HCT116 cells were subcutaneously injected into the flanks of 5-week old mice. When solid tumors were established (the average tumor volume by 100 mm^3, 6 days after cell implantation), in vivo-jetPEI/miRNA complexes were treated. Treatment with in vivo-jetPEI/miRNA complexes was done by intratumoral injection with 3 times at 3 day intervals. In vivo-jetPEI was used as a delivery agent (miRNA 10 μg and in vivo-jetPEI reagent 1.2 µl in 100 µl of 5% glucose for one injection).

Depending on the function of their target genes, microRNAs (miRNAs) act as either tumor suppressors or oncogenes. Therefore, miRNAs represent a novel therapeutic strategy for prevention and management of cancer by targeting of onco-miRNAs or mimicking of tumor suppressor miRNAs. Herein, we identified novel tumor suppressor miRNAs and investigated their molecular mechanisms. To identify novel tumor suppressor miRNAs, we used 532 human miRNA mimic libraries and measured cell viability using MTS assays. The function of miR-4779 was then analyzed using cell cycle analyses and apoptosis, colony forming, and soft agar assays. Target genes of miR-4779 were predicted using TargetScan and miRDB databases and were confirmed using luciferase assays. Levels of miR-4779 and target genes in colon cancer tissue samples from patients were evaluated using qRT-PCR and western blotting analyses. Finally, in vivo tumor suppressive effects of miR-4779 were evaluated in HCT116 xenografts. In this study, miR-4779 inhibited cancer cell growth by inducing apoptosis and cell cycle arrest, and the putative survival factors PAK2 and CCND3 were identified as direct targets of miR-4779. In subsequent experiments, PAK2 knockdown induced cell cycle arrest and CCND3 knockdown induced cell cycle arrest and apoptosis. In addition, miR-4779 suppressed tumor growth and tumorigenesis in an in vivo HCT116 xenograft model. Finally, miR-4779 expression was low in 9 of 10 colon cancer tissues, whereas PAK2 and CCND3 expressions were significantly high in colon cancer tissues. The novel tumor suppressor miR-4779 inhibits cancer cell growth via cell cycle arrest and apoptosis by directly targeting PAK2 and CCND3. The present data indicate the potential of miR-4779 as a therapeutic target for miRNA-based cancer therapy.