Long non-coding RNA CASC7 Contributes to the Progression of Sepsis-Induced Liver Injury by Targeting miR-217/TLR4 Axis

Background: Sepsis is a system inammation disease that can lead to liver injury. Long non-coding RNAs as crucial regulators participate in the regulation of sepsis-induced liver injury. However, the role of lncRNA CASC7 (CASC7) in the modulation of sepsis-induced liver injury remains elusive. Here, we aimed to explore the effect of CASC7 on the sepsis-induced liver injury. Methods: The sepsis mouse model was established in BALB/c mice by the treatment of lipopolysaccharide (LPS). The effect of CASC7 on sepsis-induced liver injury was analyzed by Hematoxylin and Eosin (HE) staining, ELISA assays, TUNEL detection kit, CCK ‐ 8 assays, and Annexin V-FITC Apoptosis Detection Kit in vivo or in vitro. The mechanism investigation was performed using RNA pull-down, luciferase reporter gene assays, qPCR assays, and Western blot analysis. Results: The expression of CASC7 was elevated in a time-dependent manner in the liver tissues of the sepsis mice and LPS-treated LO2 cells. The depletion of CASC7 decreased the LPS treatment-induced liver injury in the sepsis mice. The treatment of LPS enhanced the apoptosis in the sepsis mice, while the depletion of CASC7 blocked this enhancement in the system. The CASC7 knockdown inhibited the LPS-enhanced expression of TNF-α and IL-1β in the mice. CASC7 served as a sponge for the miR-217 in the liver cells. CASC7 promoted the progression of sepsis-induced liver injury by sponging miR-217. MiR-217 attenuated sepsis-induced liver injury by targeting TLR4. Conclusions: Thus, we conclude that CASC7 contributes to the progression of sepsis-induced liver injury by targeting miR-217/TLR4 axis.

cardiovascular diseases, and liver disease [10,11]. The aberrant regulation of lncRNAs have been observed in sepsis, and these lncRNAs may be linked with the development of sepsis-induced organ damage. It has been reported that LncRNA NEAT1 increases the in ammation response in sepsis-caused liver injury by the Let-7a/TLR4 signaling [12]. LncRNA MALAT1 modulates sepsis-produced cardiac dysfunction and in ammation by interacting with miR-125b and p38/MAPK/NF-κB [13]. LncRNA H19 serves as a competitive endogenous RNA of Aquaporin 1 to mediate the expression of miR-874in LPSinduced sepsis [14]. LncRNA HOTAIR elevates TNF-α production by the activation of the NF-κB pathway in the cardiomyocytes from the LPS-produced sepsis mouse model [15]. LncRNA CASC7 serves as a wellstudied lncRNA in multiple disease models, such as cancer and spinal cord ischemia-reperfusion injury [16,17]. However, the role of lncRNA CASC7 in the modulation of the progression of sepsis-induced liver injury remains unclear.
MicroRNAs (miRNAs) are identi ed as short non-coding RNAs with a length of approximately [20][21][22][23][24][25] nucleotides, which exert signi cant impacts on numerous biological processes [18]. MiRNAs are able to control gene expression in the post-transcriptional levels by pairing with target mRNAs at the 3′ untranslated region (3′ UTR) [19]. A substantial number of investigations have revealed that miRNAs are involved in the progression of sepsis-induced liver injury. For example, it has been reported that miR-155 aggravates the liver injury by regulating mitochondrial dysfunction and oxidative stress-mediated ER stress through targeting Nrf-2 [20]. Increased serum levels of miR-122 serve as an independent biomarker of liver injury in in ammatory disorders [21]. Meanwhile, the role of miR-217 in the in ammation-related damage has been reported [22]. Moreover, Toll-like receptor 4 (TLR4), as an essential regulator in multiple physiological and pathological processes, including liver disease, has been identi ed to participate in the regulation of sepsis-related liver injury [23]. In addition, miR-217 is able to target TLR4 to modulate podocyte apoptosis [24]. However, the correlation of lncRNA CASC7 with miR-217 and TLR4 in the modulation of sepsis-induced liver injury is still elusive.
Lipopolysaccharide (LPS) serves as the endotoxin that is issued by bacterial membranes and interacts with receptors on the surface of endothelial cells, whereby working as a noxious factor that produces acute in ammation [25]. LPS has been identi ed to cause sepsis by modulating the oxidative stress or in ammatory factors, and the growth of endothelial cells [26]. In this study, we aimed to explore the function of lncRNA CASC7 in the development of sepsis-induced liver injury. The sepsis mouse model was established in BALB/c mice by the treatment of lipopolysaccharide (LPS). The effect of CASC7 and the underlying mechanism on the sepsis-induced liver injury were investigated in the sepsis mouse model and LPS-treated liver cells. We identi ed that CASC7 contributed to the progression of sepsis-induced liver injury by targeting miR-217/TLR4 axis.

