LncRNA X Inactive Specific Transcript Exerts a Protective Effect on High Glucose-Induced Podocytes by Promoting the Podocyte Autophagy via miR-30d-5p/BECN-1 Axis

Inhibiting podocyte autophagy promotes the development of diabetic nephropathy (DN). This study aims to explore the upstream regulatory mechanism of the autophagy-related gene BECN1 in high glucose (HG)-induced podocytes. C57BL/6 mice were treated with 50 mg/kg streptozotocin to construct a DN model. Biochemical indexes, pathological morphology of renal tissue, the morphology of renal podocytes, and the expressions of autophagy-related proteins in DN mice and normal mice were detected. The upstream miRNAs of BECN1 and the upstream long noncoding RNAs (lncRNAs) of miR-30d-5p were predicted by bioinformatics analysis and verified by dual-luciferase reporter assay. Mouse podocyte clone 5 (MPC5) cells were exposed to HG to construct a DN cell model. The levels of miR-30d-5p, X inactive specific transcript (XIST), and BECN1 in mouse kidney and MPC5 cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The regulation of XIST/miR-30d-5p on the viability, apoptosis as well as proteins related to apoptosis, epithelial-mesenchymal transition (EMT), and autophagy in MPC5 cells were determined by rescue experiments. The levels of glucose, urinary protein, serum creatinine, and blood urea nitrogen were upregulated, but the kidney tissues and podocytes were damaged in DN mice. XIST targeted miR-30d-5p to promote viability while suppressing the apoptosis of HG-induced MPC5 cells. In kidney tissues or HG-induced MPC5 cells, the expressions of Beclin-1, light chain 3 (LC3) II/I, XIST, B-celllymphoma-2 (Bcl-2), and E-cadherin were downregulated, while the expressions of P62, miR-30d-5p, Bcl-2-associated X protein (Bax), cleaved-caspase-3, vimentin, and alpha-smooth muscle actin (α-SMA) were upregulated, which were reversed by XIST overexpression. The reversal effect of XIST overexpression was offset by miR-30d-5p mimic. Collectively, XIST promotes the autophagy of podocytes by regulating the miR-30d-5p/BECN1 axis to protect podocytes from HG-induced injury.


Introduction
Diabetic nephropathy (DN) is the main cause of end-stage renal disease (ESRD) worldwide, and hyperglycemia is the main factor driving DN into ESRD [1]. Te typical symptom of DN is proteinuria, and podocyte injury is closely related to proteinuria [2]. Podocytes are highly diferentiated terminal glomerular epithelial cells, which play an important role in regulating glomerular function. Podocyte injury is the basic feature of glomerular diseases, including podocyte fusion, podocyte hypertrophy, podocyte number reduction, podocyte apoptosis, podocyte epithelial-mesenchymal transition (EMT), and so on [3]. Terefore, protecting podocytes from high glucose (HG)-induced injury may be a new strategy to treat DN.
Increasing research has shown that the defciency of autophagy is the key cause of podocyte injury [4,5]. Autophagy is the process of transporting damaged organelles and aging proteins in cells to lysosomes for degradation [6]. Terefore, podocytes need to maintain intracellular homeostasis through autophagy, and the lack of autophagy will lead to podocyte injury, proteinuria, and glomerulosclerosis [5]. Beclin-1 encoded by BECN1 is an indispensable protein in autophagy and is the key factor of autophagy initiation. Te activation of autophagy mediated by Beclin-1 is an important mechanism to reduce podocyte injury induced by HG [7]. Terefore, it is our focus to promote the autophagy of podocytes by activating the expression of Beclin-1 in podocytes.
Epigenetic mechanism exerts an important efect on the regulation of malignant tumors, immune diseases, and metabolic diseases [8]. Noncoding RNA, an epigenetic mechanism, including long noncoding RNA (lncRNA) and microRNA (miRNA), has been proven to be involved in the development of DN and has been suggested as a diagnostic marker or therapeutic target for DN [9,10]. miRNA can inhibit the translation of mRNA or promote its degradation by binding to downstream mRNA [11]. Trough an online database, in this research, the possible target miRNAs of the BECN1 gene were predicted, among which miR-30d-5p has been proven to suppress the autophagy of renal cell carcinoma cells [12]. Nevertheless, whether miR-30d-5p is also involved in the regulation of podocyte autophagy is yet to be elucidated, which is thus discussed in this research. Given that lncRNAs often participate in the disease progression by sponging miRNA to inhibit the function of miRNA, the upstream lncRNAs of miR-30d-5p were predicted in this research, of which lncRNA XIST was reported to participate in the protection of podocytes against HG-induced cell injury by regulating the miR-30/AVEN axis [13]. Terefore, we speculated that XIST may engage in the modulation of podocyte autophagy via the miR-30d-5p/BECN1 axis, thereby exerting a protective efect on HG-induced podocytes.

