Silencing of Long non-coding RNA Plasmacytoma variant translocation 1 inhibits the proliferation, migration, invasion and fibrosis of high glucose-induced mouse mesangial cells via targeting microRNA-93-5p

Background: This study aimed to investigate the regulatory role of Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) on the biological processes of high glucose (HG)-induced mouse mesangial cells (MMCs). Methods: PVT1 expression in diabetic nephropathy (DN) mice and HG-induced MMCs was detected by qRT-PCR. PVT1 was silenced in HG-induced MMCs by the transfection of PVT1 siRNA (si-PVT1). EdU and Colony formation, Annexin V-PI staining, Muse cell cycle, Scratch, and Transwell assay were performed to detect the cell proliferation, apoptosis, cell cycle, migration, and invasion, respectively. The contents of fibrosis factors in cell-culture supernatants were detected by ELISA. Western blot was performed to detect the expression of factors involved in apoptosis, cell cycle, migration and invasion, fibrosis, and PI3K/Akt/mTOR pathway. In addition, the targeting relation between miR-93-5p and PVT1 was predicted by StarBase3.0 and identified by Dual-luciferase reporter gene assay. Results: PVT1 was overexpressed in DN kidney tissues and HG-induced MMCs. HG-induced MMCs exhibited significantly increased EdU positive cells, cell colonies, S and G2/M phase cells, migration and invasion ability, and contents of fibrosis factors, as well as significantly decreased apoptosis rate compared with NG-induced MMCs. HG significantly up-regulated Bcl-2, CyclinD1, CDK4, N-cadherin, vimentin, Col. IV, FN, TGF-β1 and PAI-1, and down-regulated Bax, cleaved caspase-3, cleaved PARP, and E-cadherin in MMCs. Silencing of PVT1 eliminated the effects of HG in MMCs and blocked PI3K/Akt/mTOR pathway. MiR-93-5p was a target of PVT1, which eliminated the effects of PVT1 on HG-induced MMCs. Conclusions:


Introduction
Background: Diabetic nephropathy (DN), characterized by decreased glomerular filtration, proteinuria, and renal fibrosis is a common diabetes-associated disease [1]. DN affects more than 40% diabetic patients worldwide, and leads to millions of deaths due to end-stage kidney disease [2]. The occurrence of DN attributes to diverse factors, such as hyperglycemia, accumulation of advanced glycosylation products, and activation of cytokines [3]. Although many attempts have been made to the clinical treatment of DN [4,5], the modern medical treatment is still unable to completely prevent the development and deterioration of DN due to the insufficient understanding of the pathological mechanisms involving DN [6].
Long non-coding RNAs (lncRNAs) are important transcripts that take part in the regulation of disease pathways [7][8][9][10]. The dys-regulation of lncRNAs has been widely reported in various diseases, including DN [11,12]. Plasmacytoma variant translocation 1 (PVT1) is an important lncRNA regulator in diabetes [13]. It has proved that autophagy ameliorates cognitive impairment in diabetic mice through activating PVT1 [14]. Type 1 diabetesinduced end-stage renal disease is closely associated with the variants in PVT1 [15]. PVT1 mediates the accumulation of extracellular matrix in DN [16]. LncRNAs can modulate the translation and degradation of mRNAs through interacting with microRNAs (miRs) [17]. The biological function of PTV1 during the disease process is realized by targeting specific miRs [18]. Six locations on PVT1 are revealed on miR-1204, -1205, -1206, 1207-3p, -1207-5p, and -1208 [19]. The mediation effect of PTV1 during extracellular matrix accumulation in kidney cells of DN is realized by targeting miR-1207-5p [20]. MiR-93 is an onco-miR in cancer [21], that is also a regulator in DN [22]. It has proved that the serum miR-93 is low-expressed in DN patients [23]. Comparative miR expression profile arrays showed that miR-93 is a signature miR in hyperglycemic condition [24]. Up-regulation of miR-93 4 inhibits epithelial mesenchymal transformation and renal fibrogenesis in TGF-β1-induced HK2 cells [25]. Although the biological functions of PTV1 and miR-93 in DN have been mentioned by previous studies, the detailed molecular mechanism of PTV1 involving miR-93 in DN remains unclear.
Objectives: In this study, the regulatory role of PVT1 on the proliferation, apoptosis, cell cycle, migration, invasion, and fibrosis of high glucose (HG)-induced mouse mesangial cells (MMCs) was evaluated. The regulatory mechanism of PVT1 involving miR-93 was analyzed. This study may lay a novel theoretical basis for the treatment of DN.

