MiR-30c-5p/ROCK2 axis regulates cell proliferation, apoptosis and EMT via the PI3K/AKT signaling pathway in HG-induced HK-2 cells

Abstract Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus. Increasing evidence suggests that microRNA-30c-5p (miR-30c-5p) participates in the pathogenesis of DN, but the mechanism has not been clearly understood. Therefore, this study aimed to investigate the biological role of miR-30c-5p in human DN progression in vitro. Compared with the controls, DN tissues and high glucose-induced HK-2 cells had significantly reduced miR-30c-5p levels, while ROCK2 expression was prominently elevated. Additionally, the miR-30c-5p mimic distinctly facilitated cell proliferation and blocked cell apoptosis and epithelial–mesenchymal transition (EMT). However, ROCK2 was a target gene of miR-30c-5p, and the effects of miR-30c-5p mimic on cell proliferation, apoptosis and EMT were reversed by ROCK2 upregulation in vitro. Furthermore, the pathogenesis of DN was regulated by the miR-30c-5p/ROCK2 axis via the PI3K/AKT pathway. MiR-30c-5p regulating cell proliferation, apoptosis and EMT through targeting ROCK2 via the PI3K/AKT pathway provides the novel potential target for clinical treatment of DN.


Introduction
Diabetic nephropathy (DN) is a major complication of diabetes mellitus, as well as the predominant cause of advanced renal disease [1]. Around 10-40% of patients with type 2 diabetes eventually develop DN in urban China [2]. However, the pathogenesis of DN is complex, influenced by multiple factors such as nonenzymatic saccharification, renal hemodynamic changes, hypertension, dyslipidemia, oxidative stress, protein kinase C activation, vasoactive substances and cytokines, as well as other genetic factors [3]. It seems that different types of kidney cells are sensitive to hyperglycemia in varying degrees [4]. Research on the pathogenesis of DN is particularly important to better understand the mechanism and thus develop more effective therapies.
MicroRNAs (miRNAs) have been shown to be a type of non-coding RNA with a length of 20-24 nucleotides (nts) [5]. MiRNAs are involved in a number of processes in the development of various diseases such as heart failure and cancer as well as diabetes [6][7][8]. Studies have reported that miRNAs are associated with some renal diseases. For example, miR-29c is a signature miRNA in high glucose (HG)-induced conditions, targeting sprouty homolog 1, and reduced expression of miR-29c prevents DN progression [9]. MiR-193a appears to induce focal segmental glomerulosclerosis by inhibiting the expression of Wilms' tumor protein [10]. MiR-192 exerts its role in diabetic kidney glomeruli via inhibition of E-box repressors [11]. Moreover, miR-29s and miR-let-7s have been confirmed to function as key antifibrotic players in DN [12]. MiR-30c-5p in serum acts as a potential biomarker in multiple system atrophy [13]. Furthermore, miR-30c-5p has been revealed to be involved in reducing renal ischemia-reperfusion via macrophages [14]. In this study, the biological role of miR-30c-5p in DN was investigated using HG-induced HK-2 cells in vitro, which uncovered the possible mechanisms of miR-30c-5p's effects on the progression of human DN.
Rho-associated coiled coil-containing protein kinase 2 (ROCK2) belongs to the Rho-associated kinase (ROCK) family. The ROCK family includes ROCK1 and ROCK2 members, and the high similarity of their amino acid sequences suggests that these two members perform many of the same functions [15]. Particularly, ROCK2 downregulation blocks HG-induced hyperpermeability in kidney glomerular endothelium [16]. Previous research indicates that ROCK2 is a critical regulator of axonal degeneration and neuronal death, as well as axonal regeneration in the central nervous system [17]. Thus, ROCK2 could be closely related to the development of multiple types of cancer, as well as diseases of the central nervous system and kidney dysfunction.
The underlying mechanism of ROCK2 in the progression of kidney diseases needs further exploration. Since phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) play vital roles in several cellular processes, including proliferation, apoptosis, migration and glucose metabolism [18,19], we hypothesized that the PI3K/ AKT signaling pathway participates in the progression of DN. Herein, we measured the level of miR-30c-5p and ROCK2 expression in DN tissues and in an HG-induced DN cell model. The regulatory mechanism of miR-30c-5p and ROCK2 in the pathogenesis of DN in vitro was investigated.

