Kanchana Ayyar1, Kudumula Venkata Rami Reddy1. 1. Division of Molecular Immunology and Microbiology (MIM), National Institute for Research in Reproductive Health (NIRRH), Mumbai, India.
Abstract
PROBLEM: In endocervical epithelial cells (End1/E6E7), miRNA let-7f plays an important role in the control of innate immunity. The underlying molecular mechanism for let-7f regulation in these cells remains largely unclear. METHODS OF STUDY: let-7f was knocked down in End1/E6E7 cells using siRNA, and differential gene expression was analyzed by microarray. Differentially expressed genes were validated by qPCR and Western blot. Expression of let-7f was studied by qPCR after inhibition of C/EBPβ with betulinic acid (BA) and pCMVβ plasmid and after overexpression of C/EBPβ with pCMVβ+ plasmid. ChIP assay was performed to confirm binding of C/EBPβ to let-7f promoter. Levels of Lin28A/B were checked by qPCR after similar treatment. RESULTS: let-7f knockdown (KD) affects the expression of many transcription factors (eg, C/EBPβ) which are important regulators of immune responses. We observed let-7f-1 promoter to contain 6 C/EBPβ binding sites. KD of C/EBPβ led to decreased let-7f expression while overexpression of C/EBPβ increased its expression. Treatment of End1/E6E7 cells with TLR-3 ligand, poly(I:C) increased binding of C/EBPβ at binding sites 3, 5, and 6. Expression of Lin28A/B also changed upon inhibition and overexpression of C/EBPβ. Its expression is opposite to that of let-7f in End1/E6E7 cells. CONCLUSION: let-7f-1 is a direct target of transcription factor, C/EBPβ in End1/E6E7 cells.
PROBLEM: In endocervical epithelial cells (End1/E6E7), miRNA let-7f plays an important role in the control of innate immunity. The underlying molecular mechanism for let-7f regulation in these cells remains largely unclear. METHODS OF STUDY: let-7f was knocked down in End1/E6E7 cells using siRNA, and differential gene expression was analyzed by microarray. Differentially expressed genes were validated by qPCR and Western blot. Expression of let-7f was studied by qPCR after inhibition of C/EBPβ with betulinic acid (BA) and pCMVβ plasmid and after overexpression of C/EBPβ with pCMVβ+ plasmid. ChIP assay was performed to confirm binding of C/EBPβ to let-7f promoter. Levels of Lin28A/B were checked by qPCR after similar treatment. RESULTS: let-7f knockdown (KD) affects the expression of many transcription factors (eg, C/EBPβ) which are important regulators of immune responses. We observed let-7f-1 promoter to contain 6 C/EBPβ binding sites. KD of C/EBPβ led to decreased let-7f expression while overexpression of C/EBPβ increased its expression. Treatment of End1/E6E7 cells with TLR-3 ligand, poly(I:C) increased binding of C/EBPβ at binding sites 3, 5, and 6. Expression of Lin28A/B also changed upon inhibition and overexpression of C/EBPβ. Its expression is opposite to that of let-7f in End1/E6E7 cells. CONCLUSION:let-7f-1 is a direct target of transcription factor, C/EBPβ in End1/E6E7 cells.