Long Noncoding RNA SNHG10 Inhibits Renal Fibrosis By Negatively Regulating MiR-378b Expression

Background: LncRNA have been increasingly shown that plays pivotal roles in the development of various diseases, including renal brosis. Nevertheless, the pathological function of Long non-coding RNA SNHG10 (SNHG10) in the renal brosis remains obscure. Methods: We detected the expression levels of SNHG10 in the tissue samples and cell lines via RT-qPCR analysis. The functions of SNHG10 on the progression of renal brosis were examined by CCK-8, EdU, dual luciferase reporter and immunouorescence analyses. Results: In the present study, SNHG10, production of extracellular matrix (ECM), including α-SMA and Fibronectin levels were signicantly increased in HK-2 cells after TGF-β stimulation. Ectopic of SNHG10 inhibited cell proliferation and inhibits theα-SMA and Fibronectin expression of TGF-β1-induced HK-2 cells. In addition, bioinformatics analysis and dual luciferase reporter assay indicated that miR-378b was a target gene of SNHG10. Mechanistically, miR-378b overexpression abolished the repressive effects of SNHG10 on TGF-β1-induced HK-2 cells. Conclusion: SNHG10 plays an anti-brotic effect through suppression of miR-378b expression in renal brosis, which provides a promising therapeutic target for the treatment of renal brosis.


Introduction
Chronic kidney disease (CKD) is a type of progressive diseases and a critical global public health issue which leading contributions to death globally with a prevalence rate of 10% to 12% in the world [1][2][3][4].
Owing to lack of renal reserve function and clinical features in the early onset, CKD generally progresses to the middle and late stages when obvious symptoms were appeared [5]. Fibrosis is a hallmark and common pathway leading to end-stage organ dysfunction [6]. Renal interstitial brosis, as the nal pathological hallmark of CKD, irreversibly damages of renal function, and the nal convergent pathway of renal disease [7][8][9].
Renal brosis is the usually ultimate manifestation of various kidney diseases including glomerulonephritis and diabetic nephropathy [10]. In histological features, renal brosis manifests the excessive deposition of extracellular matrix (ECM) [11], which is always companied by various pathological alterations in tubular epithelial cells including epithelial-to-mesenchymal transition (EMT), broblast activation, immune cell in ltration, and apoptosis [12,13].
Despite that extensive research exists on the pathogenesis and molecular mechanisms underlying of renal brosis, the effective intervention for this disease is still lacking. In view of that renal brosis can lead to scar formation and even renal failure, further exploring the novel mechanisms of renal brosis, as well as searching for novel CKD therapeutic targets is compellingly needed, which is of great signi cance for the development of effective therapeutic strategies and prevention of patients with renal diseases.
Long non-coding RNAs (lncRNAs) are a type of RNAs whose transcripts are more than 200 nucleotides in length, and have no/little potentials in coding proteins [14,15]. A growing body of evidence indicates that lncRNAs play extensive regulatory roles in life activities and the development of many diseases and exert vital roles in the regulation of cell proliferation, migration, invasion and apoptosis [16][17][18][19]. In recent years, researchers have proved lncRNAs act as important regulators in the progress of brosis. For instance, silencing lnc-Hser aggravated liver brosis through inducing the epithelial-mesenchymal transition (EMT) and apoptosis [20]. Liu et al proved that lnc-PCF can accelerate pulmonary brogenesis by directly targeting miR-344a-5p to regulate map3k11 [21]. Moreover, study has demonstrated that the liver-enriched lncRNA Lfar1 could promote hepatic brosis through inducing hepatic stellate cells activation and hepatocytes apoptosis [22]. These ndings showed that lncRNAs played an important role in brosis progression. However, to date, the roles of lncRNA SNHG10 in renal brosis progression was not illuminated.
In this study, experiments were implemented to shed light on the potential role of SNHG10 in renal brosis progression, providing novel prognostic biomarkers for the development of patients with renal diseases.

