| Renal clear cell carcinoma(RCC)accounts for about 2-3% of all malignant diseases in adult patients,and kidney cancer is the third leading cause of death among urinary tract tumors.Worldwide,it is the 9th most common cancer among female patients and 7th among male patients.As research in recent years has shown that the incidence and mortality of RCC have been rising,and its mortality is usually closely related to the metastatic disease of this disease,and clear cell carcinoma is the most common tissue type of malignant tumors of the kidney.Current research believes that partial nephrectomy or radical nephrectomy is considered to be the best treatment for clear cell carcinoma of the kidney,but after the removal of the primary tumor,the recurrence rate of RCC patients is about 20-30% If a tumor metastasizes,its five-year survival rate is still less than 10%.Sunitinib has been used as the main treatment for metastatic clear cell renal cell carcinoma(cc RCC)because it can effect by inhibiting tumor growth and angiogenesis.However,due to the development of sunitinib resistance,most cc RCC tumors may still re-grow,and the detailed mechanism remains to be further studied.From the currently reported research on factors related to tumor angiogenesis,the Angiopoietin family includes Angiopoietin-1 and Angiopoietin-2(ANGPT-1 and-2).In the experimental model of stroke,estradiol can regulate the expression of ANGPT-1 through the estrogen receptor ?(ER?),but not the expression of ANGPT-2.To date,no research pathways have been found on regulating ANGPT-2 and E2 / ER signaling.Therefore,this study explores how estrogen receptor ?(ER?)upregulates ANGPT-2 in RCC cells,and how ANGPT-2 with increased ER? promotes the formation of HUVEC tubes and reduces the sensitivity of sunitinib.The research mechanism shows that ER? can function through the transcriptional regulation of the cytokine ANGPT-2 in cc RCC cells.At the same time,it was found that the upregulation of ANGPT-2 in RCC cells can increase the phosphorylation of Tie-2 in HUVEC cells,thereby promoting angiogenesis and increasing sunitinib sensitivity to vascular endothelial cells.Furthermore,using endothelial cell tube formation and aortic ring assay,the preclinical mouse RCC model also confirmed this finding.We use the FDA approved anti-estrogen fulvestrant in combination with sunitinib.Through this new ER? /ANGPT-2/Tie-2 signaling pathway,it may help to develop a new combination of sunitinib Therapy to better inhibit cc RCC progression.Part One ER? promotes angiogenesis and sunitinib inhibits renal cell carcinoma angiogenesisObjective: To explore the effect of ER? on HUVEC angiogenesis and the effect of sunitinib on HUVEC phenotype after co-culture with RCC and HUVEC.Methods: 1.Detection of overexpressed or knocked down ER? protein expression in two different renal tumor cells.2.Co-culture of renal tumor cells and vascular endothelial cells,after overexpression or knockdown of ER? in renal tumor cells,through the vascular tube formation experiment,after detecting different stimuli,we observe the phenotype of vascular endothelial cells.3.After intervention with different concentrations of sunitinib in vascular endothelial cells,we observe the changes in vascular endothelial cells angiogenic phenotype,and select the appropriate concentration of sunitinib for subsequent experiments.4.Co-culture of renal tumor cells and vascular endothelial cells,after overexpression or knockdown of ER? in renal tumor cells,add sunitinib drug to vascular endothelial cells,through the vascular tube test,detect after giving different stimuli,observe changes in phenotype of vascular endothelial cells.5.Arterial ring experiment,when co-culture renal tumor cells and vascular endothelial cells,after overexpression or knockdown of ER? in renal tumor cells,collect the medium after co-culture of the two cells,without adding or adding sunitinib drugs,observe the changes of vascular synapse phenotype in vascular arterial ring.6.Query the database to count the survival rate of ER? in renal tumor patients and the survival period of male and female patients.Results: 1.Overexpression or knockdown of ER? protein expression in two different renal tumor cells.In the current study,the content of ER? in kidney tumor cells A498 was low,while the content of ER? in kidney tumor cells 786-O was high.Overexpression of ER? in kidney tumor cells A498 and knockdown of ER? in kidney tumor cells showed that changed in ER? protein;2.Vascular tube