Sepsis mouse model
The sepsis mouse model was established in BALB/c mice by the treatment of lipopolysaccharide (LPS, sigma, USA). The BALB/c mice (male, four-week-old) were purchased from the Academy of Military Medical Sciences (Beijing, China). Brie y, the BALB/c mice (male, four-week-old) (n=5) were saved in a humidity-and temperature-regulated place under the 12-hours usual dark/light circle with water and food. The mice were intraperitoneally injected with LPS (20 mg/kg) or the equal volume of saline. The mice were injected with the lentivirus carrying the CASC7 shRNA or corresponding control through tail vein. The lentivirus carrying the CASC7 shRNA corresponding control were obtained (GenePharma, China). The mice were euthanatized by cervical dislocation (CD) and harvest liver tissue and plasma samples for further analysis. The liver injury was analyzed by the Hematoxylin and Eosin (HE) staining in the mice. The levels of TNF-α and IL-1β were measured by the ELISA assays in the mice. Animal care and method procedure were authorized by the Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences Animal Ethics Committee.

Cell culture and treatment
The human liver LO2 cells were purchased in American Type Tissue Culture Collection. The cells were cultured in the medium of DMEM (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 0.1 mg/mL streptomycin (Solarbio, China) and 100 units/mL penicillin (Solarbio, China) at a condition of 37°C with 5% CO 2 . The lentivirus carrying the CASC7 shRNA, miR-217 mimic, CASC7 overexpression vector, TLR4 overexpression vector and corresponding control were obtained (GenePharma, China). The transfection in the cells was performed by Liposome 3000 (Invitrogen, USA) according to the manufacturer's instructions. The levels of TNF-α and IL-1β were measured by the ELISA assays in the cells.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) The apoptosis was analyzed by using the TUNEL detection kit (Roche, Germany) in the liver tissues of the mice according to the product's guidance. After the staining of TUNEL, the ventricular samples were dyed by DAPI (Sigma, USA) to stain nuclear. Fluorescence was observed using a confocal microscope (Olympus Fluoview1000, Tokyo, Japan).

CCK-8 assays
The cell viability was analyzed by the CCK-8 assays. About 5×10 3 cells were put into 96 wells and cultured for 12 hours. Then the cells were used for the transfection or treatment. After 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours, the cells were added with a CCK-8 solution (KeyGEN Biotech, China) and culture for another 2 hours at 37°C. The ELISA browser was applied to analyze the absorbance at 450nm (Bio-Tek EL 800, USA).

Analysis of cell apoptosis
Approximately 2×10 5 cells were plated on 6-well dishes. Cell apoptosis was analyzed by using the Annexin V-FITC Apoptosis Detection Kit (CST, USA) according to the manufacture's instruction. Brie y, the cells were collected and washed by binding buffer (BD Biosciences, USA) and were dyed at 25 ℃, followed by the ow cytometry analysis.

Quantitative reverse transcription-PCR (qRT-PCR)
The total RNAs were extracted by TRIZOL (Invitrogen, USA) from the tissues of the mice and cells. The rst-strand cDNA was synthesized using Stand cDNA Synthesis Kit (Thermo, USA) as the manufacturer's instruction. The qRT-PCR was carried out by applying SYBR Real-time PCR I kit (Takara, Japan). The standard control for mRNA/lncRNA and miRNA was GAPDH and U6, respectively. Quantitative determination of the RNA levels was conducted by SYBR GreenPremix Ex TaqTM II Kit (TaKaRa, Japan). The primer sequences are as follows: Same concentration of protein was divided by SDS-PAGE (12% polyacrylamide gels), transferred to PVDF membranes (Millipore, USA) in the subsequent step. The membranes were hindered with 5% milk and hatched overnight at 4°C with the primary antibodies for PARP TLR4 (1:1000) (Abcam, USA), cleaved PARP (1:1000) (Abcam, USA), caspase3 (1:1000) (Abcam, USA), cleaved caspase3 (1:1000) (Abcam, USA) and GAPDH (1:1000) (Abcam, USA), in which GAPDH served as the control. Then, the corresponding second antibodies (1:1000) (Abcam, USA) were used for hatching the membranes 1 hour at room temperature, followed by the visualization by using an Odyssey CLx Infrared Imaging System. The results of Western blot analysis were quanti ed by ImageJ software.
Luciferase reporter gene assay The luciferase reporter gene assays were conducted by applying the Dual-luciferase Reporter Assay System (Promega, USA). Brie y, the cells were treated with the miR-21 mimic or control mimic, the vector containing CASC7, CASC7 mutant, TLR4, and TLR4 mutant fragment were transfected in the cells by using Lipofectamine 3000 (Invitrogen, USA), followed by the analysis of luciferase activities, in which Renilla was applied as a normalized control.

