# Zinc Oxide Nanoparticle Inhibits Tumorigenesis of Renal Cell Carcinoma by Modulating Lipid Metabolism Targeting miR-454-3p to Repressing Metabolism Enzyme ACSL4

Authors: Xudong Zhou; Tingting Cao
Journal: Journal of Oncology
 (2022)

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## Abstract

Background. Renal cell carcinoma (RCC) affects the life quality of patients with advanced diseases despite good prognosis and exhibits abnormal lipid metabolism. Zinc oxide nanoparticles (ZONs) are metal oxide nanoparticles that are regarded as promising therapeutic candidate for multiple diseases. This study was for exploring the function of ZONs in RCC. Methods. We established in vitro cell model and in vivo xenograft model to determine the antitumor effect of ZONs. Cell viability and proliferation were evaluated via the cell counting kit-8 (CCK-8), colony formation, and 5-ethynyl-2’-deoxyuridine (EDU) assay. Protein and RNA levels were checked by using immunohistochemistry (IHC) and qRT-PCR assay. ROS, malondialdehyde (MDA), triglyceride, and total cholesterol were quantified to assess lipid oxidation and synthesis. Oil red O staining was performed to check lipid droplets accumulation. The ACSL4 and miR-454-3p expression in tumor samples and normal tissues were evaluated. The luciferase reporter gene assay was performed for checking the interaction between miR-454-3p and ACSL4 3’UTR region. Results. ZONs suppressed the proliferation and viability of RCC cells both in vitro and in vivo. ZONs suppressed accumulation of ROS, MDA, triglyceride, total cholesterol, and lipid droplets in RCC cells, along with upregulated miR-454-3p. miR-454-3p targeted the 3’UTR region to suppress its expression. In patient samples, ACSL4 expression was notably elevated and indicated poor prognosis of RCC patients. Conclusion. ZONs treatment notably impeded proliferation, lipid accumulation, and oxidation in RCC cells, through upregulating miR-454-3p to suppression the function of ACSL4. Our data suggested that ZONs are promising and effective agent for RCC treatment.


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## Body

## 1. Introduction

Renal cell carcinoma (RCC) is the most frequently occurred malignancy globally, with a steadily increased incidence [1]. Despite the development of therapeutic methods, the prognosis of RCC remains unsatisfactory [2]. Therefore, exploring the underlying mechanisms and developing efficient therapeutic manners is imperative for RCC treatment. Accumulating evidences have indicated the important role of fatty acid metabolism during cancer progression, and reprogramming of lipid metabolism is recently regarded as hallmark of malignancy [3, 4]. The abnormal metabolism of fatty acid would affect energy store, influence drug resistance, regulate survival and proliferation of cells, and stimulate the tumor microenvironment [5]. Several studies have explored regulatory factors and signaling pathways that are associated with de novo lipid metabolism are involved in the transformation and malignancy of tumor cells [6]. Thus, targeting lipid biosynthesis may be a novel strategy to manage the development of malignant tumors [7].MicroRNAs (miRNAs) are a group of short-length noncoding RNA (about 20 nucleotides long). miRNAs commonly modulate protein synthesis through directly binding with the 3’ untranslated region (3’UTR) of target mRNAs [8]. Various studies have verified a pivotal role of miRNAs in both tumor initiation and progression [9]. miR-454-3p is reported to act as tumor suppressor or activator in diverse cancer context [10–12]. For example, miR-454-3p downregulated RPRD1A to activate the function of Wnt/β-catenin signaling and promoted the metastasis of breast cancer cells [11]. In glioma, miR-454-3p could be delivered by exosomes and suppresses the proliferation, autophagy, invasion, and migration in glioma cells [13].Zinc oxide nanoparticles (ZONs) are nanoparticles that contain metal oxide and are capable of exhibiting multiple regulatory functions such as anti-inflammatory, antimicrobial, antidiabetic, anticancer, antiaging functions, and wound healing [14]. With high biocompatibility and low toxicity, ZONs are recognized as the most promising drug delivery manner in disease therapy [15]. In this work, we explored the function of ZONs on RCC, determined that ZONs inhibited growth of RCC cells and suppressed the lipid accumulation and toxicity in RCC cells, via upregulating miR-454-3p to repress the function of ACSL4. Our data suggested that ZONs may be promising therapeutic agent for RCC therapy.


