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. 2022 May 19:2022:4213401.
doi: 10.1155/2022/4213401. eCollection 2022.

MicroRNA-4735-3p Facilitates Ferroptosis in Clear Cell Renal Cell Carcinoma by Targeting SLC40A1

Cairong Zhu  1 , Zhiquan Song  2 , Zhen Chen  3 , Tianqi Lin  4 , Haili Lin  4 , Zhenqiang Xu  4 , Fen Ai  3 , Shijiao Zheng  1
Affiliations

MicroRNA-4735-3p Facilitates Ferroptosis in Clear Cell Renal Cell Carcinoma by Targeting SLC40A1

Cairong Zhu et al. Anal Cell Pathol (Amst). .

Abstract

Objective: Clear cell renal cell carcinoma (ccRCC) is the major histopathological subtype of renal cancer, and ferroptosis is implicated in the pathogenesis of ccRCC. The present study was aimed at investigating the role and underlying mechanisms of microRNA-4735-3p (miR-4735-3p) in ccRCC.

Methods: Human ccRCC cell lines were transfected with the miR-4735-3p mimic or inhibitor to manipulate the expression of miR-4735-3p. Cell proliferation, colony formation, cell migration, cell invasion, cell death, oxidative stress, lipid peroxidation, and iron metabolism were determined. To validate the necessity of solute carrier family 40 member 1 (SLC40A1), human ccRCC cell lines were overexpressed with SLC40A1 using adenoviral vectors.

Results: miR-4735-3p expression was reduced in human ccRCC tissues and cell lines but elevated upon ferroptotic stimulation. The miR-4735-3p mimic increased, while the miR-4735-3p inhibitor decreased oxidative stress, lipid peroxidation, iron overload, and ferroptosis of human ccRCC cell lines. Mechanistic studies identified SLC40A1 as a direct target of miR-4735-3p, and SLC40A1 overexpression significantly attenuated iron overload and ferroptosis in the miR-4735-3p mimic-treated human ccRCC cell lines.

Conclusion: miR-4735-3p facilitates ferroptosis and tumor suppression in ccRCC by targeting SLC40A1.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Expression of miR-4735-3p in human ccRCC tissues and cell lines. (a) Relative expression of miR-4735-3p in human ccRCC tissues and adjacent normal tissues. (b) Relative expression of miR-4735-3p in human ccRCC tissues of different histological grades. (c) Relative expression of miR-4735-3p in human ccRCC tissues of different clinical TNM stages. (d) Relative expression of miR-4735-3p in human ccRCC cell lines or normal kidney cell line. (e and f) Relative expression of miR-4735-3p in human ccRCC cell lines upon different ferroptotic stimulations. N = 6 per group. All data were expressed as the mean ± SD; ∗P < 0.05 was considered significant.
Figure 2
Figure 2
Overexpression of miR-4735-3p induces ferroptosis of human ccRCC cell lines. (a) Relative expression of miR-4735-3p in human ccRCC cell lines treated with the miR-4735-3p mimic or mimic control. (b) Cell viability was examined by the MTT method. (c) Relative LDH level in the medium from the miR-4735-3p mimic or mimic control-treated ccRCC cells. (d and e) Relative ROS and MDA levels. (f and g) Relative intracellular iron and Fe2+ levels. (h) Cell viability in the miR-4735-3p mimic-treated ccRCC cells in the presence of various cell death inhibitors. N = 6 per group. All data were expressed as the mean ± SD; ∗P < 0.05 was considered significant.
Figure 3
Figure 3
Suppression of miR-4735-3p reduces ferroptosis of human ccRCC cell lines. (a) Relative expression of miR-4735-3p in human ccRCC cell lines treated with the miR-4735-3p inhibitor or inhibitor control. (b) Cell viability was examined by the MTT method. (c) Relative LDH level in the medium from the miR-4735-3p inhibitor or inhibitor control-treated ccRCC cells. (d and e) Relative ROS and MDA levels. (f and g) Relative intracellular iron and Fe2+ levels. N = 6 per group. All data were expressed as the mean ± SD; ∗P < 0.05 was considered significant.
Figure 4
Figure 4
Overexpression of miR-4735-3p induces ferroptosis through downregulating SLC40A1. (a) The complementary sequences of miR-4735-3p in SLC40A1 3′-UTR. (b) Relative SLC40A1 protein level in human ccRCC tissues and adjacent normal tissues. (c and d) Relative SLC40A1 protein and mRNA levels in 786-O cells treated with the miR-4735-3p mimic, inhibitor, or respective controls. (e) Relative luciferase activity in HEK293T cells transfected with the miR-4735-3p mimic and SLC40A1 3′-UTR. (f) Relative viability in SLC40A1-overexpressed 786-O cells treated with the miR-4735-3p mimic or mimic control. (g) Relative LDH releases in 786-O cells. (h) ROS level and MDA content in 786-O cells. (i) Relative iron level in 786-O cells. N = 6 per group. All data were expressed as the mean ± SD; ∗P < 0.05 was considered significant.
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