Circular RNA CircHMGCS1-016 Remodels The Immune Microenvironment Of Intrahepatic Cholangiocarcinoma By Up-regulating GAL-8 And CD73 Expression Via Sponge Adsorption Of MiR-1236-3p

Epidemiological studies suggest that the incidence rate of intrahepatic cholangiocarcinoma(ICC)is on the rise,which is about 5%new cases/years.Radical surgical resection is the only effective way to cure ICC.However,there are no typical symptoms in the early stage of ICC.Most ICC patients are already in the advanced stage at the time of diagnosis and have lost the opportunity for surgery.Moreover,they are prone to early metastasis and recurrence after surgical resection.These are the key factors that contribute to the poor prognosis of ICC.In the past decade,immunotherapy has made important progress,and it has been applied to the treatment of advanced cancer,such as the clinical application of a variety of PD1 antibodies.At present,the number of clinical trials of immunotherapy in ICC is still small,but the results suggest that immunotherapy can benefit some patients and improve the survival rate of patients.The only clinical case reports also suggest that most patients are not sensitive to immunotherapy.This may be due to the lack of research on tumor immune microenvironment(time)in ICC,which leads to the lack of effective biomarkers.Therefore,it is still an important work for relevant practitioners to further elaborate the time characteristics of ICC and the detailed mechanism of ICC immune tolerance and put forward more reasonable and effective strategies for ICC immunotherapy.Circular RNAs(circRNAs)are newly discovered single stranded circular RNA molecules,which are usually produced by the reverse splicing process of pre-mRNA.Because they have a unique structure different from linear RNA,they are stable and cannot be cut by exonuclease,and are highly conserved among species.And studies have confirmed that most circRNAs expression has strict tissue specificity,cell specificity and developmental specificity,which suggests that circRNAs have essential functions and their abnormalities may be related to the occurrence and development of many diseases.Recent studies have confirmed that their abnormal expression is related to neurological diseases,cardiovascular diseases and tumors,especially tumors.For example,the down-regulation of circTRIM33-12 expression promotes liver cancer progression,while circSMARCA5 is related to the clinicopathological characteristics and prognosis of HCC,and it's up-regulation increases the sensitivity of HCC chemotherapy.Studies have confirmed that circRNA regulates gene expression mainly by acting as a miRNA molecular sponge.For example,circMET up-regulates snail by adsorbing miR-30-5p to promote liver cancer progression and immune tolerance.As a newly discovered RNA molecules class,circRNAs have been rarely studied in ICC.In this study,the expression of circRNAs in 5 surgically resected ICC tumors and corresponding adjacent tissues were sequenced.Choose hsa_circ₀008621(circHMGCSl-016)with apparent differences and apply in situ hybridization,and qRT-PCR will be further verified in ICC and related adjacent tissues.Interference and overexpression of circHMGCS1 in ICC cells were conducted to study the effect of circHMGCS 1-016 on ICC cells' biological functions.The potential miRNAs adsorbed by circHMGCS 1-016 and miRNA's targeted genes were determined through RIP-Seq,proteomics and network prediction.Finally,the function and mechanism of circHMGCS 1-016 in ICC were verified by humanized mice.PART ONE Abnormal expression of circHMGCSl-016 in ICC tissues and its' role in the prognosis of ICC patientsPurposes:Screen the differentially expressed circRNA in ICC tissues;study the expression of circRNA molecules with the most significant differences in expression in surgically resected tissues of ICC patients,and explore its relationship with ICC patients' clinical prognosis.Methods:Sequencing circRNAs from 4 pairs of ICC surgically removed tumors and corresponding adjacent tissues.According to the difference between the abundance of sequenced circRNAs(FPKM value