SNHG1-miR-186-5p-YY1 feedback loop alleviates hepatic ischemia/reperfusion injury

ABSTRACT

As a common cause of liver injury, hepatic ischemia/reperfusion injury (HIRI) happens in various clinical conditions including trauma, hepatectomy and liver transplantation. Since transcription factor Yin Yang 1 (YY1) was reported to be downregulated after ischemia/reperfusion (I/R) injury, we focused on YY1 to explore its function in HIRI by functional assays like Cell Counting Kit-8 (CCK-8) assays and flow cytometry assays. The RT-qPCR assay revealed that YY1 was downregulated in hepatocytes after I/R injury. The function assays disclosed that YY1 facilitated cell viability and proliferation, but hindered cell apoptosis in hepatocytes after I/R injury. Through mechanism assays including luciferase reporter assay, RIP and RNA pulldown assay, miR-186-5p was found to bind with YY1 and promote hepatocyte apoptosis by targeting YY1. Subsequently, we verified that small nucleolar RNA host gene 1 (SNHG1) could sponge miR-186-5p to upregulate YY1. Importantly, we figured out that YY1 had a positive regulation on SNHG1. Along the way, YY1 was identified as the upstream transcription factor for SNHG1. In conclusion, our study unveiled a novel competing endogenous RNA (ceRNA) pattern of SNHG1/miR-186-5p/YY1 positive feedback loop in hepatic I/R injury, which might provide new insight into prevention of HIRI during liver transplantation or hepatic surgery.

Introduction

Hepatic ischemia/reperfusion injury (HIRI) has been confirmed to affect the life of thousands of people every year [1]. Most HIRI happened after liver transplantation and hemorrhagic shock [1]. It meant a pathological phenomenon in which liver blood supply was blocked during a period of liver surgery, and blood supply was restored after a period of time, resulting in liver dysfunction and structural damage [2]. The viability of normal liver cell lines has been verified as an important marker in the response of HIRI [3,4]. The gene regulation has been identified to play a crucial role in the progression of HIRI. In this study, we aimed to verify the molecular mechanism involved in HIRI [5,6].

In recent years, non-protein coding RNAs (ncRNAs) have been carefully studied and researchers have found the important regulatory roles of ncRNAs in the progression of various human diseases, including cancers and organ dysfunction [7–9]. Long noncoding RNAs (lncRNAs) are longer than 200 nts and lack the potential of coding proteins [10,11]. lncRNAs have been reported to have significant regulatory functions in the development of many diseases. For example, lncRNA HOTAIR expression was activated in breast cancer, thus functioning as a positive regulator in the proliferation, migration and invasion of breast cancer cells [12]. lncRNA PFL enhanced the progression of cardiac fibrosis via regulating the expression of miRNA miR-let-7d [13]. Small nucleolar RNA host gene 1 (SNHG1) is a lncRNA that has been widely reported as a crucial regulator in various human cancers [14,15]. The role of SNHG1 has been revealed in osteoarthritis [16]. It has also been reported to play crucial roles in the progression of oxygen-glucose deprivation/reperfusion in brain microvascular endothelial cells [17]. However, the function of SNHG1 in HIRI is still not clear. In this study, we aimed to explore the function and mechanism of SNHG1 in HIRI.

lncRNA has been proven to function as competing endogenous RNAs (ceRNA) to sponge microRNAs (miRNAs) for modulating the expression of target messenger RNAs (mRNAs) [18]. CeRNA hypothesis reveals a new mechanism for RNA-to-RNA interactions, which plays a regulatory role in various diseases [19].

MiRNAs are small noncoding RNAs with the length of 20–24 nts, which are considered as significant participants in the regulation of mRNA expression [20,21]. Based on bioinformatics analysis, miR-186-5p had potential binding sites with SNHG1. It has been reported that upregulated miR-186-5p could improve neurological outcomes induced by spinal cord I/R injury. Additionally, miR-186-5p might restrain neuroinflammation via Wnt5a-, TLR3- or CXCL13-mediated pathways in spinal cord I/R injury [22].