## 2. Materials and Methods

### 2.1. Patient Samples

This research was performed with approved from the Ethics Committee of Cangzhou Central Hospital (no. CCH972-19) and following the government policies and the Declaration of Helsinki. Tumor tissues and paired adjacent normal ones were acquired from 42 patients diagnosed with RCC and hospitalized at Cangzhou Central Hospital. Histological and pathological diagnoses of the specimens were confirmed according to the 2016 World Health Organization Consensus Classification and Staging System of Renal Tumor and Fuhrman grade by two experienced pathologists. The inclusion criteria for the study subjects were as follows: (1) pathologically confirmed RCC; (2) no prior adjuvant treatment before radical resection; and (3) complete follow-up data. The exclusion criterion was a diagnosis of RCC with prior adjuvant treatment before radical resection or incomplete follow-up data. The patients are divided into two groups (ACSL4-high,n = 21 and ACSL4-low, n = 21) according to the relative expression of ACSL4. Patients were divided into high- and low-expression groups based on the median cutoff values of ACSL4. Samples were frozen in liquid nitrogen for further assays. The characteristics of 42 RCC patients were shown in Table S1.


### 2.2. Cell Culture and Transfection

RCC cell lines 786-O and 769P brought from Shanghai Cell Bank of Chinese Science Academy (China) were subjected to culturing in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) that contains 10% fetal bovine serum (Gibco, USA).The ACSL4 overexpressing vector (pcDNA-ACSL4), small interfering RNA (siACSL4), and miR-454-3p mimics and inhibitors were purchased from GenePharma (China). Cells were transfected with oligonucleotides by Lipofectamine 2000 reagent (Invitrogen, USA). ZONs used in this study were purchased from Sigma-Aldrich Chemical Co. (Sigma-Aldrich Co., St Louis, MO, USA).


### 2.3. Quantitative Real-Time PCR (qRT-PCR)

Total RNA extraction was conducted using Trizol reagent (Thermo Fisher Scientific, USA). For cDNA synthesis, 2μg RNA was treated by an M-MLV RTase cDNA synthesis kit (Takara, Japan). The following thermocycling conditions were used for cDNA synthesis: 30°C for 10 minutes, 42°C for 45 minutes, and 95°C for 5 minutes. To detect RNA expression, Superscript II enzyme (Takara, Japan) was adopted for qRT-PCR. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec; followed by 39 cycles of 95°C for 5 sec and 60°C for 30 sec; and a final extension at 72°C for 5 min. Gene quantification was calculated with 2−ΔΔCq means and normalized to the GAPDH level.


### 2.4. Cell Counting Kit-8 (CCK-8) Assay

Cells were transferred to a 96-well plate after transfection and incubated for indicated time periods (0, 24, 48, and 72 hours). After incubation for 0, 24, 48, and 72 hours, CCK-8 reagent was then added to the medium, followed by 2 hours incubation. The absorbance (450 nm) was metered by using one microplate reader (Thermo Fisher Scientific, USA).


### 2.5. Colony Formation Assay

Cells were transferred to 12-well plates at 500 cells/well, followed by 2-week incubation. The colonies were stained by crystal violet (Sigma-Aldrich, USA), captured under a dissection microscope (Nikon, Japan), and counted.


### 2.6. 5-Ethynyl-2’-Deoxyuridine (EDU) Assay

Cells were fixed and permeabilized, then hatched with EDU (50µM) for 3 hours. The cell nuclei were treated with 10 min staining with DAPI (1 µg/ml). The positive staining was checked using fluorescence microscopy (Leica, Germany).


### 2.7. Oil Red O Staining

For determination of lipid droplet, cells were fixed in 4% PFA and prewetted with 60% isopropanol, then incubated with Oil Red O for 15 min. The images were captured under optical microscope (Leica, Germany).


### 2.8. Lipid Peroxidation (MDA) Assay

The intracellular level of malondialdehyde (MDA) was checked via a lipid peroxidation MDA kit (Beyotime, China, S0131S) under the instruction of the manufacturer.


### 2.9. Detection of ROS

To detect the change of ROS, cells were hatched (37°C) with 5µM DCFDA in dark for 30 min. Cells were collected and suspended in phenol red free medium, followed by analysis via flow cytometry (FACSCalibur, BD Biosciences, USA).


### 2.10. Detection of Lipid Accumulation

The levels of triglyceride (TG) and total cholesterol (TC) in cell culture medium were assessed by using the GPO-PAP and COD-PAP enzymatic colorimetric assay kits (Thermo Fisher Scientific, USA) following the protocols.