Kidney tissue samples
Human DN tissue samples (n = 40) and the adjacent normal kidney tissues (n = 40) were donated by DN patients at Weihai Central Hospital. The DN patients included 18 males and 22 females, ranging from 40 to 65 years. All the patients were diagnosed and classified according to the World Health Organization diagnostic criteria for diabetes [20]. The DN patients underwent a negative urine protein test, with 30-300 mg/24 h urine albumin, indicating early renal damage. Moreover, the DN patients were diagnosed with the presence of specific nodular glomerulosclerosis of diabetes mellitus (the Kimmelstiel-Wilson lesion). We did not find other primary and secondary factors or complications causing renal damage.
Informed consent: Informed consent has been obtained from all individuals included in this study.
Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration and has been approved by the Ethics Committee of Weihai Central Hospital.
To establish the DN cell model, HK-2 cells were trypsinized, and 2 mL of cell suspension was added to each well of a six-well culture plate at a density of 1 × 10 6 cells/mL. Then, the cells were incubated in serumfree DMEM for 12 h until 70-80% confluence. HK-2 cells were treated with normal glucose (NG; final concentration of D-glucose 5.6 mmol/L), HG (final concentration of D-glucose 30 mmol/L) and high osmotic pressure control group (HO; 5.6 mmol/L D-glucose + 24.4 mmol/L D-mannitol).

Western blot assay
HK-2 cells and tissues were harvested and lysed in RIPA lysis buffer (Millipore, Bedford, MA, USA) containing protease and phosphatase inhibitors. Then, total protein concentration was evaluated with a BCA Protein Assay Kit (Sangon Biotech, Shanghai, China), and loading buffer was used for modulating the volume of protein solution. Briefly, 20 µL of solution containing 30 µg of protein was added to each well of a 10% sodium dodecyl sulfate-polyacrylamide gel. The separated protein was electro-transferred onto polyvinylidene difluoride membranes (Millipore), which were then blocked with 5% bovine serum albumin (Sangon Biotech). Then, the membranes were incubated with primary antibodies: ROCK2

Flow cytometry for analyzing cell apoptosis
An Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) was used to detect cell apoptosis. HK-2 cells treated under different treatment conditions and in different transfected groups were washed twice with phosphate buffered saline on ice (Gibco), and then diluted with a 1× binding buffer to a final concentration of approximately 1 × 10 6 cells/mL. Subsequently, 5 µL of Annexin V-FITC and propidium iodide (PI) was added to the labeled cell tubes, and the cells were stained for 15 min at room temperature in the dark. Finally, cell apoptosis was detected using flow cytometer (BD Biosciences, San Jose, CA, USA).

Dual-luciferase reporter assay
TargetScan predicted that ROCK2 might be a target of miR-30c-5p. Thus, a dual-luciferase reporter assay was conducted to confirm the direct interaction between miR-30c-5p and ROCK2. Common fragments of the synthetic wild-type (ROCK2 3′-UTR WT) or mutant (ROCK2 3′-UTR MUT) of ROCK2 3′-UTR were introduced into a pGL3-Basic vector (Promega, Madison, WI, USA). Subsequently, the correctly sequenced ROCK2 3′-UTR WT and ROCK2 3′-UTR MUT were co-transfected into HK-2 cells, and the cells were collected after transfection for 48 h. Then, luciferase assay reagent and Renilla assay buffer were dissolved with Dual-Luciferase Reporter Assay System Kit reagent (Promega). Ten microliters of cell lysate were added to each well of an opaque 96-well plate before the measurement of the luciferase and Renilla activities. Meanwhile, the firefly activities, as the indicator of the reporter gene, were identified by Luciferase Assay Reagent II, and the internal reference of Renilla activity was measured using Stop&Glo Reagent. The reporter gene was described via counting the ratio of firefly activity/Renilla activity.