RNA pull-down
Biotin-marked RNAs were transcribed by using biotin-UTP of MEGAscript T7 Kit (Thermo, USA) in vitro and puri ed by MEGAclear Kit (Thermo, USA) according to manufacturer's guidance, and then incubated with entire cell lysates. Biotin-labeled transcripts and interacted RNAs were isolated with streptavidin beads and then subjected to qPCR analysis Statistical analysis Data were presented as mean ± SD, and the statistical analysis was performed by GraphPad Prism 7 software. The unpaired Student's t-test was applied for comparing two groups, and the one-way ANOVA was applied for comparing among multiple groups. P < 0.05 were considered as statistically signi cant.

Results
The expression of lncRNA CASC7 is positively correlated with the sepsis-induced liver injury To evaluate the correlation of lncRNA CASC7 with the sepsis-induced liver injury, the sepsis mouse model was established in BALB/c mice by the treatment of lipopolysaccharide (LPS). Hematoxylin and Eosin (HE) staining revealed the symptoms of liver injury in a time-dependent manner, such as disorder of liver structure, in ltration of neutrophils into the portal area and hepatic sinusoid, cytoplasm rarefaction, nodular necrosis, and karyopyknosis (Fig. 1A). Meanwhile, the levels of TNF-α and IL-1β were increased in a time-dependent manner in the in ltration of in ammatory cells (P < 0.01) (Fig. 1B). Moreover, the expression of lncRNA CASC7 was elevated by the treatment of LPS in a time-dependent manner in the liver tissues of the mice (P < 0.01) (Fig. 1C). Similarly, the treatment of LPS time-dependently up-regulated the expression of CASC7 in the LO2 cells (P < 0.01) (Fig. 1C). Together these data suggest that the expression of lncRNA CASC7 was positively correlated with the sepsis-induced liver injury.
The depletion of lncRNA CASC7 relieves sepsis-induced liver injury in vivo Then, we investigated the effect of lncRNA CASC7 on the progression of sepsis-induced liver injury in vivo. To this end, the sepsis mice were injected with the lentivirus carrying the CASC7 shRNA or corresponding control shRNA. The e ciency of CASC7 shRNA was validated in the liver tissues of the mice (P < 0.05) ( Fig. 2A). The depletion of CASC7 signi cantly decreased the LPS treatment-induced liver injury in the sepsis mice (Fig. 2B). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis showed that the treatment of LPS enhanced the TUNEL-positive cells while the depletion of CASC7 could block this enhancement in the system, indicating that the CASC7 depletion attenuated LPS-induced apoptosis (P < 0.05) (Fig. 2C). In addition, the CASC7 knockdown inhibited the LPS-enhanced expression of TNF-α and IL-1β in the mice (P < 0.05) (Fig. 2D). Together these suggest that the depletion of lncRNA CASC7 relieves sepsis-induced liver injury in vivo.
LncRNA CASC7 serves as a sponge for the miR-217 in the liver cells Next, we tried to explore the mechanism of lncRNA CASC7 in LPS-induced sepsis liver injury. The expression of miR-217 was reduced by the treatment of LPS in a time-dependent manner in the liver tissues of the mice (P < 0.05) (Fig. 3A). Similarly, the treatment of LPS time-dependently down-regulated the expression of miR-217 in the LO2 cells (P < 0.05) (Fig. 3B). Meanwhile, the depletion of lncRNA CASC7 signi cantly enhanced the expression of miR-217 in the LO2 cells (P < 0.05) (Fig. 3C). The bioinformatic analysis identi ed the potential interaction between lncRNA CASC7 and miR-217 by using Starbase 3.0v software (Fig. 3D). The e ciency of miR-217 mimic was validated in the LO2 cells (P < 0.05) (Fig. 3E).
The miR-217 mimic attenuated the luciferase activities of CASC7 but failed to affect the CASC7 with the miR-217-binding site mutant in the LO2 cells (P < 0.05) (Fig. 3F). RNA pull-down assays showed that wildtype Bio-miR-217, but not mutant Bio-miR-217, could interact with CASC7 (P < 0.01) (Fig. 3G). Together these results suggest that lncRNA CASC7 serves as a sponge for the miR-217 in the liver cells.
LncRNA CASC7 promotes the progression of sepsis-induced liver injury by targeting miR-217 We then evaluated whether CASC7 promoted the progression of sepsis-induced liver injury by targeting miR-217 in the liver cells. The overexpression of CASC7 inhibited the cell viability, while miR-217 mimic could rescue the cell viability in the LPS-treated LO2 cells (P < 0.05) (Fig. 4A). The treatment of miR-217 mimic was able to reverse the CASC7 overexpression-enhanced levels of TNF-α and IL-1β in the LPStreated LO2 cells (P < 0.05) (Fig. 4B). The CASC7 overexpression-induced apoptosis was attenuated by the miR-217 mimic in the LPS-treated LO2 cells (P < 0.05) (Fig. 4C). The expression of cleaved PARP (c-PARP) and cleaved caspase3 (c-caspase3) elevated by CASC7 overexpression was reduced by the treatment of miR-217 mimic in the LPS-treated LO2 cells (P < 0.05) (Fig. 4D). Together these data indicate that lncRNA CASC7 promotes the progression of sepsis-induced liver injury by sponging miR-217 in vitro.