### 2.11. Luciferase Reporter Gene Assay

The wild type and mutated sequences of the ACSL4 3’UTR were inserted into the pmirGLO vectors (Promega, USA) to obtain ACSL4-WT and ACSL4-Mut vectors. Cells were cotransfected with the ACSL4-WT and ACSL4-Mut vectors and miR-454-3p mimics or the negative control, and incubated for 24 hours. The pRLTK renilla luciferase reporter vector (Promega, USA) was cotransfected as the internal reference. Cells were then lysed and checked with the luciferase viability detection kit (Promega, USA).


### 2.12. Xenograft Mouse Model

All animal assays were performed under the authorization of Ethics Committee of Cangzhou Central Hospital (no. CCH/IACUC972-19). Female BALB/c nude mice were offered by Charles River Laboratory (USA). Cells (5 × 106) were suspended in 100 μL PBS, followed by injection into the right fat pad of mice. For treatment, ZONs (5 mg/kg) were injected subcutaneously around the tumor site every two days. When the average tumor volume reached about 100 mm3. There were no mice dead during the injection. Tumor volume was measured and calculated: Volume = length × (width)2/2.


### 2.13. Immunohistochemistry (IHC) Analysis

Tumor tissues were isolated, fixed, dehydrated, embedded in paraffin, and sectioned into 5μM slices. The samples were then deparaffined and treated with overnight incubation (4°C) with anti-ACSL4 antibody (Proteintech, China). Next, the samples were subjected to incubation with anti-rabbit horseradish peroxidase (Thermo Fisher Scientific, USA) and detected with a DAB kit (Beyotime, China).


### 2.14. Statistical Analyses

Data presented by mean ± S.E.M were processed using SPSS (version 19.0). Intergroup or multigroup comparison was analyzed using student’st-test or one-way ANOVA test. Pearson’s chi-squared test was adopted for understanding associations between categorical variables. P<0.05 implies a notable difference.


## 2.1. Patient Samples

This research was performed with approved from the Ethics Committee of Cangzhou Central Hospital (no. CCH972-19) and following the government policies and the Declaration of Helsinki. Tumor tissues and paired adjacent normal ones were acquired from 42 patients diagnosed with RCC and hospitalized at Cangzhou Central Hospital. Histological and pathological diagnoses of the specimens were confirmed according to the 2016 World Health Organization Consensus Classification and Staging System of Renal Tumor and Fuhrman grade by two experienced pathologists. The inclusion criteria for the study subjects were as follows: (1) pathologically confirmed RCC; (2) no prior adjuvant treatment before radical resection; and (3) complete follow-up data. The exclusion criterion was a diagnosis of RCC with prior adjuvant treatment before radical resection or incomplete follow-up data. The patients are divided into two groups (ACSL4-high,n = 21 and ACSL4-low, n = 21) according to the relative expression of ACSL4. Patients were divided into high- and low-expression groups based on the median cutoff values of ACSL4. Samples were frozen in liquid nitrogen for further assays. The characteristics of 42 RCC patients were shown in Table S1.


## 2.2. Cell Culture and Transfection

RCC cell lines 786-O and 769P brought from Shanghai Cell Bank of Chinese Science Academy (China) were subjected to culturing in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) that contains 10% fetal bovine serum (Gibco, USA).The ACSL4 overexpressing vector (pcDNA-ACSL4), small interfering RNA (siACSL4), and miR-454-3p mimics and inhibitors were purchased from GenePharma (China). Cells were transfected with oligonucleotides by Lipofectamine 2000 reagent (Invitrogen, USA). ZONs used in this study were purchased from Sigma-Aldrich Chemical Co. (Sigma-Aldrich Co., St Louis, MO, USA).


## 2.3. Quantitative Real-Time PCR (qRT-PCR)

Total RNA extraction was conducted using Trizol reagent (Thermo Fisher Scientific, USA). For cDNA synthesis, 2μg RNA was treated by an M-MLV RTase cDNA synthesis kit (Takara, Japan). The following thermocycling conditions were used for cDNA synthesis: 30°C for 10 minutes, 42°C for 45 minutes, and 95°C for 5 minutes. To detect RNA expression, Superscript II enzyme (Takara, Japan) was adopted for qRT-PCR. The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 30 sec; followed by 39 cycles of 95°C for 5 sec and 60°C for 30 sec; and a final extension at 72°C for 5 min. Gene quantification was calculated with 2−ΔΔCq means and normalized to the GAPDH level.


