Interference with Tim-3 protein expression attenuates the invasion of clear cell renal cell carcinoma and aggravates anoikis

Abstract

Tumor cells resistant to anoikis are considered to be candidates for metastasis. In the present study, the role of Tim‑3 in anoikis and its influence on the invasion of clear cell renal cell carcinoma (ccRCC) was investigated. Here, polyhydroxylethylmethacrylate (poly‑HEMA) was applied to two ccRCC cell lines, 786‑O and Caki‑2, to induce detachment from the extracellular matrix (ECM). Tim‑3 mRNA and protein expression levels were assayed by reverse transcription-quantitative polymerase chain reaction (RT‑qPCR) and western blot, respectively. Anoikis was measured by Ho33342/PI double staining, acridine orange staining, and further determined using the CytoSelect™ 24‑well Anoikis Assay kit. Apoptosis was measured using flow cytometry, E‑cadherin and N‑cadherin protein expression were determined using western blotting and a Chemicon cell invasion assay kit was used to quantify the invasive capacity of 786‑O and Caki‑2 cells. It was demonstrated that detachment from the ECM decreases transcription and the protein expression level of Tim‑3 in 786‑O and Caki‑2 cells compared with control cells. Interference with Tim‑3 expression using small interfering RNA exacerbated anoikis in 786‑O and Caki‑2 cells induced by poly‑HEMA treatment. E‑cadherin upregulation, N‑cadherin downregulation, and ECM detachment‑induced reduction in invasion ability were all exacerbated by knockdown of Tim‑3. In conclusion, interference with Tim‑3 expression may attenuate the invasion of renal cell carcinoma by aggravating anoikis, indicating Tim‑3 as a potential therapeutic target for treating ccRCC.

Figure 1.

Poly-HEMA-induced detachment from the extra cellular matrix decreases mRNA and protein expression levels of Tim-3 in 786-O and Caki-2 cells. (A) Reverse transcription-quantitative polymerase chain reaction analysis of Tim-3 levels. (B) The results of western blotting to determine Tim-3 protein expression levels. (C) Quantification of Tim-3 protein levels by denisometric analysis of western blots. Results are presented as the mean ± standard deviation. *P<0.05 vs. control cells. Poly-HEMA, polyhydroxylethylmethacrylate.

Figure 2.

Knockdown of Tim-3 expression reduces ccRCC cell activities. (A) Western blot analysis confirmed the downregulation of Tim-3 expression in both 786-O and Caki-2 cells following transfection with Tim-3 siRNA. Tim-3 protein levels were quantified by densitometric analysis. (B) The semi-quantification based on Image J software showed significant decrease of Tim-3 expression under Tim-3 siRNA in both 786-O (P=0.009) and Caki-2 cells (P=0.024). (C) Detachment from the extracellular matrix (poly-HEMA plates) reduced ccRCC activities, which meant an increase to anoikis. Activity was reduced further by knockdown of Tim-3 expression. Results are presented as the mean ± standard deviation. *P<0.05 vs. control cells. ccRCC, clear cell renal cell carcinoma; siRNA, small interfering RNA; poly-HEMA, polyhydroxylethylmethacrylate.

Figure 3.

Morphological observation of cell anoikis in 786-O and Caki-2 cells treated with poly-HEMA or poly-HEMA + Tim-3 siRNA. (A) Hoechst 33342/propidium iodide double staining under fluorescence microscopy (x200). (B) Acridine orange staining under fluorescence microscopy (x200). Poly-HEMA, polyhydroxylethylmethacrylate; siRNA, small interfering RNA.

Figure 4.

Flow cytometry analyses of 786-O and Caki-2 cell apoptosis. A total of 30,000 cells were collected in each group for flow cytometry analyses. The number of cells in Q4 and Q2 quadrant reflects early and late apoptotic cells, respectively. Both the Q2 and Q4 cells were counted as apoptosis here. The number of apoptotic 786-O cells in control, Poly-HEMA, and Poly-HEMA + siRNA groups were 591±63, 1,611±201 and 2,949±279, respectively. Meanwhile, the number of apoptotic Caki-2 cells in control, Poly-HEMA, and Poly-HEMA + siRNA groups were 690±60, 1,821±210 and 3,051±357, respectively. Interference with Tim-3 expression using a small interfering RNA significantly increased anoikis in 786-O (P=0.015) and Caki-2 (P=0.003). Poly-HEMA, polyhydroxylethylmethacrylate; siRNA, small interfering RNA.

Figure 5.

Knockdown of Tim-3 expression attenuates extracellular matrix detachment-induced E-cadherin upregulation and N-cadherin downregulation in clear cell renal cell carcinoma cells. (A) Expression levels of E-cadherin and N-cadherin were analyzed by western blot. Quantification of (B) E-cadherin protein expression and (C) N-cadherin protein expression level by densitometric analysis using Image J software. Results are presented as the mean ± standard deviation. *P<0.05 vs. control cells. Poly-HEMA, polyhydroxylethylmethacrylate; siRNA, small interfering RNA.

Figure 6.

Knockdown of Tim-3 expression and detachment from the extracellular matrix significantly decreases the invasive ability of 786-O and Caki-2 cells. The absorbance of 560 nm represents the invasive ability. Results are presented as the mean ± standard deviation. *P<0.05 vs. control cells. Poly-HEMA, polyhydroxylethylmethacrylate; siRNA, small interfering RNA.