Focused screening reveals functional effects of microRNAs differentially expressed in colorectal cancer

Author(s)

Sastre, Danuta ^{[1, 2]} ; Baiochi, Joao ^[1] ; de Souza Lima, Ildercilio Mota ^[1] ; de Souza, Felipe Canto ^[1] ; Corveloni, Amanda Cristina ^[1] ; Thome, Carolina Hassib ^[3] ; Faca, Vitor Marcel ^[3] ; dos Santos Schiavinato, Josiane Lilian ^[1] ; Covas, Dimas Tadeu ^[1] ; Panepucci, Rodrigo Alexandre ^[1]

Affiliation

^[1] Univ Sao Paulo, Reg Blood Ctr, Ribeirao Preto Med Sch, Lab Funct Biol LFBio, Ctr Cell Based Therapy CTC, R Ten Catao Roxo 2501, BR-14051140 Ribeirao Preto, SP - Brazil

^[2] Fed Univ Para, Lab Human & Med Genet, Rua Augusto Correa 01, BR-66075110 Belem, Para - Brazil

^[3] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, Av Bandeirantes 300, BR-14049900 Ribeirao Preto, SP - Brazil

• Document type: Journal article
• Source: BMC CANCER; v. 19, n. 1 DEC 21 2019.
• Web of Science Citations: 0

Abstract

Background Colorectal cancer (CRC) is still a leading cause of death worldwide. Recent studies have pointed to an important role of microRNAs in carcinogenesis. Several microRNAs are described as aberrantly expressed in CRC tissues and in the serum of patients. However, functional outcomes of microRNA aberrant expression still need to be explored at the cellular level. Here, we aimed to investigate the effects of microRNAs aberrantly expressed in CRC samples in the proliferation and cell death of a CRC cell line. Methods We transfected 31 microRNA mimics into HCT116 cells. Total number of live propidium iodide negative (PI-) and dead (PI+) cells were measured 4 days post-transfection by using a high content screening (HCS) approach. HCS was further used to evaluate apoptosis (via Annexin V and PI staining), and to discern between intrinsic and extrinsic apoptotic pathways, by detecting cleaved Caspase 9 and 8, respectively. To reveal mRNA targets and potentially involved mechanisms, we performed microarray gene expression and functional pathway enrichment analysis. Quantitative PCR and western blot were used to validate potential mRNA targets. Results Twenty microRNAs altered the proliferation of HCT116 cells in comparison to control. miR-22-3p, miR-24-3p, and miR-101-3p significantly repressed cell proliferation and induced cell death. Interestingly, all anti-proliferative microRNAs in our study had been previously described as poorly expressed in the CRC samples. Predicted miR-101-3p targets that were also downregulated by in our microarray were enriched for genes associated with Wnt and cancer pathways, including MCL-1, a member of the BCL-2 family, involved in apoptosis. Interestingly, miR-101-3p preferentially downregulated the long anti-apoptotic MCL-1 L isoform, and reduced cell survival specifically by activating the intrinsic apoptosis pathway. Moreover, miR-101-3p also downregulated IL6ST, STAT3A/B, and MYC mRNA levels, genes associated with stemness properties of CRC cells. Conclusions microRNAs upregulated in CRC tend to induce proliferation in vitro, whereas microRNAs poorly expressed in CRC halt proliferation and induce cell death. We provide novel evidence linking preferential inhibition of the anti-apoptotic MCL-1 L isoform by miR-101-3p and consequent activation of the intrinsic apoptotic pathway as potential mechanisms for its antitumoral activity, likely due to the inhibition of the IL-6/JAK/STAT signaling pathway. (AU)

• FAPESP's process: 13/08135-2 - CTC - Center for Cell-Based Therapy
• Grantee: Dimas Tadeu Covas
• Support type: Research Grants - Research, Innovation and Dissemination Centers - RIDC