formation experiment,co-cultured of renal tumor cells and vascular endothelial cells,after giving renal tumor cells overexpression or knocking down ER?,observe the phenotype of vascular endothelial cells.After transfecting the overexpressed or knocked-down ER? plasmid into 293 T tool cells,transfected renal tumor cells and co-cultured with vascular endothelial cells for 72 hours.After taking the co-culture medium and fresh medium for 1: 1 mixing,do vascularization experiments.Compared with the control group,the number of vascular tubes in the ER? overexpression group increased(P<0.01),while the number of vascular tubes in the ER? knockdown group decreased,indicating that ER? has a significant effect on angiogenesis of vascular endothelial cells(P<0.01);3.Effect of different concentrations of sunitinib on vascular tube formation of vascular endothelial cells.When the concentration of sunitinib drug was selected as the preliminary experiment,the concentration of sunitinib drug was 6 concentrations from DMSO,0.5?M,1?M,2?M,5?M to 10?M,when the drug concentration was 1?M,2?M,5?M and at these four concentrations,the tube formation test was inhibited by the drug at 10 ?M,and no angiogenesis was observed(P>0.05).When the drug concentration was 0.5 ?M and 1 ?M,the tube formation of vascular endothelial cells was statistically different (P<0.05)Therefore,in the follow-up experiment,we chose the sunitinib drug concentration of 1?M;4.Vascular tube formation experiment,co-culture of renal tumor cells and vascular endothelial cells,after overexpression or knockdown of ER? in renal tumor cells,at the same time,add sunitinib drug to vascular endothelial cells to observe the phenotype of vascular endothelial cells the change.After transfecting the overexpressed or knocked-down ER? plasmid into 293 T tool cells,transfected renal tumor cells and co-cultured with vascular endothelial cells for 72 hours.After taking the co-culture medium and fresh medium for 1: 1 mixing,add 1 ?M sunitinib to the pre-mixed medium.Compared with the control group,the number of vascular tubes in the ER? overexpression group increased(P<0.001),while the number of vascular tubes in the ER? knockdown group decreased,indicating that ER? has a significant effect on angiogenesis of vascular endothelial cells(P<0.05);5.Arterial ring experiment: Co-culture of renal tumor cells and vascular endothelial cells,after overexpression or knockdown of ER? in renal tumor cells,collect the medium after co-culture of the two cells,without adding or adding sunitinib drugs,observe the changes of vascular synapse phenotype in vascular arterial ring.After transfecting the overexpressed or knocked-down ER? plasmid into 293 T tool cells,transfected renal tumor cells and co-cultured with vascular endothelial cells for 72 hours.After taking the co-culture medium and fresh medium for 1: 1 mixing,No or 20 n M sunitinib drug was added to the pre-mixed medium for the arterial ring experiment,and the number of synapses formed by blood vessels was observed within 7-9 days.Compared with the control group,the number of vascular tubes increased in the overexpressed ER? group without the sunitinib drug(P<0.01),while the number of vascular tubes decreased in the ER? group without the sunitinib drug,indicating ER? has a significant effect on the angiogenesis of vascular endothelial cells(P<0.05);compared with the control group,the number of vascularization in the ER? group with 1 ?M of sunitinib over-expressing group increased(P<0.001),while adding 1?M sunitinib drug knocked down the number of vascularization in the ER? group,indicating that ER? has a significant effect on angiogenesis of vascular endothelial cells(P<0.01);6.Query the database to count the survival rate of ER? in renal tumor patients and the survival period of male and female patients.With the extension of follow-up time,compared with patients with low expression of ER?,the survival rate of patients with high expression of ER? in all renal tumor patients is poor,the difference is statistically significant(P<0.001);for male patients,male patients with high expression of ER? have a poor survival rate in all renal tumor patients,and the difference is statistically significant(P = 0.007);compared with female patients with low expression of ER?,females with high expression of ER? the survival rate of patients in all patients with renal tumors was poor,and the difference was statistically significant(P= 0.012).Therefore,the above data can indicate that