU-87 cells;5,After 48 hours of transfection of the lentiviral vector CXCR7-shRNA-1,the number of transmembrane cells in group B was 292.157±19.069,and the number of perforating cells in group A was 103.874±11.253.The two groups were statistically significant(p<0.05).It can inhibit the transmembrane process of 64.45%cells,and the ability of cells to invade distantly is inhibited after silencing the expression of CXCR7 gene in BIU-87;6,After 24 hours of scratching,the cell migration index(MI)of BIU-87 cells was(49.27±9.58)%and(26.14±7.26)%,respectively,statistically significant(p<0.05)After 48h,the MI of the control group and the experimental treatment group were(91.05±17.22)%and(59.3 6±11.29)%,respectively,which were statistically significant(p<0.05).It is suggested that CXCR7-shRNA-1 can decrease the migration index of BIU-87 cells and further reduce the migration ability of bladder urothelial carcinoma cells.Analysis conclusions:1,The expression levels of CXCR7 mRNA in urothelial carcinoma BIU-87,5637,J82 and EJ-1 cells were detected.It was found that CXCR7 mRNA was highly expressed in bladder urothelial carcinoma cells BIU-87 and J82.The expression level was lower in the 5637 and EJ-1 cell lines,and the CXCR7 mRNA was expressed at a higher level in BIU-87 cells,which was used in subsequent experiments2,The CXCR7-shRNA recombinant lentiviral vector was successfully constructed,which can effectively silence the CXCR7 gene.It was found that the recombinant lentivirus CXCR7-shRNA-1 has the highest silencing efficiency and was used in subsequent experiments3,Silencing CXCR7 gene by CXCR7-shRNA-1 can significantly inhibit the expression and proliferation of bladder urothelial carcinoma cell line BIU-87.4,The recombinant lentiviral vector CXCR7-shRNA can significantly inhibit the local invasion and distant metastasis of urothelial carcinoma cell line BIU-87. |
PDF Full Text Request