SSRP1 promotes colorectal cancer progression and is negatively regulated by miR-28-5p

Abstract

In this study, microarray data analysis, real-time quantitative PCR and immunohistochemistry were used to detect the expression levels of SSRP1 in colorectal cancer (CRC) tissue and in corresponding normal tissue. The association between structure-specific recognition protein 1 (SSRP1) expression and patient prognosis was examined by Kaplan-Meier analysis. SSRP1 was knocked down and overexpressed in CRC cell lines, and its effects on proliferation, cell cycling, migration, invasion, cellular energy metabolism, apoptosis, chemotherapeutic drug sensitivity and cell phenotype-related molecules were assessed. The growth of xenograft tumours in nude mice was also assessed. MiRNAs that potentially targeted SSRP1 were determined by bioinformatic analysis, Western blotting and luciferase reporter assays. We showed that SSRP1 mRNA levels were significantly increased in CRC tissue. We also confirmed that this upregulation was related to the terminal tumour stage in CRC patients, and high expression levels of SSRP1 predicted shorter disease-free survival and faster relapse. We also found that SSRP1 modulated proliferation, metastasis, cellular energy metabolism and the epithelial-mesenchymal transition in CRC. Furthermore, SSRP1 induced apoptosis and SSRP1 knockdown augmented the sensitivity of CRC cells to 5-fluorouracil and cisplatin. Moreover, we explored the molecular mechanisms accounting for the dysregulation of SSRP1 in CRC and identified microRNA-28-5p (miR-28-5p) as a direct upstream regulator of SSRP1. We concluded that SSRP1 promotes CRC progression and is negatively regulated by miR-28-5p.

Keywords: SSRP1; colorectal cancer; microRNA; progression.

Figure 1

SSRP1 expression is upregulated in CRC. A, SSRP1 expression levels in CRC tissue and normal tissue in two independent cohorts (GSE32323 and GSE4107). B, SSRP1 mRNA expression levels in 10 paired tissue tumour samples and normal tissue samples. C, SSRP1 protein expression levels in five CRC cell lines and the normal colon NCM460 cell line. D and E, Representative photographs of IHC staining for SSRP1 in normal and CRC tissue. F, SSRP1 expression was up‐regulated in 85% of CRC patients according to IHC. *P < 0.05, **P < 0.01, and ***P < 0.001. IHC: immunohistochemical staining

Figure 2

SSRP1 modulates CRC cell proliferation and the cell cycle in HCT116 cells. A, SSRP1 knockdown or overexpression reduced or accelerated the proliferation rate of cells, respectively. B, Representative data show that the overexpression of SSRP1 significantly promoted tumour growth in a nude mouse xenograft model (n = 6). C, Tumours were dissected, and tumours from the two groups are shown. D, The effects of SSRP1 knockdown on the cell cycle were determined. The percentages of cells in the G1, S and G2/M phases of the cell cycle are presented. The bars represent the mean values of six independent tests (mean ± SD). E, The effects of SSRP1 overexpression on the cell cycle were determined. F, Cell cycle‐related molecules were screened by Western blot analysis, and SSRP1 expression levels altered the expression of cell‐cycle‐related proteins in HCT116 cells. *P < 0.05, and **P < 0.01. p21: cyclin‐dependent kinase inhibitor 1A; p27: Cyclin‐dependent kinase inhibitor 1B; 14‐3‐3: YWHAS, epithelial cell marker protein 1

Figure 3

SSRP1 modulates cell motility and phenotype‐related molecules in CRC. A, Cell migration was assessed by transwell migration assay in SSRP1 knockdown cells. B, Cell invasion was assessed by transwell invasion assay in SSRP1 knockdown cells. C and D, Cell migration and invasion were assessed by transwell assay in SSRP1 overexpressing cells. E, Phenotype‐related molecules were screened by Western blot analysis

Figure 4

SSRP1 modulates the sensitivity of CRC cells to chemotherapeutic drugs. A and B, SSRP1 knockdown promotes cell apoptosis. C, siRNA‐mediated SSRP1 knockdown increases the sensitivity of CRC cells to chemotherapeutics

Figure 5

Identification of SSRP1 as a novel target for miR‐28‐5p. A, The miR‐28‐5p target sites in the 3’UTR of SSRP1 are shown as a schematic representation. B, miR‐28‐5p expression levels in CRC tissue and adjacent/normal tissue from the mRNA microarray data obtained from GSE35834. C, Low miR‐28‐5p levels were associated with decreased disease‐free survival in CRC patients in the GSE29623 dataset. D, Western blotting showed the expression of SSRP1 protein in SW480 and HCT116 cells transfected with miR‐28‐5p. E, Wild‐type or mutant 3’UTR constructs of SSRP1 were cloned into a pMIR‐REPORT luciferase vector and cotransfected with miR‐28‐5p mimics into HEK293 cells. Renilla luciferase activity was normalized to firefly luciferase activities. All assays were performed in triplicate and repeated at least three times. F, An inverse correlation was found between miR‐28‐5p expression and SSRP1 expression in CRC samples (GSE29623 dataset) (Spearman’s correlation, P < 0.0001, R = −0.731). The data are expressed as the mean ± SD of three independent experiments; **P < 0.01, and ***P < 0.001