in the survival period of patients with renal tumors,patients with high expression of ER? have lower survival rate and poorer prognosis than those with low expression of ER?.Conclusions: 1.ER?can promote angiogenesis and increase resistance to sunitinib drugs in renal tumor cells.2.Research through the database shows that in the survival period of patients with renal tumors,patients with high expression of ER? have lower survival rate and poorer prognosis than those with low expression of ER?.Part Two The mechanism of ER? targeted regulation of angiopoietin-2/Tie-2 signaling pathway in renal cell carcinoma angiogenesisObjective: To explore the changes of Angiopoietin-2/Tie-2 signaling pathway and angiogenesis after co-culture of RCC and HUVEC.Methods: 1.Detect the protein expression of two different sequences of knockdown ER? in renal tumor cells 786-O,at the same time,transfect the two knockdown ER? into 786-O cells and co-culture with vascular endothelial cells,add or add 1?M sunitinib drug and observe the change of phenotype.2.In renal tumor cells,after overexpression or knockdown of ER? in renal tumor cells,q RT-PCR was used to detect genes related to meaningful angiogenesis.3.Caki-1 knocked down ER? in the third kidney tumor cell line,and used q RT-PCR to detect the downstream gene changes,and at the same time verified the protein changes in the kidney tumor cells knock down by ER?.4.Screen out the downstream genes HGF and ANGPT-2,knock them down and transfect them into kidney tumor cells,and co-culture with vascular endothelial cells,observe the change of cell phenotype,through the database,find ANGPT-2 in kidney tumor.The effect of intracellular expression on the survival rate of patients.5.Detection of ER? and ANGPT-2 protein expression in A498 and 786-O cells during overexpression and knockdown.6.Transfection using ER? and ANGPT-2 knockdown plasmids into A498 cells and ER? knockdown and ANGPT-2 plasmids into 786-O cells,respectively,in renal tumor cells Changes in protein in and within vascular endothelial cells;phenotypic changes in vascular endothelial cell were observed after no and added sunitinib drugs were added.7.Search the database to study how ER? regulates the downstream gene ANGPT-2 in kidney tumor cells.8.Use q RT-PCR methods to detect the content of A498 and 786-O in ANGPT-1 and ANGPT-2 in kidney tumor cells.The protein content of ANGPT-2 in renal tumor cells 786-O,Caki-1,A498 and HUVEC cel ls was detected.Results: 1.Choose two different sequences of knockdown ER? for protein expression detection and phenotypic changes.The protein expression of two different sequences of knockdown ER? in renal tumor cells 786-O,compared with the control group,the number of vascularization in the ER? group with and without the addition of sunitinib and two different sequences of knockdown was reduced(* P<0.05;** P<0.01),but after two different sequences of knockdown ER? were co-cultured with renal tumor cells and vascular endothelial cells,there was no statistical difference in the number of vascular tubes(P> 0.05).From the perspective of protein expression and the decrease in the number of vascular tubes,the first knocked down ER? sequence was selected for subsequent experiments;2.Search the database and use q RT-PCR to detect genes related to kidney tumors.In kidney tumor 786-O cells,knock down ER?,and use q RT-PCR method to detect the changed genes are ANGPT-2,b FGF,HBEGF,HGF,LEP,VEGFA;in kidney tumor A498 cells,overexpress ER?,use q RT-PCR method to detect the changed genes are ANGPT-2,HGF;3.Knock down ER? in renal tumor cells Caki-1 to detect changes in q RT-PCR and ER?.Caki-1 knocked down ER? in the third kidney tumor cell line,and detected the changes of downstream genes by q RT-PCR.It was found that q RT-PCR of ANGPT-2 and HGF genes changed,and the knockdown of ER? sequence was confirmed in the kidney.Proteins in tumor cells can be reduced;4.Knock down the two downstream genes of HGF and ANGPT-2,observe the phenotypic changes of vascular endothelial cells,and obtain the survival rate of downstream genes in renal tumor patients through the database.Through the above studies,the downstream genes HGF and ANGPT-2 were screened,transfected into renal tumor cells after knockdown,and co-cultured with vascular endothelial cells,and the changes in cell phenotype were observed.It was concluded that compared with the control group,the knockdown of HGF was transfected into renal tumor cells,and there was no statistical difference in