## 2.4. Cell Counting Kit-8 (CCK-8) Assay

Cells were transferred to a 96-well plate after transfection and incubated for indicated time periods (0, 24, 48, and 72 hours). After incubation for 0, 24, 48, and 72 hours, CCK-8 reagent was then added to the medium, followed by 2 hours incubation. The absorbance (450 nm) was metered by using one microplate reader (Thermo Fisher Scientific, USA).


## 2.5. Colony Formation Assay

Cells were transferred to 12-well plates at 500 cells/well, followed by 2-week incubation. The colonies were stained by crystal violet (Sigma-Aldrich, USA), captured under a dissection microscope (Nikon, Japan), and counted.


## 2.6. 5-Ethynyl-2’-Deoxyuridine (EDU) Assay

Cells were fixed and permeabilized, then hatched with EDU (50µM) for 3 hours. The cell nuclei were treated with 10 min staining with DAPI (1 µg/ml). The positive staining was checked using fluorescence microscopy (Leica, Germany).


## 2.7. Oil Red O Staining

For determination of lipid droplet, cells were fixed in 4% PFA and prewetted with 60% isopropanol, then incubated with Oil Red O for 15 min. The images were captured under optical microscope (Leica, Germany).


## 2.8. Lipid Peroxidation (MDA) Assay

The intracellular level of malondialdehyde (MDA) was checked via a lipid peroxidation MDA kit (Beyotime, China, S0131S) under the instruction of the manufacturer.


## 2.9. Detection of ROS

To detect the change of ROS, cells were hatched (37°C) with 5µM DCFDA in dark for 30 min. Cells were collected and suspended in phenol red free medium, followed by analysis via flow cytometry (FACSCalibur, BD Biosciences, USA).


## 2.10. Detection of Lipid Accumulation

The levels of triglyceride (TG) and total cholesterol (TC) in cell culture medium were assessed by using the GPO-PAP and COD-PAP enzymatic colorimetric assay kits (Thermo Fisher Scientific, USA) following the protocols.


## 2.11. Luciferase Reporter Gene Assay

The wild type and mutated sequences of the ACSL4 3’UTR were inserted into the pmirGLO vectors (Promega, USA) to obtain ACSL4-WT and ACSL4-Mut vectors. Cells were cotransfected with the ACSL4-WT and ACSL4-Mut vectors and miR-454-3p mimics or the negative control, and incubated for 24 hours. The pRLTK renilla luciferase reporter vector (Promega, USA) was cotransfected as the internal reference. Cells were then lysed and checked with the luciferase viability detection kit (Promega, USA).


## 2.12. Xenograft Mouse Model

All animal assays were performed under the authorization of Ethics Committee of Cangzhou Central Hospital (no. CCH/IACUC972-19). Female BALB/c nude mice were offered by Charles River Laboratory (USA). Cells (5 × 106) were suspended in 100 μL PBS, followed by injection into the right fat pad of mice. For treatment, ZONs (5 mg/kg) were injected subcutaneously around the tumor site every two days. When the average tumor volume reached about 100 mm3. There were no mice dead during the injection. Tumor volume was measured and calculated: Volume = length × (width)2/2.


## 2.13. Immunohistochemistry (IHC) Analysis

Tumor tissues were isolated, fixed, dehydrated, embedded in paraffin, and sectioned into 5μM slices. The samples were then deparaffined and treated with overnight incubation (4°C) with anti-ACSL4 antibody (Proteintech, China). Next, the samples were subjected to incubation with anti-rabbit horseradish peroxidase (Thermo Fisher Scientific, USA) and detected with a DAB kit (Beyotime, China).


## 2.14. Statistical Analyses

Data presented by mean ± S.E.M were processed using SPSS (version 19.0). Intergroup or multigroup comparison was analyzed using student’st-test or one-way ANOVA test. Pearson’s chi-squared test was adopted for understanding associations between categorical variables. P<0.05 implies a notable difference.


## 3. Results

### 3.1. ZONs Suppress RCC Cell Proliferation

We first determined the function of ZONs in RCC cellsin vitro. RCC cells were treated by 20 μg/ml ZONs for 24, 48, and 72 hours. Results from CCK-8 indicated that ZONs notably suppressed the viability of 786-O and 769P cells in a time-dependent manner (Figures 1(a) and 1(b)). Cell proliferation was determined through the colony formation and EDU assay. ZON treatment significantly decreased the percentage of RCC cell colonies (Figures 1(c) and 1(d)). The reduced EDU-positive staining under ZON treatment suggested depressed cell proliferation (Figures 1(e) and 1(f)).Figure 1
ZONs suppress RCC cell proliferation. (a, b) CCK-8 assay to determine RCC cell viability. (c, d) Colony formation to determine proliferation of RCC cells. (e, f) EDU assay to detect proliferation of RCC cells.P∗∗<0.01 vs control group.


