MicroRNA-8073: Tumor suppressor and potential therapeutic treatment

Abstract

The comprehensive screening of intracellular and extracellular microRNAs was performed to identify novel tumor suppressors. We found that miR-8073 was present in exosome and predominantly exported from colorectal cancer cells. Treatment with a synthetic miR-8073 mimic resulted in a dramatic decrease in the proliferation of various types of cancer cells, which was not observed in similarly treated normal cells. As little is known about the biological functions of miR-8073, its target mRNAs were analyzed by both mRNA expression and in silico sequence analyses, leading to five probable target candidates (FOXM1, MBD3, CCND1, KLK10, and CASP2) that enhance survival during the regulation of the cell cycle, cell proliferation, and apoptosis. We experimentally confirmed that miR-8073 binds the 3'-UTR of each of these mRNA target candidates and that the introduction of a synthetic miR-8073 mimic into cancer cells reduced levels of protein expression. Finally, the antiproliferative effects of miR-8073 were validated in vivo: the subcutaneous injection of a synthetic miR-8073 mimic suppressed colorectal tumor volume to 43% in tumor-bearing xenografted mice. These results suggest that because miR-8073 binds, and thus reduces the levels of, these oncogenic targets, cancer cells must actively downregulate miR-8073 as a survival mechanism. The introduction of miR-8073 into tumors could thus inhibit tumor growth, indicating its great potential for cancer therapeutics.

Fig 1

Intracellular, extracellular (A) or exosomal (B) miR-8073 expression in 6x10⁴ colorectal cancer cell HCT116 and normal cell HCoEpic. In Fig 1B, exosomes that were extracted from the same amount of the cell cultured supernatant used in Fig 1A, were assayed for microRNA expression profiling. MiR-8073 was one of microRNAs that was most exported to extracellular space in the cancer cells but much less in the normal cells. The error bar indicates standard deviation. The star indicates p<0.05 in student’s t-test.

Fig 2. Viability of human colorectal cancer cells HCT116, and other types of cancer cells or normal cells.

Viability of human colorectal cancer cells HCT116, and (B) other types of cancer cells or normal cells at fifth day from transfection with miR-8073 mimic or negative control sequence (miR-NC). Concentrations of the synthetic nucleic acids used for the transfection were either 0.03nM to 30nM (A) or 30nM (B). In Fig 2B, cells tested were colorectal (HCT116, HT29), pancreatic (Panc-1, Panc0.05), breast (MCF7), lung (A549) cancer cells, and normal cells derived from microvascular endothelium (HMVEC) or mammary epithelium (184B5). The error bar indicates standard deviation. The star indicates p<0.05 in student’s t-test.

Fig 3. Apoptotic activity induced by miR-8073 mimic in colorectal cancer cell HCT116.

Caspase-3/7 activity was measured after transfection for 72h, 30nM of miR-8073 mimic or a negative control sequence (miR-NC). The level of caspase-3 and caspase-7 activity in the mock treated cells was set at 100%. The error bar indicates the standard deviation.

Fig 4. Potential mRNA targets of miR-8073 and their predicted binding sites.

Fig 5. miR-8073 targeted FOXM1, MBD3, CCND1, KLK10 and CASP2 in HCT116 cells.

Relative luciferase activity of HCT116 cells co-transfected with either 3nM of miR-8073 mimic or microRNA negative control (miR-NC) and with the luciferase reporter construct in which the 3’-UTR of each gene of interest was inserted. The control indicates the cells transfected with the same luciferase reporter plasmid without the added 3’UTRs, and the activity level of the control with miR-NC was set as 1.0. The error bars indicate the standard error of triplicate samples. The star indicates p<0.05 in student’s t-test.

Fig 6. Western blot analysis of FOXM1, MBD3, CCND1, KLK10 and CASP2 expression in the HCT116 cells.

Protein expression levels of FOXM1, MBD3, CCND1, KLK10 and CASP2 in HCT116 cells transfected with either 30nM of miR-8073 mimic or microRNA negative control (miR-NC) detected by Western blots. The images were numerated by software and ratios of the protein expression signal of miR-8073 treated cells over miR-NC treated cells were calculated.

Fig 7

Administration of miR-8073 mimic significantly reduced colorectal tumor volume (A) and weight (B) in xenografted mice. BALB/c nude mice was transplanted with 5x10⁶ HCT116 cells two days before the treatment. The mice were treated with either 2nmol of miR-8073 mimic or microRNA negative control (miR-NC) mixed with atelocollagen at the day 0, 2, 5 (arrows). The tumor weight (B) was measured at the day 13. The error bars indicate the standard error in five mice per treatment group. The star indicates p<0.05 in student’s t-test.