Yin Yang 1 (YY1) is a transcription factor that can interact with histone acetyltransferase and deacetylase to activate or repress the transcription of genes. A recent study has revealed that YY1 is involved in the acute I/R-mediated hippocampal injury [23]. The oxidative stress-elicited YY1 could potentiate antioxidative response by enhancing NRF2-driven transcriptional activity in I/R cerebral injury [24]. Nevertheless, the function of YY1 in HIRI is still unclear.

In this study, the main purpose was to investigate the impacts of SNHG1/miR-186-5p/YY1 on HIRI, which might provide new insight into prevention of HIRI during liver transplantation or hepatic surgery.

Materials and methods

Cell culture and treatment

The AML12 mouse hepatocytes were procured from the American Type Culture Collection (ATCC) Bank (Manassas, VA) and maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) medium (Gibco, Rockville, MD) with 10% fatal bovine serum (FBS, Gibco). H₂O₂ at various concentrations was procured from Sigma-Aldrich (Miamisburg, OH) and used as guided.

Total RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR)

The total RNA was isolated from AML-12 cells using TRIzol Reagent (Invitrogen, Carlsbad CA) and then converted into cDNA using a Reverse Transcriptase Kit (Takara, Shiga, Japan). Gene expression was examined using a SYBR Green PCR Kit (Takara) and calculated by the 2^−ΔΔCt method. U6 or GAPDH served as endogenous control.

Cell transfection

Confluent AML-12 cells were collected for 48 h transfection, in the presence of Lipofectamine 3000 (Invitrogen). The shRNAs targeting YY1 or SNHG1 and negative control shRNA (sh-NC) were procured from Genechem (Shanghai, China). Similarly, pcDNA3.1 vectors used for augment of YY1 or SNHG1 and the corresponding empty vectors were synthesized and purchased from Genechem (Shanghai, China). The miR-186-5p mimics and NC mimics were designed at GenePharma (Shanghai, China).

Cell counting kit-8 (CCK-8) assay

The CCK-8 assay kit was acquired from Dojindo (Osaka, Japan) to undertake cell proliferation assay. Transfected AML-12 cells were placed in 96-well plates, and cell ability was assessed at 0, 24, 48 and 72 h according to the absorbance at 450 nm using a spectrophotometer.

Colony formation assay

The processed AML-12 cells were added to 6-well plates for 14 days, with 500 cells per well. The resulting colonies were then fixed in 4% paraformaldehyde (PFA), stained in 0.5% crystal violet solution and pictured. The number of colonies was counted manually.

5-Ethynyl-2-deoxyuridine (EdU) assay

EdU assay of transfected AML-12 cells was undertaken by the use of a BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China) with Alexa Fluor 594. Cell nucleus was treated with 4’,6-diamidino-2-phenylindole (DAPI) solution for counterstaining. All samples were observed using an inverted microscope (Olympus, Tokyo, Japan).

TdT-mediated dUTP Nick-End labeling (TUNEL) assay

The processed AML-12 cells were prepared using phosphate-buffered saline (PBS) washing buffer and fixed in 4% PFA. After being permeabilized with 0.5% Triton X-100, a TUNEL assay kit (Merck KGaA, Darmstadt, Germany) was used for treating the apoptotic cells for 1 h. After DAPI staining, samples were analyzed under an optical microscope (Olympus).

Flow cytometry

Apoptosis of processed AML-12 cells was studied with the application of a flow cytometer (BD Biosciences, Franklin Lakes, NJ) and the Annexin V/PI double staining method (Invitrogen). Cells were collected and washed in PBS before being stained in 6-well plates for 15 min in the dark. Then, cell samples were assayed using flow cytometry.

Caspase-3/Caspase-9 activity assay

The caspase-3/caspase-9 activities in processed AML-12 cells were examined by the use of Caspase-3/9 activity assay kits (Beyotime, Beijing, China), as per the instructions. Total proteins were extracted with lysis buffer. The absorbance at 405 nm was finally determined by the use of a spectrophotometer.