### 3.2. ZONs Alleviates Lipid Accumulation in RCC Cells

We next detected the oxidative stress and lipid accumulation in RCC cells under ZON treatment. The ROS level was significantly decreased by ZON treatment (Figure2(a)). MDA is the end product of lipid peroxidation, and ZON treatment notably suppressed the intracellular MDA level (Figure 2(b)). The secreted levels of triglyceride (TG) and total cholesterol (TC) in cell culture medium were downregulated by ZONs, manifesting the inhibited lipid accumulation (Figures 2(c) and 2(d)). Besides, Oil red O staining demonstrated that ZON treatment lessened the lipid drop in RCC cells (Figure 2(e)).Figure 2
ZONs alleviate lipid accumulation in RCC cells. (a) ROS level determined by DCFDA staining. (b) The intracellular MDA levels. (c, d) The levels of triglyceride and total cholesterol in culture medium. (e) Oil red O staining of lipid drop.P∗∗<0.01 vs control group.


### 3.3. miR-454-3p Negatively Regulates ACSL4 in RCC Cells

Subsequently, we probed into the mechanisms under the therapeutic effect of ZONs. We observed a remarkably elevated level of miR-454-3p in RCC cells under ZON administration (Figure3(a)). Prediction by online tool indicated the potential binding site of miR-454-3p on 3’UTR region of ACSL4 (Figure 3(b)). The luciferase reporter gene experiment demonstrated that miR-454-3p interact with the wild type ACSL4 3’UTR rather than the mutated sequences (Figures 3(c) and 3(d)), which confirmed the direct interaction between miR-454-3p and ACSL4 3’UTR. Moreover, analysis on collected RCC tumor samples and healthy tissues also showed elevated expression of ACSL4 in RCC tumors (Figure 4(a)). Noteworthy, the level of miR-454-3p was negatively correlated with level of ACSL4 (Figure 4(b)). Survival analysis further indicated that higher level of ACSL4 is associated with poor prognosis of RCC patients (Figure 4(c)).Figure 3
miR-454-3p interacts with the 3’UTR region of ACSL4. (a) Quantification of miR-454-3p in RCC cells by qRT-PCR. (b) Prediction of miR-454-3p binding site on ACSL4 3’UTR region. (c) Luciferase reporter gene assay to determine luciferase activity of wild type (WT) and mutated (MUT) ACSL4 3’UTR promoter vectors. (d) Detection of ACSL4 level in RCC cells by qRT-PCR.∗∗P<0.01 vs control group.Figure 4
Patient sample analysis. We collected RCC tumor tissues (n = 42) and healthy thyroid tissues (n = 42) to analyze gene expression and patient survival. (a) ACSL4 RNA level in tissues detected by qRT-PCR. (b) Comparison of miR-454-3p and ACSL4 levels in tissues. (c) Survival analysis of RCC patients. ∗∗P<0.01 vs control group.
(a)(b)(c)


### 3.4. ZONs Affect RCC Cells Proliferation via miR-454-3p/ACSL4 AxisIn Vitro

To confirm the function of the miR-454-3p/ACSL4 axis in the regulation of ZON on RCC cell proliferation, we conducted transfection of miR-454-3p mimics and overexpression of ACSL4 in RCC cells before ZON treatment. We observed that overexpressed ACSL4 and inhibited miR-454-3p could notably reverse the ZON-suppressed RCC cell viability (Figures5(a) and 5(b)). The results of the EDU assay also implied that ZON decreased the portion of proliferative cells, whereas miR-454-3p mimics and ACSL4 overexpression abolished this effect (Figures 5(c)–5(e)2.Figure 5
ZONs affect RCC cells proliferation via the miR-454-3p/ACSL4 axisin vitro. (a, b) CCK-8 assay to detect RCC cell viability. (c–e) EDU assay to detect proliferation of RCC cells. Histogram to calculate EDU-positive cells. ∗∗P<0.01 vs control or the ZON group.


