MicroRNA-140-5p Suppresses Hypoxia-induced Nephrotoxicity and Renal Fibrogenesis in in vitro Conditions

MicroRNAs (miRNAs) are responsible for inducing mRNA degradation or blocking translation (Romaine et al., 2015). MiRNAs participate in a broad array of cellular processes, including cell growth, differentiation and death in part, by regulating multiple genes and their downstream networks (Yang et al., 2015). In recent years, miRNAs have been recognized as important modulator of diabetic nephropathy (DN) pathophysiology, including extracellular matrix protein production (Wang et al., 2008), cell death (Kato and Natarajan, 2015), glomerulosclerosis and tubulointerstitial fibrosis (Kato and Natarajan, 2015). MiR-140-5p is frequently dysregulated in different tumor cells, including colorectal cancer and ovarian cancer (Zhai et al., 2015a). Earlier, miR-140-5p has been found to suppress inflammation through TLR4 in lung injury (Yang et al., 2018).
       
Renal cells are prone to injury associated with hypoxia due to their more oxygen demand high metabolic activity (Louis and Hertig, 2015). In response to persistent hypoxia insult, tubular epithelial cells initiate an inflammatory and fibrogenic cytokine response, ultimately activating fibroblasts and tubular epithelial cells. Fibroblasts produce excessive extracellular matrix proteins (ECM), while activated tubular epithelial cells undergo apoptosis. Furthermore, transition of epithelial cells from epithelial-to-mesenchymal stage contributes to tubular atrophy. On the other hand, excessive production of ECM results in endothelial dysfunction and promotes hypoxia. Taken together, all these changes in response to persistent hypoxia causes tissue destruction (Zafrani and Ince, 2015).
       
Toll-like receptor 4 (TLR4) encodes a transmembrane protein that contains leucine-rich repeat (LRR)-motifs in its extracellular portions involved in recognizing various biological molecules (Seifert et al., 2015). TLR4 regulates complex cellular processes like apoptosis, proliferation, Wnt signaling pathway and immunosuppressive cytokines (Yi et al., 2012). A previous study strongly implicates TLR4 in protecting against pulmonary fibrosis (Liang et al., 2016). Experimental evidence also supports the role of TLR4 in fibrosis of the kidney, skin and lungs (Bhattacharyya et al., 2018). Importantly, TLR4 can promote fibrosis and inhibits tubular damage in the context of renal injury (Pulskens et al., 2010a). A good number of previous studies have reported an association between TLR4 and miR-140-5p (Wang et al., 2019). Additionally, targeting TLR4 by miR-140-5p has been demonstrated to be linked with the pathogenesis of atrophic nonunion (Guo et al., 2019), acute lung injury (Yang et al., 2018), respiratory syncytial virus disease (Zhang and Shao, 2018), intervertebral disc degeneration (Zhang et al., 2018), and pulmonary arterial hypertension (Li et al., 2017).

Cell culture and treatment
 
HK-2 cell line was provided by ATCC and grown in DMEM (GBICO, USA) with 10% FBS and antibiotic in CO2 incubator at 37°C overnight. For hypoxia studies, HK-2 cells were stimulated with CoCl₂ (200 µM) for 12 h, as described by Waza and colleagues (Waza et al., 2018).
       
All the experimental work was carried out in Ningbo Yinzhou No.2 Hospital, Ningbo, (China) from 2019 to 2021.
 
Cell transfection
 
The miR-140-5p mimic, si-TLR4, si-NC and miR-NC vectors were used. The coding sequence of TLR4 was amplified from HK-2 cells and inserted in pcDNA3.1 expression vector (pcDNA3.1-TLR4). The empty pcDNA3.1 plasmid was used as negative control (NC). Transfection of different vectors was performed using Lipofectamine. After transfection, HK-2 cells were induced with hypoxia for 12 h. Transfection efficiency was determined by qRT-PCR.
 
qRT-PCR
 
HK-2 cells were lysed to obtain total RNA using QIAzol Lysis Reagent and then reversed to cDNA. It was followed by mRNA quantification using SYBRGreen (Takara) with appropriate primers against MiR-140-5p, Bad, Bax, Bcl-2, CTGF, Fibronectin, Collagen I, TLR4 and GAPDH. The mRNA level of the target gene was determined by 2^-DDCT, using GAPDH as control.
 