Mir-217 Attenuates Sepsis-induced Liver Injury By Targeting Tlr4
Next, we explored the downstream target of miR-217 in the liver cells. The bioinformatic analysis identi ed the miR-217-targeted site in TLR4 3' UTR by using miRDB and miRmap software (Fig. 5A). The miR-217 mimic attenuated the luciferase activities of TLR4 but failed to affect the TLR4 with the miR-217-binding site mutant in the LO2 cells (P < 0.01) (Fig. 5B). The protein expression of TLR4 was reduced by the treatment of miR-217 mimic in the cells (P < 0.01) (Fig. 5C). The depletion of CASC7 downregulated while the overexpression of CASC7 up-regulated the protein levels of TLR4 in the cells (P < 0.01) (Fig. 5D). The depletion of CASC7 reduced the LPS-elevated expression of TLR4 in the cells (P < 0.01) (Fig. 5E). The e ciency of TLR4 overexpression was validated in the cells (P < 0.01) (Fig. 5F). The overexpression of TLR4 was able to block the miR-217 mimic-increased cell viability of the LO2 cells (P < 0.05) (Fig. 5G). The miR-217 mimic-attenuated cell apoptosis was enhanced by the TLR4 overexpression in the cells (P < 0.05) (Fig. 5H). Together these data indicate that miR-217 attenuates sepsis-induced liver injury by targeting TLR4 in vitro.