### 3.5. ZONs Affect RCC Cells Lipid Metabolism via miR-454-3p/ACSL4 AxisIn Vitro

To more deeply determine the function of the miR-454-3p/ACSL4 axis in ZON-suppressed RCC cell proliferation, we also evaluated the oxidative stress lipid accumulation. As shown in Figures6(a) and 6(b), ZON suppressed ROS and MDA levels, whereas miR-454-3p mimics and ACSL4 overexpression reversed this phenotype. The ZON-decreased levels of triglyceride, total cholesterol, and lipid drop accumulation were also recovered by inhibited miR-454-3p and overexpressed ACSL4 (Figures 6(c)–6(e)).Figure 6
ZONs affect RCC cells lipid metabolism via the miR-454-3p/ACSL4 axisin vitro. (a) ROS level determined by DCFDA staining. (b) The intracellular MDA levels. (c, d) The levels of triglyceride and total cholesterol in culture medium. (e) Oil red O staining of lipid drop. ∗∗P<0.01 vs control or the ZON group.


### 3.6. ZONs RepressIn Vivo RCC Tumorigenesis via miR-454-3p/ACSL4 Axis

Consistent with the results from cellular experiments,in vivo xenograft study indicated that administration of ZON suppressed RCC tumor growth, tumor size, and weight (Figures 7(a) and 7(b)). The expression of ACSL4 and miR-454-3p was altered by ZON treatment and could be reversed by overexpressed ACSL4 and miR-454-3p mimics (Figures 7(c) and 7(d)). The regulated expression of ACSL4 by treatment of ZON and miR-454-3p mimics was also verified by IHC staining (Figure 7(e)).Figure 7
ZONs repressin vivo RCC tumorigenesis via the miR-454-3p/ACSL4 axis. Xenograft tumor model was constructed for determining the in vivo effect of ZONs. Tumor growth cure (a) and tumor weight (b) were detected. (c, d) The RNA levels of miR-454-3p and ACSL4 were detected by qRT-PCR. (e) IHC staining of ACSL4 protein. ∗∗P<0.01 vs control or the ZON group.


## 3.1. ZONs Suppress RCC Cell Proliferation

We first determined the function of ZONs in RCC cellsin vitro. RCC cells were treated by 20 μg/ml ZONs for 24, 48, and 72 hours. Results from CCK-8 indicated that ZONs notably suppressed the viability of 786-O and 769P cells in a time-dependent manner (Figures 1(a) and 1(b)). Cell proliferation was determined through the colony formation and EDU assay. ZON treatment significantly decreased the percentage of RCC cell colonies (Figures 1(c) and 1(d)). The reduced EDU-positive staining under ZON treatment suggested depressed cell proliferation (Figures 1(e) and 1(f)).Figure 1
ZONs suppress RCC cell proliferation. (a, b) CCK-8 assay to determine RCC cell viability. (c, d) Colony formation to determine proliferation of RCC cells. (e, f) EDU assay to detect proliferation of RCC cells.P∗∗<0.01 vs control group.


## 3.2. ZONs Alleviates Lipid Accumulation in RCC Cells

We next detected the oxidative stress and lipid accumulation in RCC cells under ZON treatment. The ROS level was significantly decreased by ZON treatment (Figure2(a)). MDA is the end product of lipid peroxidation, and ZON treatment notably suppressed the intracellular MDA level (Figure 2(b)). The secreted levels of triglyceride (TG) and total cholesterol (TC) in cell culture medium were downregulated by ZONs, manifesting the inhibited lipid accumulation (Figures 2(c) and 2(d)). Besides, Oil red O staining demonstrated that ZON treatment lessened the lipid drop in RCC cells (Figure 2(e)).Figure 2
ZONs alleviate lipid accumulation in RCC cells. (a) ROS level determined by DCFDA staining. (b) The intracellular MDA levels. (c, d) The levels of triglyceride and total cholesterol in culture medium. (e) Oil red O staining of lipid drop.P∗∗<0.01 vs control group.


