The Preliminary Study On The Regulation Of IL-6 Expression By Super-enhancer And Its Role In The Occurrence And Development Of Pancreatic Cancer

Pancreatic cancer is one of the most malignant solid tumors with a very poor prognosis,with a 5-year survival rate of less than 5%,and currently commonly used tumor treatment options(such as surgery、radiotherapy、chemotherapy and immunotherapy,etc.)have not shown satisfactory efficacy,and there is also lack of effective molecular targeted therapy.Understanding the pathogenesis of pancreatic cancer is of great significance for futher development or improvement of therapeutic methods.IL-6(Interleukin 6)is an important pro-inflammatory factor,and its coding gene is located the P15.3 region on chromosome 7(7P15.3).Studies have shown that IL-6 is highly expressed in a variety of tumors,including pancreatic cancer,and IL-6 plays an important role in the occurrence and development,vascular formation,inflammatory microen-vironment and metastasis of pancreatic cancer.The expression level of IL-6 in pancreatic cancer tissue may be related to the prognosis of patients,furthermore,the molecular regulation mechanism of IL-6 expression remains to be further studied.Part Ⅰ:Expression of IL-6 in pancreatic cancer tissues and cells and its clinical significanceObjectiveThe expression level of IL-6 in pancreatic cancer tissues and pancreatic cancer cells was preliminarily studied.To analyze the correlation between IL-6 expression in pancreatic cancer tissues and clinicopathological factors in patients with pancreatic cancer,and to further analyze the relationship between IL-6 and prognosis of patients with pancreatic cancer.Methods1.The expression level of IL-6-mRNA in various tumors including pancreatic cancer、the expression level of IL-6-mRNA in pancreatic cancer tissues and the correlation between the expression level of IL-6-mRNA and prognosis of pancreatic cancer patients were analyzed by biological information analysis techniques;2.Quantitative real-time PCR(qRT-PCR)was used to detect the expression of IL-6-mRNA in normal pancreatic ductal epithelial cells(HPDE6-C7)and four pancreatic cancer cells(human in situ pancreatic adenocarcinoma BXPC-3,human metastatic pancreatic adenocarcinoma ASPC-1,human pancreatic ductal cancer MIAPaCa-2 and CFPAC-1);3.Immunohistochemistry was used to detect the expression of IL-6 protein in paraffin sections of 68 patients with pancreatic cancer after operation and in paraffin sections of 33 paracancer tissues,and the relationship between IL-6 protein expression and clinicopathological factors was analyzed.The survival data of patients with pancreatic cancer were followed up,and the survival curve was drawn by Kaplan-Meier analysis to analyze the correlation between IL-6 expression and survival of patients.Result1.According to the analysis of IL-6-mRNA sequencing results of 31 types of tumors in TCGA database,the expression level of IL-6-mRNA in pancreatic cancer、esophageal cancer、glioblastoma and other tumors was significantly higher than that of corresponding normal tissue;IL-6-mRNA sequencing analysis of 179 pancreatic cancer tissues and 171 control paracancer tissues showed that the expression of IL-6-mRNA in pancreatic cancer tissues was significantly higher than that in para-cancer tissues,and the high expression of IL-6 was negatively correlated with the prognosis of patients(P<0.05);2.Compared with normal pancreatic ductal epithelial cells(HPDE6-C7),the expression of IL-6-mRNA in four pancreatic cancer cells was significantly increased(P<0.05);3.Immunohistochemical staining results showed that the positive IL-6 signal was mainly located in the cytoplasm of pancreatic cancer cells.The high expression rate of IL-6 protein in pancreatic cancer tissues was 33/68(48.5%),and the high expression rate of IL-6 protein in paracancer tissues was 8/33(24.2%),with statistical significance(P<0.05).The expression of IL-6 was correlated with the degree of