RNA pulldown

RNA pulldown assay was performed in the processed AML-12 cells as per the protocol of Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA). Cell lysates were mixed with biotinylated miR-186-5p probes and control probes, following addition of magnetic beads. RNAs precipitated in the complexes were purified and then analyzed via RT-qPCR.

Luciferase reporter assay

The SNHG1 or YY1 fragments covering wild-type (WT) and mutant (Mut) miR-186-5p binding sites were used for constructing SNHG1 WT/Mut and YY1-WT/Mut vectors, using the pmirGLO luciferase plasmids (Promega, Madison, WI). They were cotransfected to AML-12 cells with miR-186-5p mimics or NC mimics for 48 h. In addition, cells were cotransfected with YY1 silencing vectors and the pGL3-basic vector (Promega) with SNHG1 promoter-WT/Mut for promoter analysis. All luciferase intensities were studied using a luciferase reporter assay system (Promega).

Subcellular fractionation

Cytoplasmic and nuclear RNAs of AML-12 cells were separately isolated and purified by the use of a Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Belmont, CA), as instructed. The SNHG1 content was assayed in cell cytoplasm and cell nucleus via RT-qPCR.

RNA immunoprecipitation (RIP)

RIP assay was conducted in AML-12 cells by using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit, as requested by the provider (Millipore, Bedford, MA, USA). The human Ago2 antibody (TS-10X10ML-U, Millipore) and normal mouse IgG antibody (control; M4155, Millipore) were bound to magnetic beads for RIP assay. The duplex was then cocultivated with cells, and the enrichments of SNHG1, miR-186-5p and YY1 in the immunoprecipitation were all assayed using RT-qPCR.

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed in AML-12 cells in line with the instruction of the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore). Cells were fixed by 15 min of crosslink and sonicated to shear DNA to fragments of 500-bp. Immunoprecipitation was conducted for 6 h with YY1 antibody (ab109237, Abcam, Cambridge, MA, UK) or IgG antibody, and magnetic beads. The conjugated DNAs were subjected to RT-qPCR analysis.

Fluorescence In Situ Hybridization (FISH)

RNA FISH KIT designed for SNHG1 was bought from RiboBio (Guangzhou, China). This SNHG1 probe was hybridized with the air-dried cell samples and then treated with Hoechst staining solution. At length, samples were imaged and observed using an Olympus fluorescence microscope.

Western blot

RIPA buffer was first applied for acquisition of the protein lysates. Subsequent to extraction of total protein and measurement of the protein concentration by means of a Bradford Protein Assay Kit, the proteins were treated with 12% SDS-PAGE and transferred to the PVDF membranes. The membranes blocked by 5% skimmed milk were subsequently cocultivated with specific primary antibodies targeting YY1 and GAPDH (Ab8245, Abcam) overnight at 4°C and then subjected to cultivation with secondary antibodies. An enhanced chemiluminescence (ECL) detection system was eventually applied for quantification of the proteins.

Statistical analyses

Each experiment in our study was repeated at least three times. Data were statistically exhibited as the mean ± standard deviation (SD) of three separately conducted assays. The difference in groups was compared in the form of Student’s t-test or one-way analysis of variance (ANOVA) employing GraphPad Prism 7® (GraphPad Software, Inc., La Jolla, CA). The p-value below 0.05 was set as the threshold of significant data.

