LRG1 promotes proliferation and inhibits apoptosis in colorectal cancer cells via RUNX1 activation

Abstract

Leucine-rich-alpha-2-glycoprotein 1 (LRG1) has been shown to be involved in various human malignancies. Whether it plays a role in colorectal cancer (CRC) development remains unclear. Here, we investigated whether and through what mechanism LRG1 functions in human CRC cells. The plasma level of LRG1 was significantly increased in CRC patients, but it was remarkably decreased in patients with resected colorectal cancers. Meanwhile, both mRNA and protein levels of LRG1 were remarkable overexpressed in CRC tissues than normal tissues. The knockdown of LRG1 significantly inhibited cell proliferation, induced cell cycle arrest at the G0/G1 phase, and promoted apoptosis in SW480 and HCT116 cells in vitro. In addition, LRG1 silencing led to the downregulation of the levels of key cell cycle factors, such as cyclin D1, B, and E and anti-apoptotic B-cell lymphoma-2(Bcl-2). However, it up-regulated the expression of pro-apoptotic Bax and cleaved caspase-3. Furthermore, RUNX1 could be induced by LRG1 in a concentration-dependent manner, while the knockdown of RUNX1 blocked the promotion of the proliferation and inhibition of apoptosis induced by LRG1. Collectively, these findings indicate that LRG1 plays a crucial role in the proliferation and apoptosis of CRC by regulating RUNX1 expression. Thus, LRG1 may be a potential detection biomarker as well as a marker for monitoring recurrence and therapeutic target for CRC.

Fig 1. The plasma and tissue level of LRG1 was increased in human CRCs.

(a) Plasma samples from 30 normal patients, 30 adenoma patients, 30 CRC patients and the same 30 CRC patients after surgery were obtained, and the secretion of LRG1 was quantified by an ELISA. (b) Real-time RT-PCR and western blot were performed to assess LRG1 expression in 60 cases of CRC tissues and matched normal tissues. **P<0.01, and ***P<0.001.

Fig 2. The knockdown of LRG1 inhibited CRC cell proliferation and induced cell cycle arrest.

(a) RT-PCR and Western blot analyses showed that LRG1 was effectively down-regulated by siRNA transfection. (b) A CCK-8 assay was performed to evaluate cell proliferation in vitro. (c-d) SW480 and HCT116 cells were fixed, stained with PI, and subjected to flow cytometry analysis for cell cycle. (e–f) The expression levels of cyclin D1, B, and E in SW480 and HCT116 cells transfected with LRG1 siRNA or control siRNA were determined by RT-PCR and Western blot analysis. This figure shows representative images of repeated experiments, and the data are presented as the mean±standard deviation. Compared with NC, *P<0.05, **P<0.01, and ***P<0.001.

Fig 3. The knockdown of LRG1 promoted CRC cell apoptosis.

(a-b) SW480 and HCT116 cells were double stained with an FITC-conjugated anti-Annexin V antibody and PI and then subjected to flow cytometry for cell apoptosis analysis. The cells in the upper right and lower right quadrants were considered late and early apoptotic cells, respectively. (c-d) The expression levels of Bcl-2, Bax, and cleaved caspase-3 in SW480 and HCT116 cells transfected with LRG1 siRNA or control siRNA were determined by RT-PCR and Western blot analysis. This figure shows representative images of repeated experiments, and the data are presented as the mean±standard deviation. Compared with NC, **P<0.01 and ***P<0.001.

Fig 4. LRG1 promoted the expression of RUNX1 and CRC cell proliferation.

(a) SW480 and HCT116 cells were transfection with LRG1 siRNA, and the expression of the tested RUNXs was quantified by RT-PCR. (b) Cells were stimulated with rLRG1 (0–1,000 ng/ml) for 24 h, and the RUNX1 expression was determined by RT-PCR. (c) Cells were stimulated with rLRG1 (0–1000 ng/ml), and a CCK-8 assay was performed to evaluate cell proliferation. (d) The protein levels of cyclin D1, B, E, Bcl-2, Bax, and cleaved caspase-3 in response to LRG1 treatment in SW480 cells were analysed by Western blot. This figure shows representative images of repeated experiments, and the data are presented as the mean±standard deviation. Compared with NC, *P<0.05, **P<0.01, and ***P<0.001.

Fig 5. The role of RUNX1 in LRG1-induced cell proliferation and anti-apoptosis.

(a) RUNX1 expression was knocked down in siRNA-transfected SW480 cells at both the mRNA and protein levels. (b) SW480 cells were transfected with RUNX1 or control siRNA with or without rLRG1 (500 ng/ml) stimulation, and a CCK-8 assay was performed to evaluate cell proliferation. (c-d) SW480 cells were transfected with RUNX1 or control siRNA for 24 h and treated with or without rLRG1 (500 ng/ml) for an additional 24 h. Then, the SW480 cells were fixed, stained with PI, and subjected to flow cytometry analysis to probe the cell cycle. (e-f) SW480 and HCT116 cells were harvested and double stained with an FITC-conjugated anti-Annexin V antibody and PI. Then, the cells were subjected to flow cytometry for cell apoptosis analysis. (g) The protein levels of Runx1,cyclin D1, B, E, Bcl-2, Bax, and cleaved caspase-3 in response to RUNX1 or control siRNA with or without rLRG1 treatment in SW480 cells, which were analysed by Western blot. This figure shows representative images of repeated experiments, and the data are presented as the mean±standard deviation. Compared with NC, *P<0.05, **P<0.01, and ***P<0.001.