MCOLN1 Promotes Proliferation and Predicts Poor Survival of Patients with Pancreatic Ductal Adenocarcinoma

Abstract

Background: MCOLN1 (mucolipin subfamily, member 1) was first identified as an autophagic regulator, which was essential for efficient fusion of both autophagosomes and late endosomes with lysosomes. This study is aimed at investigating the role of MCOLN1 in the development of pancreatic ductal adenocarcinoma (PDAC).

Methods: Immunohistochemistry (IHC) assay was conducted to evaluate the expression level of MCOLN1 in 82 human PDAC tumor tissues. Overall survival (OS) and recurrence-free survival (RFS) analysis was performed to assess the prognosis of patients. Colony formation and MTT assays [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] were performed to measure the proliferation capacity of tumor cells. The expression level of related genes was measured by RT-PCR (reverse transcription polymerase chain reaction) and western blot assays. The animal model was used to examine the effects of indicated protein on tumorigenesis in vivo.

Results: The results of IHC showed that a high level of MCOLN1 expression was associated with the poor clinical characteristics of PDAC patients. OS and RFS were significantly worse in patients with high MCOLN1 expression. Silencing of MCOLN1 dramatically blocked the proliferation of PDAC cells. Mechanism studies confirmed that knockdown of MCOLN1 decreased the expression of Ki67 and PCNA (proliferating cell nuclear antigen), two markers of cell proliferation. In vivo, MCOILN1 depletion reduced the formation and growth of tumors in mice.

Conclusion: The high level of MCOLN1 expression was associated with poor clinical outcomes of PDAC patients. MCOLN1 ablation could inhibit PDAC proliferation of both in vitro and in vivo, which provide a new insight and novel therapeutic target for the treatment of PDAC.

Figure 1

The expression of MCOLN1 in lentivirus-transfected cell lines. The mRNA (a) and (b) protein level expression of MCOLN1 was dramatically decreased in both MCOLN1 shRNA cell lines compared to controls. ^∗ P < 0.05.

Figure 2

The influence of MCOLN1 on the proliferation of PANC-1 and BxPC-3 cell lines by regulating Ki67 and PCNA. (a) The colony formation array showed that the colonies were inhibited in the MCOLN1 shRNA group. (b) The results of MTT showed that the OD value in the MCOLN1 group was lower than that in the control group. (c, d) The expression of Ki67 and PANC was significantly inhibited in the MCOLN1 shRNA group compared to control. ^∗ P < 0.05.

Figure 3

In vivo animal experiments. (a) At the 14th day after injection, the tumor volume was measured every day. The growth curve showed that the tumors in the MCOLN1 group grew more slowly than controls. After inoculation of 29 days, the final tumor tissues were obtained. The tumors in the shRNA group were smaller than controls. (b, c) The expression of MCOLN1 in tumors of mice was detected by western blot or IHC. MCOLN1 expression was dramatically decreased in the shRNA group, which confirmed the effective silencing of MCOLN1 in tumors of mice in the shRNA group. “∗” represents the significant association (P < 0.05 for the difference was significant).

Figure 4

The expression of MCOLN1 in tumor tissues and the associations of MCOLN1 with the prognosis of PDAC patients. (a, b) The staining of high and low expressions of MCOLN1 by IHC in PDAC tissues and paracarcinoma tissues. (c) OS and RFS rates with MCOLN1 expression in the 82 PDAC patients: shorter OS or RFS was significantly observed in high MCOLN1 expression than in low MCOLN1 expression in the 82 PDAC patients (P < 0.05, respectively).