Discussion
Sepsis is a systemic in ammatory disease caused by severe trauma, burns and postoperative infections, leading to multiple organ failure, including liver injury [27]. The incidence of occurrence and death of sepsis-induced liver injury is high [4]. Nevertheless, the mechanism of sepsis-induced liver injury is still elusive. In the present study, we identi ed that lncRNA CASC7 contributed to the progression of sepsisinduced liver injury by modulating miR-217/TLR4 axis.
The pathogenesis of sepsis is complex, and lncRNAs have been well-recognized to participate in the modulation of sepsis development. It has been well-identi ed that multiple lncRNAs are involved in the development of sepsis-induced liver injury. For example, lncRNA colorectal neoplasia, differentially displayed, relieves sepsis-caused liver damage by targeting miR-126-5p [28]. Circulating lncRNA NEAT1 is correlated with an unfavorable prognosis, high severity, and increased risk in sepsis patients [29]. LncRNA SNHG16 changes the consequences of miR-15a/16 on the LPS-produced in ammation pathway [30]. LPS increases sepsis development by stimulating the lncRNA HULC/miR-204-5p/TRPM7 axis in the HUVECs [31]. LncRNA TapSAKI increases in ammatory injury and urine-derived sepsis injury [32]. The impact of lncRNA HOTAIR on serious organ injury in the sepsis rat model by mediating miR-34a/Bcl-2 axis has been reported [33]. LncRNA NEAT1 plays an essential role in the sepsis-produced severe injury by mediating miR-204 and changing the NF-κB signaling [34]. Enhanced expression of lncRNA HULC and UCA1 is needed for the pro-in ammatory response of the LPS-induced sepsis in the endothelial cells [35]. In the present study, we identi ed that the expression of lncRNA CASC7 was elevated in the sepsis mice. It presents a novel function of lncRNA CASC7 in the modulation of the progression of sepsis-induced liver injury, providing valuable evidence for the fundamental role of lncRNAs in the development of sepsisinduced liver injury.
As a primary component of non-coding RNA and the signi cant interplay factors with lncRNAs in the physiological and pathological processes, miRNAs are also involved in the modulation of sepsis-induced liver injury. It has been reported that the repression of miRNA 155 inhibits sepsis-caused liver damage by the inactivation of the JAK/STAT signaling [36]. Paclitaxel alleviates liver damage of the septic mouse by relieving in ammation response through microRNA-27a/TAB3/NF-κB axis [37]. MCPIP1 alleviates lipopolysaccharide-produced liver injury by controlling the expression of SIRT1 by modulation of miR-9 [38]. MiR-103a-3p is able to inhibit sepsis-produced liver injury by mediating HMGB1 [39]. MiR-21 is needed to remote and local ischemic preconditioning in the protection of various organs against sepsis [40]. The restraint of miRNA 195 inhibits multiple organ injury and apoptosis in the sepsis mouse models [41]. MicroRNA-30e represses apoptosis and increases hepatocyte proliferation in puncture and cecal ligation-induced sepsis by the modulation of JAK/STAT signaling through interacting with FOSL2 [42]. Puncture and cecal ligation-induced sepsis is correlated with the inhibited expression of adenylyl cyclase nine and enhanced expression of miR142-3p [43]. Our data demonstrated that lncRNA CASC7 served as a sponge for miR-217 in the liver cells and lncRNA CASC7 promoted the progression of sepsis-induced liver injury by sponging miR-217. It presents the valuable information that miR-217 is involved in the CASC7-mediated sepsis-induced liver injury progression, providing another evidence that miR-217 participate in the modulation of sepsis-induced liver injury development.
TLR4 has been involved in the development of sepsis-induced liver injury. TLR4 antagonist eritoran tetrasodium inhibits liver ischemia and reperfusion injury by the repression of high-mobility group box protein B1 (HMGB1) signaling [44]. Dexmedetomidine-regulated the attenuation against sepsis liver damage rely on the downregulation of TLR4 and MyD88/NF-κB signaling, partly through anti-in ammatory cholinergic mechanisms [45]. Trichostatin preserves the liver against sepsis damage by the inhibition of the TLR4 signaling [46]. The treatment of Green Tea extract in the obese mice with nonalcoholic steatohepatitis reclaims the liver metabolome in the correlation with inhibiting endotoxemia/TLR4/NFκBregulated in ammation [47]. Experimental sepsis-produced mitochondria biogenesis is reliant on TLR9, TLR4, and signaling in the liver [48]. The inhibition of TLR4 reduces bacterial clearance in murine abdominal sepsis and in a corrective setting [49]. Leukadherin-1-regulated stimulation of CD11b represses LPS-produced pro-in ammation response in the macrophages and preserves mice upon endotoxic shock by pressing the interaction of LPS-TLR4 [50]. Extracellular histones serve as the mediators of destruction by TLR4 and TLR2 in the fatal liver injury mouse model [51]. The protecting impacts of apilarnil against lipopolysaccharide related liver damage in rats by TLR4/ HMGB-1/NF-κB signaling have been reported [52]. TLR4 controls platelet capacity and leads to organ injury and coagulation abnormality in hemorrhagic resuscitation and shock [53]. In the present study, we revealed that miR-217 attenuated sepsis-induced liver injury by targeting TLR4. It indicates that TLR4 plays a crucial role in the modulation of sepsis-induced liver injury.

Conclusion
In conclusion, we discovered that lncRNA CASC7 contributed to the progression of sepsis-induced liver injury by targeting miR-217/TLR4 axis. Our nding provides new insights into the mechanism by which CASC7 modulates sepsis-induced liver injury development. LncRNA CASC7, miR-217, and TLR4 may serve as potential targets for the treatment of sepsis-induced liver injury.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no con ict of interest.

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

Authors' contributions
XQZ were dedicated to the study concepts, study design, de nition of intellectual content, and statistical analysis; TZ carried out the literature research; YHT, WYJ, and WWY were involved in the experimental studies and data acquisition; HW carried out the data analysis; XQZ and GL were dedicated to the manuscript preparation, manuscript editing, and manuscript review. All authors have read and approved this article.  presented as mean ± SD. Statistic signi cant differences were indicated: * P < 0.05, ** P < 0.01. Figure 5 MiR-217 attenuates sepsis-induced liver injury by targeting TLR4. (A) The interaction of miR-217 and TLR4 3' UTR was identi ed by bioinformatic analysis using miRDB and miRmap software. (B and C) The

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