Small Nuclear Ribonucleoprotein Polypeptide N Accelerates Malignant Progression and Poor Prognosis in Colorectal Cancer Transcriptionally Regulated by E2F8

Abstract

Colorectal cancer is a major cause of death worldwide, and the identification of new diagnostic and prognostic biomarkers is crucial to develop new strategies to avoid colorectal cancer-related deaths. Small nuclear ribonucleoprotein polypeptide N (SNRPN) is an imprinted gene that plays an important role in various neurodevelopmental disabilities. In this study, SNRPN was highly expressed in colorectal cancer tissues and involved in the progression of this disease. Immunohistochemistry analysis of 1,310 colorectal cancer tissue samples showed that SNRPN highly expressed in cancer tissues than in adjacent tissues and was mainly localized in the nucleus. Clinical pathological factor analysis demonstrated that higher expression of SNRPN was significantly associated with larger tumor size, location of the tumor on the left-sided colon, neural invasion, and distant metastasis. Univariate and multivariate analyses showed that SNRPN expression was an independent risk factor for survival, with high expression levels indicating worse overall survival. Both in vitro and in vivo experiments confirmed that high expression of SNRPN was associated with tumor proliferation, cell cycle, and metastasis. Knocking down SNRPN blocked the cell cycle at the G2/M phase transition and promoted tumor cell apoptosis, inhibiting the progression of colorectal cancer. To explore the up-steam of SNRPN, we found by luciferase reporter assay and chromosomal immunoprecipitation assay that E2F8 was a transcriptional regulator up-steam of SNRPN in colorectal cancer. Systematic studies of SNRPN will help us discover new regulatory molecules and provide a theoretical basis for finding new molecular targets for this disease.

Keywords: E2F8; SNRPN; colorectal cancer; metastasis; proliferation.

Figure 1

SNRPN is highly expressed in colorectal cancer tumor tissues. Immunohistochemistry (IHC) staining of SNRPN in colorectal cancer tissues. (A) Representative images of IHC staining intensity: normal—no staining, grade 1—weak staining, grade 2—moderate staining, and grade 3—strong staining. (B) IHC staining score of SNRPN was significantly higher in tumor tissues than in normal tissues. (C) IHC staining score of SNRPN in four stages. ^*P < 0.05; ^****P < 0.001. P value: Wilcoxon signed rank test (matched pairs).

Figure 2

Downregulation of SNRPN in tumor tissues indicates poor overall survival. Kaplan–Meier survival curve with expression levels of SNRPN in tumor tissues for colorectal cancer patients after primary tumor resection. HR, hazard ratio; CI, confidence interval; P value, log-rank test.

Figure 3

Downregulation of SNRPN suppresses malignant cell proliferation in colorectal cancer cell. (A) RT-PCR analysis of SNRPN mRNA levels in common colorectal cancer cell lines. (B) RT-PCR analysis of SNRPN mRNA levels after transduction with lentivirus containing shSNRPN(S1) and shSNRPN(S2) in SW1116 cells and shSNRPN(S2) in HCT116 cells. ^**P < 0.01. (C) Western blotting analysis of SNRPN protein levels after transduction with lentivirus containing shSNRPN(S1) and shSNRPN(S2). (D) Growth curves of shCon, shSNRPN(S1), and shSNRPN(S2)-transduced cells determined using MTT assays. ^***P < 0.001. (E) Representative images of colony formation assays. (F) Graph of colony numbers in the shCon, shSNRPN(S1), and shSNRPN(S2) groups. ^*P < 0.05; ^***P < 0.001.

Figure 4

Downregulation of SNRPN suppresses metastasis in colorectal cancer cells. (A) Wound healing experiment assays showing migration of SW1116 cells after downregulation of SNRPN. Area of occupation shown by representative images and graphs. ^**P < 0.01; ^***P < 0.001. (B) Migration assay of SW1116 cells after downregulation of SNRPN using transwell experiments. ^***P < 0.001. (C) Invasion assay of SW1116 cells after downregulation of SNRPN using transwell experiments with Matrigel. ^**P < 0.01; ^***P < 0.001.

Figure 5

SNRPN suppresses cell cycle and promotes apoptosis. (A) Representative picture of the cell cycle assayed using flow cytometry with PI staining. (B) Histogram of cell number in G0/G1, S, and G2/M phases. ^**P < 0.01; ^***P < 0.001. (C) Histogram of cell number in the sub G1 phase. (D) Representative picture of apoptosis assayed using flow cytometry with 7-ADD and Annexin V double staining. (E) Percentage of apoptotic cells. (F) Western blotting analysis of apoptosis markers. (G) Western blotting analysis of markers involved in the cell cycle.

Figure 6

Downregulation of SNRPN impairs growth of human colorectal cancer xenografts. (A) Effect of SNRPN on tumor formation in a nude mouse xenograft model. Pictures of tumors were taken after sacrifice on day 19. (B) Western blotting analysis of SNRPN and p-CDC2 expression levels. (C) The tumor size was smaller compared with the control group measured twice a week from day 5 after injection. ^***P < 0.001.

Figure 7

E2F8 is one of the transcriptional regulators up-steam of SNRPN. (A) Relative Luciferase activity of SNRPN promoter. (B) ChIP-qPCR assay for E2F8's occupancy on the SNRPN promoter. (C) Correlation analyses of E2F8 expression with expression of SNRPN using IHC staining including 1,310 colorectal cancer tissue samples. (D) IHC staining score of E2F8 was significantly higher in tumor tissues than in normal tissues. (E) Kaplan–Meier analysis of the association between E2F8 expression and overall survival. ^*P < 0.05; ^***P < 0.001.