the number of angiogenesis(P>0.05);compared with the control group,the knockdown of ANGPT-2 was transfected.When stained into kidney tumor cells,the number of angiogenesis formed was statistically significant(P<0.001).Through database analysis,the high or low expression of ANGPT-2 gene in renal tumor cells has no statistical significance for the survival rate of patients(P=0.26);5.Detection of overexpression or knockdown of ER? in renal tumor cells,ER? and ANGPT-2 protein level changes.In the kidney tumor cell A498 cells,after overexpression of ER?,it can be seen that the protein expression of ER? and ANGPT-2 in kidney tumor cells is increased;while in the kidney tumor cells 786-O cells,after knocking down ER?,kidney tumor cells can be seen Both ER? and ANGPT-2 protein expression decreased;6.In kidney tumor cells A498 and 786-O,after overexpression of ER?,knockdown of ANGPT-2 and ER? knockdown,and overexpression of ANGPT-2,changes in the number of angiogenesis and protein expression in both cells the change.Transfected with plasmids that overexpress ER? and knock down ANGPT-2 into A498 cells,and detect the ANGPT-2 protein in kidney tumor cells.After ER? and knockdown of ANGPT-2,the protein can be reversed.The changes of ANGPT-2 and Tie-2 phosphorylated proteins in vascular endothelial cells were detected.The expression of phosphorylated proteins of ANGPT-2 and Tie-2 overexpressing ER? was increased,and the ANGPT-2 and Tie-2 proteins were knocked down.The expression of phosphorylated protein is reduced,and after overexpression of ER? and knockdown of ANGPT-2,the protein can be reversed.However,when transfected with plasmids overexpressing ER? and knocking down ANGPT-2 into A498 cells and co-cultured with vascular endothelial cells,with or without the addition of sunitinib drugs,it can be seen that the overexpression of ER? group increased vascular tube formation number,while knocking down ANGPT-2 can reduce the number of vascular tubes,after adding overexpression of ER? and knocking down ANGPT-2,it can be seen that the number of vascular endothelial cells is reversed,the difference is statistically significant(* P<0.05;** P<0.01).The same result is that in 786-O of renal tumor cells,using ER? knockdown and overexpressed ANGPT-2 plasmid transfection in 786-O cells,the changes in protein expression and the number of vascular tubes have changed,the difference is statistical difference(* P<0.05;** P<0.01);7.Search the database to make sure that the downstream gene ANGPT-2 is regulated by the transcription level in kidney tumor cells.Through the database,find the ERE region of ANGPT-2 regulated by ER?,a total of 6,through the Chip experiment,detect the binding sites of ERE # 1, ERE # 2,ERE # 3,ERE # 4,ERE # 5 and ERE # 6.It is concluded that ERE # 5 is its binding site and performs transcriptional regulation.The wild type of the binding site was transformed into a mutant type by Bam H1 enzyme.The wild-type and mutant plasmids were transfected into kidney tumor cells A498 and 786-O cells,respectively.The regulation of wild-type in A498 and 786-O cells was changed,and the difference was statistically significant(P <0.01);while the mutant type.There was no change in the regulation in A498 and 786-O cells,and the difference was not statistically significant(P> 0.05).It can be concluded that ER? can directly regulate the downstream gene ANGPT-2 through transcriptional regulation;8.Detection of m RNA changes in ANGPT-1 and 2 is the content of kidney tumor cells,ANGPT-2 protein content in kidney tumor cells.Using q RT-PCR method,the m RNA content of ANGPT-1 and ANGPT-2 in kidney tumor cells A498 and 786-O was detected.It was concluded that in 786-O and A498 cells,the m RNA content of ANGPT-2 was much higher the m RNA content of ANGPT-1.The protein content of ANGPT-2 of three kidney tumor cells Caki-1,786-O,A498 and vascular endothelial cells was determined,and the ANGPT-2 content of vascular endothelial cells was much lower than that of three kidney tumor cells.Conclusions: In renal tumor cells,ER? directly transcriptional regulates Angiopoi etin-2 and secretes ANGPT-2 to the Tie-2 signaling pathway of vascular endothelial cells,thereby mediating the mechanism of angiogenesis in renal cancer and the drug sensitivity of sunitinib.Part Three The FDA approved anti-estrogen drug Faslodex combined with sunitinib targeted inhibition of ER?