Animal
A total of 10 SPF DN mice (4-week-old, Strain: BKS.Cg-+Leprdb/Leprdb/J, db/db, 20-30 g) and 10 normal mice (4-week-old, db/m, 20-30 g) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). Mice were fed in a standalone environment at 22°C and 50% relative humidity under the alternating day and night of 12 h/12 h. After 4 days of feeding with standard diet (acclimatization), mice were anesthetized by intraperitoneal injection of 50 mg/kg sodium pentobarbital, and then sacrificed by cervical dislocation. The kidney tissues were stripped and frozen in liquid nitrogen. This study was approved by the local ethics committee of our hospital. All animal experiments were performed in accordance with the care and use of laboratory animals (National Institutes of Health, USA).

Cell culture
MMCs (SV40 MES13, established from the kidney of a mouse transgenic for the SV40 early region) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MMCs were cultured in a mixture of DMEM containing 10% FBS and Ham's F12 medium (3: 1) containing 14 mM HEPES, and maintained in a humidified incubator (MCO-15AC, SANYO) with 5% CO 2 at 37°C. The medium was refreshed every 48 h, and cells were passaged at 80% confluence. Logarithmic growth phase cells at the third passage were used for transfection.

Cell treatment and transfection
PVT1 siRNA (si-PVT1) and siRNA negative control (si-NC) were synthesized by Shenggong Bioengineering Co., Ltd. (Shanghai, China) ( Table 1). MiR-93-5p mimics, miR-93-5p inhibitor, mimics negative control (mimics-NC) and inhibitor negative control (INC) were purchased from Jima Pharmaceutical Technology Co., Ltd. (Shanghai, China). Cells were transfected with the above agents by using a Lipofectamine kit (Invitrogen). Cells without transfection were considered as the control. The transfection efficiency was identified by qRT-PCR. After the transfection for 48 h, cells were incubated in DMEM containing 5.55 mmol/L (normal physiological environment, NG group) or 30 mmol/L D-Glucose (diabetic physiological environment, HG group) for another 48 h. Cells were collected for the following assay.

qRT-PCR
Total RNA was extracted from MMCs by using TRIZOL reagent (Invitrogen), and then reverse-transcribed on a SimpliAmp PCR instrument (Applied Biosystems, Foster City, CA, USA). The qRT-PCR was performed on an ABI7500 Sequence Detection System (Applied Biosystems). The PCR program included 40 cycles of 95°C for 10 min, 95°C for 10 s, 60°C for 20 s and 72°C for 34 s. The relative mRNA expression level was analyzed by the 2 -ΔΔCt method [26]. Oligonucleotide primers were synthesized by Biotechnology Bioengineering Co., Ltd. (Shanghai, China), and the primer sequences were shown in Table 1.

Western blot
Total proteins were extracted from cells using lysis buffer, and quantified using a BCA kit 6 (Invitrogen). Then, 20 μg protein samples were run in a 10% SDS-PAGE, and transferred onto PVDF membrane. Afterwards, the membrane was blocked with 5% skim milk in TBST solution for 2 h, and incubated with primary antibody overnight at 4°C. The antibodies

EdU and Colony formation assay
An EdU cell proliferation detection kit (Ruibo Biotechnology Co., Ltd., Guangzhou, China) was used for EdU assay (cell proliferation). Briefly, cells were labeled with EdU, fixed with 4% paraformaldehyde, and then stained with DAPI. The EdU fluorescence (red) was detected by a laser confocal microscope (NIKON A1, Japan).
For Colony formation assay, cells were seeded into 6-well plates (300 cells/well) and cultured for 2 weeks. Cells were then fixed with 4% paraformaldehyde for 10 min and stained with crystal violet for 15 min. The stained colonies were observed under an inverted microscope (Olympus Ckx53, Japan), and counted using an ImageJ (1.48 V) software.

