MicroRNA-216a-5p Alleviates Acute Kidney Injury of Mice via Suppressing FAS Ligand Expression

Acute kidney injury (AKI) is clinically defined as a reduction in kidney function, which is manifested in elevated serum creatinine or declined urine output [1]. Ischemia/reperfusion (I/R)-induced AKI is the most common cause of AKI, characterized by damage to vascular endothelial cells and tubular epithelial cells (TECs) and strong inflammatory responses, such as upregulation of chemokines, cytokines, and leukocyte infiltration in the kidney [2]. It is linked to high mortality and morbidity rates, as well as financial burdens on account of potential need for hemodialysis and prolonged hospitalization [3]. In clinical practice, the criteria for evaluation of kidney function are urea levels and determination of creatinine and balanced diuresis [4]. Current treatments mainly include supportive care rather than curative treatments or effective prevention [5]. The tough situation of AKI treatment makes it necessary to further explore the mechanism and find a new therapeutic strategy.

MicroRNAs (miRs) are endogenous noncoding RNAs of about 23 nucleotides in length [6]. Serving as pivotal modulators of cellular homeostasis, the dysregulation of miRNAs is a vital component of both cell and organ injury [7]. The contribution of miRs to AKI pathophysiology has been highlighted together with their potential for therapeutic applications [8]. Previous articles have disclosed that miR-216a-5p is dysregulated in multiple malignancies, such as liver cancer, colorectal cancer, as well as pancreatic cancer [9‒11]. Especially, miR-216a-5p has been revealed to be expressed in the glomerulus and could participate in diabetic nephropathy progression [12]. Moreover, miR-216a-5p carried in urine-derived stem cells exosomes could transfer to kidney TECs and alleviate cell damage in HK-2 cell exposed to hypoxia/reoxygenation (H/R) and kidney I/R injury rat model [13]. It is displayed in a study that FAS ligand (FASL) is directly targeted by miR-204-5p in kidney I/R injury [14], while its relationship with miR-216a-5p has not been discussed. The FASL (CD178) is located on the human chromosome 1q23 and is the key to induce apoptosis of susceptible cells [15]. FASL is expressed by kidney cells and its expression elevates during kidney damage, and the activation of the Fas receptor triggers apoptosis of non-stimulated or cytokine-primed kidney cells in culture [16]. A study has purported that induction of matrix metalloproteinase-7 has a protective effect in AKI through mobilizing β-catenin and degrading FASL, thereby initiating kidney tubules to promote survival and regeneration [17]. According to Soni et al. [18], cisplatin causes nephrotoxicity via promoting FASL/Fas-dependent death of oxidative kidney tubular cells. Nevertheless, the interaction of miR-216a-5p and FASL in AKI is seldom discussed. Therefore, we aimed to investigate the mechanism of the miR-216a-5p/FASL axis in AKI.

All animal experiments were ratified by the Institutional Animal Care Use Committee of The Affiliated Suzhou Hospital of Nanjing Medical University (approval number: 20210615).

Male C57BL/6 mice aged 6–8 weeks and weighed 22–25 g were supplied by the animal center of Nanjing Medical University. The mice were fed in a laboratory of specific pathogen-free grade at 22–25°C with water and food available ad libitum.

C57BL/6 mice were allocated into sham group (n = 15) and I/R group (n = 60). Then, the I/R group was sub-grouped with 10 mice in each group: I/R group (mice were only modeled), agomir negative control (ago-NC) group (mice were intravenously injected with miRNA agomir NC and modeled), ago-miR-216a-5p group (mice were intravenously injected with miR-216a-5p agomir and modeled), anti-NC + si-NC group (mice were intravenously injected with miRNA antagomir NC and siRNA NC and modeled), anti-miR-216a-5p + si-NC group (mice were intravenously injected with miR-216a-5p antagomir and siRNA NC and modeled), and anti-miR-216a-5p + si-FASL group (mice were intravenously injected with miR-216a-5p antagomir and FASL siRNA and modeled). The above miRNA agomir, antagomir, siRNA were purchased from RiboBio (Guangzhou, China). The miRNA agomir, antagomir, siRNA, and corresponding NCs were injected into the mice 24 h before ischemia modeling through the tail vein at a dose of 40 mg/kg bodyweight [19, 20].

