The online version of this article (https://doi.org/10.1186/s12943-017-0751-3) contains supplementary material, which is available to authorized users.
Colorectal cancer (CRC) is a severe health problem worldwide. Clarifying the mechanisms for the deregulation of oncogenes and tumour suppressors in CRC is vital for its diagnosis, treatment, prognosis and prevention. Hu antigen R (HuR), which is highly upregulated in CRC, functions as a pivotal oncogene to promote CRC progression. However, the underlying cause of its dysregulation is poorly understood.
In CRC tissue sample pairs, HuR protein levels were measured by Western blot and immunohistochemical (IHC) staining, respectively. HuR mRNA levels were also monitored by qRT-PCR. Combining meta-analysis and microRNA (miRNA) target prediction software, we predicted miRNAs that targeted HuR. Pull-down assay, Western blot and luciferase assay were utilized to demonstrate the direct binding of miR-22 on HuR’s 3’-UTR. The biological effects of HuR and miR-22 were investigated both in vitro by CCK-8, EdU and Transwell assays and in vivo by a xenograft mice model. JASPAR and SABiosciences were used to predict transcriptional factors that could affect miR-22. Luciferase assay was used to explore the validity of putative Jun binding sites for miR-22 regulation. ChIP assay was performed to test the Jun’s occupancy on the C17orf91 promoter.
We observed a significant upregulation of HuR in CRC tissue pairs and confirmed the oncogenic function of HuR both in vitro and in vivo. We found that an important tumour-suppressive miRNA, miR-22, was significantly downregulated in CRC tissues and inversely correlated with HuR in both CRC tissues and CRC cell lines. We demonstrated that miR-22 directly bound to the 3’-UTR of HuR and led to inhibition of HuR protein, which repressed CRC proliferation and migration in vitro and decelerated CRC xenografted tumour growth in vivo. Furthermore, we found that the onco-transcription factor Jun could inhibit the transcription of miR-22.
Our findings highlight the critical roles of the Jun/miR-22/HuR regulatory axis in CRC progression and may provide attractive potential targets for CRC prevention and treatment.
Additional file 1: Table S1. Clinical features of colorectal cancer patients. (DOCX 17 kb)12943_2017_751_MOESM1_ESM.docx
Additional file 2: Table S2. Sequences of siRNAs, probe and primers. (DOCX 16 kb)12943_2017_751_MOESM2_ESM.docx
Additional file 3: Figure S1. HuR protein is significantly upregulated in CRC tissues and negatively correlated with CRC patient survival. (a) HuR levels in normal colon, normal rectum, colon adenocarcinoma and rectal adenocarcinoma in the TCGA dataset analysed by Oncomine. (b) Kaplan-Meier curve showing the negative correlation of HuR level and CRC patients’ survival. (TIFF 167 kb)12943_2017_751_MOESM3_ESM.tif
Additional file 4: Figure S2. The efficiencies of siRNA and overexpression vector of HuR (a) or Jun (b). (TIFF 377 kb)12943_2017_751_MOESM4_ESM.tif
Additional file 5: Figure S3. HuR functions as an oncogene in CRC. (a-c) HuR promoted SW480 proliferation. a: CCK-8 assays; b and c: EdU assays. (d and e) HuR promoted SW480 migration. (f-h) HuR accelerated CRC xenografted tumour growth. f: Photos of CRC tumours; g: Tumour volume curves; h: Tumour weights. (i) Western blot analysis of HuR levels in CRC xenografted tumours. (j and k) HE staining and IHC staining for HuR and Ki-67 in xenografted tumours. **P < 0.01; ***P < 0.001. (TIFF 5991 kb)12943_2017_751_MOESM5_ESM.tif
Additional file 6: Figure S4. (a and b) The Kaplan-Meier curve revealed the positive correlation of miR-22 (a) or miR-129 (b) level and CRC patients’ survival. (c and d) The transfection efficiencies of miR-22 (c) or miR-129 (d) mimics or inhibitors in 3 CRC cell lines. ***P < 0.001. (TIFF 1147 kb)12943_2017_751_MOESM6_ESM.tif
Additional file 7: Figure S5. Pri-miR-22 is downregulated in CRC tissues compared with that in normal adjacent tissues. *P < 0.05. (TIFF 186 kb)12943_2017_751_MOESM7_ESM.tif
Edwards BK, Ward E, Kohler BA, Eheman C, Zauber AG, Anderson RN, Jemal A, Schymura MJ, Lansdorp-Vogelaar I, Seeff LC, et al. Annual report to the nation on the status of cancer, 1975-2006, featuring colorectal cancer trends and impact of interventions (risk factors, screening, and treatment) to reduce future rates. Cancer. 2010;116:544–73. CrossRefPubMedPubMedCentral
Corcoran RB, Ebi H, Turke AB, Coffee EM, Nishino M, Cogdill AP, Brown RD, Della Pelle P, Dias-Santagata D, Hung KE, et al. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov. 2012;2:227–35. CrossRefPubMedPubMedCentral
Embade N, Fernandez-Ramos D, Varela-Rey M, Beraza N, Sini M, Gutierrez de Juan V, Woodhoo A, Martinez-Lopez N, Rodriguez-Iruretagoyena B, Bustamante FJ, et al. Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation. Hepatology. 2012;55:1237–48. CrossRefPubMedPubMedCentral
Alvarez-Diaz S, Valle N, Ferrer-Mayorga G, Lombardia L, Herrera M, Dominguez O, Segura MF, Bonilla F, Hernando E, Munoz A. MicroRNA-22 is induced by vitamin D and contributes to its antiproliferative, antimigratory and gene regulatory effects in colon cancer cells. Hum Mol Genet. 2012;21:2157–65. CrossRefPubMed
Tu M, Li H, Lv N, Xi C, Lu Z, Wei J, Chen J, Guo F, Jiang K, Song G, et al. Vasohibin 2 reduces chemosensitivity to gemcitabine in pancreatic cancer cells via Jun proto-oncogene dependent transactivation of ribonucleotide reductase regulatory subunit M2. Mol Cancer. 2017;16:66. CrossRefPubMedPubMedCentral
- The Jun/miR-22/HuR regulatory axis contributes to tumourigenesis in colorectal cancer
- BioMed Central
Neu im Fachgebiet Onkologie
Mail Icon II