Mechanism Of Transcriptional Regulation Of IL-10 Gene In Cervical Tumor Associated Macrophages

Objectives The incidence of cervical cancer has shown younger.Interleukin 10(IL-10)is a kind of has obvious inhibitory effects on cell immune cytokines,when the synthesis of a large number of regulation to suppress the immune system in the human body caused by HPV virus,leading to cervical cancer.In tumor microenvironment,tumor associated associated(TAMs)is involved in tumor growth,invasion and migration through complex autocrine and paracrine cytokines.As one of the main sources of expression of IL-10,the molecular mechanism of IL-10 expression in cervical carcinogenesis has not been reported by TAMs.Previous studies have found that cervical tumor tissue IL-10 expression was significantly increased,and the gene promoter methylation of the low expression,suggesting that DNA methylation plays an important role in the changes in response to infection and inflammation in the process of environment.Bioinformatics analysis showed that the transcription factor E26-1(Ets1)and specific protein 1(Sp1)combined with IL-10 gene promoter cis element containing CG sites,and reported that the expression of Ets1 and Sp1 is related to cervical cancer,but whether it is involved in regulation of IL-10 TAMs and its role in cervical cancer has not been reported.Therefore,human monocyte macrophages,human cervical cancer cells and different pathological cervical tissues used in this study,to explore the molecular mechanism of TAMs in Ets1 and DNA methylation or Sp1 mediated transcriptional regulation of IL-10 gene and its role in cervical carcinogenesis,designed for cervical cancer with TAMs as target point to provide new ideas to treat.Methods Select patients who are September 2014 to March 2016 in Tangshan Worker's Hospital to cervical biopsy and surgical treatments,who have complete clinical data and did not receive preoperative radiotherapy and chemotherapy,a total of 160 cases,including47 cases of normal cervical(control group),cervical intraepithelial neoplasia grade 27cases(CIN?group),cervical intraepithelial neoplasia and 46 cases of grade ?-?(CIN?-?group),40 cases of cervical squamous carcinoma(CSCC group).The basic information of the patients was collected through the online electronic patient records.The co-expression of IL-10,Ets1 and Sp1 with CD68 in different pathological cervical tissues were detected by immunofluorescence double staining;the relationship between IL-10+TAMs,Ets1+ TAMs,Sp1+ TAMs clinical analysis level and clinical pathological features of cervical squamous cell carcinoma;Human monocyte cell line THP-1 was cultured in vitro to induce macrophages to form TAMs;Analysis of the impact of the level of transcription factor Ets1 and Sp1 in TAMs on IL-10 gene expression by si RNA interference;Bioinformatics analysis of transcription factor Sp1 and Ets1 in the IL-10 promoter proximal possible binding sites within 2000 bp.The binding sites of Sp1 binding sites were found to play an important role in the regulation of IL-10 promoter activity by site directed mutagenesis and deletion mutation;By molecular biological methods of TAMs methylation intervention,luciferase reporter gene,CHIP and MSP,analysis of transcription factors Sp1,Ets1 and IL-10 promoter DNA methylation in TAMs and its transcriptional activity.Results 1 Compared with the patients' age,Obstetrical history,age at menarche,menstrual cycle,menstrual days,the number of abortions,smoking,drinking,the difference was not statistically significant(P>0.05).2 Double immunofluorescence staining showed that the expression of IL-10+ TAMs,Ets1+ TAMs and Sp1+ TAMs in cervical tissue were gradually increased with the progress of cervical lesions in TAMs.3 The levels of IL-10+ TAMs,Ets1+ TAMs and Sp1+ TAMs in CSCC group were correlated with the degree of differentiation,FIGO stage and lymph node metastasis(TAMs)(P<0.05).4 In TAMs using si RNA interference knockdown of Ets1 and Sp1 at the same time or knockdown of Ets1 gene expression and Sp1,q RT-PCR and Western blot results showed that knockdown of Sp1 or knockdown of Ets1 and Sp1 gene can downregulate the expression of the IL-10,and the double knock inhibition of its expression is more obvious,but only knockdown Ets1 protein of IL-10 little effect.5 Methylation intervention experiment and MSP sequencing and Ch IP,according to the results of experiment in TAMs IL-10 gene promoter region methylation get Sp1 transcription factors and their combination and increase the expression of IL-10.6 Further CHIP and immunofluorescence staining the experimental results show that the Ets1 in Sp1 regulation of IL-10 gene promoter activity of the synergy.Conclusions 1 TAMs source IL-10 expression was positively correlated with cervical cancerous.2 The expression of Ets1 and Sp1 in cervical cancerous process was upregulated and correlated with the expression of IL-10 in TAMs.3 DNA methylation of Sp1 binding sites regulates IL-10 gene promoter activity and plays an important role in cervical cancerous happened,and promote the Sp1 and Ets1 expression regulation IL-10 gene promoter transcriptional activity in TAMs.