Long non-coding RNA TUG1/microRNA-29/PTEN axis in ischemia-reperfusion in a rat model

Objective: Long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) is increased under ischemia. This study intended to identify the potential competing endogenous RNA network involving TUG1 in renal ischemia-reperfusion (I/R). Methods: A rat model of acute renal injury induced by I/R was established, and the differentially expressed genes were analyzed by microarray. The levels of blood urea nitrogen (BUN), serum creatine (SCr), methylenedioxyphetamine (MDA) and superoxide dismutase (SOD) in serum of rats were measured. HE staining evaluated the pathological damage of renal tissues, western blot analysis detect the levels of apoptosis-and autophagy-related proteins, immuno�uorescence staining detected LC3 �uorescence intensity, and transmission electron microscope observed autophagosomes. Pull-down assay and dual luciferase reporter gene assay were used to verify the targeting relationship among TUG1, miR-29 and PTEN. The effects of TUG1 on biological behaviors of renal tubular cells were evaluated by simulating the acute renal injury induced by I/R in vitro. Results: In vivo, the levels of BUN, SCr and MDA in serum of I/R-treated rats were increased, SOD level and autophagosomes were reduced, tubule epithelial cells were necrotic, and TUG1 was upregulated in renal tissues of I/R-treated rats, which were reversed by TUG1 knockdown. Autophagy inhibition attenuated the protective effect of TUG1 knockdown on I/R-treated rats. TUG1 could competitively bind to miR-29 to promote PTEN expression. In vitro, low expression of TUG1 promoted proliferation and autophagy of renal tubular cells and inhibited apoptosis. Conclusion: TUG1 knockdown promotes autophagy and improves acute renal injury in I/R-treated rats by binding to miR-29 to silence PTEN.


Introduction
Ischemia/reperfusion (I/R) injury is a process when a blood supply returns after hypoxia to tissue, causing ischemia and induces a cascade of events related to oxidative damage and dysfunction, which is responsible for most cardiovascular diseases worldwide [1].Serious clinical manifestations such as acute heart failure, myocardial infarction, cerebral and gastrointestinal disorder, systemic in ammatory response and multiple organ dysfunction are critical medical conditions of I/R injury [2].What is worse, clinical symptoms are often subtle at rst, making it impossible to identify the exact time of the onset of ischemia; if it is diagnosed within 24 hours after symptoms appear, the survival rate of acute ischemia is about 50%, but this rate decreases to 30% or lower in case of delayed diagnosis [3].I/R injury is the main negative factor in uencing the outcome after kidney transplantation and is associated with organ rejection [1].Post-ischemic AKI is characterized by decreased glomerular ltration rate and high renal vascular resistance with endothelial activation and dysfunction, a process of critical importance that is followed by a reduction in microvascular blood ow mainly affecting the renal outer medulla [4].I/R injury of the kidney is a leading cause of acute kidney injury, and it may result in worsening or even loss of organ function and is also a common and unavoidable phenomenon in kidney transplantation [5].
Tubular cells are critical targets of I/R injury in renal transplantation [6].If we want to de ne biomarkers or develop targeted therapeutic interventions, it is urgent to comprehend the mechanism of renal tubular cells on I/R response.
Emerging studies have implicated a fundamental role for non-coding RNAs, such as microRNAs (miRs), and more recently long non-coding RNAs (lncRNAs) in acute I/R injury [7,8].LncRNA taurine upregulated gene 1 (TUG1) is essential for retinal development in the developing mouse eye, and it is abnormally regulated in tumorigenesis, either as a potential tumor suppressor or oncogene [9].TUG1 has been identi ed to protect mouse livers against cold-induced liver damage in liver transplantation via inhibiting apoptosis and in ammation [10].In addition, TUG1 protects renal tubular epithelial cells from lipopolysaccharide-induced damage by regulating miR-223 [11].It has been documented that hypoxia treatment signi cantly increases expression and overexpression of TUG1 aggravates hypoxia-induced injury in H9c2 cells [12].LncRNA TUG1 may function as a competing endogenous RNA (ceRNA) for miR-145 to upregulate aquaporin-4 to induce cell damage, possibly providing a new target in cerebral I/R injury [13].In light of these references, we hypothesize there is an underlying ceRNA network involving TUG1 in renal I/R injury.Therefore, we carried out in vivo and in vitro experiments to gure out the protective roles of TUG1 in renal I/R injury through which pathway.