Animal model preparation: according to Supavekin et al. [21], a unilateral kidney ischemia model was established. Mice were anesthetized, and core body temperature was maintained at 37°C and then fixed on the dissecting table in a prone position. The skin was disinfected with iodine after preparation, a dorsal longitudinal incision of 1.5 cm was made into the abdominal cavity, the muscle was bluntly separated, and the left kidney pedicle was exposed and dissociated. The kidney artery was separated, and the blood flow was blocked by a clamp. The change in the left kidney from red to dark purple indicated successful ischemia. After ischemia for 45 min, the artery clamp was released, the abdominal cavity was closed when the left kidney returned to perfusion, and I/R model was established. Mice in the sham group were only treated with abdominal cavity opening and kidney exposure for 45 min.

Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (200 mg/kg) 24 h after surgery. Blood was collected from the eyeball and placed in a centrifuge tube to separate the serum. Mice were euthanized by neck dislocation to collect the kidney. The kidney was dissected sagittal and rinsed by phosphate buffered saline. The kidney tissues were weighed and added with precooled normal saline or radioimmunoprecipitation assay cell lysis buffer and centrifuged to obtain the supernatant. The supernatant was preserved at −80°C. The residual tissues were fixed in 4% paraformaldehyde and placed in 4°C refrigerator for 24 h to make paraffin slices.

Kidney tissues fixed with 4% paraformaldehyde were dehydrated by alcohol, embedded by paraffin and sliced (5 μm), then heated at 60°C for 12 h, immersed in turpentine for 1 h, soaked in gradient alcohol, dyed with hematoxylin, differentiated in 1% hydrochloric acid alcohol, returned to blue in saturated lithium carbonate, and stained in eosin. Finally, tissues were dehydrated by alcohol, dried, and sealed by neutral gum. The tissue slices were observed under a light microscope in 5 fields to observe the pathological damage characteristics of tissues and scored. The score was performed by the nephrologist with blind method. Semiquantitative analysis of tubulointerstitial damage: Jablonski method [22] was used to score the pathological damage of tubulointerstitial. 0 point, normal; 1 point, tubulointerstitial area of damaged kidney <25%; 2 points, 25–50%; 3 points, 50–70%; 4 points, 75%. The average value was reckoned, and high score indicated severe damage.

The content of serum creatinine (SCr) of mice was determined by picric acid method. Serum (0.2 mL) was supplemented with 2 mL tungstate protein precipitator, centrifuged for 10 min at 3,500 r/min, and the supernatant was taken to mix with preconfigured related standard liquid, then bathed for 10 min. The optical density (OD) value at 510 nm was tested by a spectrophotometer. The content of blood urea nitrogen (BUN) was tested by urease method: the sample was mingled with the standard liquid and bathed for 10 min, and the OD value at 640 nm was detected by a spectrophotometer. The SCr and BUN kits were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Xanthinoxidase method, myeloperoxidase (MPO) method, and thiobarbituric acid were applied for detection of superoxide dismutase (SOD), MPO activities, and malondialdehyde (MDA) content with the SOD, MPO, and MDA detection kits (Nanjing Jiancheng Bioengineering Institute). The OD value was tested by spectrophotometer, then the SOD (550 nm) and MPO (450 nm) activities as well as MDA content (532 nm) were counted.

Enzyme-linked immunosorbent assay was utilized to test the contents of interleukin (IL)-1β and tumor necrosis factor-α (TNF-α). The OD value at 450 nm was gauged by spectrophotometer. TNF-α and IL-1β kits were provided by R&D Systems (Minneapolis, MN, USA).

The tissue paraffin sections were dewaxed and hydrated, and then incubated with proteinase K and 1% Triton-X-100. The endogenous catalase was inactivated. The sections were successively reacted with 20 μL TUNEL mixture, 20 μL converter-peroxidase, and 50–100 μL DAB. Afterward, the sections were stained with hematoxylin, dehydrated with gradient alcohol, cleared by xylene, sealed in neutral gum, and observed under the light microscope. Ten nonoverlapping fields of kidney cortex and medulla area were selected for each section, and 100 cells were counted in each field, a total of 1,000 cells. Apoptotic cells were the positive cells with brownish yellow nucleus and the percentage of apoptotic cells was calculated.