Animals and Drug
Administration. C57BL/6 mice (n � 10, 18-22 g) were supplied by Shanghai Slake Animal Laboratory Co. Ltd. Te room temperature was maintained at 22 ± 2°C and humidity was set at 50-60% with a normal period (12 hours (h) light/12 h dark). Te research was approved by the Ethics Committee of Zhejiang Baiyue Biotech Co., Ltd. for Experimental Animals Welfare (approval number: ZJBYLA-IACUC-20210818).
Te mice were randomly divided into two groups, namely, the DN group (n � 5) and the control group (n � 5). Subsequently, the DN model was constructed as previously described [14]. Briefy, mice in the DN group were treated with 50 mg/kg streptozotocin (STZ, S0130, Merck, Germany) in citrate bufer (C2488, Merck, Germany) for 5 consecutive days, and those merely treated with citrate bufer were applied as the control group.

Biochemical Parameters and 24 h Urine Protein
Determination. Blood samples were collected from the tail of mice, and glucose (GLU) was tested every week by a blood glucose meter (Accu-Chek, Roche, Switzerland) to ensure the successful construction of the diabetes model. Four weeks after the injection of STZ, the 24-h urine of mice was collected, and the urine protein was determined by the urine protein test kit (C035-2-1, Nanjing Jiancheng, China). Te establishment of the DN model was considered to be successful, as obvious albuminuria was detected in mice. Blood from the tail vein of mice was collected and centrifuged at 7000 r/min for 10 minutes at 4°C to obtain plasma samples. Te levels of serum creatinine (Scr) and blood urea nitrogen (BUN) in plasma were detected by an automatic biochemical analyzer (AU680, Beckman, USA).

Histopathological Examination.
Te mice were sacrifced by cervical dislocation and their renal tissues were collected and fxed in 4% paraformaldehyde. Te fxed kidney tissues were embedded in parafn and made into 4 μm sections. After being dewaxed with xylene and rehydrated with gradient ethanol, the sections were subjected to staining with the hematoxylin and eosin (H&E) kit (G1003, Servicebio, China) and Periodic Acid-Schif (PAS) staining kit (G1008, Servicebio, China), respectively. Tereafter, the sections were dehydrated and transparentized. Finally, the sections were sealed with glycerol gelatin aqueous (S2150, Solarbio, China), and the results were observed under a microscope (AE2000, Motic, China).