Results

YY1 could improve viability and proliferation of hepatocytes after HIRI

It has been reported that YY1 is downregulated after I/R injury [25]. Thus, we first detected the expression of YY1 in the mouse normal liver cell line (AML-12) via RT-qPCR and Western blot analysis and AML-12 cells were treated with different concentrations of H₂O₂. We found that the expression of YY1 was reduced more significantly with the increase in the H₂O₂ concentration, and when the concentration reached 200 uM, the expression of YY1 was dramatically decreased (Figure 1(a) &S1A). Next, we used a series of functional assays to verify the role of YY1 in AML-12 cells treated with 200uM H₂O₂ for six hours. First, we knocked down YY1 expression in AML-12 cells and the inhibition efficiency of sh-YY1 was assessed via RT-qPCR and Western blot (Figure 1(b) &S1B). The results displayed that YY1 expression was visibly suppressed in cells transfected with sh-YY1. Then, CCK-8 assay was conducted to detect the impact of silencing YY1 on cell viability. We found that the OD 450 values were lessened when YY1 was inhibited in AML-12 cells, which indicated that cell viability was suppressed by YY1 depletion (Figure 1(c)). Next, colony formation was implemented to measure cell proliferation and the results revealed that the number of colonies was reduced in the group of cells transfected with sh-YY1, indicating that cell proliferation capability could be repressed by knockdown of YY1 (Figure 1(d)). Then, the results of EdU assay further suggested that silenced YY1 could restrain cell proliferation since the rate of EdU positive cells was decreased after YY1 was knocked down (Figure 1(e)). Furthermore, we assessed the cell apoptosis of AML-12 cells. TUNEL assay revealed the promoting impacts of YY1 depletion on cell apoptosis since YY1 silence resulted in an increase in the TUNEL-positive cells (Figure 1(f)). Moreover, flow cytometry experiments also indicated that cell apoptosis was elevated in response to transfection of sh-YY1 (Figure 1(g)). Also, the spectrophotometer tested the activity of caspase-3 and caspase-9, and results showed that their activities were both elevated by silenced YY1, which further validated that cell apoptosis was accelerated by YY1 reduction (Figure 1(h)). In a word, YY1 could improve viability and proliferation of hepatocytes after HIRI.

Figure 1.

YY1 could improve viability and proliferation of hepatocytes after HIRI. a. Expression of YY1 in the mouse normal liver cell line (AML-12) was tested via RT-qPCR after the treatment with different doses of H₂O₂. b. YY1 inhibition efficiency was assessed via RT-qPCR assay in AML-12 cells. c. Proliferation of AML-12 cells with or without YY1 silence was investigated via CCK-8 assay. d. Colony formation assay was conducted to test cell proliferation after YY1 was silenced. e. EdU assay was further utilized to evaluate cell proliferation after knockdown of YY1. f. TUNEL assay was carried out for testing cell apoptosis in cells transfected with sh-YY1. g. Flow cytometry analysis was conducted to estimate cell apoptosis in response to YY1 inhibition. h. A pectrophotometer was used for testing the activities of caspase-3 and caspase-9 for probing the apoptosis of AML-12 cells with YY1 depletion. *P < 0.05, **P < 0.01.

MiR-186-5p could bind to YY1 in hepatocytes

