Huang Luxin, Duan Feng, Dong Xianning, Zhang Zengzhen
Gynecology Department, Jinan Maternity and Child Care Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250002, China.
Department of Oncology, Qingdao Municipal Hospital, Qingdao, Shandong, 266071, China.
Cent Eur J Immunol. 2024;49(4):393-403. doi: 10.5114/ceji.2024.145307. Epub 2024 Nov 8.
The study was designed to determine whether and how sevoflurane (Sev) regulates tumor associated macrophage (TAM) polarization and cervical cancer (CC) cell progression.
The M2 polarized THP-1 was treated with 3%Sev. The culture supernatant of M2 polarized THP-1 was co-cultured with the CC cell line Hela. The NF-B activity was determined by luciferase reporter assay. The key genes dis-regulated by 3%Sev were determined by RNA sequencing (RNA-seq) followed by real-time reverse transcription PCR (qRT-PCR) assay. Luciferase reporter assay was used to analyze the function of 3%Sev based on N6-methyladenosine (m6A) site activity on MAP3K7 3 untranslated regions (3 UTRs). RNA immunoprecipitation (IP) using an anti-m6A antibody (anti-m6A RNA-IP) was performed to determine the m6A levels at MAP3K7 3 UTR.
3%Sev treatment significantly up-regulated the M2 polarization markers and down-regulated the NF-B activity of THP-1. Meanwhile, 3%Sev treated macrophages could enhance the migratory and invasive potential of CC cells. Further, 3%Sev significantly regulated the NF-B pathway, including MAP3K7 inhibition. MAP3K7 overexpression reversed the 3%Sev-regulated NF-B activity and M2 polarization. 3%Sev treatment increased m6A levels in the 3 UTR of MAP3K7. Mutational analysis of potential m6A sites within MAP3K7 3 UTR revealed that these sites were required for 3%Sev regulation. In conclusion, the m6A pattern of MAP3K7 mediates the effects of 3%Sev on macrophage M2 polarization and cervical cancer progression.
3%Sev enhanced TAMs M2 polarization through regulating the m6A pattern of MAP3K7, and therefore enhanced the stimulatory effect of M2 TAMs on the migration and invasion of CC cells.
本研究旨在确定七氟醚（Sev）是否以及如何调节肿瘤相关巨噬细胞（TAM）极化和宫颈癌（CC）细胞进展。
用3%的七氟醚处理M2极化的THP-1细胞。将M2极化的THP-1细胞的培养上清液与CC细胞系Hela共培养。通过荧光素酶报告基因检测法测定核因子-κB（NF-κB）活性。通过RNA测序（RNA-seq），随后进行实时逆转录PCR（qRT-PCR）检测，确定受3%七氟醚失调的关键基因。基于丝裂原活化蛋白激酶激酶激酶7（MAP3K7）3'非翻译区（3'UTR）上的N6-甲基腺苷（m6A）位点活性，使用荧光素酶报告基因检测法分析3%七氟醚的功能。使用抗m6A抗体进行RNA免疫沉淀（IP）（抗m6A RNA-IP），以确定MAP3K7 3'UTR处的m6A水平。
3%七氟醚处理显著上调M2极化标志物，并下调THP-1细胞的NF-κB活性。同时，3%七氟醚处理的巨噬细胞可增强CC细胞的迁移和侵袭能力。此外，3%七氟醚显著调节NF-κB信号通路，包括抑制MAP3K7。MAP3K7过表达逆转了3%七氟醚调节的NF-κB活性和M2极化。3%七氟醚处理增加了MAP3K7 3'UTR中的m6A水平。对MAP3K7 3'UTR内潜在m6A位点的突变分析表明，这些位点是3%七氟醚调节所必需的。总之，MAP3K7的m6A模式介导了3%七氟醚对巨噬细胞M2极化和宫颈癌进展的影响。
3%七氟醚通过调节MAP3K7的m6A模式增强TAMs的M2极化，并因此增强M2 TAMs对CC细胞迁移和侵袭的刺激作用。