The rest 5 mice in the sham group were euthanized by neck dislocation. The kidney was perfused by NaCl solution (0.9%) through the lower abdominal aorta, and the kidney medulla was cut into 1 mm³ and immersed in Dulbecco’s Modified Eagle Medium (DMEM)/F12 solution containing 1 g/L collagenase and fetal bovine serum for 30 min. Cell single suspension was centrifuged, and the precipitate was cultured in DMEM/F12 solution containing 10% fetal bovine serum. After 4–5 days, the sterilized cover glass was put into the 24-well plate. Cell suspension was dripped on the surface of the cover glass and then incubated. When the cells covered with the entire cover glass, it was observed under the microscope.

Cell grouping: control group (cells were cultured routinely), H/R group (H/R cell model was established), mimic NC group (transfected with miRNA mimic NC and H/R), miR-216a-5p mimic group (transfected with miR-216a-5p mimic and H/R), inhibitor NC+ si-NC group (transfected with miRNA inhibitor NC, siRNA NC, and H/R), miR-216a-5p inhibitor + si-NC group (transfected with miR-216a-5p inhibitor, siRNA NC, and H/R), and miR-216a-5p inhibitor + si-FASL group (transfected with miR-216a-5p inhibitor, FASL siRNA, and H/R).

Establishment of the cell model: H/R cell model was established by mineral oil covering cell method to simulate kidney I/R injury. TEC suspension was prepared with 5 × 10⁵ cells/mL by complete medium, seeded in a 6-well plate with 2 mL/well, and then cultured. The culture solution was discarded when the confluence reached about 70–80%. Each well was added with mineral oil until the cells were completely covered, then cells were cultured for 1 h. Next, each well was supplemented with 2 mL serum-free DMEM/F12 and cultured in a 5% CO₂ incubator.

Cells were seeded in the 96-well plate (1 × 10⁴ cells/well). Each well was added with 20 μL MTT solution (5 mg/mL) and cultured for 4 h. Afterward, each well was supplemented with 150 μL dimethyl sulfoxide oscillation until the formazan inside the cell was fully dissolved. The OD_{570 nm} value was measured by a microplate reader.

The total RNA in tissues and cells were extracted by miRNeasy Mini Kit (Qiagen Company, Hilden, Germany). RNA was reverse-transcribed into complementary DNA by PrimeScript RT reagent Kit (Takara, Kyoto, Japan). Bulge-loop^™ U6 and miR-216a-5p reverse transcription quantitative polymerase chain reaction (RT-qPCR) Primer Sets (one for reverse transcription and its pair for amplifying each target gene) were synthesized by RiboBio (Guangzhou, China). The target genes primers are exhibited in online supplementary Table 1 (for all online suppl. material, see https://doi.org/10.1159/000539980). The ViiA 7 system (Applied Biosystems, Carlsbad, CA, USA) was used for RT-qPCR. U6 was the loading control of miR-216a-5p, and β-actin was internal reference of FASL. The data were analyzed by the 2^−ΔΔCt method.

Bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was implemented to assess the protein concentration. Proteins were separated on a 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA) that was blocked and incubated overnight at 4°C with primary antibodies against β-actin (1:2000, Cell Signaling Technology, Beverly, MA, USA) and FASL (1:1000, Santa Cruz Biotechnology, CA, USA), then incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000, Cell Signaling Technology), and observed by ChemiDox XRS gel imager (Bio-Rad). The gray value of protein was determined by ImageJ software.

Both wild-type (Wt) and mutant (Mut) FASL 3′- untranslated regions were amplified by PCR and cloned into the pmiR-RB-REPORT^™ luciferase reporter vector (RiboBio). The kidney TECs were seeded in the 6-well plate (4 × 10⁵ cells/well), and the transfection was implemented when cells reached 70% confluence. Cells were grouped into FASL-Wt + miR-216a-5p mimic group, FASL-Wt + mimic NC group, FASL-Mut + miR-216a-5p mimic group, and FASL-Mut + mimic NC group. Cells in each well were added with 100 μL passive lysis buffer for 15 min, oscillated for 15 s, and centrifuged at 4°C, 12,000 r/min for 30 s. The supernatant (20 μL) was mixed with 100 μL luciferase detection reagent II to measure firefly luciferase. Then, the firefly luciferase was quenched and the Renilla luciferase was measured. The ratio of firefly luciferase and Renilla luciferase was the relative luciferase activity.