Materials And Methods
Cell culture and treatment Human nephric proximal tubular epithelial cell line HK-2 was purchased from American Tissue Culture Collection (ATCC; Rockville, USA) and grown in DMEM complemented with 10% FBS with 5% CO 2 in an incubator at 37°C. Cells were starved for 24 h before any stimulation. For establishment of renal brosis, HK-2 cells were stimulated with different doses of transforming growth factor β1 (TGF-β1) for different hours and then subjected to further analysis.

Cell transfection
The mimic of miR-378b as well as negative control (NC mimic) were purchased from Invitrogen After 48 h, cells were harvested for following experiments.

Quantitative real-time PCR (qRT-PCR) ananlysis
The total RNA from cells was extracted with Trizol reagent (Invitrogen) and subsequently reversed transcribed into cDNA by utilizing PrimeScript RT reagent Kit (Takara, Ohtsu, Japan). Real-time PCR was carried out with SYBR Premix EX Taq™ II kit (TaKaRa, Dalian, China) on the PCR detection instrument (Opticon CFD-3200; MJ Research, Waltham, MA, USA). U6 and GAPDH were used as internal reference.

Western blot assay
Cells were lysed with RIPA Buffer (cat: 89900; Thermo Fisher Scienti c, USA), and the protein concentration was detected with the BCA Kit (cat: P0012S; Beyotime, Beijing, China). The equivalent amount of proteins were segregated on 12% SDS-PAGE and subsequently transferred to PVDF membranes (Millipore, Billerica, MA, USA). Blocking the lm with BSA and sealed with 5% skim milk, then the membranes was incubated with the primary antibody at 4℃ overnight, followed by incubation with secondary antibody at room temperature for 1 h. Finally, the bands were measured with enhanced chemiluminescence reagent ECL detection kit (Thermo Fisher Scientifc, Inc.) on ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA).

Cell proliferation assay
Cell visibility was assessed using the Cell Counting Kit-8 (CCK-8) assay. In details, cells were plated in 96well plates at a density of 1×10 5 cells/well and cultivated at 37℃ in the presence of 5% CO 2 . At 0, 24,48 and 72 h post incubation, cells were processed with CCK-8 reagent (Dojindo, Tokyo, Japan), followed by incubation for additional 4 h at 37℃. At last, absorbance was examined at wave length of 450 nm by a microplate reader (BioTek, Winooski, VT, USA).

Immuno uorescence assay
Transfected cells were cultured for 24 h at 37°C on a Petri dish and xed in 4% formaldehyde. After PBS washes, cells were penetrated and blocked with 5% BSA for 2 h. Then, the cells were incubated with the primary antibody overnight at 4°C and then incubated with the secondary antibody for 30 min. DAPI (1: 1000) was used to stain the nuclei. The laser scanning uorescence microscope was used to visualize uorescence.

Luciferase reporter assay
The WT and MUT 3′-UTR sequences of SNHG10 were synthesized by Sangon Biotech. The 3'UTR of SNHG10 containing or without binding sites for miR-378b were cloned into pGL3 reporter vector (Promega, Madison, WI, USA) to shape SNHG10-WT or SNHG10-MUT, respectively. Cells were cotransfected with indicated vectors and miR-378b mimic or NC mimic by using Lipofectamine 2000 (Thermo Fisher Scienti c, Waltham, MA, USA) according to the product instructions. After 48 h, the luciferase activity was examined using Dual-Luciferase Reporter Assay System (Promega).

Statistical analysis
All data were shown as means ± SD via three parallel experiments. Statistical analysis was conducted by employment of SPSS 21.0 software. Student's t test and one-way ANOVA were applied for comparison between two or more groups. Differences were de ned as statistically signi cant when P < 0.05.

