Fig 1: CTHRC1 is a direct target of let-7c-5p. (A) WT and Mut sequences of the putative let-7c-5p target sequences of CTHRC1 3’-UTR. (B) Dual-luciferase activity assay showing the effect of let-7c-5p on CTHRC1 3’-UTR luciferase activity in HEK-293T cells. (C) TE-1 and KYSE150 cells were transfected with let-7c-5p mimics and mRNA levels of CTHRC1 were assessed by qRT-PCR. (D) TE-1 and KYSE150 cells were transfected with let-7c-5p inhibitor and mRNA levels of CTHRC1 were examined by qRT-PCR. (E) TE-1 and KYSE150 cells were transfected with let-7c-5p mimics or inhibitor and protein levels of CTHRC1 were analyzed by Western blot. (F) The CTHRC1/GAPDH ratio was quantified. All results are shown as the mean ± SD of three separate experiments (n=3). Statistical analysis was performed with Student’s t-test. *p < 0.05 and **p < 0.01.
Fig 2: CTHRC1 reversed let-7c-5p-mediated AKT and ERK signaling pathways. (A) mRNA levels of CTHRC1 were measured after transfection with siCTHRC1 in TE-1 cells by qRT-PCR. (B) mRNA levels of CTHRC1 were examined after transfection with siCTHRC1 in KYSE150 cells by qRT-PCR. (C) Protein levels of CTHRC1 were detected after transfection with siCTHRC1 in TE-1 and KYSE150 cells by Western blot. (D) The CTHRC1/GAPDH ratio was quantified. (E) Protein levels of AKT, p-AKT, ERK and p-ERK were measured after transfection with siCTHRC1-2 in TE-1 and KYSE150 cells by Western blot. (F) The p-AKT/AKT and p-ERK/ERK ratio were quantified. (G) Protein levels of AKT, p-AKT, ERK and p-ERK were measured after cotransfection with siCTHRC1-2 and let-7c-5p inhibitor in TE-1 and KYSE150 cells by Western blot. (H) The p-AKT/AKT and p-ERK/ERK ratio were quantified. All results are shown as the mean ± SD of three separate experiments (n=3). Statistical analysis was performed with Student’s t-test. *p < 0.05 and **p < 0.01.
Fig 3: Let-7c-5p inhibits ESCC progression in vivo. (A) Images of nude mice tumor specimen. (B) The growth curves of TE-1 cells xenograft tumors after injection with let-7c-5p agomir and let-7c-5p antagomir. Tumor volumes are shown as the mean±SD for each group of three mice. (C) Protein levels of CTHRC1 in tumor tissues were detected by Western blot. (D) The CTHRC1/GAPDH ratio was quantified. All results are shown as the mean ± SD of three separate experiments (n=3). Statistical analysis was performed with Student’s t-test. **p < 0.01.
Fig 4: Expression of CTHRC1 in ESCC and its correlation with let-7c-5p. (A) Higher mRNA expression of CTHRC1 in 50 pairs of ESCC tissues compared to adjacent normal tissues by qRT-PCR. (B) Higher mRNA expression of CTHRC1 in five ESCC cell lines (TE-1, TE-10, TE-11, KYSE140 and KYSE150) compared to Het-1A by qRT-PCR. (C) Higher protein expression of CTHRC1 in ESCC tissues compared to adjacent noncancerous tissues by Western blot. (D) Higher protein expression of CTHRC1 in TE-1 and KYSE150 cells compared to Het-1A by Western blot. (E) Negative correlation between CTHRC1 and let-7c-5p in ESCC tissues. Statistical analysis was performed with Pearson’s correlation analysis (r =-0.418; p = 0.009). All results are shown as the mean ± SD of three separate experiments (n=3) **p < 0.01.
Fig 5: CTHRC1 knockdown inhibited EMT in ATC in vitro and in vivo.(A) Western blotting was used to analyze the E-Cadherin, Vimentin, and Snail expression in CTHRC1 knockdown and control group. (B) IHC staining was used to analyze the E-Cadherin, Vimentin, and Snail expression in CTHRC1 knockdown and control group tumors, brown staining represented the expression of the target protein. **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Anti-CTHRC1 antibody [EPR22851-145]