Knockdown of lncRNA MALAT1 ameliorates acute kidney injury by mediating the miR‐204/APOL1 pathway

Abstract Background Acute kidney injury (AKI) was characterized by loss of renal function, associated with chronic kidney disease, end‐stage renal disease, and length of hospital stay. Long non‐coding RNAs (lncRNAs) participated in AKI development and progression. Here, we aimed to investigate the roles and mechanisms of lncRNA MALAT1 in AKI. Methods AKI serum samples were obtained from 129 AKI patients. ROC analysis was conducted to confirm the diagnostic value of MALAT1 in differentiating AKI from healthy volunteers. After hypoxic treatment on HK‐2 cells, the expressions of inflammatory cytokines, MALAT1, miR‐204, APOL1, p65, and p‐p65, were measured by RT‐qPCR and Western blot assays. The targeted relationship between miR‐204 and MALAT1 or miR‐204 and APOL1 was determined by luciferase reporter assay and RNA pull‐down analysis. After transfection, CCK‐8, flow cytometry, and TUNEL staining assays were performed to evaluate the effects of MALAT1 and miR‐204 on AKI progression. Results From the results, lncRNA MALAT1 was strongly elevated in serum samples from AKI patients, with the high sensitivity and specificity concerning differentiating AKI patients from healthy controls. In vitro, we established the AKI cell model after hypoxic treatment. After experiencing hypoxia, we found significantly increased MALAT1, IL‐1β, IL‐6, and TNF‐α expressions along with decreased miR‐204 level. Moreover, the targeted relationship between MALAT1 and miR‐204 was confirmed. Silencing of MALAT1 could reverse hypoxia‐triggered promotion of HK‐2 cell apoptosis. Meanwhile, the increase of IL‐1β, IL‐6, and TNF‐α after hypoxia treatment could be repressed by MALAT1 knockdown as well. After co‐transfection with MALAT1 silencing and miR‐204 inhibition, we found that miR‐204 could counteract the effects of MALAT1 on HK‐2 cell progression and inflammation after under hypoxic conditions. Finally, NF‐κB signaling was inactivated while APOL1 expression was increased in HK‐2 cells after hypoxia treatment, and lncRNA MALAT1 inhibition reactivated NF‐κB signaling while suppressed APOL1 expression by sponging miR‐204. Conclusions Collectively, these results illustrated that knockdown of lncRNA MALAT1 could ameliorate AKI progression and inflammation by targeting miR‐204 through APOL1/NF‐κB signaling.


| INTRODUC TI ON
Sepsis was caused by a fatal systemic inflammatory syndrome that can lead to shock, multiple organ dysfunction syndromes, and even death. 1 Globally, the incidence of sepsis increased dramatically, with more than 30 million patients and nearly 6 million deaths each year. 2 Sepsis may be caused by a variety of infections and other noninfectious diseases, such as ischemia, trauma, drug reactions, or even cancer, but the underlying pathogenetic mechanisms remained unclear. 3 Acute kidney injury (AKI), a severe syndrome associated with renal insufficiency, was one of the most serious complications of sepsis. 3 It can trigger the development of organ dysfunction, which led to high morbidity and mortality in most patients with sepsis. 4 Studies found that severe sepsis can result in approximately 50% of AKI cases. 5 Therefore, exploring the mechanisms of pathophysiological alterations of sepsis-induced AKI patients was a challenging and necessary task.
There was strong evidence that renal tubular epithelial cell injury, inflammation, vascular dysfunction, and fibrosis were involved in the pathology and physiology of AKI. 6 Sepsis promoted the release of inflammatory factors from renal tissues, resulting in apoptosis of renal cells, which led to AKI. 7 Therefore, improving the level of inflammation and apoptosis in patients with a renal injury would be of great benefit to patients with AKI. Despite advances in therapeutic approaches, the mortality rate of sepsis-induced AKI remained high. Therefore, it is necessary and urgent to explore new therapeutic targets to improve the survival of patients with septic AKI.
Long non-coding RNAs (lncRNAs), located in the nucleus or cytoplasm, were a class of ncRNAs with more than 200 nucleotides. 8 LncRNAs have been verified to exert significant roles in biological processes such as cell differentiation, cell proliferation, apoptosis, and tumorigenesis. 9,10 Recent evidence suggested that lncRNAs may also be involved in the pathophysiology of AKI. For instance, downregulation of LncRNA MEG3 may protect against AKI induced by hypoxia/reoxygenation in HK-2 cells by regulating the miR-129-5p/ MHGB1 axis. 11 LINC00520 may promote the development of AKI by regulating miR-27b-3p/OSMR/PI3K/AKT signaling pathway. 12 Silencing of lncRNA NEAT1 can target miR-125-5p and regulate TRAF6/TAK1 signaling to protect against sepsis-induced AKI. 13 LncRNA MALAT1 exerted either oncogenetic or tumor-suppressive roles in various cancers, including lung, breast, gastric, gallbladder, and colorectal cancers. [14][15][16][17][18] In AKI, lncRNA MALAT1 was proved to be upregulated in AKI patients. 19 However, the role of lncRNA MALAT1 in the pathology and physiology of sepsis still needed to be further explored.
In this study, we investigated the expression of lncRNA MALAT1 in the serums of AKI patients and its clinical diagnostic value.