Statistical analysis
The data were analyzed using SPSS 19.0 software and presented as mean ± standard deviation of three independent experiments. Student's t-test and one-way analysis of variance were employed to examine the difference of pair groups and multiple groups, respectively. Statistical significance was considered at P < 0.05.

Results
3.1 Level of miR-30c-5p was effectively decreased, whereas ROCK2 expression was significantly increased in DN tissues In order to investigate the biological roles of miR-30c-5p and ROCK2 in DN development, the levels of miR-30c-5p and ROCK2 were analyzed by qRT-PCR. The results demonstrated that miR-30c-5p was greatly downregulated, while ROCK2 was notably upregulated in DN tissues (Figure 1a and b). Furthermore, the ROCK2 protein level was increased in the DN group (Figure 1c). Moreover, there was a negative linear correlation between miR-30c-5p level and ROCK2 expression Figure 1: Level of miR-30c-5p was effectively decreased, whereas ROCK2 expression was significantly increased in DN tissues. (a and b) qRT-PCR was carried out to analyze the miR-30c-5p level and the mRNA level of ROCK2 in DN tissues compared with the paired control.
(c) Protein expression of ROCK2 was measured by a western blot assay. (d) Spearman's correlation analysis showed an inverse correlation between miR-30c-5p and ROCK2 mRNA levels. *P < 0.05.
( Figure 1d). In brief, the data suggested that miR-30c-5p and ROCK2 may play vital roles in the progression of DN.

HG conditions induced ROCK2 but suppressed miR-30c-5p expression in HK-2 cells
To determine whether the expression of miR-30c-5p and ROCK2 in the HG-induced DN cell model was in line with the tissues, HK-2 cells were treated with NG, HG and HO. qRT-PCR results suggested that the level of miR-30c-5p was significantly reduced, while the ROCK2 mRNA level was increased in the HG group compared with that in NG and HO groups (Figure 2a and b). Western blot assay showed that the protein expression of mature ROCK2 was enhanced in the HG group ( Figure 2c). These data show that the expression trend of miR-30c-5p and ROCK2 in HGstimulated HK-2 cells is in line with that in DN tissues.

MiR-30c-5p promoted cell proliferation and repressed cell apoptosis and epithelial-mesenchymal transition (EMT) in HG-induced HK-2 cells
Because of the low expression of miR-30c-5p in DN tissues, the assay aimed to investigate its potential biological role. An MTT assay was performed to detect cell proliferation of HK-2 cells under exposure to HG, and the result revealed that the proliferation of HK-2 cells in mock and NC groups was hindered with time, and there was no marked difference between these two groups. Simultaneously, the miR-30c-5p mimic dramatically promoted cell proliferation, whereas the miR-30c-5p inhibitor conspicuously restrained cell proliferation in HG-induced HK-2 cells (Figure 3a). As shown in Figure 3b, there was a distinct repression of cell apoptosis in HK-2 cells with the miR-30c-5p mimic, while the miR-30c-5p inhibitor prominently expedited the apoptosis rate in HK-2 cells. Furthermore, the boosted expression of E-cadherin, snail1 and TGFB1, as well as the inhibited levels of vimentin and α-SMA indicated that EMT was efficiently constrained by the miR-30c-5p mimic, while there was an opposite result caused by the miR-30c-5p inhibitor (Figure 3c). All evidence indicates that the miR-30c-5p mimic strikingly accelerates cell proliferation, while obviously impeding apoptosis and EMT in HG-stimulated HK-2 cells, while the miR-30c-5p inhibitor exerts an inverse effect.

ROCK2 was a target gene of miR-30c-5p
TargetScan predicted the short binding sites shared by miR-30c-5p and ROCK2 (Figure 4a). Subsequently, a dual-luciferase reporter assay was conducted to confirm the interaction between miR-30c-5p and ROCK2, and the results uncovered that luciferase activity is efficiently inhibited by ROCK2 3′-UTR WT, while no evident difference was seen in the ROCK2 3′-UTR MUT group (Figure 4b). Additionally, qRT-PCR and western blot assays were performed to investigate the regulatory  mechanism between miR-30c-5p and ROCK2, and the expression of ROCK2 was markedly suppressed by miR-30c-mimic but apparently induced by the miR-30c-5p inhibitor (Figure 4c and d). In short, these data suggest that ROCK2 is a direct downstream gene of miR-30c-5p.