greater than 10)and the difference between the tumor and adjacent tissues,the target circRNA is selected.qRT-PCR detection 30 was used to verify ICC and corresponding adjacent tissues.In situ hybridization was used to detect the expression of target circRNA in 108 cases of ICC tissue microarray.Survival analysis was used to analyze the expression of target circRNA in ICC tissues and its relationship with patients' clinicopathological characteristics and prognosis.Results:We found that 21 circRNAs were up-regulated in ICC tissues,while 17 circRNAs were down-regulated(the difference is more than 2-fold).Among them,circHMGCS1-016 was the most up-regulated in ICC organizations.It was found by qRT-PCR that the expression of circHMGCS1-016 in ICC was significantly higher than that in the corresponding adjacent tissues(p<0.001).We also found that the expression of circHMGCS1-016 in most ICC tissues was higher than that in the affiliated adjacent tissues through in situ hybridization,and the expression of circHMGCS1-016 was related to the TNM stage and tumor size in ICC patients.The prognosis of liver cancer patients with high expression of circHMGCS1-016 is worse than that of patients with low expression;multivariate analysis showed that circHMGCS1-016 is an independent prognostic factor for ICC patients' tumor-free survival rate.Conclusions:The expression of circHMGCS1-016 is significantly increased in ICC tissues,which is related to the malignant phenotype of ICC,and its high expression is associated with the poor prognosis of ICC patients.PART TWO The roles of circHMGCS1-016 in ICC invasion and metastasisPurposes:To study the expression difference of circHMGCS1-016 in ICC cell lines with different metastatic potential and study its effect on the invasion and metastasis ability of ICC cells by up-regulating the expression of circHMGCS1-016.Methods:qRT-PCR was used to detect the expression of circHMGCS1-016 in ICC cells,including RBE,HuCCTl,SSP-25,HCCC9810 and QBC939.Transfection interference and overexpression of circHMGCS1-016 lentivirus in ICC corresponding high expression and low expression of circHMGCS1-016 cells,after screening stable cell lines,qRT-PCR verified the interference and overexpression of circHMGCS1-016.The effects of circHMGCS1-016 on tumor formation and metastasis of ICC cells were studied through cell invasion experiments,CCK8,clone formation,and nude mouse subcutaneous tumor model.Results:qRT-PCR indicated that circHMGCS1-016 was highly expressed in QBC939 cells,while it was the lowest expressed in RBE cells.The expression of circHMGCS1-016 was up-regulated in RBE cells,while the expression of circHMGCS1-016 was disturbed in QBC939 cells.Invasion experiments confirmed that ICC cells' invasion ability is affected by the expression of circHMGCS1-016.High expression of circHMGCS1-016 means strong cell invasiveness.The CCK8 experiment showed that the cells with high expression of circHMGCS1-016 have intense proliferation and clonal formation ability.The cells' tumor formation rate and metastasis rate in the circHMGCS1-016 high expression group in vivo were higher than those in the control group.Conclusions:The high expression of circHMGCS1-016 enhances the invasion,metastasis and proliferation of ICC cells.PART THREE Mechanism of circHMGCS1-016 to promote ICC invasion,metastasis and immune tolerancePurposes:Explore the specific mechanism of circHMGCS1-016 in promoting the progress of ICC and explain the miRNA and key downstream target genes that circHMGCS1-016 affects in ICC.Methods:Four ICC cell lines expressing different levels of circHMGCS1-016,QBC939-Mock,QBC939-vshcircHMGCS1-016,RBE-Mock and RBE-circHMGCS1-016,were constructed by lentiviral vectors.Using RIP-Seq to enrich the miRNA adsorbed by circHMGCS1-016.After using the network base pairing prediction tool to determine the miRNA adsorbed by circHMGCS1-016,it was verified by reporter gene assays.The protein expression differences of circHMGCS1-016 differentially expressed cells were studied through proteomics,and multiple miRNA target gene prediction software was used to determine the direct target