Ethics statement
This study was rati ed and supervised by the ethics committee of Hainan Medical University.We made signi cant efforts to minimize animals used and their suffering.
Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (50 mg/kg).Then the right kidney of each rat was removed and the left renal artery was exposed and clamped for 45 minutes.Subsequently, the clamp was removed and the kidney was observed for 4-5 minutes to assure reperfusion was established successfully.After 24 hours of reperfusion, the left kidney of each rat was removed and rats were euthanized.The serum of 10 rats in each group was applied for detecting serum index, the kidney tissues of 3 rats was used for tissue homogenate, and the kidney tissues of 6 rats was used for tissue section detection.

Microarray analysis
Total RNA of kidney tissues in rats from the sham and I/R groups was extracted by a TRIzol kit (Invitrogen Inc., Carlsbad, CA, USA).Double strand cDNA was synthesized by SuperScript Doublestranded cDNA synthesis kit (Invitrogen), and labeled and hybridized to an lncRNA expression microarray (12 × 135K, Arraystar Inc., Rockville, MD, USA).After hybridization and washing, processed slides were scanned by an Axon GenePix 4000B scanner (Molecular Devices Inc., Sunnyvale, CA, USA).Raw data were extracted as pair les using NimbleScan software (Version 2.5; Roche).The threshold for up-and downregulated genes was set as fold change > = 1.5 and p value < = 0.05.All the above works were completed by Shanghai Sensichip Hightech Co., Ltd.(Shanghai, China).Hierarchical cluster analysis was done by Shanghai Novel Bioinformatics Company (Shanghai, China).

Detection of renal function
Peripheral blood samples obtained from each group were centrifuged at 3000 r/min for 10 minutes.Then 500 µL serum was taken for determination of serum creatinine (SCr) and blood urea nitrogen (BUN) using an automatic biochemical analyzer (Roche Diagnostics, Indianapolis, Indiana, USA) with an AU2700 Analyzer (Olympus, Tokyo, Japan).

Detection of contents of renal malondialdehyde (MDA) and superoxide dismutase (SOD)
The MDA content in the rat kidneys was measured using an MDA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) and the thiobarbituric acid method [16].The renal SOD content in rats was detected using a SOD kit (Nanjing Jiancheng Bioengineering Institute) [17].

Histological examination
Kidney tissues were xed in 10% buffered formalin, embedded in para n and cut at 4-µm.After depara nization and rehydration, sections were stained with hematoxylin and eosin (HE) for the quantitative evaluation of renal injury score.A histologic score of tissue damage was estimated by two external pathologists according to the following criteria: tubular dilatation, cast deposition, brush border loss and necrosis in 25 randomly chosen non-overlapping elds.Lesions were graded on a scale from 0 (normal) to 5 (extensive damaged).

TUNEL assay
TUNEL assay was performed with an In situ cell death detection kit ( uorescein, Roche).Para ned sections were depara nized, sectioned at 4-µm, treated with 20 mg/mL proteinase K, and treated with 3% hydrogen peroxide.Subsequently, the sections were treated with a mixture of nucleotides and TdT enzyme at 37 °C for 1 hour, and then treated with the converter conjugated with horseradish peroxidase at 37 °C for 30 minutes.Under the uorescence microscope (Carl Zeiss, Jena, Germany), cells with green stained nuclei were regarded as TUNEL-positive and expressed as the percentage of total cells.Finally, TUNEL-positive cells were counted in 10 randomly selected elds (× 200) in a blinded manner.

Electronic microscopy
The renal samples at 1 mm 3 were xed with 2.5% glutaraldehyde overnight at 4 °C, and treated with osmium tetroxide, embedded and sliced.Autophagosome ultrastructure was observed under the transmission electron microscope (Olympus).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA of renal tissues or tubular cells from each group of rats was extracted by TRIzol reagent (Invitrogen).After the concentration and purity of RNA were determined, cDNA was synthesized by a reverse transcription kit, ampli ed by PCR instrument, and the expression of each primer in (Table 1) was detected by a SYBR PCR Master Mix kit (Bio-Rad, Inc., Hercules, CA, USA), with U6 or β-actin as an internal reference.All primers used in the experiment were designed by Primer 3Plus website and synthesized by Genewiz Biotechnology Co., Ltd.(Suzhou, Jiangsu, China).The experiment was repeated 3 times to calculate the average CT value, and the concentration of each sample was calculated according to 2 −△CT to detect the relevant RNA level in cells and tissues.