/Angiopoietin-2/Tie-2 signaling mediated by renal cell carcinoma,the mechanism of angiogenesis and animal experimental researchObjective: To explore the use of the anti-estrogen drug fulvestrant combined with sunitinib to inhibit ER?/Angiopoietin-2/Tie-2 signal-media ted angiogenesis and animal experiments after co-culture of RCC and H UVEC.Methods: 1.After adding estrogen and estrogen inhibitors to renal tumor cells 786-O,the protein expression of renal tumor cells and vascular endothelial cells was detected.At the same time,after adding or not adding sunitinib drug,it was also observed Phenotypic changes of vascular endothelial cells forming tubes.2.After renal tumor cells 786-O and Caki-1,after over-expression of ANGPT-2 and estrogen inhibitors,the protein expression of renal tumor cells and vascular endothelial cells was detected,and at the same time,sunitinib was not added or added After the drug,the phenotypic changes of vascular endothelial cells forming tubes were also observed.3.Animal model test,divided into 4 groups,blank group,estrogen inhibitor group,sunitinib group,estrogen inhibitor + sunitinib group,mice were injected with tumor under the kidney capsule,14 days later,given the mice were injected intraperitoneally with estrogen inhibitors and / or sunitinib drugs.After the mice were sacrificed,they were examined for tumor metastasis,the size and length of tumors in each group,and the immunohistochemical results in tumor tissue sections.Results: 1.After adding estrogen and estrogen inhibitor to renal tumor cells 786-O,changes in intracellular protein level and vascular endothelial cell tube formation phenotype can be detected.After adding estrogen and estrogen inhibitor in renal tumor cells 786-O,it can be seen that estrogen can increase the phosphorylation expression of ANGPT-2 and Tie-2 in renal tumor cells and vascular endothelial cells,and increase the formation of vascular endothelial cells number,while estrogen inhibitors reduce the phosphorylated expression of ANGPT-2 and Tie-2 in renal tumor cells and vascular endothelial cells,and reduce the number of vascular endothelial cells into tubes,indicating that administration of estrogen and estrogen inhibitors to renal tumor cells angiogenesis is related and the difference is statistically significant(*,P<0.05;**,P<0.01);2.In renal tumor cells 786-O and Caki-1,after overexpression of ANGPT-2 and estrogen inhibitors,with or without the addition of sunitinib drugs,changes in cellular protein levels and vascular tube formation can be seen.In 786-O and Caki-1 renal tumor cells,after overexpression of ANGPT-2 and estrogen inhibitors,with or without the addition of sunitinib drug,it can be seen that overexpression of ANGPT-2 can increase renal tumor cells and vascular endothelial cells The phosphorylated expression of ANGPT-2 and Tie-2 can also increase the number of vascular endothelial cells,while estrogen inhibitors reduce the phosphorylated expression of ANGPT-2 and Tie-2 in renal tumor cells and vascular endothelial cells,while Reducing the number of vascular endothelial cells into tubes,such as the simultaneous addition of overexpression of ANGPT-2 and estrogen inhibitors,can partially reverse the function of estrogen inhibitors.Angiogenesis is related,and the difference is statistically significant(*,P<0.05;**,P<0.01,***,P<0.001);3.Animal model experiments show that estrogen inhibitor combined with sunitinib can better inhibit tumor growth.The mice were randomly divided into 4 groups,1.blank group,2.estrogen inhibitor group,3.sunitinib group,4.estrogen inhibitor + sunitinib group,after the mice were injected with tumor under the kidney capsule.After 14 days,the mice were intraperitoneally injected with estrogen inhibitors and / or sunitinib drugs.After the mice were sacrificed,the mice were metabolized in vivo,and the tumor metastasis,tumor length,tumor weight,and pathological sections of tumor specimens were obtained.Compared with the control group,both the estrogen inhibitor group and the sunitinib group can inhibit tumor growth,while the estrogen inhibitor combined with sunitinib group has a better tumor suppression effect than the other two groups.Conclusions: 1.In renal tumor cells,estrogen inhibitors combined with sunitinib increase the ability to inhibit angiogenesis of vascular endothelial cell.2.In animal experiments,both estrogen inhibitors and sunitinib have the ability to inhibit renal tumor angiogenesis,and the combination of estrogen inhibitors and sunitinib drugs can better inhibit tumor blood vessel growth.. |
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