Effects of miR-330-3p on Invasion, Migration and EMT of Gastric Cancer Cells by Targeting PRRX1-Mediated Wnt/β-Catenin Signaling Pathway

Abstract

Background: miRNA, as a biological marker, had more and more attention in recent years due to the important role it plays in cancer. Currently, there are extensive studies on miRNAs, among which miR-330-3p is reported to be implicated in the pathophysiological processes of various cancers. However, little progress has been made in the mechanism of miR-330-3p in gastric cancer.

Objective: To explore the expression and relevant mechanism of miR-330-3p and PRRX1 in gastric cancer (GC).

Methods: Forty-five GC patients (study group), from whom paired GC and paracancerous tissues were collected, and another 45 healthy subjects (control group) who underwent physical examination during the same period were enrolled. In addition, GC cells and human gastric mucosa cells were purchased, and miR-330-3p-mimics, miR-330-3p-inhibitor, miR-NC, si-PRRX1, and sh-PRRX1 were transfected into MKN45, SGC7901 cell. QRT-PCR was employed to assess the miR-330-3p and PRRX1 expressions in the samples, and the cell expressions of PRRX1, GSK-3β, p-GSK-3β, β-catenin, p-β-catenin, cyclin D1, N-cadherin, E-cadherin and vimentin were evaluated by Western blot (WB). MTT, Transwell and wound-healing experiments were adopted to detect cell proliferation, invasion and migration.

Results: MiR-330-3p was under-expressed, while PRRX1 was highly expressed in the serum of patients, both of which had an area under the curve (AUC) of more than 0.9. MiR-330-3p and PRRX1 were associated with tumor diameter, TNM staging, lymph node metastasis and differentiation of GC patients. Overexpression of miR-330-3p and inhibition of PRRX1 expression could suppress epithelial-mesenchymal transition (EMT), proliferation, invasion and apoptosis of cells. What is more, WB assay showed that overexpressed miR-330-3p and inhibited PRRX1 could inhibit the expression levels of p-GSK-3β, β-catenin, cyclin D1, N-cadherin and vimentin proteins, while elevating GSK-3β, p-β-catenin and E-cadherin protein expressions. Dual-luciferase reporter assay confirmed that there was a targeting relation between miR-330-3p and PRRX1. Furthermore, rescue experiments revealed that the cell proliferation, invasion, migration did not differ significantly between co-transfected miR-330-3p-mimics+sh-PRRX1, miR-330-3p-inhibitor+si-PRRX1 groups of MKN45 and SGC7901 and the miR-NC group (without transfected sequences).

Conclusion: Overexpressed miR-330-3p can promote cell EMT, proliferation, invasion and apoptosis through inhibiting PRRX1-mediated Wnt/β-catenin signaling pathway, which is expected to be a potential therapeutic target for GC.

Keywords: GC; PRRX1; Wnt/β-catenin signaling pathway; biological function; miR-330-3p.

Figure 1

Expression and clinical value of serum RNA-330-3p and PRRX1 in GC patients. (A) The expression of miR-330-3p was low while PRRX1 was high in the serum of GC patients. (B) The serum expression of miR-330-3p and PRRX1 presented a negative correlation in GC patients. (C) MiR-330-3p was lowly expressed while PRRX1 was highly expressed in GC tissues. (D) The positive rate of immunohistochemical detection of PRRX1 in GC tissues was significantly higher than in paracancerous tissues. (E) The AUC of miR-330-3p curve was 0.944, and that of the PRRX1 was 0.920.**Indicates P <0.05.