Next, we researched the upstream of YY1 for investigating possible RNA regulation mechanisms in hepatocytes. The bioinformatics tool was applied to screen out the possible miRNAs. It was obtained on ENCORI (http://starbase.sysu.edu.cn/index.php) that miR-186-5p potentially bound to YY1 under the specific conditions (CLIP-Data ≥ 5, Degradome-Data ≥ 3, pan-Cancer ≥ 8, programNum ≥ 5, program: TargetScan). Thence, RNA pulldown assay investigated the binding correlation of YY1 and miR-186-5p (Figure 2(a)). We found that YY1 was enriched in the pulldown of biotinylated miR-186-5p-WT but not in that of biotinylated miR-186-5p-Mut, indicating that miR-186-5p could bind to YY1. Then, possible binding sites of miR-186-5p and YY1 were predicted from ENCORI (Figure 2(b)). Afterward, we upregulated miR-186-5p expression in AML-12 cells by transfecting miR-186-5p mimics into cells and miR-186-5p overexpression efficiency was examined through RT-qPCR assay (Figure 2(c)). After transfection, miR-186-5p expression was effectively enhanced in AML-12 cells. Next, expression of YY1 was tested in cells transfected with miR-186-5p mimics through RT-qPCR and Western blot analysis (Figure 2(d) &S1C). We found that the expression of YY1 was inhibited by overexpressed miR-186-5p. Additionally, luciferase reporter assay was further implemented to verify the interaction of miR-186-5p and YY1, and we noticed that the relative luciferase activity of YY1-WT was inhibited by overexpressed miR-186-5p, while that of YY1-Mut was almost unchanged when miR-186-5p was upregulated compared with negative control (Figure 2(e)). It has been reported that miR-186-5p could regulate spinal cord ischemia-reperfusion injury [22]. Thence, functional assays were implemented to investigate the function of miR-186-5p in HIRI. All cells used in the functional experiments were treated with 200uM H₂O₂ for six hours. Through CCK-8 assay, we found that cell viability was inhibited by overexpressed miR-186-5p based on the decline in the OD value at 450 nm as a result of the transfection with miR-186-5p mimics (Figure 2(f)). Then, it was indicated through colony formation assay that the colonies were significantly reduced after miR-186-5p was upregulated (Figure 2(g)). Additionally, EdU assay results also unclosed that the ratio of EdU-positive cells was decreased in response to miR-186-5p overexpression (Figure 2(h)). These results demonstrated that cell proliferation could be suppressed by miR-186-5p overexpression. Then, cell apoptosis was also measured. We carried out TUNEL assay and discovered that TUNEL positive cell percent was elevated on account of the transfection with overexpressed miR-186-5p (Figure 2(i)), suggesting that cell apoptosis could be accelerated by miR-186-5p upregulation. In addition, flow cytometry analysis further validated that the cell apoptosis rate was promoted after miR-186-5p was overexpressed (Figure 2(j)). Finally, the spectrophotometer was utilized for activity detection of caspase-3 and caspase-9, and the results verified that cell apoptosis capability was exactly enhanced as a result of transfection with miR-186-5p mimics since the activities of both caspase-3 and caspase-9 were elevated (Figure 2(k)). In a word, miR-186-5p could bind to YY1 and restrain the growth of hepatocytes.