All data were interpreted by SPSS 24.0 software (IBM, Armonk, NY, USA). Measurement data were indicated as mean ± standard deviation. Comparisons between two groups were performed by independent sample t test. Comparisons among multiple groups were assessed by one-way analysis of variance (ANOVA) with Tukey's multiple comparisons test. The p value <0.05 was regarded as significant.

MiR-216a-5p and FASL in kidney tissues of mice were tested by RT-qPCR and Western blot analysis, and it was unveiled that versus the sham group, miR-216a-5p was decreased while FASL was increased in the I/R group (all p < 0.05) (Fig. 1a, b). Under the light microscope, it was observed that the glomerulus and tubules were clear and well-arranged with no inflammatory cell infiltration in the mesenchyme in the sham group. In the I/R group, the glomeruli epithelial cells and kidney TECs showed obvious edema, necrosis, and exfoliation, the brush border of some kidney TECs was eluted, and the transparent tube type and a large number of epithelial cell fragments could be seen in the lumen. There was a large amount of inflammatory cell infiltration in the mesenchyme in the I/R group relative to that in the sham group (p < 0.05) (Fig. 1c, d). The cell apoptosis in kidney tissues was tested by TUNEL staining, and the results demonstrated that in relation to the sham group, the apoptotic index was heightened in the I/R group (p < 0.05) (Fig. 1e, f). The results of detecting SCr and BUN levels in the serum of mice in the sham and I/R groups revealed that the levels of SCr and BUN were raised in the I/R group compared to the sham group (both p < 0.05) (Fig. 1g, h). Enzyme-linked immunosorbent assay was utilized to test inflammatory factors in kidney tissues of mice, and it was displayed that TNF-α and IL-1β levels were enhanced in the I/R group versus the sham group (all p < 0.05) (Fig. 1i, j). Detection of oxidative stress revealed that in comparison to the sham group, MPO activity and MDA content were enhanced while SOD activity was decreased in the I/R group (all p < 0.05) (Fig. 1k–m).

Western blot analysis and RT-qPCR were implemented to test miR-216a-5p and FASL expression in kidney tissues of mice, and the results unveiled that in relation to the ago-NC group, miR-216a-5p was enhanced while FASL was reduced in the ago-miR-216a-5p group (all p < 0.05) (Fig. 2a–b). It was observed under the light microscope that versus the ago-NC group, the damage of kidney tissues was mitigated in the ago-miR-216a-5p group (p < 0.05) (Fig. 2c, d). TUNEL staining revealed that in relation to the ago-NC group, the apoptotic index in kidney tissues was decreased in the ago-miR-216a-5p group (p < 0.05) (Fig. 2e, f). The findings of measuring SCr and BUN levels in the serum of mice in the ago-NC and ago-miR-216a-5p group indicated that the levels of SCr and BUN were reduced in the ago-miR-216a-5p group versus the ago-NC group (both p < 0.05) (Fig. 2g, h). Measurements of inflammatory factors levels and oxidative stress indices in kidney tissues signified that in contrast with the ago-NC group, TNF-α and IL-1β levels, MPO activity, and MDA content were decreased while SOD activity was raised in the ago-miR-216a-5p group (all p < 0.05) (Fig. 2i–m).

FASL expression levels were tested by RT-qPCR and Western blot analysis, and it was displayed that compared to the anti-NC + si-NC group, the anti-miR-216a-5p + si-NC group showed reduced miR-216a-5p and elevated FASL (both p < 0.05); in relation to the anti-miR-216a-5p + si-NC group, FASL was decreased in the anti-miR-216a-5p + si-FASL group (p < 0.05) (Fig. 3a, b). It was observed under the light microscope that in the anti-miR-216a-5p + si-NC group, most kidney TECs showed patchy necrosis, exfoliation, and increased infiltration of kidney mesenchyme focal inflammatory cells. Compared to the anti-NC + si-NC group, the anti-miR-216a-5p + si-NC group showed increased damage score of kidney tubule (p < 0.05); versus the anti-miR-216a-5p + si-NC group, the damage score of kidney tubule was decreased in the anti-miR-216a-5p + si-FASL group (p < 0.05) (Fig. 3c, d). TUNEL staining and detection results of other biochemical factors revealed that in comparison to the anti-NC + si-NC group, the anti-miR-216a-5p + si-NC group exhibited elevated apoptotic index, serum SCr and BUN levels, TNF-α and IL-1β levels, as well as MPO activity and MDA content, and reduced SOD activity (all p < 0.05); by comparison with the anti-miR-216a-5p + si-NC group, the apoptotic index, serum SCr and BUN levels, TNF-α and IL-1β levels, as well as MPO activity and MDA content were decreased while SOD activity was enhanced in the anti-miR-216a-5p + si-FASL group (all p < 0.05) (Fig. 3e-m).

