MicroRNA-551b-3p inhibits tumour growth of human cholangiocarcinoma by targeting Cyclin D1

Abstract

MicroRNAs (miRNAs) are powerful regulators in the tumorigenesis of cholangiocarcinoma (CCA). Previous studies report that miR-551b-3p acts as an oncogenic factor in ovarian cancer, but plays a tumour suppressive role in gastric cancer. However, the expression pattern and potential function of miR-551b-3p were still unclear in CCA. Therefore, this study aimed to explore the expression of miR-551b-3p and its role as well as molecular mechanism in CCA. Analysis of TCGA dataset suggested that miR-551b-3p was under-expressed in CCA tissues compared to normal bile duct tissues. Furthermore, our data confirmed the decreased levels of miR-551b-3p in CCA samples and cell lines. Interestingly, TCGA data suggested that low miR-551b-3p level indicated reduced overall survival of CCA patients. Gain- and loss-of-function experiments found that miR-551b-3p inhibited the proliferation, G1-S phase transition and induced apoptosis of CCA cells. In vivo experiments revealed that ectopic expression of miR-551b-3p inhibited tumour growth of CCA in mice. Further investigation demonstrated that miR-551b-3p directly bond to the 3'-UTR of Cyclin D1 (CCND1) mRNA and negatively regulated the abundance of CCND1 in CCA cells. An inverse correlation between miR-551b-3p expression and the level of CCND1 mRNA was detected in CCA tissues from TCGA dataset. Notably, CCND1 knockdown showed similar effects to miR-551b-3p overexpression in HuCCT-1 cells. CCND1 restoration rescued miR-551b-3p-induced inhibition of proliferation, G1 phase arrest and apoptosis in HuCCT-1 cells. In summary, miR-551b-3p inhibits the expression of CCND1 to suppress CCA cell proliferation and induce apoptosis, which may provide a theoretical basis for improving CCA treatment.

Keywords: Cyclin D1; cholangiocarcinoma; microRNA-551b-3p; tumour growth.

Figure 1

The expression of miR‐551b‐3p in CCA. A, CCA samples (n = 36) and normal bile duct tissues (n = 9) in TCGA dataset from starBase V3.0 platform showed that miR‐551b‐3p expression was significantly down‐regulated in tumour tissues. B, The expression of miR‐551b‐3p was detected in CCA tissues (n = 15) and normal bile duct tissues (n = 15) using qRT‐PCR. C, The levels of miR‐551b‐3p between CCA cell lines (HuCCT‐1, RBE, HCCC‐9810 and QBC939) and HiBEC cells were analysed by qRT‐PCR. *P < 0.05. C, TCGA data from starBase V3.0 platform indicated that CCA patients with low miR‐551b‐3p level had an obvious poorer overall survival compared to miR‐551b‐3p high‐expressing patients

Figure 2

miR‐551b‐3p overexpression suppresses proliferation, cell cycle progression and induces apoptosis of CCA cells. A, Vectors containing miR‐551b‐3p or control mimics were transfected into HuCCT1 and QBC939 cells and detected by qRT‐PCR for miR‐551b‐3p expression. B and C, CCK‐8 and EdU assays demonstrated that miR‐551b‐3p overexpression inhibited CCA cell proliferation. D, miR‐551b‐3p overexpression induced the apoptosis of CCA cells as suggested by flow cytometry analysis. E, The G1 to S phase transition was blocked by miR‐551b‐3p overexpression in HuCCT1 and QBC939 cells. *P < 0.05

Figure 3

miR‐551b‐3p knockdown enhances proliferation, cell cycle progression and inhibits apoptosis of RBE cells. A, Vectors containing miR‐551b‐3p inhibitors or negative control (NC) were transduced into RBE cells and detected for miR‐551b‐3p expression using qRT‐PCR. B and C, CCK‐8 and EdU assays demonstrated that miR‐551b‐3p knockdown enhanced CCA cell proliferation. D, miR‐551b‐3p knockdown inhibited the apoptosis of RBE cells as suggested by flow cytometry analysis. E, miR‐551b‐3p knockdown facilitated the G1 to S phase transition in RBE cells. *P < 0.05

Figure 4

miR‐551b‐3p restoration restrains tumour growth of HuCCT‐1 cells. A, HuCCT‐1 cells that were transfected with vectors containing miR‐551b‐3p or control mimics were subcutaneously implanted into nude mice . Both tumour volume and tumour weight in miR‐551b‐3p group were obviously less than those in control group. B, The expression of miR‐551b‐3p in xenograft tumour tissues from miR‐551b‐3p group was significantly higher than that in control group. C, IHC analysis showed that strong staining of Ki‐67 was observed in most of tumour cells in control group, whereas only several tumour cells showed weak staining of Ki‐67 in miR‐551b‐3p group. *P < 0.05

Figure 5

CCND1 is recognized as a target of miR‐551b‐3p in CCA cells. A, Bioinformatic analysis in starBase V3.0 platform indicated the potential binding sequences between miR‐551b‐3p and 3′UTR of CCND1. B, TCGA data from starBase V3.0 platform indicated that the expression of miR‐551b‐3p was negatively correlated with the level of CCND1 mRNA in CCA tissues. C, miR‐551b‐3p or control mimics were transfected into HuCCT‐1 and QBC939 cells and detected for CCND1 expression using qRT‐PCR and immunoblotting. D, miR‐551b‐3p inhibitors or negative control (NC) was transduced into RBE cells and detected for CCND1 expression using qRT‐PCR and immunoblotting. E, The expression of CCND1 protein in xenograft tumour tissues from miR‐551b‐3p group was significantly lower than that in control group. (F) Co‐transfection of miR‐551b‐3p mimics/inhibitors and wild‐type (wt) or mutated (mt) 3′UTR of CCND1 was performed in HuCCT‐1 cells and the relative luciferase activity was measured. *P < 0.05

Figure 6

CCND1 knockdown suppresses the growth of HuCCT‐1 cells. A, CCND1 siRNA or non‐targeting (NT) siRNA was transfected into HuCCT‐1 cells and detected for CCND1 protein using immunoblotting. B, CCK‐8, (C) EdU, (D) flow cytometry apoptosis and (E) cell cycle analysis were carried out to detect the proliferation, apoptosis and cell cycle progression of HuCCT‐1 cells. *P < 0.05

Figure 7

CCND1 restoration rescues miR‐551b‐3p induced growth arrest and apoptosis of HuCCT‐1 cells. A, CCND1 expression was rescued by transfecting vectors in miR‐551b‐3p overexpressing HuCCT‐1 cells as detected by immunoblotting. B, CCK‐8, (C) EdU, (D) flow cytometry apoptosis and (E) cell cycle analysis were carried out to detect the proliferation, apoptosis and cell cycle progression of HuCCT‐1 cells. CCND1 restoration abrogated the effects of miR‐551b‐3p on HuCCT‐1 cells. *P < 0.05