ROCK2 inhibited cell proliferation and enhanced cell apoptosis and EMT in HGinduced HK-2 cells
To assess the therapeutic potential of ROCK2 in DN, si-ROCK2 and pcDNA-ROCK2 were transfected into HGinduced HK-2 cells. MTT assay results indicate that ROCK2 is an inhibitory factor in cell proliferation, and knockdown of ROCK2 evidently contributes to cell proliferation, as upregulation causes decreased cell proliferation in HGinduced HK-2 cells (Figure 5a). Moreover, cell apoptosis was dramatically repressed in the si-ROCK2 group, but apparently reinforced after transfection of pcDNA-ROCK2 in HG-treated HK-2 cells (Figure 5b). The EMT level was also determined through measuring the expression of vimentin, E-cadherin, α-SMA, snail1 and TGFB1, and the efficiently increased expression of E-cadherin, snail1 and TGFB1, and the conspicuously constrained expression of vimentin and α-SMA in the si-ROCK2 group suggest that knockdown of ROCK2 suppresses EMT in HG-induced HK-2 cells. The results of the pcDNA-ROCK2 group also support this conclusion (Figure 5c). These data reveal that knockdown of ROCK2 induces cell proliferation and suppresses apoptosis and EMT, whereas the function of ROCK2 overexpression in cell proliferation, apoptosis and EMT was opposite to that of ROCK2 silencing.
miR-30c-5p mimic + pcDNA-ROCK2 were transfected into HG-stimulated HK-2 cells, respectively. An MTT assay discovered that the promotion effect of miR-30c-5p mimic on cell proliferation was abrogated by pcDNA-ROCK2 (Figure 6a). Synchronously, cell apoptosis suppressed by miR-30c-5p was reversed via pcDNA-ROCK2 in vitro (Figure 6b). In addition, the inhibitory effect of miR-30c-5p mimic on EMT was restrained by overexpression of ROCK2 in HG-induced HK-2 cells (Figure 6c). In brief, the effects of miR-30c-5p mimic on cell proliferation, apoptosis and EMT were relieved by upregulation of ROCK2 in the HG-induced DN cell model.

3.7
The effects of miR-30c-5p and ROCK2 were exerted on DN progression via the PI3K/AKT signaling pathway In order to investigate the regulatory mechanism of miR-30c-5p and ROCK2 in the progression of DN, we detected the expression of p-PI3K, PI3K, p-AKT and AKT after transfection with miR-30c-5p mimic, miR-30c-5p mimic + vector and miR-30c-5p mimic + pcDNA-ROCK2 in HK-2 cells treated with HG. The results showed that the expressions of both p-PI3K and p-AKT were remarkably decreased in the miR-30c-5p mimic group, and they were improved after co-transfection with miR-30c-5p and pcDNA-ROCK2 in vitro (Figure 7). This indicates that the roles of miR-30c-5p and ROCK2 are carried out via the PI3K/AKT signaling pathway.

