MicroRNA-340 inhibits the proliferation and promotes the apoptosis of colon cancer cells by modulating REV3L

Abstract

DNA Directed Polymerase Zeta Catalytic Subunit (REV3L) has recently emerged as an important oncogene. Although the expressions of REV3L are similar in normal and cancer cells, several mutations in REV3L have been shown to play important roles in cancer. These mutations cause proteins misfolding and mislocalization, which in turn alters their interactions and biological functions. miRNAs play important regulatory roles during the progression and metastasis of several human cancers. This study was undertaken to determine how changes in the location and interactions of REV3L regulate colon cancer progression. REV3L protein mislocalization confirmed from the immunostaining results and the known interactions of REV3L was found to be broken as seen from the PLA assay results. The mislocalized REV3L might interact with new proteins partners in the cytoplasm which in turn may play role in regulating colon cancer progression. hsa-miR-340 (miR-340), a microRNA down-regulated in colon cancer, was used to bind to and downregulate REV3L, and found to control the proliferation and induce the apoptosis of colon cancer cells (HCT-116 and DLD-1) via the MAPK pathway. Furthermore, this down-regulation of REV3L also diminished colon cancer cell migration, and down-regulated MMP-2 and MMP-9. Combined treatment of colon cancer cells with miR-340 and 5-FU enhanced the inhibitory effects of 5-FU. In addition, in vivo experiments conducted on nude mice revealed tumor sizes were smaller in a HCT-116-miR-340 injected group than in a HCT-116-pCMV injected group. Our findings suggest mutations in REV3L causes protein mislocalization to the cytoplasm, breaking its interaction and is believed to form new protein interactions in cytoplasm contributing to colon cancer progression. Accordingly, microRNA-340 appears to be a good candidate for colon cancer therapy.

Keywords: REV3L; colon cancer; interactions; miR-340; mislocalization.

Figure 1. Immunostaining analysis of the location of REV3L in normal colon and colon cancer cell lines

Normal colon cell line CCD-18-Co shows REV3L nuclear location, coloncancer cell lines HCT-116 and DLD-1 shows mislocalisation of the protein upon immunostaining with REV3L antibody and counter staining with DAPI. The images were analyzed using an LSM 710 confocal microscope (Carl Zeiss). Scale bar 200μm.

Figure 2. Protein-Protein interaction of REV3L in Normal colon and Colon cancer cell lines

(A) PLA assay for REV3L interaction with REV1 and MAD2L2 in CCD-18-Co normalcolon and HCT-116, DLD-1 colon cancer cells. The red blobs indicate the protein interactions. The images were analyzed using an LSM 710 confocal microscope (Carl Zeiss). (B) The number of interactions between REV3L-REV1, REV3L-MAD2L2, REV1-MAD2L2 in CCD-18-Co normal colon and HCT-116, DLD-1 colon cancer cells plotted as graph. The data are presented as means with SDs for three independent experiments. ^*p<0.05; ^**p<0.01; ^***p<0.001. Scale bar 20μm.

Figure 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.

Figure 4. 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.

Figure 5. Ectopic expression of miR-340 suppresses colon cancer cell proliferation and induces apoptosis in vitro

(A, B) The short term effects of ectopic expression of miR-340 on theproliferation of HCT-116 and DLD-1 colon cancer cells examined by cell proliferation assay (MTS assay). Graph represents the percentage of viable cells in control and treated groups. (C) Effects of ectopic expression of miR-340 on the apoptosis of HCT-116 and DLD-1 colon cancer cells examined by TUNEL assay. The number of TUNEL positive cells were counted and made as graph. (D, E) Effects of overexpression of miR-340 on the colony formation or clonogenic ability of HCT-116 and DLD-1 colon cancer cells. Culture dishes represents the number of colonies formed upon transfection with pCMV and pCMV-miR-340. Graph represents the number of colonies formed in pCMV-miR-340 transfected cells normalized to the number of colonies formed by the pCMV transfected cells. The data are presented as means with SDs for three independent experiments. ^*p<0.05; ^**p<0.01; ^***p<0.001. Scale bar 20μm.

Figure 6. 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.

Figure 7. 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.

Figure 8. 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.