Western blot analysis
The renal tissues were homogenized centrifuged at 12,000 g for 10 minutes at 4 °C.Then proteins were collected and quanti ed using a bicinchoninicacid kit (Thermo Fisher Scienti c, Rockford, IL, USA), and then separated by electrophoresis and transferred onto nitrocellulose membranes.The blots were blocked with 5% non-fat dry milk in tris-buffered saline-tween (TBST) at 37 °C for 2 hours, followed by incubation with primary antibodies at (Table 2) 4 °C overnight.After TBST washes, the membranes were incubated for 1 hour with horseradish-peroxidase conjugated secondary antibody.After TBST washes, protein bands were detected by an enhanced chemiluminescence system and visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).Target protein/glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as relative protein expression.RNA pull-down TCMK-1 cells were transfected with biotinylated miR, and were collected after 48-hour transfection.The cell lysates were incubated with M-280 streptavidin magnetic beads (Invitrogen).The bound RNAs were puri ed using TRIzol reagent (Invitrogen) for further RT-qPCR analysis.
Cell culture and grouping Cell counting kit-8 (CCK-8) Cell viability was detected by a CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the instructions.The absorbance at a wavelength of 450 nm was measured using a microplate reader.The percentage of living cells was calculated by the ratio of absorbance of the H/R group to that of the blank group.

Flow cytometry
Cells were planted into 6-well plates at 5 × 10 5 /mL cells/well and 1 mL/well.After cell adherence for 24 hours, cells were stimulated by hypoxia and H/R in refreshed medium.Cells in each group were collected into ow tubes, and each tube was added with 5 uL uorescein isothiocyanate-labeled Annexin-V buffer and 100 uL 1 × loading buffer, followed by 30-nimute incubation without light exposure.Cell apoptosis was detected by a ow cytometer (Beijing YourHope Medical Equipment Co., Ltd., Beijing, China).

Statistical analysis
Statistical analysis was conducted by SPSS 21.0 (IBM Corp. Armonk, NY, USA).All the data were in normality distribution checked by the Kolmogorov-Smirnov test.Measurement data were expressed as mean ± standard deviation.The t test processed comparisons between two groups, while one-way or twoway analysis of variance (ANOVA) processed comparisons among multiple groups, and Tukey's multiple comparisons test or Sidak's multiple comparisons test was used for post-hoc test.The Kaplan-Meier method was used to draw the survival curve.The p value was obtained by a two-tailed test and p < 0.05 indicated a statistical difference.

TUG1 is upregulated in renal I/R-treated rats
To understand the renal injury of rats after I/R treatment, the levels of serum BUN, SCr, MDA and SOD in sham-operated rats and I/R-treated rats.Compared with the sham-operated rats, the levels of BUN, SCr and MDA in the serum of I/R-treated rats were increased, and the levels of SOD was decreased signi cantly (all p < 0.01) (Fig. 1A).Then, HE staining evaluated the pathological damage of renal tissues.
The results showed that the structure of renal tubules in sham-operated rats was clear and complete, the renal tissues in I/R-treated rats were seriously damaged, the epithelial cells of renal tubules were degenerated and necrotic, part of basement membrane was exposed, and the lumen of tubules was dilated (Fig. 1B).In addition, lncRNA microarray analysis was performed in two groups of rats.The results showed multiple differentially expressed lncRNAs in the two groups of rats, while in I/R-treated rats, TUG1 was signi cantly upregulated (p < 0.01) (Fig. 1C).To verify the most signi cant upregulation of TUG1 in clinical practice, we selected the rst six lncRNAs with the most signi cant difference in microarray analysis for clinical validation.The results of RT-qPCR showed that compared with other lncRNAs, TUG1 was signi cantly upregulated (all p < 0.01) (Fig. 1D).