Annexin V-PI assay
An Annexin V-PI kit (Invitrogen) was used for the detection of cell apoptosis. Briefly, cells were stained with Annexin V-EGFP and PI for 15 min under darkness at 25°C. The apoptosis rate was then analyzed on a MUSETM cytometer (EMD Millipore, USA).

Cell cycle analysis
A MuseTM Cell Cycle Reagent (Invitrogen) was used for the detection of cell cycle. Briefly, cells were fixed in 70% ethanol, and incubated with MuseTM Reagent in the dark for 30 min at 25°C. The cell cycle was then analyzed on a MUSETM cytometer (EMD Millipore).

Scratch assay
Cells were seeded into 6-well plate, and cultured in serum-free DMEM until 90% confluence. A scratch wound was then made in each well with a pipet tip. Cell debris was removed by washing with PBS. After 48 h of culturing in serum-free DMEM, the scratch width was photographed under an inverted microscope (Olympus Ckx53). The migration rate = (1 -scratch width at measurement/initial scratch width) × 100%.

Transwell assay
The cell invasion was detected by using transwell chambers (

ELISA for fibrosis factors
Cells were cultured for 48 h after transfection, and the cell-culture supernatants were collected. The contents of Col. IV, PAI-1, FN and TGF-β1 in the supernatants were detected 8 by using ELISA kits (BioSource International, Camarillo, CA, USA) in accordance with the manufacturers' instructions.

Dual-Luciferase reporter gene (DLR) assay
The binging site of PVT1 in miR-93-5p was predicted by StarBase3.0 software. A DLR kit (Promega, USA) was used to identify the relationship between PVT1 and miR-93-5p. The mutant sequence (MT) and the wild sequence (WT) of PVT1 were amplificated according to the binging sequences, and then cloned into the pmirGLO luciferase vector. HEK-293T cells were then co-transfected with WT/MT and miR-93-5p mimics/miR-93-5p mimics NC (Shanghai Jima Pharmaceutical Technology Co., Ltd.), and grouped as MT + mimics, MT + NC, WT + mimics, and WT + NC group. After 48 h of transfection, the fluorescence was measured on a Microplate Reader (Invitrogen).

Statistical analysis
All assays were performed in triplicate. Data were expressed as mean ± standard deviation (SD). All statistical analyses were performed by SPSS 20.0 and GraphPad.Prism 5.01 statistical software. The data among multi-groups were analyzed by One-Way ANOVA, followed by Tukey's test (between two groups). P < 0.05 represented statistically significant.

PVT1 was up-regulated in DN mice and HG-induced MMCs
The expression of PVT1 was detected in kidney tissues of DN mice and HG-induced MMCs by qRT-PCR. PVT1 expression was significantly higher in kidney tissues of db/db mice (DN) than that in tissues of db/m mice (Normal) (P < 0.05) ( Figure 1A). In addition, PVT1 was significantly up-regulated in HG-induced MMCs compared with NG-induced MMCs (P < 0.05) ( Figure 1B).

Silencing of PVT1 inhibited the proliferation of HG-induced MMCs
PVT1 was silenced in HG-induced MMCs by the transfection of siPVT1. The mRNA expression of PVT1 in the HG + siPVT1 group was significantly lower than that in the HG group (P < 0.05). The expression of PVT1 was not significantly different between the HG group and the HG + siNC group (Figure 2A). EdU assay showed that the percentage of EdU cells in the HG group was significantly higher than that in the NG group (P < 0.05). The percentage of EdU cells was significantly decreased in the HG + siPVT1 group compared with that in the HG group (P < 0.05) ( Figure 2B). Colony formation assay showed consistent results with EdU assay (P < 0.05) ( Figure 2C). In addition, the cell apoptosis was opposite to the cell proliferation detected above (P < 0.05) ( Figure 2D). The expression of Bax, cleaved caspase-3, and cleaved PARP in the HG group was significantly lower than that in the NG group. The expression of Bax, caspase-3, and PARP was significantly increased in the HG + siPVT1 group compared with that in the HG group (P < 0.05). The expression change of Bcl-2 was opposite to that of Bax (P < 0.05) ( Figure 2E).