Ultrastructural Changes under Transmission Electron
Microscope. Te renal tissues were washed in phosphatebufered saline (PBS) and then fxed in a fxing solution (G1102, Servicebio, China). After dehydration by acetone, the tissues were embedded in resin and sectioned with an ultrathin microtome (Leica EM UC7, Germany). Te sections were stained with uranyl acetate and lead citrate, and the morphological changes of renal podocytes were observed by the transmission electron microscope (HITACHI HT7700, Japan).
2.6. Western Blot. Kidney tissues or mouse podocyte clone 5 (MPC5) cells were subjected to the extraction of total proteins using the protein extraction kit (W034, Nanjing Jiancheng, China). Next, the protein concentration was quantifed with the BCA protein assay kit (P1511, Applygen, China). Subsequently, the proteins were separated by SDS-PAGE gel (W003, Nanjing Jiancheng, China), blocked with BSA, and successively incubated using primary antibodies and secondary antibodies. Te details of the antibodies are shown in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal reference. Eventually, the protein bands were visualized using an electrochemical luminescence reagent (W028, Nanjing Jiancheng, China) with a Tanon 5200 Imaging System (Shanghai, China).

qRT-PCR.
Total RNA was extracted from MPC5 cells using Trizol reagent (15596026, Invitrogen, USA) and miR-Neasy mini kit (217004, Qiagen, German). Te cDNA synthesis was performed using the frst strand cDNA synthesis kit (K1612 or B532453, Sangon, China). Te qPCR was conducted on a real-time PCR system (CFX Connect, Bio-rad, USA) using SYBR Green Abstract PCR Mix (B110031) and MicroRNAs qPCR Kit (B532461) obtained from Sangon (China). Te relative values were calculated by the 2 −ΔΔCT method [16] and normalized to GAPDH or U6. Te sequences of the primers are listed in Table 2.

Cell Apoptosis Assay. Annexin V-FITC/PI Apoptosis
Detection Kit (556547, BD, USA) was utilized to evaluate the cell apoptosis. MPC5 cells were digested and prepared into 1 × 10 6 /mL cell suspension in binding bufer, and the treated cell suspension was incubated with 5 μL of Annexin V-FITC and 10 μL of PI for 20 minutes in the dark. In the end, the cell apoptosis was detected by an Accuri C6 fow cytometer (BD Biosciences, USA).

Statistical Analysis.
Te measurement data were presented as mean ± standard deviation. Te results between the two groups were analyzed by an independent sample t test. One-way analysis of variance (ANOVA) was adopted for the comparison among multiple groups. Te experiments were repeated three times. All statistical analyses were implemented with GraphPad 8.0 software, and P value less than 0.05 was considered to be statistically signifcant.

Autophagy Inhibition was Observed in the Kidney Tissue of DN Mice.
We detected the levels of GLU, urinary protein, Scr, and BUN in two groups of mice, and found that the levels of these indexes in DN mice were higher than those in control mice (Figures 1(a)-1(d), P < 0.01). Histological staining results showed that compared with those of the control mice, the glomerular structure of DN mice was disordered and the basement membrane was thickened (Figures 1(e) and 1(f )). Trough transmission electron microscope, we observed the disorganized podoid processes, exfoliated podocytes, and thickened basement membrane in DN mice (Figure 1(g)). In addition, the expression of LC3 in the kidney tissue of DN mice was decreased (Figure 1(h)). Meanwhile, the results of Western blot showed that the levels of Beclin1 and LC3 II/LCE I were lower yet the P62 level was higher in the DN group than those in the control group (Figures 1(i)-1(l), P < 0.001).

MiR-30d-5p
Targeted BECN1 and XIST. We predicted the possible target miRNAs of BECN1 through the online website and then obtained fve candidate miRNAs by the intersection of the results (Figure 2(a)). Based on the literature retrieval results, miR-30d-5p was fnally selected as the target miRNA of BECN1 and XIST for subsequent studies. According to the predicted sequences in Figures 2(b) and 2(d), we construct the recombinant plasmid and transfect it with miR-30d-5p mimic into 293T cells. Te results indicated that the luciferase activity of 293T cells transfected with miR-30d-5p mimic and BECN1-WT plasmid or XIST-WT was reduced (Figures 2(c) and 2(e), P < 0.001).

