| Purpose: Bladder cancer is one of the most common malignant disease all over the world,associating with serieses of pathological lesions of the urothelium.It is considered to be mainly caused by environmental factors,including smoking.Basal cells can be the cells originating from bladder cancer and develop into bladder cancer under various stimuli related to the tumor microenvironment.Although the first-line treatment methods for bladder cancer(such as transurethral resection,intravesical injection of Bacillus Calmette-Guerin(BCG),chemotherapy or targeted drugs(e.g.bevacizumab,cetuximab,sunitinib,etc.)for bladder treatment,etc.)have been clinically proven to be able to control bladder cancer to a certain extent.However,in terms of basic research,researches on the mechanism of the occurrence and development of bladder cancer are still significantly insufficient.This article mainly studies the hypoxic environment of the tumor microenvironment by down-regulating mi RNA and its downstream proteins to promote the tumor immune escape mechanism of bladder cancer.Methods: Phenotypic research: In order to verify the effect of abnormal mi RNA production on the immune escape of bladder cells under the hypoxic environment of bladder cancer,we first treated PBMC with hypoxia,and then detected the m RNA expression levels of Drosha and Dicer.Furthermore,flow cytometry and western blot were used to detect the expression of Drosha and Dicer.At the same time,western blot was used to detect the expression of hypoxia-inducible factor-1?(HIF-1?).In order to further study whether the hypoxic environment can affect the immune escape of bladder cancer cells,flow cytometry was used to detect the activity of immune cells and the expression of immune checkpoint programmed death receptor-1(PD-1).At the same time,enzyme-linked immunosorbent assay(Elisa)was used to further detect the cellular immune factors interleukin-2(IL-2)and tumor necrosis factor-?(TNF-?).The apoptosis of tumors cells was then detected using flow cytometer.The expression of the Cassitas B lymphoma b(Cbl-b)protein(may be involved in the immune escape of cells)in PBMC cells was detected using western blot.Mechanism research: To further verify the role of Cbl-b in tumor immune escape,the overexpression vector of Cbl-b was constructed and transfected into cells,and its expression efficiency was detected by western blot.Subsequently,under hypoxic conditions,Cbl-b si RNA interference was added,western blot was used to test the protein expression level of Drosha and Dicer.Then,the positive expression of immune cells CD3,CD4 and CD8 after Cbl-b overexpression/hypoxia conditions were added Cbl-b si RNA interference.At the same time,we used Elisa to further detect the levels of cellular immune factors IL-2 and TNF? after Cbl-b overexpression/hypoxic conditions were added Cbl-b si RNA interference.In addition,flow cytometry was used to detect the apoptosis status of tumor cells after Cbl-b overexpression/hypoxic conditions were added Cbl-b si RNA interference.Next,the binding sites of mi R-30 c,mi R-135 a,mi R-27 a and their target genes were predicted by bioinformatics analysis,and constructed a luciferase reporter plasmid containing Cbl-b 3'UTR sequence or mutant sequence.Luciferase activity experiment was used for further verification.Then under the action of mi R-30 c,mi R-135 a,and mi R-27 a inhibitor,the expression changes of Cbl-b protein were detected by western blot.After the si Drosha,si Dicer si RNA,and si Drosha+si Dicer double interference group(Both-si)model were established,the expression level of various mi RNAs were detected by q PCR,and the expression level of Cbl-b protein were detected by western blot.At the same time,we use flow cytometry to interfere with the positive expression of CD3,CD4 and CD8 after the expression of Drosha and Dicer,and Elisa to detect the content of cytokines.Animal research: We first build an animal tumor model,inoculate the transfected tumor cells of different groups into mice,and regularly measure the tumor volume.Then,the serum cytokines were detected,and the tumor tissue was stripped,fixed,and pathologically sectioned,and then HE,Tunel staining,immunohistochemistry(IHC)and other experimental methods were performed to verify the in vitro experimental results.Results: Phenotypic research: We found that Drosha and Dicer m RNA decreased significantly after hypoxia treatment,and the protein abundance also decreased significantly.This result suggested that hypoxic treatment can reduce the expression of Drosha and Dicer in PBMC cells.In the detection of immune cell activity,we found that the immune cell CD3+/CD4+ double positive increased under hypoxic conditions,while the CD3+/CD8+ double positivity was significantly reduced.In addition,the PD-1expressed in CD8 T cells was up-regulated,and the content of cellular immune factors IL-2 and TNF-? were significantly reduced under hypoxic conditions.The detection