Cell viability assay
 
Cell viability was analyzed in HK-2 cells using CCK-8 assay. In brief, HK-2 cells (3×10³ cells per well) were grown in 96-well plates for 24 h at the normal conditions. It was followed by transfection and hypoxia studies, as described above. After completion of experimental time, 10 μl of CCK-8 was added to each well. Absorbance was taken at 450 nm, followed by cell viability calculations.
 
Cell apoptosis assay
 
HK-2 cells were grown in 96-well plates and were either treated with hypoxia alone or transfections followed by hypoxia. After completion of experimental time, ELISA-based cell death assay kit (Roche, Germany) was used to determine apoptosis by following manufacturer’s instructions.
 
Luciferase reporter assay
 
3¢UTR sequences of TLR4 containing miR-140-5p target site [wild or mutant type] were cloned in pmir-GLO report luciferase vector to generate TLR4-WT or TLR4-MUT reporter plasmid, respectively. Subsequently, cells were grown and co-transfected with either miR-140-5p mimic with WT TLR4 or MUT TLR4 reporter plasmid. Calculation of relative luciferase activities was performed post-transfection (48 h).
 
Protein extraction
 
Preparation of HK-2 cell lysate was achieved using lysis buffer (NP-40). To prevent proteolysis of cell lysate, Halt Protease Inhibitor Cocktail (Thermo Scientific) was used. Centrifugation (3000 rpm for 10 min) of cell lysate was performed to obtain supernatant. Bradfords assay was used to determine protein concentration.
 
Western blotting
 
Preparation of protein samples was carried out as performed by (Waza et al., 2012). Detection of target proteins was determined using specific antibodies; anti-Bad, anti-Bax, anti-Bcl-2, anti-CTGF, anti-Fibronectin, anti-Collagen I, anti-TLR4 and anti-GAPDH. LI-COR system was used for secondary detection.

Decreased expression of miR-140-5p in hypoxia-insulted HK-2 cells
 
Initially, miR-140-5p levels in hypoxia-induced HK-2 cells were determined using qRT-PCT. The induction of hypoxia with CoCl₂ and expression levels of miR-140-5p in hypoxia stimulated HK-2 cells is depicted in Fig 1A and Fig 1B, respectively. Tissue/cell-type specific expression of miR-140-5p has been reported earlier (Zhang et al., 2015). MiR-140-5p is frequently upregulated in mesenchymal tissues (Hwang et al., 2014). MiR-140-5p expression has been reported to decrease in primary colorectal carcinomas tissues and liver metastatic tissues (Zhai et al., 2015b). In addition, downregulation of miR-140-5p has been observed in rat cerebral tissues following middle cerebral artery occlusion (Sun et al., 2016).
 

 
Effects of miR-140-5p on hypoxia-stimulated cell viability and apoptosis
 
Considering miR-140-5p down-regulation in hypoxia-induced HK-2 cells, we performed transfection assays to determine its role in hypoxia-stimulated HK-2 cell toxicity and fibrogenesis. We transfected miR-140-5p mimic and then stimulated with hypoxia for 12 h. miR-140-5p overexpression was successfully constructed in HK-2 cells by miR-140-5p mimic transfection, as demonstrated by quantitative real-time PCR (Fig 2A). CCK-8 assay showed miR-140-5 pup-regulation decreases cell viability in hypoxia-treated cells (Fig 2B).
 

       
Similarly, miR-140-5p up-regulation increased apoptosis in hypoxia treated cells (Fig 2C). Moreover, we analyzed the impact of miR-140-5p on key mediators of apoptosis. We found that hypoxia-stimulation significantly elevated mRNA (Fig 2D) and protein (Fig 2E) levels of pro-apoptotic (Bad, Bax) and reduced anti-apoptotic Bcl-2 were notably attenuated by miR-140-5p overexpression.
 