Figure 2.

MiR-186-5p could bind to YY1 in hepatocytes. a. RNA pulldown assay investigated the binding of YY1 and miR-186-5p. b. Possible binding sites of YY1 and miR-186-5p were found from ENCORI. c. RT-qPCR was applied for determining miR-186-5p overexpression efficiency. d. Expression of YY1 was tested in miR-186-5p overexpressed cells via RT-qPCR assay. e. Luciferase reporter assay detected the correlation of YY1 and miR-186-5p in AML-12 cells. f. Proliferation of AML-12 cells was investigated via CCK-8 assay when miR-186-5p was overexpressed. g. Colony formation assay was performed for testing cell proliferation after overexpressing miR-186-5p. h. EdU assay tested the influence of overexpressing miR-186-5p on cell proliferation. i. TUNEL assay was conducted to measure cell apoptosis in response to the transfection with miR-186-5p mimics. j. Flow cytometry analysis was conducted to evaluate cell apoptosis after overexpressing miR-186-5p in cells. k. A spectrophotometer was utilized to test the activities of caspase-3 and caspase-9 for measuring the apoptosis of AML-12 cells when miR-186-5p was overexpressed. **P < 0.01.

lncRNA SNHG1 could bind to miR-186-5p in hepatocytes

In recent years, the ceRNA mechanism was confirmed to take part in the regulation of assorted diseases progression [26]. We proved that miR-186-5p could bind to YY1 through the above experiments. Thus, we hypothesized the involvement of the ceRNA mechanism in YY1 regulation in HIRI. It was reported that lncRNA could sponge miRNA to release mRNA expression by acting as a ceRNA at the post-transcriptional regulation level [18]. For further searching the ceRNA mechanism in HIRI, we focused on the SNHG family and implemented RNA pulldown assay to screen out the most suitable lncRNAs, which could bind to miR-186-5p in AML-12 cells (Figure 3(a)). We found that only SNHG1 was enriched by the miR-186-5p biotin probe. Next, subcellular fraction assay found that SNHG1 was mainly located in cytoplasm of AML-12 cells (Figure 3(b)). In addition, FISH assay also indicated that SNHG1 was distributed in the cytoplasm, implying the regulation at the post-transcriptional level (Figure S1D). Then, RT-qPCR assay assessed SNHG1 inhibition and overexpression efficiency in AML-12 cells. After transfection of sh-SNHG1 in cells, the expression of SNHG1 was visibly decreased (Figure 3(c)). Moreover, SNHG1 expression was elevated in AML-12 cells transfected with pcDNA3.1/SNHG1 (Figure 3(d)). Subsequently, expression of YY1 was tested in SNHG1 silenced or overexpressed AML-12 cells (Figure 3(e-f)and S1E-F). The results indicated that YY1 expression was reduced by silenced SNHG1 and increased by overexpressed SNHG1, suggesting the positive correlation between SNHG1 and YY1 expression. Next, RIP assay probed the connection of SNHG1, miR-186-5p and YY1 in AML-12 cells (Figure 3(g)). As a result, we found that SNHG1, miR-186-5p and YY1 were all substantially enriched in the Ago2 group, indicating their coexistence in the RISC complex. Moreover, the binding site of SNHG1 and miR-186-5p was presented after searching on ENCORI (Figure 3(h)). Then, RNA pulldown assay was conducted, and we discovered that SNHG1 was enriched in the pulldown of biotinylated miR-186-5p-WT (Figure 3(i)). Luciferase reporter assay was utilized to further detect the interaction of SNHG1 and miR-186-5p (Figure 3(j)). Results showed that overexpressed miR-186-5p hampered the luciferase activities of SNHG1-WT but could not affect that of SNHG1-Mut, which indicated that SNHG1 could bind to miR-186-5p. Additionally, results of RT-qPCR and Western blot analysis in a rescue manner demonstrated that the augment in YY1 expression caused by SNHG1 overexpression was reversed in response to miR-186-5p upregulation (Figure 3(k) and S1G). In brief, lncRNA SNHG1 could bind to miR-186-5p to positively regulate YY1 in hepatocytes.

Figure 3.

lncRNA SNHG1 could bind to miR-186-5p in hepatocytes. a. RNA pulldown assay was adopted to screen out the most suitable lncRNAs, which could bind to miR-186-5p in AML-12 cells. b. Subcellular fraction assay was conducted to test the position of SNHG1 in AML-12 cells. c and d. RT-qPCR assay assessed SNHG1 inhibition and overexpression efficiency in AML-12 cells. e and f. Expression of YY1 was tested in AML-12 cells with SNHG1 silence or overexpression. g. RIP assay was implemented for verifying the connection of SNHG1, miR-186-5p and YY1 in AML-12 cells. h. Binding site of SNHG1 and miR-186-5p was predicted on ENCORI. i. RNA pulldown assay detected the correlation of SNHG1 and miR-186-5p. j. Luciferase reporter assay further verified the interaction of SNHG1 and miR-186-5p. k. RT-qPCR rescue assay tested the expression of YY1 in cells transfected with different plasmids. **P < 0.01.

lncRNA SNHG1 could alleviate HIRI in hepatocytes

Next, we further studied the function of SNHG1 in AML-12 cells with six-hour H₂O₂ (200uM) treatment. CCK-8 assay indicated that overexpressed SNHG1 could accelerate the cell viability, while the cell viability was repressed as a result of SNHG1 inhibition (Figure 4(a)). Then, colony formation assay displayed that the number of colonies declined by SNHG1 knockdown but increased by SNHG1 overexpression (Figure 4(b)). Furthermore, EdU assay also showed that the ratio of EdU-positive cells was suppressed by silenced SNHG1 and elevated by upregulated SNHG1 (Figure 4(c)). These experimental results demonstrated that cell proliferation could be accelerated by SNHG1 upregulation and reduced by SNHG1 inhibition after HIRI. Next, TUNEL assay was conducted to evaluate cell apoptosis, and we discovered that SNHG1 inhibition caused a notable increase in the rate of TUNEL-positive cells (Figure 4(d)). Then, flow cytometry analysis revealed that the cell apoptosis rate could be accelerated after transfecting with sh-SNHG1 (Figure 4(e)). In the end, it was indicated through the detection of caspase-3 and caspase-9 activities that SNHG1 silence could overtly enhance cell apoptosis after HIRI (Figure 4(f)). Thence, we concluded that lncRNA SNHG1 could alleviate HIRI in hepatocytes.