results of tumor cell apoptosis showed that cell apoptosis was significantly reduced under hypoxic conditions.The above results all suggest that the hypoxic environment can promote the immune escape of bladder cancer cells.Mechanism research: The detection results of Cbl-b expression showed that the expression of Cbl-b protein in PBMC cells increased significantly under hypoxic conditions.After the Cb1-b transfection efficiency verified,the subsequent experiment showed that,the double positivity of CD3+/CD4+ increased after Cbl-b overexpression,while the double positivity of CD3+/CD8+ was significantly reduced,and the expression of CD8+/PD-1 was up-regulated,indicating that the tumor cells had immune escape after overexpression of Cbl-b;and after adding Cbl-b si RNA interference under hypoxic conditions,CD3+/CD4+ double positivity decreased,while CD3+/CD8+double positivity increased,and CD8+/PD-1 expression returned to normal,which suggests that hypoxia conditions interfere with Cbl-b expression.The results of Elisa showed that the levels of cellular immune factors IL-2 and TNF-? were significantly reduced after Cbl-b over expression,but recovery occurred after the interference of Cbl-b si RNA was added under hypoxic conditions.The subsequent flow cytometry results showed that cell apoptosis was significantly decreased after Cbl-b overexpression,and cell apoptosis was significantly increased when Cbl-b si RNA was added under hypoxia.The above results all suggest that Cbl-b overexpression is involved in the occurrence of immune escape of bladder cancer cells.The analysis and prediction of bioinformatics software revealed that Cbl-b is the common target of mi R-30 c,mi R-135 a,and mi R-27 a.The luciferase activity test results alao showed that,compared with the control group,co-transfection in Cbl-b 3'UTR and mimics the luciferase activity was significantly reduced;while there was no significant difference in luciferase activity of cells co-transfected with Cbl-b 3'UTR MUT plasmid and mimics.The above results suggest that Cbl-b is the common targe.In addition,the expression of Cbl-b protein in the mi RNA inhibitor group was significantly increased,while the Cbl-b protein in the mi R-30 c,mi R-135 a,and mi R-27 a inhibitor mixed group was significantly increased.In the si Drosha and si Dicer groups,the expression levels of a variety of mi RNAs were significantly reduced,among which the Both-si double interference group significantly decreased;the si Drosha and si Dicer groups,the Cbl-b protein expression increased,and the both-si group,the expression increased significantly.In addition,the CD3+/CD4+ double positivity after interfering with the expression of Drosha and Dicer increased,among which the Both-si group increased the most,while the CD3+/CD8+ double positivity decreased significantly,the expression of CD8+/PD-1 increased inversely,and the both-si group decreased the most.After interfering with the expression of Drosha and Dicer,the cytokine concentration decreased,and the increase in Both-si group was the most obvious.These results suggest that we interfered with the expression of Drosha and Dicer to promote the immune escape of tumor cells.Animal research: The results in vivo verified that compared with the control group of PBMC,the tumor volume and growth curve of the experimental group was increased.Among them,the PBMC-si Both group had the largest tumor volume and the fastest tumor growth curve.In the detection of cytokine content,compared with the control group PBMC,the cytokine IL-2 and TNF-? content in the interference group were reduced,and the PBMC-si Both group decreased the most.The results of HE staining of tumor histopathological sections showed that,compared with the control group of PBMC,the inflammatory infiltration of the tumor tissue in the experimental group was significantly reduced,and the PBMC-si-both group was the most obvious.Similarly,Tunel staining results also showed that compared with the control group PBMC,the tumor tissue apoptosis of the experimental group was significantly reduced,and the PBMC-si Both groups was the most obvious.IHC results also showed that the positive expression of CD68 in the tumor tissues of the experimental group was significantly reduced.Conclusion: This article,PBMC was used as the research object.To explored the mechanism of mi RNA production under hypoxic conditions on the immune escape of tumor cells during the development of bladder cancer through molecular biology,cell biology and proteomics.The results showed that under hypoxic conditions,the expression of Drosha and Dicer were downregulated in PBMC cells,and promoted tumor immune escape in bladder cancer.Further mechanism studies and in vivo experiments found that under hypoxic conditions,Hypoxia-mediated downregulation of mi RNA biosynthesis promotes tumor immune escape in bladder cancer through upregulated of cbl-b. |
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