Silencing of PVT1 influenced the cell cycle of HG-induced MMCs
The percentages of cells in S and G2/M phase were significantly higher and the percentage of cells in G0/G1 phase was significantly lower in the HG group than that in the NG group (all P < 0.05). The transfection of siPVT1 eliminated the effects of HG on the cell cycle of MMCs (all P < 0.05), suggesting that PVT1 silencing might block the cell cycle of HGinduced MMCs in G0/G1 phase ( Figure 3A

Silencing of PVT1 inhibited the migration and invasion of HG-induced MMCs
Scratch and Transwell assay showed that the cell migration and invasion ability were significantly enhanced in the HG group compared with the NG group (all P < 0.05). The transfection of siPVT1 inhibited the migration and invasion ability of HG-induced MMCs (all P < 0.05) ( Figure 4A-B). Western blot showed that the expression of E-cadherin was significantly lower, and the expression of N-cadherin and vimentin was significantly higher in the HG group than that in the NG group (all P < 0.05). The transfection of siPVT1 significantly reversed the expression of E-cadherin, N-cadherin and vimentin in HGinduced MMCs (all P < 0.05) ( Figure 4C).

Silencing of PVT1 inhibited the fibrosis of HG-induced MMCs
The
Because miR-93 plays an important role in the progression of DN, the targeting relation between miR-93-5p and PVT1 was further analyzed. DLR assay showed that the luciferase activity in the WT + mimics group was significantly lower than that in the WT + NC group (P < 0.05) ( Figure 6B). The mRNA expression of PVT1 was significantly higher in the inhibitor group and significantly lower in the mimics group than that in the Mock group (all P < 0.05). The expression of PVT1 was not significantly influenced by the transfection of mimics-NC and INC. In addition, the expression of miR-93-5p in the HG group was significantly lower than that in the NG group (P < 0.05). The transfection of siPVT1 significantly up-regulated miR-93-5p in HG-induced MMCs (P < 0.05) ( Figure 6C and D).

MiR-93-5p eliminated the role of PVT1 in HG-induced MMCs
MiR-93-5p inhibitor was used to silence miR-93-5p in HG-induced MMCs. The mRNA expression of miR-93-5p in the inhibitor group was significantly down-regulated compared with that in the Mock group (P < 0.05). The expression of miR-93-5p was not significantly different between the INC group and the Mock group ( Figure 7A). The miR-93-5p inhibitor and siPVT1 were then con-transfected into HG-induced MMCs. The cell proliferation, migration, invasion, and fibrosis ability were promoted by the transfection of miR-93-5p inhibitor, and inhibited by the transfection of siPVT1 (all P < 0.05). The transfection of miR-93-5p inhibitor significantly eliminated the effects of siPVT1 on the above biological processes of HG-induced MMCs (all P < 0.05) ( Figure 7B-E).

Silencing of PVT1 blocked PI3K/Akt/mTOR pathway in HG-induced MMCs
The expression of PI3K/Akt/mTOR pathway related proteins was detected by Western blot.
The expression of p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR was higher in the siNC + miR-93-5p inhibitor group and was lower in the siPVT1 + INC group than that in the siNC + INC group (all P < 0.05). The blocking effect of siPVT1 on PI3K/Akt/mTOR pathway was eliminated by miR-93-5p inhibitor (all P < 0.05) (Figure 8).

Discussion
DN is one of the most common conditions found in patients with renal disease. Although PVT1 has been proved to be associated with DN, the detailed molecular mechanism of PVT1 in DN progression remains unclear. Here, PVT1 was found to be overexpressed in

Consent to publish
All contributing authors have agreed to submit this manuscript and all authors approved to publish this study.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Tables Table 1 The  #, P < 0.05 compared with the HG and HG + siPVT1 group. The data among multigroups were analyzed by One-Way ANOVA, followed by Tukey's test (between two groups).

Figure 5
Effect of lncRNA-PVT1 on the fibrosis of mouse mesangial cells (MMCs) under high glucose (HG) condition. A-D, ELISA was used to detect the contents of fibrosis proteins (Col. IV, FN, TGF-β1, and PAI-1). E, Western blot was used to detect the expression of fibrosis proteins. *, P < 0.05 compared with the NG group; #, P < 0.05 compared with the HG and HG + siPVT1 group. The data among multi-groups were analyzed by One-Way ANOVA, followed by Tukey's test (between two groups).

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