Overexpression of Myosin Phosphatase Target Subunit 1 (MYPT1) Inhibits Tumor Progression and Metastasis of Gastric Cancer

Abstract

BACKGROUND Myosin phosphatase target subunit 1 (MYPT1) serves as a subgroup of myosin phosphatases, and is frequently low-expressed in human cancers. However, little is known about the effects of MYPT1 in gastric cancer (GC). MATERIAL AND METHODS In our study, MYPT1 expression was detected by quantitative real-time reverse transcription PCR (qRT-PCR) in GC tissues, different advanced pathological stages of GC tissues, and preoperative and postoperative patients. Kaplan-Meier analysis was used to measure the overall survival of GC patients. MYPT1 expression was analyzed by qRT-PCR and Western blot assays in GES-1 cells and GC cells. Cell proliferation, cycle, and migration and invasion abilities were detected by CCK-8, flow cytometry, and Transwell assays. E-cadherin, TIMP-2, MMP-2, MMP-9 RhoA, and p-RhoA expressions were assessed by qRT-PCR and Western blot assays in treated SNU-5 cells. RESULTS Our results indicated that MYPT1 was down-regulated in GC tissues and cells, and is related to clinical stages and overall survival of GC. Functional research demonstrated that overexpression of MYPT1 can inhibit cell proliferation, cell cycle progression, and migration and invasion of GC cells. Many studies on mechanisms reported that overexpression of MYPT1 dramatically improved the expression levels of cell cycle-related genes (Cyclin D1 and c-myc), significantly increased epithelial marker (E-cadherin) expression, and decreased invasion-associated genes (TIMP-2 and MMP-2) expressions in SNU-5 cells. In addition, we found that MYPT1 suppressed RhoA phosphorylation. CONCLUSIONS We verified that MYPT1 inhibits GC cell proliferation and metastasis by regulating RhoA phosphorylation.

Figure 1

Relative MYPT1 expression in GC and the relationship with overall survival of GC. (A) The mRNA expression level of MYPT1 was detected by qRT-PCR assay in GC tissues and corresponding non-tumor tissues (n=68, *** P<0.001). (B) MYPT1 expression was measured by qRT-PCR assay in different advanced pathological stages normal (N0, n=43), I & II phase (N1 and N2, n=33), III and IV phase (N3 and N4, n=35), * P<0.05, ** P<0.01. (C) Based on MYPT1 expression in GC tissues, the overall survival of GC patients was calculated by Kaplan-Meier analysis (P=0.009). (D) MYPT1 expression was analyzed by qRT-PCR assay in preoperative and postoperative patient serum (n=43, P=0.0032).

Figure 2

Overexpression of MYPT1 inhibits GC cell proliferation. MYPT1 expression was analyzed by qRT-PCR (A) and Western blot (B) assays in gastric epithelial cell (GES-1) and GC cell lines (SNU-5, MKN-45, BGC-823, SGC-7901, AGS, and HGC-27), ** P<0.01, *** P<0.001 vs. GES-1 group. (C) SNU-5 cells were treated with PBS (Blank), pcDNA3.1, pcDNA3.1-MYPT1 for 48 h, respectively. MYPT1 mRNA expression level was evaluated by qRT-PCR assay (*** P<0.001). (D) MYPT1 protein expression level was measured by Western blot assay in treated SNU-5 cells. (E) CCK-8 assay was performed to detect SNU-5 cell proliferation (* P<0.05, *** P<0.001).

Figure 3

Overexpression of MYPT1 induces SNU-5 cell cycle arrest in G1 phase. (A) The cell cycle was analyzed flow cytometry in treated SNU-5 cells, and the values of G1, S, and G2 were shown in the bar graphs (* P<0.05, ** P<0.01). (B) qRT-PCR and (C) Western blot assays were performed to analyze the mRNA and protein expression levels of Cyclin D1 and c-myc in treated SNU-5 cells (* P<0.05, ** P<0.01, *** P<0.001).

Figure 4

Overexpression of MYPT1 inhibits SNU-5 cell migration and invasion. The migration (A) and invasion (B) abilities of the treated SNU-5 cells were measured by Transwell assays (** P<0.01, *** P<0.001). (C) qRT-PCR and (D) Western blot assays were performed to detect the mRNA and protein expression levels of E-cadherin, TIMP-2, MMP-2 and MMP-9 in treated SNU-5 cells (* P<0.05, ** P<0.01, *** P<0.001).

Figure 5

Overexpression of MYPT1 suppresses RhoA phosphorylation. RhoA and p-RhoA expressions were detected by Western blot assay in treated SNU-5 cells, and the relative expression levels were counted from 3 independent experiments (*** P<0.001).