Figure 4.

lncRNA SNHG1 could alleviate HIRI in hepatocytes. a. Proliferation of AML-12 cells was investigated via CCK-8 assay in response to SNHG1 upregulation or downregulation. b. Colony formation assay was performed in order to test cell proliferation under different conditions. c. EdU assay was further utilized to evaluate cell proliferation in response to transfection with different plasmids. d and e. Apoptotic abilities of cells with SNHG1 knockdown were examined via TUNEL and flow cytometry assays. f. A spectrophotometer was used for testing the activities of caspase-3 and caspase-9 for probing the apoptosis of SNHG1-silenced AML-12 cells. *P < 0.05, **P < 0.01.

SNHG1/YY1 axis could regulate hepatocyte growth

Furthermore, we wondered whether the SNHG1/YY1 axis could exert the regulatory function in HIRI. First, we overexpressed YY1 in AML-12 cells and the overexpression efficiency of pcDNA3.1/YY1 was tested via RT-qPCR and Western blot (Figure 5(a) and S1H). As depicted in Figure S1I, CCK-8 revealed that the inhibited cell proliferation, resulting from SNHG1 depletion, was reversed by overexpressed YY1. From the colony formation assay, the decline in the colony number induced by SNHG1 knockdown was rescued after cotransfection of pcDNA3.1/YY1 (Figure 5(b)). Similarly, it was demonstrated in EdU assay that the reduction in EdU-positive cells was reversed as a result of YY1 overexpression (Figure 5(c)). Additionally, it was displayed in TUNEL assay that the augment in TUNEL-positive cells, resulting from SNHG1 silence, was recovered by upregulated YY1 (Figure 5(d)). Moreover, in flow cytometry assay, we observed that the cell apoptosis rate was also accelerated by SNHG1 inhibition, which was fully offset by overexpressed YY1 (Figure 5(e)). In the end, we discovered that the caspase-3 and caspase-9 activities were elevated by suppressed SNHG1, which then declined in response to overexpression of YY1 (Figure 5(f)). These experiments proved that the elevated cell apoptosis by SNHG1 deficiency could be recovered by YY1 upregulation. In a word, the SNHG1/YY1 axis could regulate the growth of hepatocytes.

Figure 5.

The SNHG1/YY1 axis could regulate the growth of hepatocytes. a. YY1 overexpression efficiency was tested via RT-qPCR assay in AML-12 cells. b. Colony formation assay was performed for cell proliferation examination under different transfection conditions. c. EdU assay was performed for further evaluating cell proliferation after transfection of different plasmids. d and e. TUNEL and flow cytometry assays were jointly applied for estimating cell apoptosis in different groups. f. A spectrophotometer was utilized to test the activities of caspase-3 and caspase-9 for detecting the apoptosis of AML-12 cells in different groups. **P < 0.01.

YY1 could activate the expression of SNHG1 by binding to the SNHG1 promoter

There are existing studies reporting the transcriptional activating role of YY1 in the expression of lncRNA [27]. Thence, we verified the function of YY1 in SNHG1 expression. We first searched the possible binding site in YY1 and SNHG1 promoter via the JASPAR website (http://jaspar.genereg.net/) (Figure 6(a)). It was demonstrated in RT-qPCR results that YY1 depletion could result in the decline in SNHG1 expression, while YY1 upregulation led to the opposite consequence, reflecting that YY1 could positively modulate SNHG1 expression (Figure 6(b,c)). Moreover, ChIP assay investigated the binding relationship of YY1 and SNHG1 promoter, and the abundant enrichment of SNHG1 promoter could be discovered in the anti-YY1 group (Figure 6(d)). Luciferase reporter assay further indicated that the relative luciferase activity of SNHG1 promoter-WT was obviously inhibited by silenced YY1, while that of SNHG1 promoter-Mut was hardly affected (Figure 6(e)). The above results proved that YY1 could activate the expression of SNHG1 by binding to SNHG1 promoter.

Figure 6.

YY1 could activate the expression of SNHG1 by binding to SNHG1 promoter. a. Binding site of YY1 and SNHG1 promoter was predicted according to the information from JASPAR. b and c. RT-qPCR quantified SNHG1 in cells with YY1 depletion or augment. d. ChIP assay investigated the correlation of YY1 and SNHG1 promoter. e. Luciferase reporter assay testified the interaction of YY1 and SNHG1 promoter. **P < 0.01.

