Fig 1: miR-340 targets the 3’-UTR of REV3L(A) Binding site of miR-340 to the 3’UTRregion of REV3L. miR-340 binds with 7 complimentary bases at the seed sequence and deletion of the miR-340 binding seed sequence in the 3’UTR region of REV3L. (B) Sequencing result of 3’UTR of REV3L in CCD-18-Co normal colon cell line and HCT-116, DLD-1 colon cancer cell lines at miR-340 binding site. (C) HCT-116 and DLD-1 colon cancer cells were transfected with 50nM miR-340 mimics. mRNA expression of REV3L upon miR-340 transfection were profiled by qPCR. GAPDH gene was used as endogenous control. (D) The relative miR-340 expression level in miR-340 mimic transfected HCT-116 and DLD-1 colon cancer cell lines compared to the not transfected cells and positive control miR transfected cells. U6 expression was used as endogenous control. (E) The 3’ UTR of REV3L cloned into psi-CHECK Luciferase vector with XhoI and NotI digestion sites. (F, G) Luciferase reporter assay showed that miR-340 directly targets the REV3L 3’-UTR-luciferase reporter (wild type or deleted miR-340 binding seed sequence), in HCT-116 and DLD-1 cells incubated with the miR-340 mimic for 48h before analysis. The firefly luciferase activity of the reporter was normalized to the internal Renilla luciferase activity. The data are presented as means with SDs for three independent experiments. *p<0.05; **p<0.01; ***p<0.001.
Fig 2: miR-340 inhibits tumorigenicity of colon cancer cells in vivo(A) Control and miR-340 overexpressing stable HCT-116 colon cancer cell line was made by transfecting pCMV and pCMV-miR-340 in HCT-116 colon cancer cells. 48 h after transfection, the cells were treated with desired concentrations of G418. mRNA expression of REV3L in not treated, pCMV and pCMV-miR-340 transfected HCT-116 cells selected with G418 was analyzed by qPCR. GAPDH gene was used as endogenous control. (B) The relative miR-340 expression level in pCMV-miR-340 transfected G418 selected HCT-116 stable colon cancer cell line compared to pCMV transfected G418 selected cells was analyzed by qPCR. U6 expression was used as endogenous control. (C) The number of colonies and the size of the colonies formed by the HCT-116-pCMV-miR-340 was less compared to HCT-116-pCMV stable cell line as analyzed by the soft agar colony formation assay. (D) The HCT-116-pCMV and HCT-pCMV-miR-340 stable colon cancer cells were subcutaneously injected into nude mice model (n=3). The tumor volume was calculated from the measured width and length. Graph represents smaller tumor volume in HCT-116-pCMV-miR-340 injected groups compared to HCT-116-pCMV injected groups. The data are presented as means with SDs for three independent experiments. *p<0.05; **p<0.01; ***p<0.001. (E) Schematic model of the REV3L location and interactions and the regulatory pathways involving miR-340 and REV3L in colon cancer. REV3L, a nuclear bound protein upon mutation breaks its known protein interactions and is mislocalized to the cytoplasm in colon cancer. miR-340 targets and downregulates REV3L thereby regulating the proliferation and apoptosis of colon cancer cells.
Fig 3: mRNA expression pattern of REV3L, miR-340 and RNF-130 in normal colon and colon cancer cell lines(A) mRNA expression levels of REV3L in CCD-18-Co normal coloncell line and HCT-116, DLD-1 colon cancer cells. Cell lines were analyzed for REV3L mRNA levels by qPCR. GAPDH gene was used as endogenous control. (B) miR-340 expression levels were quantified in CCD-18-Co normal colon cell line and HCT-116, DLD-1 colon cancer cell lines by qPCR. Expression of miR for each colon cancer cell line was compared with the normal cell line CCD-18-Co. U6 expression was used as endogenous control. (C) mRNA expression levels of RNF-130, the intronic region of the gene that codes for miR-340 in CCD-18-Co normal colon cell line and HCT-116, DLD-1 colon cancer cell lines. GAPDH gene was used as endogenous control. Consecutive to miR-340 expression, RNF-130 mRNA expression levels decreases in HCT-116, DLD-1 colon cancer cells compared to CCD-18-Co normal colon cell line. The data are presented as means with SDs for three independent experiments. *p<0.05; **p<0.01; ***p<0.001.
Fig 4: miR-340 regulates colon cancer via MAPK pathway and downregulation of REV3L sensitizes cancer cells to 5-Flurouracil in vitro(A) Cells were transfected with pCMV, pCMV-miR-340 plasmids or not transfected (control); 48 h after transfection, protein samples were used to perform western blot analysis. The downstream targets of MAPK pathway total ERK, p-ERK, total p38, p-p38, total JNK 1/2, p-JNK 1/2, was analyzed. β-Actin was used as a loading control. (B, C) The relative protein expressions of the control, pCMV and pCMV-miR-340 transfected HCT-116 and DLD-1 colon cancer cells were compared and represented as graph. (D) The sensitization of miR-340 treated HCT-116 and DLD-1 colon cancer cells to 5-FU was analyzed by cell proliferation MTS assay. HCT-116 and DLD-1 colon cancer cells were transfected with pCMV and pCMV-miR-340 plasmids followed by 5-FU treatment at concentration of 15μg/ml and incubated for 48 h. The cell viability in pCMV-miR-340 and 5-FU treated cells was compared to pCMV and 5-FU treated cells. The data are presented as means with SDs for three independent experiments. *p<0.05; **p<0.01; ***p<0.001.
Fig 5: Transfection of colon cancer cells with miR-340 suppresses migration and metastatic properties of cells in vitro(A, B) HCT-116 and DLD-1 colon cancer celllines that were transfected with pCMV-miR-340, or pCMV were assessed for migration with the wound healing assay. The area of the wound was measured at 0, 24 and 48 h. Graph represents the relative percentages of wound closure of the pCMV and pCMV-miR-340 transfected cells. (C, D) HCT-116 and DLD-1 colon cancer cells were transfected with pCMV and pCMV-miR-340 plasmids. mRNA expression of the markers for metastasis MMP2 and MMP-9 upon miR-340 transfection were profiled by qPCR. GAPDH gene was used as endogenous control. The data are presented as means with SDs for three independent experiments. *p<0.05; **p<0.01;***p<0.001.
Supplier Page from OriGene Technologies for MIR340 Human MicroRNA Expression Plasmid (MI0000802)