Results
The SNHG10 expression was decreased in TGF-β-induced HK-2 cells We investigated the role of SNHG10 in renal brosis using HK-2 cells in vitro. After incubation with the pro-brotic cytokine TGF-β1, the expression of SNHG10 was markedly decreased at 48 h and 72h (Fig. 1A). Moreover, the main components of ECM including α-SMA and Fibronectin were signi cantly induced in HK-2 cells after TGF-β stimulation (Fig. 1B, C). These results potentially indicated that SNHG10 plays a protective role in attenuating renal brosis.
Ectopic of SNHG10 inhibited cell proliferation of TGF-β1induced HK-2 cells In order to investigate the effects of SNHG10 on renal brosis in vitro, HK-2 cells were transfected with pcDNA3.1 SNHG10 and negative control, and qRT-PCR assay was performed to detect the transfected e ciency. The results certi ed that pcDNA3.1 SNHG10 led to overexpression of SNHG10 in TGF-β1induced HK-2 cells compared to the pcDNA3.1 group (Fig. 2A). Moreover, we explore the effect of SNHG10 on the viability of TGF-β1-induced HK-2 cells by the CCK-8 assay. The data of Fig. 2B demonstrated that overexpression of SNHG10 remarkably suppressed the viability of HK-2 cells, when compared with pcDNA3.1 group. Subsequently, the role of SNHG10 in the proliferation of TGF-β1-induced HK-2 cells was determined by colony formation assay. The results veri ed that transfection with pcDNA3.1 SNHG10 signi cantly suppressed the proliferation of TGF-β1-induced HK-2 cells (Fig. 2C). The data of EdU assay revealed that the EdU-positive cells in HK-2 cells transfected with pcDNA3.1 SNHG10 were obviously reduced compared to pcDNA3.1 group (Fig. 2D). In addition, western blot assay was carried out to assess the effects of SNHG10 on the cell proliferation-related proteins. Up-regulating SNHG10 dramatically suppressed the expression levels of Ki-67 and PCNA in TGF-β1-induced HK-2 cells (Fig. 2E).

Overexpression of SNHG10 inhibits the expression of α-SMA and Fibronectin in TGF-β1-induced HK-2 cells
To further explore the roles of SNHG10 in the renal brosis, the qRT-PCR was applied to detect the relative expression of α-SMA, a marker of myo broblasts, and Fibronectin, the major components of ECM. The results of Fig. 3A shown that the expression level of α-SMA and bronectin were augmented caused by TGF-β1 treatment and decreased by transfecting with pcDNA3.1 SNHG10 in HK-2 cells. Meanwhile, western blot analysis manifested that ectopic of SNHG10 aggravated the diminution of protein levels of α-SMA and Fibronectin which increased by TGF-β1 administration (Fig. 3B).

MiR-378b is a direct target of SNHG10 in renal brosis
In order to study on the possible target genes of SNHG10 involved in the occurrence and progression of renal brosis, we used Starbase to search for genes that were directly regulated by SNHG10. Among all the targets, miR-378b was chosen for further study, since miR-378 functions as an important regulator controlling the occurrence and development of various brosis. MiR-378 has been proved that miR-378 could limit activation of hepatic stellate cells and liver brosis [23]. Furthermore, miR-378 reduces mesangial hypertrophy and kidney tubular brosis [24]. The binding site between SNHG10 and miR-378b were as shown in Fig. 4A. In addition, the miR-378b expression in TGF-β-induced HK-2 cells was analyzed from 0 h to72 h and results indicated that the expression of miR-378b was signi cantly increased at 48 h and 72h (Fig. 4B). Furthermore, we found that miR-378b was negatively regulated by SNHG10. The expression level of miR-378b was markedly decreased in pcDNA3.1 SNHG10 group compared with pcDNA3.1 group (Fig. 4C).
To further validate whether SNHG10 binding to miR-378b, luciferase reporter assay was performed. The luciferase reporter plasmids were contained with the 3' UTR of SNHG10 wild type (WT) or mutant (Mut).
As shown in Fig. 4D, the luciferase activity of SNHG10-WT was signi cantly decreased when cotransfected with miR-378b mimic, while no signi cant alterations were observed mutation at miR-378b binding site in the 3' UTR of SNHG10.

