Conclusions
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.
Material and methods
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-κB 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.
Methods
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-κB 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.
Results
3%Sev treatment significantly up-regulated the M2 polarization markers and down-regulated the NF-κB 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-κB pathway, including MAP3K7 inhibition. MAP3K7 overexpression reversed the 3%Sev-regulated NF-κB 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