Investig Clin Urol.  2018 Jul;59(4):263-274. 10.4111/icu.2018.59.4.263.

Down-regulation of transient receptor potential melastatin member 7 prevents migration and invasion of renal cell carcinoma cells via inactivation of the Src and Akt pathway

• Ha YS
• Kim YY
• Yu NH
• Chun SY
• Choi SH
• Lee JN
• Kim BS
• Yoo ES
• Kwon TG

• KMID: 2414760
• DOI: http://doi.org/10.4111/icu.2018.59.4.263

Abstract

PURPOSE
Transient receptor potential melastatin member 7 (TRPM7), an ion channel and serine/threonine protein kinase, has been linked with distinct human malignancies. However, the role of TRPM7 in renal cell carcinoma (RCC) has not been investigated. The aim of this study is to determine whether TRPM7 regulates the migration and invasion of RCC cells. Its relationship with signal transduction pathways was also studied.
MATERIALS AND METHODS
The human RCC cell lines ACHN and SN12C were chosen for this study. The molecular mechanisms of TRPM7 action were studied using Western blot analysis and small interfering RNA (siRNA)-based knockdown. The effect of TRPM7 knockdown on RCC cells was measured by using Transwell invasion and wound healing migration assays.
RESULTS
siRNA-induced silencing of TRPM7 notably decreased the migration and invasion of ACHN and SN12C RCC cells. The phosphorylation levels of Src in both cells were obviously reduced after TRPM7 silencing compared with that of the control ACHN and SN12C cells. Furthermore, the phosphorylation levels of Akt were greatly decreased in ACHN cells after siRNA-induced knockdown of TRPM7. Additionally, the treatment of cells with Src and Akt inhibitors clearly limited the migration and invasion of RCC cells.
CONCLUSIONS
Our data show that TRPM7 regulated ACHN and SN12C RCC cell invasion via the Src/Akt signaling pathway. Therefore, targeting the Src/Akt signaling pathway and/or the expression or function of TRPM7 could be a potential beneficial treatment for patients with RCC.

Keyword

Carcinoma, renal cell; Neoplasm invasiveness; Neoplasm metastasis; Signal transduction

MeSH Terms

Blotting, Western
Carcinoma, Renal Cell*
Cell Line
Down-Regulation*
Humans
Ion Channels
Neoplasm Invasiveness
Neoplasm Metastasis
Phosphorylation
Protein Kinases
RNA, Small Interfering
Signal Transduction
Wound Healing
Ion Channels
Protein Kinases
RNA, Small Interfering

Figure

• 

  Fig. 1 (A) Differentially expressed mRNA and protein levels of transient receptor potential melastatin member 7 (TRPM7) in various renal cell carcinoma lines. ACHN and SN12C cells were selected for gene silencing experiments because of the higher expression levels of TRPM7. (B) Small interfering RNA-mediated knockdown of TRPM7 in ACHN and SN12C cell lines analyzed by Western blotting. β-actin was used as an internal loading control. The band is a representative of three independent experiments. M, mock; N, negative control.

• 

  Fig. 2 Effect of the small interfering RNA (siRNA)-mediated knockdown of transient receptor potential melastatin member 7 (TRPM7) on the viability of renal cell carcinoma (RCC) cells. The viability of RCC cells was measured by MTS assay after transfection with TRPM7 siRNA. No significant effect on viability were induced by silencing of TRPM7. M, mock; N, negative control.

• 

  Fig. 3 Effect of transient receptor potential melastatin member 7 (TRPM7) small interfering RNA (siRNA) on cell migration. Wound healing assays reveal a significant decrease in migration of renal cell carcinoma cells after TRPM7 knockdown for 24 hours. (A) ACHN cells. (B) SN12C cells. M, mock; N, negative control.

• 

  Fig. 4 Effect of small interfering RNA (siRNA)-mediated knockdown of transient receptor potential melastatin member 7 (TRPM7) on the migration and invasion of ACHN and SN12C cells. (A) Effect of TRPM7 siRNA on the migration of renal cell carcinoma (RCC) cells was examined by Transwell assay. The difference in migration was measured as a fold change in absorbance as compared to mock (M) siRNA. The Transwell assay showed that knockdown of TRPM7 significantly inhibited the migration ability of ACHN and SN12C cells. (B) Invasion of RCC cells was investigated by using Matrigel-coated chambers. The number of invading cells was presented as a fold difference relative to M siRNA. The invasion rates were reduced by TRPM7 siRNA transfection, suggesting that silencing of TRPM7 inhibits the metastatic potential of RCC cells. Bar graph shows quantitative analysis of crystal violet extracted from the cells placed under the Transwell. *Significant difference at p<0.05. N, negative control.

• 

  Fig. 5 Effect of transient receptor potential melastatin member 7 (TRPM7) down-regulation on Akt and Src phosphorylation in renal cell carcinoma cells. (A) In ACHN cells, the phosphorylation levels of Akt and Src were significantly reduced by the small interfering RNA (siRNA)-mediated knockdown of TRPM7. (B) Western blot images show decreased phosphorylation of Src in SN12C cells following transfection with TRPM7 siRNA. M, mock; N, negative control.

• 

  Fig. 6 Effect of Akt and Src inhibition on cell migration. (A) Treatment with Akt or Src inhibitor of ACHN cells reduced cell migration in wound healing assays. (B) In SN12C cells, the Src inhibitor decreased the cell migration to the scratched area. M, mock; N, negative control.

• 

  Fig. 7 Effect of Akt and Src inhibition on cell migration and invasion. (A) Transwell assays show that Src inhibition significantly inhibited the migration ability of ACHN and SN12C cells. In ACHN cells, the Akt inhibitor was effective in inhibiting cell migration. (B) The effect of Src and Akt inhibition on the invasion capabilities of renal cell carcinoma (RCC) cells was also examined. Src and Akt inhibitors reduced RCC invasion rates, suggesting that transient receptor potential melastatin member 7 silencing inhibits the metastatic potential of RCC cells via inactivation of the Src and Akt signaling pathways. Bar graph shows quantitative analysis of crystal violet extracted from the cells placed under the Transwell. *Significant difference at p<0.05. M, mock; N, negative control.

• 

  Fig. 8 Effect of transient receptor potential melastatin member 7 (TRPM7) down-regulation on matrix metalloproteinase (MMP)-2 and MMP-9 phosphorylation in ACHN (A) and SN12C renal cell carcinoma (RCC) cells (B). MMP-2 and MMP-9 were not reduced by TRPM7 small interfering RNA (siRNA) transfection of RCC cells. M, mock; N, negative control.

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  Fig. 1 A diagram suggesting the potential mechanism of transient receptor potential melastatin member 7 (TRPM7) in metastatic renal cell carcinoma.

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