SNHG10 inhibits renal brosis through suppression of miR-378b expression
To further investigate the role of SNHG10/miR-378b in the regulation of renal brosis, we arti cially overexpressed miR-378b in HK-2 cells. RT-PCR assay disclosed that the expression of miR-378b was successfully enhanced by transfected with miR-378b mimic (Fig. 5A). To con rm whether the effect of SNHG10 on renal brosis was mediated by miR-378b, miR-378b mimic was co-transfected with SNHG10 overexpressed in HK-2 cells (TGF-β1 + pcDNA3.1 SNHG10 + miR-378b mimic). Proliferative capacity was determined by CCK-8 and colony formation assays. The data displayed that miR-378b mimic abolished the repressive effects of SNHG10 overexpression on TGF-β1-induced HK-2 cell viability ( Fig. 5B and 5C). Consistently, the numbers of EdU-positive cells were increased correspondingly to the pcDNA3.1 SNHG10 + miR-378b mimic-transfected HK-2 cells compared with those in pcDNA3.1 SNHG10 group (Fig. 5D). Additionally, qRT-PCR and western blot assays illustrated that miR-378b mimic abolished the inhibitory effects of SNHG10 overexpression on the mRNA and protein expression levels of α-SMA and Fibronectin in TGF-β1-induced HK-2 cells (Fig. 6).

Discussion
Renal brosis is increasingly becoming a major public health issue and considered as the common nal stage of progressive renal disease [25]. The pathogenesis of renal brosis is a progressive process that reduces the capacity for tissue repair and ultimately leads to end-stage kidney failure [13,26].
As a typical multifunctional lncRNA, SNHG10 is reported widely expressed in various diseases and plays a crucial role in multiple molecular and cellular processes. For instance, SNHG10 regulates HCC cell proliferation, invasion and epithelial-mesenchymal transition of by regulating SOX9 [33]. He et al also found that SNHG10 increases the methylation of miR-218 to facilitate cell proliferation in osteosarcoma [34]. However, explorations about the exact role of SNHG10 in the progression of renal brosis are scanty. Based on the literature, we hypothesize that SNHG10 may play regulatory function in the progression of renal brosis.
TGF-β1 is an important factor responsible for renal brosis. Herein, we used HK-2 cells induced with TGF-β1 to establish the cellular renal brosis model in vitro. In the present study, we concentrated on the potential function of SNHG10 and further investigated its latent molecular mechanism in the renal brosis progression. We found that the expression level of miR-542-3p was overtly down-regulated in TGF-β-induced HK-2 cells.
Mounting evidence has emphasized that the main pathological change of renal brosis is the deposition of ECM and TGF-β1-activated lung broblasts conceivably overlap with α-SMA positive myo broblasts, which is responsible for producing ECM components, such as Fibronectin [35,36]. Next, we performed gain-of-function experiments identi ed that ectopic of SNHG10 expression inhibits the proliferation and EMC deposition including α-SMA and Fibronectin in TGF-β1-induced HK-2 cells. Meanwhile, miR-378b is a direct target of SNHG10 in renal brosis by using multiple bioinformatic analyses. These results provided evidence that SNHG10 may function as a protector in renal brosis progression through targeting miR-378b.
In conclusion, our study uncovered the rst clue that SNHG10 plays an anti-brotic effect through the suppression of cell proliferation and down-regulation of α-SMA and Fibronectin expression by directly targeting miR-378b. On the whole, these ndings provide a deep insight into the mechanisms underlying renal brosis, which developed a new therapeutic target for clinical prevention from devastating processes of renal brosis.

Declarations
Funding: Not applicable.
Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable.
Authors' contributions: JH and DH participated in the design of the study and manuscript writing, WY performed the experiments, HL collected the data and performed the statistical analysis.
Data availability: The datasets used and/or analysed for the current study are available from the corresponding author upon reasonable request.   MiR-378b was a downstream target of SNHG10. (A) The binding site between SNHG10 and miR-378b were shown. (B, C) The expression of miR-378b was detected by RT-PCR. **P < 0.01 vs. Blank or TGF-β1.