## 3.3. miR-454-3p Negatively Regulates ACSL4 in RCC Cells

Subsequently, we probed into the mechanisms under the therapeutic effect of ZONs. We observed a remarkably elevated level of miR-454-3p in RCC cells under ZON administration (Figure3(a)). Prediction by online tool indicated the potential binding site of miR-454-3p on 3’UTR region of ACSL4 (Figure 3(b)). The luciferase reporter gene experiment demonstrated that miR-454-3p interact with the wild type ACSL4 3’UTR rather than the mutated sequences (Figures 3(c) and 3(d)), which confirmed the direct interaction between miR-454-3p and ACSL4 3’UTR. Moreover, analysis on collected RCC tumor samples and healthy tissues also showed elevated expression of ACSL4 in RCC tumors (Figure 4(a)). Noteworthy, the level of miR-454-3p was negatively correlated with level of ACSL4 (Figure 4(b)). Survival analysis further indicated that higher level of ACSL4 is associated with poor prognosis of RCC patients (Figure 4(c)).Figure 3
miR-454-3p interacts with the 3’UTR region of ACSL4. (a) Quantification of miR-454-3p in RCC cells by qRT-PCR. (b) Prediction of miR-454-3p binding site on ACSL4 3’UTR region. (c) Luciferase reporter gene assay to determine luciferase activity of wild type (WT) and mutated (MUT) ACSL4 3’UTR promoter vectors. (d) Detection of ACSL4 level in RCC cells by qRT-PCR.∗∗P<0.01 vs control group.Figure 4
Patient sample analysis. We collected RCC tumor tissues (n = 42) and healthy thyroid tissues (n = 42) to analyze gene expression and patient survival. (a) ACSL4 RNA level in tissues detected by qRT-PCR. (b) Comparison of miR-454-3p and ACSL4 levels in tissues. (c) Survival analysis of RCC patients. ∗∗P<0.01 vs control group.
(a)(b)(c)


## 3.4. ZONs Affect RCC Cells Proliferation via miR-454-3p/ACSL4 AxisIn Vitro

To confirm the function of the miR-454-3p/ACSL4 axis in the regulation of ZON on RCC cell proliferation, we conducted transfection of miR-454-3p mimics and overexpression of ACSL4 in RCC cells before ZON treatment. We observed that overexpressed ACSL4 and inhibited miR-454-3p could notably reverse the ZON-suppressed RCC cell viability (Figures5(a) and 5(b)). The results of the EDU assay also implied that ZON decreased the portion of proliferative cells, whereas miR-454-3p mimics and ACSL4 overexpression abolished this effect (Figures 5(c)–5(e)2.Figure 5
ZONs affect RCC cells proliferation via the miR-454-3p/ACSL4 axisin vitro. (a, b) CCK-8 assay to detect RCC cell viability. (c–e) EDU assay to detect proliferation of RCC cells. Histogram to calculate EDU-positive cells. ∗∗P<0.01 vs control or the ZON group.


## 3.5. ZONs Affect RCC Cells Lipid Metabolism via miR-454-3p/ACSL4 AxisIn Vitro

To more deeply determine the function of the miR-454-3p/ACSL4 axis in ZON-suppressed RCC cell proliferation, we also evaluated the oxidative stress lipid accumulation. As shown in Figures6(a) and 6(b), ZON suppressed ROS and MDA levels, whereas miR-454-3p mimics and ACSL4 overexpression reversed this phenotype. The ZON-decreased levels of triglyceride, total cholesterol, and lipid drop accumulation were also recovered by inhibited miR-454-3p and overexpressed ACSL4 (Figures 6(c)–6(e)).Figure 6
ZONs affect RCC cells lipid metabolism via the miR-454-3p/ACSL4 axisin vitro. (a) ROS level determined by DCFDA staining. (b) The intracellular MDA levels. (c, d) The levels of triglyceride and total cholesterol in culture medium. (e) Oil red O staining of lipid drop. ∗∗P<0.01 vs control or the ZON group.


## 3.6. ZONs RepressIn Vivo RCC Tumorigenesis via miR-454-3p/ACSL4 Axis

Consistent with the results from cellular experiments,in vivo xenograft study indicated that administration of ZON suppressed RCC tumor growth, tumor size, and weight (Figures 7(a) and 7(b)). The expression of ACSL4 and miR-454-3p was altered by ZON treatment and could be reversed by overexpressed ACSL4 and miR-454-3p mimics (Figures 7(c) and 7(d)). The regulated expression of ACSL4 by treatment of ZON and miR-454-3p mimics was also verified by IHC staining (Figure 7(e)).Figure 7
ZONs repressin vivo RCC tumorigenesis via the miR-454-3p/ACSL4 axis. Xenograft tumor model was constructed for determining the in vivo effect of ZONs. Tumor growth cure (a) and tumor weight (b) were detected. (c, d) The RNA levels of miR-454-3p and ACSL4 were detected by qRT-PCR. (e) IHC staining of ACSL4 protein. ∗∗P<0.01 vs control or the ZON group.


