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. 2017 Dec 26;9(4):5155–5168. doi: 10.18632/oncotarget.23703

Figure 8. miR-340 inhibits tumorigenicity of colon cancer cells in vivo.

Figure 8

(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.