Figure 2

Effects of miR-330-3p on proliferation, invasion, migration and EMT of GC cells. (A) MiR-330-3p presented low expression in GC cells. (B) The miR-330-3p expression was substantially elevated in miR-330-mimics transfected cells, and markedly abated in miR-330-3p-inhibitor-transfected cells. (C) The proliferation ability of miR-330-3p-mimics transfected cells was markedly reduced, while that of miR-330-3p-inhibitor-transfected cells was dramatically increased. (D) The invasion ability of miR-330-3p-mimics transfected cells was markedly reduced, while that of miR-330-3p-inhibitor-transfected cells was substantially increased. (E) The migration ability of miR-330-3p-mimics transfected cells was substantially reduced, while that of miR-330-3p-inhibitor-transfected cells was markedly boosted. (F) The p-GSK-3β, β-catenin and cyclinD1 expressions in miR-330-mimics cells were markedly abated, with significantly increased expressions of GSK-3β, p-β-catenin and cyclinD1, while the expressions were reversed in the miR-330-3p-inhibitor-transfected cells. (G) Western Blot. (H) The N-cadherin and vimentin expressions were substantially decreased, and expression of E-cadherin was significantly increased in miR-330-3p-mimics transfected cells, while expression of N-cadherin and vimentin was significantly increased, and the E-cadherin expression was markedly decreased in miR-330-3p-inhibitor-transfected cells. (I) Western Blot. **Indicated P<0.05, *Indicated P<0.05 in contrast with the miR-NC group. ^#Indicated P<0.05 in contrast with the miR-330-3p-inhibitor group.

Figure 3

Effects of PRRX1 on proliferation, invasion, migration and EMT of GC cells. (A) PRRX1 was highly expressed in GC cells. (B) PRRX1 expression was substantially abated in sh-PRRX1 transfected cells and markedly decreased in si-PRRX1 transfected cells. (C) The proliferation ability of si-PRRX1 transfected cells was dramatically reduced, while that of sh-PRRX1 transfected cells was substantially increased. (D) The invasion ability of si-PRRX1 transfected cells was markedly reduced, while that of sh-PRRX1 transfected cells was significantly increased. (E) The migration ability of si-PRRX1 transfected cells was substantially reduced, while that of sh-PRRX1 transfected cells was markedly increased. (F) The p-GSK-3β, β-catenin, cyclin D1 expressions were significantly reduced, GSK-3β, p-β-catenin markedly boosted in cells transfected with si-PRRX1, while reversed in cells transfected with sh-PRRX1. (G) Western Blot. (H) The N-cadherin and vimentin expressions were substantially abated, and E-cadherin markedly boosted in the si-PRRX1 transfected cells. While the N-cadherin and vimentin expressions were markedly boosted, and E-cadherin was dramatically abated in sh-PRRX1 transfected cells. (I) Western Blot. **Indicated P<0.05. *Indicated P<0.05 in contrast with the miR-NC group. ^#Indicates P<0.05 in contrast with the sh-PRRX1 group.

Figure 4

Dual-luciferase activity detection. (A) There was a binding point between miR-330-3p and PRRX1: relative luciferase activity-dual luciferase reporter assay. (B) The expression of PRRX1 in MKN-45 and MGC-803 cells after transfection. **Indicates P <0.05.

Figure 5

Rescue experiment. (A) Cell proliferation ability after transfection with miR-330-3p-mimics+sh-PRRX1 and miR-330-3p-inhibitor+si-PRRX1. (B) Cell invasion ability after transfection with miR-330-3p-mimics+sh-PRRX1 and miR-330-3p-inhibitor+si-PRRX1. (C) Cell migration ability after transfection with miR-330-3p-mimics+sh-PRRX1 and miR-330-3p-inhibitor+si-PRRX1. (D) P-GSK-3β, β-catenin, cyclin D1, GSK-3β and p-β-catenin expressions after transfection with miR-330-mimics+sh-PRRX1 and miR-330-3p-inhibitor+si-PRRX1. (E) Western Blot. (F) E-cadherin, N-cadherin and vimentin expressions after transfection with miR-330-3p-mimics+sh-PRRX1 and miR-330-3p-inhibitor+si-PRRX1. (G) Western Blot. *Indicated P<0.05 in contrast with miR-NC group, miR-330-3p-mimics+sh-PRRX1 group and miR-330-3p-inhibitor+si-PRRX1 group. ^#Represented P<0.05 in contrast with miR-330-3p-inhibitor.

Figure 6

Effects of overexpressed miR-330-3p on tumor growth in nude mice. (A) Nude mice had smaller tumors in the miR-330-3p-mimics group than those in the miR-NC group. (B) MiR-330-mimics group presented a higher expression of miR-330-3p in tumor tissues than that of the control group. (C) The tumor growth rate in the miR-330-3p-mimics group was markedly slower than that in the miR-NC group. (D) The tumors in the miR-330-3p-mimics group were markedly smaller than those in the miR-NC group. **Indicated P<0.05.