TUG1 knockdown improves renal IR injury and inhibits cell apoptosis
In the above microarray analysis, we know that lncRNA TUG1 is signi cantly upregulated in acute renal injury after I/R, so we suspected that TUG1 also had an effect on acute renal injury after I/R.To con rm this conjecture, we injected the knockdown TUG1 into I/R treated rats, and detected the levels of BUN, SCr, MDA and SOD in the serum of rats.The results showed that compared with the NC group, the serum BUN, SCr and MDA levels of rats in the ko-TUG1 group were reduced, while SOD level was signi cantly elevated (all p < 0.01) (Fig. 2A).Then pathological morphology of the rats was evaluated by HE staining, and it revealed that the in ammatory in ltration in the renal tissues was reduced and the pathological morphology was improved after the acute renal I/R injury in the ko-TUG1 group (Fig. 2B).After that, ow cytometry and TUNEL staining detected the apoptosis of renal cells.The results showed that the apoptosis and TUNEL-positive cells in the ko-TUG1 group were notably reduced (both p < 0.01) (Fig. 2C/D).
TUG1 knockdown promotes autophagy in rats with acute renal injury induced by I/R Autophagy plays an important role in acute renal injury [19].POC can reduce renal damage and epithelialmesenchymal transition after I/R injury by enhancing autophagy activation [20].Therefore, we explored the effect of TUG1 on autophagy in acute renal injury induced by I/R.The results of transmission electron microscope observation displayed that compared with the sham group, autophagosomes in renal tissue of rats in I/R group were signi cantly reduced, but notably increased after the treatment of knocking down TUG1 was (Fig. 3A).In addition, western bot analysis detected levels of autophagy-related proteins.
The results showed that relative to the sham group, light chain 3 (LC3)-II/LC3I and Becline-1 level in the I/R group were signi cantly decreased, and level of p62 was elevated.After knocking down TUG1, the autophagy-related proteins showed the opposite trends (all p < 0.01) (Fig. 3B).Immuno uorescence staining showed that relative to the I/R group, the uorescence intensity of LC3 was increased after knocking down TUG1.
Inhibition of autophagy attenuates the protective effect of TUG1 knockdown on rats with acute renal injury induced by I/R To further con rm the protective effect of TUG1 on rats with acute renal injury induced by I/R through autophagy, a functional rescue experiment was carried out by setting up a combination group of autophagy pathway inhibitor (3-mA) and knockdown TUG1, with the ko-TUG1 group as the control.The results showed that compared with the ko-TUG1 group, the serum BUN, SCr and MDA levels of rats in the ko-TUG1 + 3-mA group were signi cantly increased, while SOD was reduced, apoptosis and TUNELpositive cells were increased, LC3-II/LC3I and Becline-1 level were reduced, and p62 level was increased (all p < 0.01) (Fig. 4A-D).

TUG1 competitively binds to miR-29 to promote PTEN expression
To nd out whether there is a ceRNA network in acute renal I/R injury, we predicted that there is a targeting relationship between TUG1 and multiple miRs through bioinformatics website http://starbase.sysu.edu.cn/index.php.miR-29a overexpression can protect against I/R injury [21].As expected, we found a binding site between TUG1 and miR-29 by pull-down assay (Fig. 5A), and we predicted that there are multiple target genes in miR-29a, including PTEN.We further veri ed the relationship among TUG1, miR-29 and PTEN.Through the veri cation of dual luciferase reporter gene assay, the luciferase activity of TUG1 (WT) and miR-29 was decreased signi cantly, while the luciferase activity of PTEN (WT) and miR-29 were decreased (Fig. 5B).Compared with the sham group, miR-29 expression in the I/R group was reduced while PTEN levels were elevated; compared with the NC group, miR-29 expression in the ko-TUG1 group was signi cantly elevated while PTEN levels were downregulated (all p < 0.05) (Fig. 5C).

Low expression of TUG1 promotes proliferation and autophagy of TCMK-1 cells and inhibits apoptosis
To con rm the of TUG1 in vitro, we simulated the I/R injury of renal tubular cells (TCMK-1) by H/R, and measured the activity of TCMK-1 cells in each group by CCK-8 method.It showed that the activity of TCMK-1 cells in the H/R group was lower than that in the blank group, and that in the si-TUG1 group was higher than that in the NC group (Fig. 6A).Flow cytometry and TUNEL staining evaluated the apoptosis of TCMK-1 cells.Compared with the blank group, the apoptosis rate of TCMK-1 cells in the H/R group was signi cantly enhanced; compared with the NC group, the apoptosis rate in the si-TUG1 group was decreased (Fig. 6B-C).In addition, western blot analysis detected the levels of autophagy-related proteins, and immuno uorescence staining detected the uorescence intensity of LC3.Compared with the blank group, the autophagy of TCMK-1 cells in the H/R group was inhibited; compared with the NC group, the autophagy of TCMK-1 cells in the si-TUG1 group was promoted (Fig. 6D-E).The levels of miR-29 and PTEN were detected.The results showed that compared with the blank group, miR-29 expression in TCMK-1 cells was reduced, and PTEN levels in the H/R group were signi cantly elevated; compared with the NC group, miR-29 expression in TCMK-1 cells was upregulated, and PTEN levels in the si-TUG1 group were signi cantly decreased (Fig. 6F-G) (all p < 0.01).