Discussion
It is well known that DN leads to end-stage kidney disease and increases all-cause mortality in diabetic patients [21]. According to the previous research, renal tubular damage is distinct in DN patients [22]. Here, HK-2 cells were treated with HG to cause damage, and the study found that the level of miR-30c-5p was obviously curbed in either HG-stimulated HK-2 cells or DN tissues. In contrast, ROCK2 expression was significantly augmented in the cell model and tissue samples. The aberrant expression of miR-30c-5p and ROCK2 showed their potential functions in the pathogenesis of human DN. Emerging research indicates that miRNAs, such as miR-93 [23], miR-451 [24] and miR-23b [25], show aberrant expression in dysfunctional kidneys, including in DN. Understanding how miRNAs contribute to the progression of diseases is imminently important since several miRNAs are currently being explored as therapeutic agents for some diseases through early-stage clinical trials [26]. Moreover, miRNAs appear to be able to modify the expression of various target genes at transcription and post-transcription levels [27], and identification of classical or novel miRNA targets might provide unique therapeutic opportunities in different diseases. Based on the previous research examples, miR-27a is related to podocyte injury in DN via targeting Forkhead box protein O1 [28]. Over the past few decades, miR-30c has been proved to function as a tumor suppressor in colorectal carcinoma via regulating the target gene of B cell lymphoma 9 [29]. MiR-30c prevented diabetic cardiomyopathy by reducing the peroxisome proliferator-activated receptor alpha level [30]. Moreover, miR-30c-5p regulated neuropathic pain of rodents and was highly expressed in the spinal cord and dorsal root ganglia [31]. Furthermore, miR-30c-5p was associated with macrophage-mediated inflammation and pro-atherosclerosis signal pathways [32]. Increasing evidence reveals that EMT of glomerular endothelium is a modulation mechanism for potential cell dysfunction in endothelial injury [33]. In our study, miR-30c-5p was silent in DN tissue samples and HG-induced HK-2 cells. The miR-30c-5p mimic strikingly augmented cell proliferation, whereas it effectively hindered apoptosis and EMT in HG-induced DN cells. A previous study suggested that miR-29s and miR-let-7s exert antifibrotic effects on kidney fibrosis [12]. Consistently, miR-30c-5p has an antifibrotic effect through inhibiting EMT processes. This evidence suggested that miR-30c-5p participated in DN progression, and thus may be a novel target in clinical treatment of DN. Figure 7: Effects of miR-30c-5p and ROCK2 were exerted on DN progression via the PI3K/AKT signaling pathway. The expression of the PI3K/ AKT pathway-related proteins including p-PI3K, PI3K, p-AKT and AKT was detected using a western blot assay and quantified via Image J software. *P < 0.05.
ROCK2 knockdown, which is regulated by miR-455-3p, suppressed renal fibrosis in DN [34], but it appears that the role of ROCK2 in the regulatory mechanism of diabetic heart disease is complex, referring to the phosphoinositide-dependent kinase-1/AKT signaling pathway [35]. Two members of the ROCK family have been associated with renal injury, e.g., a mouse model of unilateral ureteral obstruction nephropathy caused by tubulointerstitial fibrosis [36]. In this study, ROCK2 as the target factor was discovered to be directly targeted by miR-30c-5p. Furthermore, the promotion effect of the miR-30c-5p mimic on cell proliferation and inhibition effects on cell apoptosis and EMT were abolished by overexpression of ROCK2 in HG-stimulated HK-2 cells. Finally, the landmark proteins of the PI3K/AKT signaling pathway were also measured by a western blot assay, and the results demonstrated that miR-30c-5p and ROCK2 exerted roles at least partially via the PI3K/AKT signaling pathway in the progression of DN.
In summary, either the miR-30c-5p mimic or ROCK2 knockdown evidently induced cell proliferation, and notably restrained apoptosis and EMT in HG-stimulated HK-2 cells. Interestingly, the biological role of miR-30c-5p in cell proliferation, apoptosis and EMT was abrogated by overexpression of ROCK2 in vitro. Simultaneously, the miR-30c-5p/ROCK2 axis modulated the development of DN through the PI3K/AKT signaling pathway.
The level of miR-30c-5p was significantly curbed, while ROCK2 expression was effectively augmented in DN tissues. Moreover, HG-treated HK-2 cells were used to simulate a DN cell model in the study. Functionally, miR-30c-5p acted as a promoter of cell proliferation, while hindering cell apoptosis and EMT in HG-induced HK-2 cells. Additionally, the function of ROCK2 was opposite to that of miR-30c-5p in cell proliferation, apoptosis and EMT. Interestingly, ROCK2 was a target gene of miR-30c-5p and its overexpression abolished the effects of the miR-30c-5p mimic on cell proliferation, apoptosis and EMT in HG-stimulated HK-2 cells. Mechanically, the miR-30c-5p/ROCK2 axis regulated the progression of DN through the PI3K/AKT signaling pathway.

Conflict of interest:
The authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.