Effects of miR-140-5p on hypoxia-stimulated fibrogenesis
 
Similarly, we analyzed miR-140-5p impact on the key mediators of fibrosis. Expression levels of pro-fibrosis, including CTGF, Fibronectin and Collagen I were all elevated after hypoxia treatment, which were partially mitigated by miR-140-5p mimic transfection. Hypoxia-stimulation significantly elevated pro-fibrosis related mRNA (Fig 3A) and protein (Fig 3B) levels and was notably attenuated by miR-140-5p overexpression. It has been shown that CTGF, Fibronectin and collagen I are substantially expressed in various forms of tissue fibrosis, including renal fibrotic diseases (Cabello-Verrugio et al., 2012).
 

 
MiR-140-5p directly targeted TLR4 in HK-2 cells
 
TargetScan was used to predict binding sites of miR-140-5p in TLR4 32 -UTR (Fig 4A). MiR-140-5p mimic decreased the luciferase activity mediated by WT TLR4 3¢-UTR, while no apparent effects were found in the miR-NC or MUT TLR4 groups (Fig 4B). Furthermore, expression levels of TLR4 were analyzed in hypoxia-stimulated HK-2 cells. MiR-140-5p suppressed the elevated protein expression of TLR4 induced by hypoxia treatment (Fig 4C). Furthermore, TLR4 mRNA was significantly upregulated in hypoxia-stimulated HK-2 cells (Fig 4D). Indeed, TLR4 mediates a range of diverse signaling pathways under apoptosis. Chen et al., (2018) found that patrinia represses apoptosis by inhibiting TLR4 associated signaling pathways in BRL-3A cells. There have been suggestions that angiotension II (Ang II) could induce mesangial cells apoptosis by activating TLR4/MyD88 signaling pathway (Lv et al., 2009). Many molecules have been shown to promote mitochondria-mediated apoptosis via upregulation of Bad/Bax and downregulation of Bcl-2 (Zhang et al., 2013).
 

 
MiR-140-5p attenuated hypoxia-stimulated toxicity and fibrogenesis of HK-2 cells by down-regulating TLR4
 
The above data indicated TLR4 was up-regulated in hypoxia-stimulated cells and miR-140-5p directly target TLR4. We thus speculated that TLR4 might be a downstream functional regulator involved in miR-140-5p regulating hypoxia-stimulated toxicity and fibrogenesis. To validate our hypothesis, transfection of si-TLR4, si-NC, miR-140-5p mimic together with TLR4 or empty vector was carried out in hypoxia-stimulated HK-2 cells (Fig 5 A, B and C). Then, we examined the effects of TLR4 knockdown or overexpression on cell viability, apoptosis and fibrogenesis. As shown in Fig 5D, TLR4 knockdown significantly increased cell viability, while TLR4 overexpression remarkably countered miR-140-5p up-regulation effects on cell viability in hypoxia-induced HK-2 cells. In line with this, TLR4 knockdown mimics, while overexpression attenuated miR-140-5p effects on hypoxia-stimulated cell apoptosis in HK-2 cells (Fig 5E). Moreover, miR-140-5p up-regulation effects on TLR-4, Bcl-2, Fibronectin and Collagen I was mimicked by TLR4 knockdown, but reversed by TLR4 overexpression (Fig 5F). The multiple lines of evidence indicate that TLR4 is also crucial for organ fibrosis (Pulskens et al., 2010b). Seki and colleagues have described that TLR4 could promote TGF-b signaling to contribute to hepatic fibrosis (Seki et al., 2007). TGF-b can induce the secretion of proteins like Collagen I and Fibronectin to promote fibrosis initiation and progression (Schnaper et al., 2003).
 

In conclusion, miR-140-5p inhibits hypoxia-associated cell toxicity and renal fibrogenesis by targeting TLR4. The detailed mechanisms underlying these processes need further investigation; however our study will deepen the understanding of hypoxia-induced nephrotoxicity and renal fibrogenesis, and open new clues into novel therapeutic treatments.

All the authors declare no competing financial interests.