| RT-qPCR analysis
Total RNAs were isolated from serum samples and cells using TRIzol
Relative luciferase activity was normalized to Renilla activity on a dual-luciferase reporter assay system (Promega) according to the manufacturer's protocol.

| CCK-8 assay
After 24 h of transfection, treated cells were inoculated in a 6-well plate at the density of 1×10 5 cells/well, cultivated in a humidified incubator with 5% CO 2 at room temperature. At 0, 24, and 48 h, 10 μl CCK-8 reagent (Dojindo, Japan) was added to each well and cultured for another 2 h. Finally, a microplate reader (Dynatech, VA) was used to detect the absorbance value at 450 nm.

| Colony formation analysis
Transfected cells of logarithmic growth phase were taken, digested with 0.25% trypsin and blown into individual cells, and the cells were suspended in RPMI-1640 medium with 10% FBS. The cell suspension was diluted in a gradient multiple, and each group of cells was inoculated in a culture dish containing 10 ml of 37°C pre-warmed culture medium. The cells were incubated for 2 weeks at 37°C with 5% CO 2 and saturated humidity in a cell incubator. 5 ml of 4% paraformaldehyde was added to fix cells for 15 min. Finally, GIMSA staining solution was added for 30 min.

| Western blot assay
Total proteins were extracted using RIPA lysis buffer (Sigma, USA) and quantified with a BCA detecting kit (Abcam, Shanghai, China) following the manufacturers' instructions. Then, proteins were separated by 10% SDS-PAGE and transferred on a PVDF membrane

| Statistical analysis
All the data in our study were presented as mean ± standard deviation (SD). Student's t test and ANOVA tests were used to distinguish differences between groups. ROC analysis was utilized to assess the diagnostic value. SPSS 22.0 (SPSS Inc, Chicago, IL, USA) and Prism 6.0 (GraphPad Software Inc.) were applied to analyze the data. A pvalue < 0.05 was considered statistically significant. All experiments were conducted at least in triplicate.

| Clinicopathological information of AKI patients
As shown in Table 1, we found that there were no significant differ-  sure to hypoxia, we found that IL-1β, IL-6, and TNFα levels were increased in a time-dependent manner ( Figure 1D-F), so as the apoptotic rate in HK-2 cells ( Figure 1G). We then uncovered that MALAT1 expression was upregulated while miR-204 was diminished after hypoxia treatment ( Figures 1H and 2C).

| miR-204 targeted MALAT1
The interaction between MALAT1 and miR-204 was displayed in Figure 3A. The luciferase reporter assay disclosed that luciferase activity in HK-2 cells after co-transfection with miR-204 mimic and  Figure 3C showed a prominently higher MALAT1 while using miR-204-bio probes than the NC-bio or miR-204 probes.

| The effects of MALAT1 on HK-2 cell progression and inflammation under hypoxic conditions were partially reversed by co-transfection with miR-204 inhibitor
To illustrate the effects of MALAT1 on the hypoxia-triggered AKI cell model, HK-2 cells were infected with LV-MALAT1 or LV-NC. As shown in Figure 4A, MALAT1 expression was found to be markedly decreased in LV-MALAT1, suggesting the transfection was successful. In addition, as evidenced by the CCK-8 and colony formation assays, LV-MALAT1 could promote HK-2 cell proliferation after hypoxia treatment ( Figure 4B,C). Meanwhile, the inflammation in HK-2 cells induced by hypoxia could be partially ameliorated after knocking down MALAT1 expression ( Figure 5). As depicted in Figure 4D,E, flow cytometry and TUNEL staining assays elucidated that hypoxiaengendered apoptosis in HK-2 cells could be rescued by transfection with LV-MALAT1 as well. Next, the loop relationship between MALAT1, miR-204, and APOL1 was confirmed. As evidenced in Figure 8A-C, we verified the targeted relationship between miR-204 and APOL1 by luciferase reporter assay and RNA pull-down analysis. Furthermore, Western blot results in Figure 8D unveiled that APOL1 expression was increased while p-p65 was decreased after hypoxia treatment in HK-2 cells. Meanwhile, under hypoxic conditions, APOL1 was reduced while p-p65 was elevated after transfection with LV-MALAT1; however, the variance induced by LV-MALAT1 could be partially rescued by a miR-204 inhibitor ( Figure 8E).