differentiation and lymph node metastasis of pancreatic cancer,but not with gender、age、tumor size、TNM stage、tumor location and whether nerve invasion.IL-6 expression level was negatively correlated with the overall survival rate of patients with pancreatic cancer.Conclusion1.IL-6 was highly expressed in pancreatic cancer cells and tissues,and the expression level of IL-6 in pancreatic cancer tissues was correlated with the differentiation degree of pancreatic cancer and lymph node metastasis;2.The expression level of IL-6 in pancreatic cancer can be used as an indicator to judge the prognosis of pancreatic cancer.PartⅡ:The studies of the molecular regulation mechanism of IL-6 expression and function of super enhancer in pancreatic cancer cellsObjectiveTo investigate whether there are super enhancers involved in the regulation of IL-6 expression in pancreatic cancer cells.And the function of super enhancer is also discussed preliminary.Methods1.Through bioinformatics analysis,the H3K27ac CHIP-Seq database was used to find out whether H3K27ac enrichment region(super enhancer region)existed around the IL-6 gene in pancreatic cancer cells;2.The super enhancer inhibitors JQ-1(0.5μmol/L and 1μmol/L)and I-BET-762(1μmol/L and 2μmol/L)were applied to pancreatic cancer cells(MIAPaCa-2 and CFPAC-1).The expression changes of IL-6-mRNA was detected by RT-PCR and the expression changes of IL-6 protein in MIAPaCa-2 cells was detected by immunofluorescence.Cell scratch assay was used to detect the migration ability of MIAPaCa-2 cells before and after the treatment with super enhancer inhibitors;3.Using CRISPR-Cas9 gene editing technology,two pairs of sgRNA primer sequences were designed and synthesized according to the location of the super enhancer presumed in the database.The constructed plasmids were transfected into MIAPaCa-2 and CFPAC-1 cells respectively.Genotyping and DNA sequencing are used to verify the successful knockout of the aim sequence;qRT-PCR was used to detect the changes of IL-6-mRNA expression level in pancreatic cancer cells after super enhancer sequence knockout;cell scratch assay and clonformation assay were used to detect the migration and clonformation of MIAPaCa-2 cells before and after super enhancer sequence knockout.Result1.Bioinformatics data showed that there was a large amount of H3K27Ac enrichment in the upper reaches of IL-6 gene in pancreatic cancer cells at chr7:22615000-CHR7:22630000,which was speculated to be the super enhancer region by the degree of acetylation and enrichment intensity;2.After the super enhancer inhibitors JQ-1 and I-BET-762 were treated on CFPAC-1 and MIAPaCa-2 cells,the mRNA expression level of IL-6 gene was significantly decreased(P<0.05),and there was no statistically significant difference between the two concentrations;the immunofluorescence intensity of MIAPaCa-2 cells was decreased、the migration ability of MIAPaCa-2 cells was significantly inhibited by super enhancer inhibitors JQ-1 and I-BET-762(P<0.05);3.In MIAPaCa-2 cells,sgRNA-Cas9 plasmid was successfully transfected,Cell lines with the targeted sequences IL-6-SE1(Chr7:22615000-Chr7:22619000)and IL-6-SE2(Chr7:22625000-Chr7:22630000)knockout were constructed,genotyping confirmed the knockout of these two sequences.DNA sequencing showed that the new DNA sequences was successfully joined after splicing;after IL-6-SE1 and IL-6-SE2 were knocked out,the IL-6-mRNA expression in MIAPaCa-2 cells was significantly decreased(P<0.05);and the migration and clonformation ability of MIAPaCa-2 cells were significantly inhibited(P<0.05).Conclusion1.IL-6-mRNA expression in pancreatic cancer cells is regulated by super enhancer;2.IL-6-SE1 and IL-6-SE2 are the key super enhancers which regulating IL-6-mRNA expression in pancreatic cancer cells;3.IL-6-SE1 and IL-6-SE2 can regulate the migration and clonformation ability of pancreatic cancer cells,which may be therapeutic targets of potential application value for pancreatic cancer.