Observed under the microscope, the cells were tightly connected, showing a typical “cobblestone paving stone shape,” with strong transparency and refractive index (Fig. 4a). MiR-216a-5 and FASL expression levels in kidney TECs were estimated by RT-qPCR and Western blot analysis, and it was signified that versus the control group, miR-216a-5p was reduced while FASL was elevated in the H/R group (both p < 0.05) (Fig. 4b, c). Cell viability, inflammatory factors, and oxidative stress indices in kidney TECs were tested upon H/R treatment, and the outcomes disclosed that in relation to the control group, cell viability and SOD activity were decreased, and TNF-α, IL-1β, and MDA levels were enhanced in the H/R group (all p < 0.05) (Fig. 4c–h). Elevation of miR-216a-5p enhances cell viability and impedes inflammation and oxidative stress response in H/R-treated TEC.

RT-qPCR was employed to test miR-216a-5p expression in kidney TECs, and it was indicated that versus the mimic NC group, miR-216a-5p was raised in the miR-216a-5p mimic group (p < 0.05) (Fig. 5a). Cell viability, inflammatory factor levels, and oxidative stress indices in kidney TECs upon transfection of miR-216a-5p mimic and H/R, and the results unearthed that by comparison with the mimic NC group, cell viability and SOD activity were raised, and TNF-α, IL-1β, and MDA levels were decreased in the miR-216a-5p mimic group (all p < 0.05) (Fig. 5b-f). Downregulating FASL reverses the impacts of depleted miR-216a-5p on H/R-treated TEC, and FASL is a target gene of miR-216a-5p.

The potential binding sites between FASL and miR-216a-5p were predicted using the bioinformatics software (Fig. 6a). Dual luciferase reporter gene assay revealed that in relation to the FASL-Wt + mimic NC group, the luciferase activity was decreased in the FASL-Wt + miR-216a-5p mimic group (p < 0.05). In contrast with the FASL-Mut + mimic NC group, the luciferase activity showed no distinct difference in the FASL-Mut + miR-216a-5p mimic group (p > 0.05) (Fig. 6b). Further RT-qPCR and Western blot analysis were implemented to assess FASL expression in cells transfected with miR-216a-5p mimic, which demonstrated that FASL was reduced in the miR-216a-5p mimic group compared with the mimic NC group (Fig. 6c, d), indicating that FASL was the target gene of miR-216a-5p. Then observe the effect of miR-216a-5p/FASL axis on H/R created TEC, miR-216a-5p and FASL expression levels were examined by RT-qPCR and Western blot analysis, and it was suggested that versus the inhibitor NC + si-NC group, the miR-216a-5p inhibitor + si-NC group presented reduced miR-216a-5p and elevated FASL (both p < 0.05); versus the miR-216a-5p inhibitor + si-NC group, FASL levels were reduced in the miR-216a-5p inhibitor + si-FASL group (p < 0.05) (Fig. 6e-f). The results of further functional experimental assays indicated that that versus the inhibitor NC + si-NC group, the miR-216a-5p inhibitor + si-NC group presented reduced cell viability and SOD activity, and elevated TNF-α, IL-1β, and MDA levels (all p < 0.05); versus the miR-216a-5p inhibitor + si-NC group, cell viability and SOD activity were raised, and TNF-α, IL-1β, and MDA levels were decreased in the miR-216a-5p inhibitor + si-FASL group (all p < 0.05) (Fig. 6g-k).

AKI is a common syndrome around the world linked to a large number of kidney and systemic morbidity and increased short- and long-term mortality [23]. Current treatment approaches for AKI are primarily supportive in nature, and so far, there is no therapeutic modality with determined efficacy [24]. In view of this, an early evaluation for AKI is imperative for eventually creating an effective intervention. The current study was designed to explore the mechanism of the miR-216a-5p/FASL axis in AKI.