Discussion
Renal ischemia, the most common cause of acute kidney injury (AKI) is related to adverse outcome and high mortality, and it is estimated that AKI occurs in about 1 of 5 hospitalizations and is associated with a more than 4-fold increased likelihood of death [22,23].Therefore, it is of prime urgency and importance to search for effective approaches for I/R injury, especially for renal I/R injury.It has been identi ed that lncRNA TUG1 was signi cantly overexpressed in oxygen-glucose deprivation/reperfusion (OGD/R)induced myocardial HL-1 cells [24].The microarray analysis and RT-qPCR also validated TUG1 upregulation in renal tissues of I/R-treated rats.We further veri ed that TUG1 knockdown could promote autophagy and improved acute renal injury in I/R-treated rats by binding to miR-29 to silence PTEN.
Initially, we carried out microarray analysis to screen out the differentially expressed lncRNAs.Among these lncRNAs, TUG1 was signi cantly upregulated in I/R-treated rats.It was also reported that TUG1 was upregulated in the brain of middle cerebral artery occlusion (MCAO) and OGD/R-treated SH-SY5Y cells, indicating the therapeutic potential of TUG1 in I/R [25].In addition, we found levels of BUN, SCr and MDA in the serum of I/R-treated rats were increased, and the level of SOD was decreased signi cantly, which were reversed after knocking down TUG1.The urine volume, BUN, SCr and MDA concentrations, LC3II/LC3I, and autophagosomes were signi cantly elevated 24 hours after renal I/R, while p62 and SOD concentrations were decreased, which were consistent with our results [17].BUN and Scr in pediatric patients with AKI were decreased in parallel during hospitalization in all age groups [26].BUN and SCr are both considered speci c markers to measure kidney function and pathological injuries after I/R [27].SOD could limits oxidative stress and renal I/R injury and is considered as the most relevant molecule against I/R-induced changes [28].Signi cantly enhanced myocardial SOD activity and reduced MDA level were bene cial for I/R-treated rats [29].Thus, our study supported the demonstration that TUG1 knockdown may be bene cial for the treatment of renal I/R injury.
After knocking down TUG1, autophagosomes in renal tissues of rats was notably increased, LC3-II/LC3I and Becline-1 level were increased, and level of p62 was reduced.Autophagy is responsible for damaged organelles, and provides energy for cell renewal and internal environment stability, and loss of autophagy is associated with I/R injury [30].LC3 and Becline-1 are speci c markers to monitor autophagy, and the amount of LC3-II is closely correlated with autophagosomes [31,32].A study supported the notion that the upregulation of autophagy was associated with a 45% reduction in infarct size [33].TUG1 knockdown reduced the infarction area and cell apoptosis in MCAO model mice, thus effectively protecting against brain I/R injury [13].Taken together, knockdown TUG1 could alleviate renal I/R injury by promoting autophagy.
Furthermore, we veri ed that TUG1 competitively binds to miR-29 to promote PTEN expression.I/Rinduced TUG1 bound to miR-132-3p to activate histone deacetylase 3 and then provoked intracellular reactive oxygen species (ROS) accumulation, and worsened the injury of acute myocardial infarction [34].
The miR sponge function of TUG1 was supported by the interaction between TUG1 and miR-145 using bioinformatic analysis and by that after knockdown of TUG1, miR-145 level was signi cantly upregulated [13].miR-29a mimic protects against cell injury and mitochondrial dysfunction after ischemia-like stresses in vitro, and increasing miR-29a expression might be a novel option for protection against I/R injury [21].A close association between TUG1 and miR-29b was veri ed in inhibiting apoptosis and in ammation in lipopolysaccharide-treated H9c2 cells [35].The directing targeting relationship between miR-29a and PTEN was previously found in osteosarcoma cells, in which miR-29 served as a tumor promoter, in osteosarcoma progression and metastasis by targeting PTEN [36].
Apoptosis is a process of programmed cell death that is activated under hypoxic stress in ischemic injury and during the production of ROS in reperfusion injury [37].In the present study, low expression of TUG1

Conclusion
To sum up, TUG1 knockdown could promote autophagy and improved acute renal injury in I/R-treated rats by binding to miR-29 to silence PTEN.In addition, studies conducted to date on animal models warrant further investigation and development because many underlying mechanisms remain unknown and need to be clari ed.Additional clinical trials are warranted to justify this approach for lncRNA TUG1 target therapy in the future.
94% N 2 and 5% CO 2 to maintain the oxygen concentration at 1%.During reoxygenation incubation, cells were removed to a normoxic chamber (21% O 2 ).Cells were allocated into blank group (in the normoxic chamber at indicated time points), hypoxia/reoxygenation (H/R) group (cells were treated with H/R), NC group (after H/R treatment, cells were treated with empty vectors), and si-TUG1 group (after H/R treatment, cells were treated with si-TUG1, at 50 nM)[18].All transfections were done with HiPerFect transfection reagent (QIAGEN, Valencia, CA, USA).