Discussion

Hepatic ischemia/reperfusion injury (HIRI) is a common tissue and organ injury in liver transplantation and liver surgery. HIRI is critical for the development of liver disease, the effects of liver surgery and the survival prognosis of patients [2]. In recent years, lncRNAs have been confirmed as the important regulators in the progression of HIRI. Previous studies have reported that lncRNA AK054386 can activate endoplasmic reticulum stress in the response of HIRI by sponging miR-199 [28]. lncRNA HOTAIR can activate the autophagy in HIRI by modulating the axis of miR-20b-5p/ATG7 [3]. lncRNA MEG3 can reduce the influence of HIRI via the miR-34a/Nrf2 pathway [29]. lncRNA MALAT1 can promote the development of HIRI to arise the apoptosis and inflammation of hepatocytes by regulating the HMGB1/TLR4 pathway [30]. In the present study, we verified the function of lncRNA SNHG1 in the progression of HIRI. Functional assays were implemented for investigating the viability and proliferation of hepatocytes by testing cell proliferation and apoptosis. It has been reported that the viability of normal liver cell lines is verified as the important marker in the response of HIRI [3,4]. Moreover, six hours of 200uM H₂O₂ treatment was implemented to hepatocytes for inducing HIRI. Experimental results found that inhibited SNHG1 could reduce the cell viability and cell proliferation and activate cell apoptosis of hepatocytes. Additionally, previous studies have proved the important regulation role of SNHG1 in oxygen-glucose deprivation of brain. It has been identified that SNHG1 plays a protective role after oxygen-glucose deprivation of brain by activating angiogenesis of brain microvascular endothelial cells [17]. Our study verified the protective role of SNHG1 in HIRI by improving the cell viability and proliferation of hepatocytes.

A previous study has reported that YY1 transcription factor (YY1) was downregulated after ischemia/reperfusion (I/R) injury [25]. We focused on YY1 to explore its function in HIRI. Functional assays found that inhibited YY1 could reduce the cell viability and proliferation of hepatocytes, which implied that YY1 could play a protective role in HIRI.

For searching the possible mechanism of lncRNA SNHG1, we further identified the location of lncRNA SNHG1 in hepatocytes. We found that SNHG1 is mainly located in cytoplasm of hepatocytes. Thence, we predicted that the ceRNA pathway of SNHG1 existed in hepatocytes. In recent years, the ceRNA mechanism was confirmed to take part in the regulation of assorted disease progression [26]. The ceRNA mechanism means that lncRNAs could sponge miRNAs for regulating the expression of mRNAs [18]. It is a post-transcriptional regulatory mechanism, meaning that cytoplasmic lncRNA can play the role of ceRNA. Thus, we deeply investigated the ceRNA pathway of cytoplasmic SNHG1. Through bioinformatics tools, miR-186-5p was found out. We proved that YY1 was the target gene of miR-186-5p through mechanism assays, such as RNA pulldown, RIP and luciferase reporter assays. A previous study has reported the role of miR-186-5p in spinal cord ischemia-reperfusion injury [22]. Also, we proved that miR-186-5p could be sponged by SNHG1 in hepatocytes. In this study, we confirmed that the SNHG1/miR-186-5p/YY1 axis could modulate the cell proliferation and apoptosis in HIRI.

Furthermore, we studied the transcriptional modulation of YY1 on lncRNA SNHG1 expression. Previous studies have reported that YY1 can activate the expression of lncRNAs to modulate the progression of human cancer [31,32]. In the present study, we found that YY1 could bind to SNHG1 promoter to activate its expression.

Taken together, in our research, we studied the function of the SNHG1/miR-186-5p/YY1 axis in HIRI. We found that SNHG1-miR-186-5p-YY1 feedback loop could alleviate HIRI, which might offer a new idea for prevention of HIRI during liver transplantation or hepatic surgery.

Funding Statement

The author(s) reported that there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed here.