MiRs serve as a novel class of diagnostic parameters for human diseases because they are stable and easily measured in body fluids [25]. MiR research first concentrated on suppression of single miRs using oligonucleotide inhibitors, but more recent methods have probed the potential of delivering oligonucleotides to mimic miR expression or using small molecules to elevate or inhibit miR function [26]. Restoring or repressing miRs expression and activity exhibits high potential in treating diseases, and some pre-clinical models have unveiled the feasibility and efficacy of miR-based therapy [27]. As previously described, miRs participate in the pathogenesis of multiple kidney diseases including AKI, and their expression changes have diagnostic values for AKI [28, 29]. It has been reported that both serum and urinary miRs could serve as effective parameter for prognosis in kidney diseases. Lorenzen et al. have supported that plasma miR-210 might be a useful and independent predictor of survival in AKI patients [30]. Another article has unveiled that the elevation of miR-452, particularly that in urine, could be an effective parameter for early measurement of AKI in sepsis patients [31]. In our work, the findings demonstrated that miR-216a-5p expression was reduced in AKI, and restoration of miR-216a-5p attenuated AKI-induced injury to mice or H/R-induced damage to TECs. Similarly, a former study has indicated miR-216a-5p from USCs-Exos could transfer to renal TECs and mitigate cell damage in H/R-exposed HK-2 cells and renal I/R injury rat models [13]. Zhang et al. [32] have stated that miR-216a-5p is downregulated in pancreatic cancer tissues and cells, and the functional assays reveal that miR-216a-5p retards pancreatic cancer cell growth and migration. Furthermore, miR-216a-5p has been demonstrated to be decreased in lipopolysaccharides (LPS)-stimulated BEAS-2B cells, and upregulated miR-216a-5p weakens LPS-evoked inflammation in BEAS-2B cells [33]. All these references highlighted the reliable effect of overexpression of miR-216a-5p in AKI.

Our research also identified FASL as a target of miR-216a-5p, and there was high level of FASL in AKI. Subsequently, we noticed that depletion of FASL exerted protective effects on mice with AKI or H/R-treated TECs. A study has reported that FASL expression is notably raised in AKI, and depleting FASL protects TEC against apoptosis in AKI [17]. Another study has purported that ischemic AKI enhances FASL gene activation relative in vitro [34]. Moreover, FASL administration has been discovered to induce cytokine/chemokine production and apoptosis of cultured TECs [35]. Ko et al. [36] have stated that depleted FASL diminishes kidney injury and declines TNF-α production in T cells after I/R injury in the kidney. It is also reported that blockage of FASL attenuates BUN, SCr, and MDA contents and increases the levels of SOD in kidney I/R injury mice [14].

Collectively, our paper underscores that upregulate miR-216a-5p mitigates the I/R-induced pathological kidney damage and impedes apoptosis of H/R-treated TECs via suppressing FASL. These findings provided a new insight in a novel target therapy for I/R-induced AKI. In this study, we used miR agomir/antagomir in vivo and found that agomir treatment alleviated kidney injury and kidney cell apoptosis in kidney I/R mice, and we used miR mimic/inhibitor in vitro and found that mimic treatment improved kidney cell viability in H/R cells. Studies on the preventive and therapeutic role of miRs in AKI are still in the animal experimental stage, and there are currently no miR-based drugs for the treatment of AKI in the clinic. Moreover, the intrinsic mechanism needs further exploration by similar experiments. The potential of miR-216a-5p as a biomarker for prognosis and clinical outcomes of AKI need be determined in future research.

The protocol was ratified by the Institutional Animal Care Use Committee of The Affiliated Suzhou Hospital of Nanjing Medical University (approval No.: 20210615).

The authors have no conflicts of interest to declare that are relevant to the content of this article.

No funds, grants, or other support was received.

Biying Zhou finished study design. Ruirui Luo finished experimental studies. Yanlin Sun finished data analysis. Biying Zhou and Aixiang Yang finished manuscript editing. All authors read and approved the final manuscript.

Biying Zhou and Ruirui Luo contributed equally to this manuscript.

All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.