genes of miRNAs,which were verified by reporter gene assays.Then Western Blot was used to detect the expression of the target gene and combined with the biological function of the target gene to further study the function of circHMGCS1-016 in ICC.Results:RIP-seq combined with network prediction suggests that miR-136-3p is adsorbed by circHMGCS1-016 in ICC cells,and the interaction between them is further verified by reporter gene assays.It was found that CD73 and GAL-8 were up-regulated in cells with high expression of circHMGCS1-016 through SILAC.It was found that CD73 and GAL-8 were enriched through Biotin-miR-136-3p enrichment of target genes.Combined with network prediction and reporter gene assays,it was found that CD73 and GAL-8 were the direct targets of miR-136-3p.It was confirmed by CD73 and GAL-8 interference that these two interferences can inhibit the invasion and metastasis of ICC induced by circHMGCS1-016.Notably,combined with the biological functions of CD73 and GAL-8,we found that the supernatant of highly expressing circHMGCS1-016 cells significantly inhibited the function of CD8+and CD4+T cells,and the levels of IL-6 and TNF-? in the supernatant were significantly reduced.Conclusions:circHMGCS1-016 induces ICC invasion,metastasis and immune tolerance through miR-136-3p/CD73 and GAL-8 axis.PART FOUR Evaluation of the correlation between the expression of circHMGCSl and the therapeutic effect of anti-PD-1 antibody on ICCPurposes:To study the correlation between the expression of circHMGCS1-016 and the levels of CD73,GAL-8 and CD8+T cells in ICC tissues.The relationship between the expression of circHMGCS1-016 and anti-PD1 treatment was studied by humanized mouse experiments.Finally,the relationship between the expression of circHMGCS1-016 and the efficacy of anti-PD1 treatment in 12 patients with ICC was evaluated.Methods:Immunohistochemical serial sections were used to detect the levels of circHMGCS1-016,CD73,GAL-8 and CD8+T cells and analyze the correlation between the above indicators.Cells differentially expressing circHMGCS1-016,CD73 and GAL-8 were inoculated into humanized mice,and the mice were treated with PD-1 antibody and IgG antibody to study the tumor size,blood CD8+T cells,IL-6 and TNF-? level.Continuous sections of mouse tumors were used to detect the levels of CD73,GAL-8 as well as CD8+T cells and analyze the correlation between the above indicators.Finally,analyze the curative effect and the expression of circHMGCS1-016 in 12 ICC patients who received PD1 antibody treatment.Results:Analyses of liver cancer and corresponding adjacent tissues by qRT-PCR revealed that the expression of CD73 and GAL-8 in most liver cancer tissues was elevated.The gene chip detection revealed that CD73 and GAL-8 were up-regulated in most ICC tissues.Correlation analysis showed that the level of circHMGCS1-016 was positively correlated with the level of CD73/GAL-8 but negatively correlated with the level of CD8+T cells.Experiments in humanized mice found that the tumor volume of PD-1 antibody treatment with low expression of circHMGCS1-016 or high expression of circHMGCS1-016 and infected with CD73 and GAL-8 was the smallest,while the tumor of circHMGCS1-016 was the largest.The blood levels of adenosine and GAL-8 were the highest in the circHMGCS1-016 high expression group,while the numbers of CD8+T and CD4+T cells were the least,which was further confirmed by mouse tumor tissue immunohistochemistry.In the tissues of 12 patients with ICC who received PD1 antibody treatment,5 cases of circHMGCS1-016 had high expression and 7 cases had low expression.In the group with high expression of circHMGCS1-016,there were 1 case of PR(partial response),1 case of SD(stable disease),and 3 cases of PD(progressive disease).In the group with low expression of circHMGCS1-016,there were 1 case of CR(complete response),2 cases of PR,3 cases of SD,and only 1 case of PD.It was confirmed by immunohistochemistry that CD8+T cells were lower in high circHMGCS1-016 tissues.Conclusion:The overexpression of circHMGCS1-016 is related to the poor efficacy of PD1 antibody treatment.