XIST Targeted miR-30d-5p to Regulate the Viability and
Apoptosis of MPC5 Cells. In addition, the expression of XIST was lower, and the expression of miR-30d-5p was higher in HG-induced MPC5 cells (Figures 3(a) and 3(b), P < 0.001). We constructed XIST overexpression plasmid and verifed its transfection efciency (Figure 3(c), P < 0.001), and found that OE-XIST can reduce the level of miR-30d-5p while increasing the level of BECN1 (Figures 3(d) and 3(e), P < 0.001). At the same time, we also tested the transfection efciency of miR-30d-5p mimic in MPC5 cells and found that miR-30d-5p mimic increased the miR-30d-5p expression while reducing the expression of BECN1 (Figures 3(f

Discussion
Inducing autophagy to protect podocytes from HG-induced injury has become a promising strategy in the treatment of DN [17]. Under normal blood glucose levels, autophagy is an important protective mechanism in renal epithelial cells, including podocytes, proximal tubular, mesangial, and endothelial cells; under hyperglycemic conditions, repression of the autophagic mechanism can contribute to the development and progression of diabetic kidney disease [18,19].
In this study, we clarifed that the mechanism of the XIST/    Figure 3: XIST targeted miR-30d-5p to regulate the viability and apoptosis of HG-induced podocytes. MPC5 cells were transfected with XIST overexpression plasmid/empty vector and or miR-30d-5p mimic/mimic control. (a-b) Te expression levels of XIST and miR-30d-5p in NG or HG-treated MPC5 cells were determined by qRT-PCR. GAPDH or U6 was used as the internal control. (c-e) Te expressions of XIST, miR-30d-5p, and BECN-1 in MPC5 cells transfected with XIST overexpression plasmid or empty vector were determined by qRT-PCR. GAPDH or U6 was used as the internal control. (f-g) Te expressions of miR-30d-5p and BECN1 in MPC5 cells transfected with miR-30d-5p mimic/mimic control were determined by qRT-PCR. GAPDH or U6 was used as the internal control. (h-j) Te frst two groups of MPC5 cells were treated with NG or HG. Te last four groups of MPC5 cells cotransfected with XIST overexpression plasmid/empty vector and miR-30d-5p mimic/mimic control were treated with HG. (h) Te viability of MPC5 cells was determined by CCK-8 assay. (i-j) Te apoptosis of MPC5 cells was determined by fow cytometry. Quantifed values were presented as mean ± standard deviation of at least three independent experiments. +++ p < 0.001 vs. vector. International Journal of Endocrinology miR-30d-5p/BECN1 axis in protecting podocytes from HGinduced injury is related to the promotion of podocyte autophagy.
Te role of XIST in DN is various. Wang reported that XIST facilitates the development of DN by afecting the biological behaviors of human mesangial cells and regulating infammation [20]. Yang et al. pointed out that XIST facilitates renal interstitial fbrosis in DN by upregulating the expressions of fbrosis-associated markers in HK-2 cells exposed to high glucose [21]. Meanwhile, Long et al. found that XISTexpression is downregulated in HG-induced podocytes, and XIST overexpression promotes viability while inhibiting the apoptosis of podocytes [13]. Consistent with those of Long et al., our results uncovered that XIST overexpression has a protective efect on podocytes induced by HG. However, this result seems to be contrary to the conclusion reported by Wang [20] and Yang et al. [21] that XIST promotes the development of DN. Tis contradictory phenomenon may be due to the diferent cells studied or the diferent functions of XIST as lncRNA [22].