| miR-204 targeted APOL1 to abolish the suppressive effects of MALAT1 silencing on HK-2 cells experiencing hypoxia by activation NF-κB
Furthermore, as depicted in Figure 9A, after hypoxia conditions, the expression of APOL1 was gradually increased with the augment of treatment time of hypoxia. Then, the RT-qPCR result in Figure 9B demonstrated the transfection efficiency of APOL1 was successful.
Then, cell proliferation and apoptosis results in Figure 9C

| DISCUSS ION
In the present study, we investigated the biological roles of lncRNA MALAT1 and miR-204 in AKI. In AKI patient serum and hypoxic HE-2 cell models, MALAT1 expression was elevated, whereas miR-204 expression was downregulated. MALAT1 knockdown reversed the effects of hypoxic treatment on HK-2 cell proliferation, apoptosis, and inflammation through sponge-mediated miR-204.
Furthermore, we observed that the NF-κB pathway was inactivated in hypoxic HK-2 cells, and miR-204 inhibition abrogated the effect of MALAT1 on HK-2 cells after hypoxic treatment by suppressing NF-κB signaling.
Sepsis was a syndrome of the systemic inflammatory response caused by microbial infection. 20 AKI, a common complication of sepsis, caused much attention due to its high mortality rate of 75% and multiple complications such as chronic kidney diseases. 21 Some studies have reported that AKI was induced in more than 50% of patients with sepsis, 5 leading to renal insufficiency and overproduction of inflammatory factors. It has been established that the pathogenesis of AKI involved multiple aspects, including renal inflammation, ischemia, acute hypoxia, oxidative stress, and microcirculatory disorders. 22 However, the specific mechanisms behind AKI remained unclear. In recent years, an increasing number of complex interactions between lncRNAs and miRNAs were involved in the development of heterogeneous diseases, including AKI. 23,24 For example, lncRNA NEAT1 mediated hypoxia-triggered apoptosis of renal tubular epithelial cells via sponging let-7b-5p. 25 Silencing of lncRNA XIST targeted miR-142-5p/PDCD4 axis to ameliorate AKI development. 26 In HK-2 cells, lncRNA PVT1 promotes LPS-induced septic AKI by regulating TNFα and JNK/NF-κB.
Numerous studies have shown that lncRNAs can be stably presented in the blood through membrane vesicles such as exosomes and microvesicles, thus becoming promising biomarkers for disease diagnosis and prognosis. 27,28 Long non-coding RNAs (lncRNAs) are a group of widely expressed non-coding RNA molecules more than 200 nucleotides in length that regulate the expression of functional genes and play a key role in a variety of pathogenic conditions such as cardiovascular diseases, 29 cancers, 30 and AKI. 31 Among them, ln-cRNA MALAT1 has recently been recognized as an essential regulator involved in cancer development, 32 innate immunity, 33 and viral infection. 34 For example, lncRNA MALAT1 promoted hepatocellular carcinoma development by regulating SRSF1 and activating mTOR signaling. 35 LncRNA MALAT1 could suppress IRF3-initiated antiviral innate immunity by inhibition of TDP43. 33 In addition, in renal cell carcinoma, MALAT1 can regulate the miR-203/BIRC5 axis to accelerate tumor progression. 36 Recently, it has been reported that lncRNA MALAT1 is an ideal marker for the diagnosis of sepsis. [37][38][39] In the present study, we found that MALAT1 expression was elevated can play a crucial role in the development of AKI as a biomarker for AKI therapy.
MicroRNAs (miRNAs) were a class of highly conserved noncoding RNAs that played key roles in cell differentiation, metabolism, proliferation, and apoptosis. 40,41 In recent years, an increasing number of studies have shown that miRNAs were involved in the progression of sepsis. 42,43 Mechanistically, lncRNAs can act as ceR-NAs to sponge miRNAs. 44 Previous studies have revealed a prevalent interaction between lncRNA MALAT1 and miR-204. 45 inhibitor had a pro-inflammatory effect on inflammation.
The NF-κB signaling pathway has been shown to be involved in the regulation of a variety of biological processes, including AKI. 65,66 It has been shown that activation of NF-κB reduced inflammation while mitigating sepsis-induced organ damage. 67 LncRNAs NEAT1 and PVT1 were found to affect LPS-triggered septic AKI by regulating the NF-κB pathway by Chen et al. 68 and Huang et al. 69 Therefore, we hypothesized that NF-κB activation may be involved in protecting sepsis-triggered AKI. Compared with the Chen's study, 68 our study further validated a downstream target of miR-204 and a upstream target of NF-κB pathway, APOL1, which participated in regulating hypoxia-induced HK-2 cell proliferation, apoptosis, and inflammation. In a word, in our study, we found that MALAT1 knockdown activated NF-κB activation, while miR-204 inhibition significantly counteracted this effect.
In some aspects, our experiments still have some limitations. Taken together, our study reveals that lncRNA MALAT1 can sponge miR-204 to regulate HK-2 cell proliferation, apoptosis, and inflammation after hypoxia treatment. More importantly, knockdown of lncRNA MALAT1/miR-204/APOL1/NF-κB axis may be a potential therapeutic target to contribute to the treatment of AKI.

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.