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Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer.

ABSTRACT: MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.

SUBMITTER: Christensen LL

PROVIDER: S-EPMC4043686 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer.

Christensen Lise Lotte LL Holm Anja A Rantala Juha J Kallioniemi Olli O Rasmussen Mads H MH Ostenfeld Marie S MS Dagnaes-Hansen Frederik F Øster Bodil B Schepeler Troels T Tobiasen Heidi H Thorsen Kasper K Sieber Oliver M OM Gibbs Peter P Lamy Philippe P Hansen Torben F TF Hansen Torben F TF Jakobsen Anders A Riising Eva M EM Helin Kristian K Lubinski Jan J Hagemann-Madsen Rikke R Laurberg Søren S Ørntoft Torben F TF Andersen Claus L CL

PloS one 20140603 6

MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs ...[more]

PMID: 24892549

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Project description:Background and aimsSporadic colorectal cancer (CRC) development is a sequential process showing age-dependency, uncontrolled epithelial proliferation and decreased apoptosis. During juvenile growth cellular proliferation and apoptosis are well balanced, which may be perturbed upon aging. Our aim was to correlate proliferative and apoptotic activities in aging human colonic epithelium and colorectal cancer. We also tested the underlying molecular biology concerning the proliferation- and apoptosis-regulating gene expression alterations.Materials and methodsColorectal biopsies from healthy children (n1?=?14), healthy adults (n2?=?10), adult adenomas (n3?=?10) and CRCs (n4?=?10) in adults were tested for Ki-67 immunohistochemistry and TUNEL apoptosis assay. Mitosis- and apoptosis-related gene expression was also studied in healthy children (n1?=?6), adult (n2?=?41) samples and in CRC (n3?=?34) in HGU133plus2.0 microarray platform. Measured alterations were confirmed with RT-PCR both on dependent and independent sample sets (n1?=?6, n2?=?6, n3?=?6).ResultsMitotic index (MI) was significantly higher (p<0.05) in intact juvenile (MI?=?0.33±0.06) and CRC samples (MI?=?0.42±0.10) compared to healthy adult samples (MI?=?0.15±0.06). In contrast, apoptotic index (AI) was decreased in children (0.13±0.06) and significantly lower in cancer (0.06±0.03) compared to healthy adult samples (0.17±0.05). Eight proliferation- (e.g. MKI67, CCNE1) and 11 apoptosis-associated genes (e.g. TNFSF10, IFI6) had altered mRNA expression both in the course of normal aging and carcinogenesis, mainly inducing proliferation and reducing apoptosis compared to healthy adults. Eight proliferation-associated genes including CCND1, CDK1, CDK6 and 26 apoptosis-regulating genes (e.g. SOCS3) were differently expressed between juvenile and cancer groups mostly supporting the pronounced cell growth in CRC.ConclusionColorectal samples from children and CRC patients can be characterized by similarly increased proliferative and decreased apoptotic activities compared to healthy colonic samples from adults. Therefore, cell kinetic alterations during colorectal cancer development show uncontrolled rejuvenescence as opposed to the controlled cell growth in juvenile colonic epithelium.

Project description:ObjectiveTo identify the expression of kinetochore scaffold 1 (KNL1) in colorectal tumor tissues and to clarify the role of this gene in the proliferation capability of colorectal cancer cells.MethodsA total of 108 paired colorectal tumor and normal tissue samples were collected from patients with colorectal cancer and subjected to quantitative polymerase chain reaction and immunohistochemistry analyses. Expression levels of KNL1 mRNA and protein were compared between tumor and normal tissues, and KNL1 levels were evaluated in relation to the patients' tumor differentiation, sex, lymph node metastasis, TNM stage, infiltration depth, age, and tumor location. Survival curves were also constructed and compared between patients with tumor samples with and without KLN1 protein expression. KNL1 was under-expressed in colorectal cancer cells in vitro using lentiviral transfection with short hairpin RNA, and its function was evaluated by proliferation, colony-formation, and apoptosis assays. Expression levels of BUB1 protein were also compared between tumor and normal tissues, and the correlation between KNL1 expression and BUB1 expression in colorectal cancer tissues was examined.ResultsKNL1 mRNA and protein were both highly expressed in colorectal tumor tissues compared with paired normal tissues. KNL1 downregulation significantly inhibited colorectal cancer cell proliferation and colony formation, and promoted apoptosis. KNL1 protein expression was significantly associated with tumor differentiation, but not with sex, lymph node metastasis, TNM stage, infiltration depth, age, or tumor location. KNL1 protein expression was also significantly associated with poorer survival. Moreover, there was a significant correlation between KNL1 and BUB1 in colorectal cancer tissues.ConclusionsKNL1 plays an effective role in decreasing apoptosis and promoting the proliferation of colorectal cancer cells, suggesting that its inhibition may represent a promising therapeutic approach in patients with colorectal cancer.

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Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer.

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Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer.

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