## 4. Discussion

RCC patients with metastatic progression and recurrence showed poor prognosis, and the life quality is severely affected [1]. Accumulating evidences have revealed the participation of de novo lipid metabolism-related elements during cell transformation and malignancy [5]. Targeting the abnormal lipid biosynthesis offers a novel insight into the exploration of management approach for malignant tumors.Lipid comprises of numerous different molecular types, such as the fatty acids, phospholipids, triglycerides, cholesterol, sphingolipids, and cholesteryl esters [16]. The excessive lipids in cells would be converted to cholesteryl esters and triglycerides and form lipid droplets [17]. The accumulation of lipid droplets has been spotted in multiple cancers, including the pancreatic cancer, glioblastoma, and prostate cancer as well as RCC [18–20]. In this work, we observed that treatment with ZONs greatly inhibited the survival and proliferation of RCC cells, and the results from Oil red O staining indicated decreased accumulation of intracellular lipid droplets under ZONs treatment. Moreover, precious studies have proved that the increased levels of total cholesterol and triglycerides in plasma facilitate the progression and aggressiveness of malignant tumors [21]. ZONs treatment reduced the secretion of total cholesterol and triglycerides by RCC cells. On the other hand, lipid peroxidation is also associated with cancer progression, via triggering production of lipid ROS and following toxic effects [21]. ZONs are also capable of alleviating the total ROS and lipid ROS production. The anticancer effect of ZONs has been widely reported. It has been reported that ZONs promote proteotoxic and oxidative stress and trigger apoptosis in ovarian cancer [22]. ZONs attenuate drug-resistant of breast cancer cells [23]. ZONs induces ferroptosis of colorectal cancer cells [24]. ZONs enhance apoptosis of epidermoid carcinoma cells by oxidative stress and DNA degradation [25]. These studies report that ZONs induce the anticancer activities by modulating multiple processes, such as drug-resistant, ferroptosis, oxidative stress, and DNA degradation. However, the function of ZONs in RCC and lipid metabolism remains unreported. Our data suggest that ZONs inhibited RCC cells proliferation, lipid synthesis, and oxidation in vitro and in vivo, providing new evidence of the anticancer function of ZONs.Subsequent analysis on molecular mechanisms discovered an elevated level of miR-454-3p after ZONs treatment. Reportedly, miR-454-3p is a tumor suppressor in several cancers like the bladder cancer, breast cancer, and non-small cell lung cancer, as well as glioblastoma [10,11,26–28]. miR-454-3p impeded breast cancer metastasis via inhibiting Wnt signaling transduction [11]. In lung cancer, miR-454-3p inhibited cancer cell migration and proliferation via targeting the expression of TGF-β [27]. In this work, we observed that miR-454-3p sponged ACSL4 and suppressed its expression in RCC cells. Analysis on RCC patient samples demonstrated elevated ACSL4 expression in RCC tumor samples, comparing with the normal tissues, and this high ACSL4 level correlated with unfavorable prognosis of RCC patients. Besides, the negative association of miR-454-3p with ACSL4 confirmed that miR-454-3p targeted ACSL4 expression. Several studies suggested the tumor-promoting role of ACSL4 in cancer development [29, 30]. Recent study mentioned that ACSL4 regulates the de novo lipogenesis in hepatocellular carcinoma cells via stimulating production of intracellular cholesterols, triglycerides, and lipid droplets [31]. Consistently, we found that overexpression of ACSL4 abolished the suppressed proliferation and lipid accumulation in RCC cells. There are some crucial issues which should be explored in future investigations, for example, whether the stability of ZONs affects the efficacy of ZONs during cell experiments. ZnO is commonly recognized as safe by the Federal Drug Administration. ZnO-NPs are more stable, easier to prepare, and significantly less expensive [32]. It has been reported that ZnO occurs in wurtzite, zinc blende, and rock salt structures. The wurtzite phase is more stable thermodynamically at ambient conditions [33]. The stability during cell experiments should be validated in future investigations. Moreover, miR-454-3p/ ACSL4 axis may be just one of the downstream mechanisms of ZONs-induced anti-RCC cancer effect and other potential targets of ZONs should be explored by more studies.


## 5. Conclusion

To summarize, our study demonstrated that ZONs inhibited RCC cells proliferation, lipid synthesis, and oxidationin vitro and in vivo. ZONs exhibit its functions by elevating the level of miR-454-3p and subsequently suppressing ACSL4 in RCC cells. Hence, ZONs is a promising agent for RCC therapy.



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*Source: 2883404-2022-03-25.xml*