Te relationship between XIST and autophagy has been reported in a variety of cells, such as hepatic stellate cells [23] and ovarian cancer cells [24]. Overexpression of XIST increases the level of LC3II/LC3I yet decreases the level of p62 to induce autophagy in nucleus pulposus cells [25]. XIST knockdown weakens the proliferation and autophagy in retinoblastoma cells [26]. In this study, we also found that XISToverexpression increased the LC3II/LC3I and Beclin1 levels but decreased p62 protein level to activate the autophagy of HG-induced podocytes. In addition, the targeting relationship between XIST and miR-30d-5p has been verifed in rat Schwann cells [27], and it has also been proved that XIST overexpression can reduce diabetic peripheral neuropathy by repressing miR-30d-5p expression to induce autophagy [27]. In addition, the inhibitory efect of miR-30d-5p on autophagy has been demonstrated in renal cell carcinoma and hypoxic-ischemic rats [12,28]. Analogous to previous studies, we found that upregulation of miR-30d-5p reversed the regulation of XIST overexpression on HG-induced podocyte autophagy, indicating that the protective efect of XIST on podocyte injury was achieved by targeting miR-30d-5p.
Classical autophagy is divided into fve stages, including the initiation process, phagocyte nucleation, phagocyte expansion, autophagosome-lysosome fusion, and lysosomal substrate degradation [29]. Beclin1 is involved in the formation of autophagic precursors, and LC3-I can bind to P62 after being cleaved into LC3-II, mediating the aggregation of ubiquitinated proteins, followed by P62 degradation with the ubiquitinated proteins [29]. Terefore, increasing the protein expression of Beclin 1 is benefcial to the promotion of autophagy. In this study, we found that BECN1 was the target gene of miR-30d-5p. Fengyan Zhao et al. found that miR-30d-5p antagonist could upregulate the Beclin1 level, promote autophagy, and then restore the neurological function of hypoxic-ischemic rats [28]. A study on DN showed that HG-induced downregulation of Beclin-1 and LC3 II/I as well as upregulation of P62 in MPC5 [30]. In our research, the same phenomenon was observed, but the efect of HG was nullifed by XIST overexpression and potentiated by miR-30d-5p upregulation, indicating that XIST boosted the expression of BECN1 by inhibiting miR-30d-5p, thus promoting autophagy. Podocyte apoptosis is one of the main causes of the decrease in the number of podocytes, and there is a crosstalk between autophagy and apoptosis of podocytes [31]. It is reported that autophagy can protect the podocytes from apoptosis [32]. Silencing SPAG5-AS1 promotes autophagy yet inhibits the apoptosis of podocytes via SPAG5/AKT/mTOR pathway to alleviate podocyte injury [33]. Jin et al. revealed that exosomes derived from adipose-derived stem cells alleviate diabetic nephropathy by promoting autophagy fux and attenuating apoptosis in podocytes [34]. Liu et al. proved that XIST targets miR-30d-5p to facilitate autophagy and thus inhibit the apoptosis of Schwann cells [27]. In our study, XIST overexpression impeded podocyte apoptosis, while miR-30d-5p reversed the efect of XIST overexpression, suggesting that XIST promoted the autophagy of podocytes by downregulating miR-30d-5p to hamper the podocyte apoptosis.
HG can induce podocyte EMT in the process of DN [35]. In this process, the great loss of E-cadherin (the landmark molecule of epithelial cells), together with abundant expression levels of vimentin and α-SMA (the landmark molecules of interstitial cells), can be observed in podocytes, which leads to the increased fusion of podocytes and the decreased adhesion to the glomerular basement membrane, fnally resulting in decreased podocytes [36]. However, EMT is a reversible process, so DN can be alleviated by inhibiting the EMT of podocytes [37]. In addition, Shi et al. found that the EMT of podocytes exposed to HG can be reduced by restoring autophagy activity [38]. Consistent with previous research results, we discovered that XIST promoted the autophagy of podocytes by modulating the miR-30d-5p/ BECN-1 axis, and then inhibited EMT of podocytes.
In summary, our data indicate that XIST promotes the autophagy of podocytes by regulating the miR-30d-5p/ BECN1 axis, thereby protecting podocytes from HGinduced injury. Tis study provides a new target for the treatment of DN.

Data Availability
Te analyzed data sets generated during the study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.