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. 2024 Feb 20;76(2):259–269. doi: 10.1007/s10616-023-00614-x

LncRNA HOTAIR accelerates free fatty acid-induced inflammatory response in HepG2 cells by recruiting SRSF1 to stabilize MLXIPL mRNA

Bo Guo 1, Shengzhe Yan 2, Lei Zhai 1, Yanzhen Cheng 2,✉
PMCID: PMC10940554  PMID: 38495293

Abstract

LncRNA HOTAIR has been reported to be associated with metabolic diseases of the liver. However, the effect of HOTAIR on non-alcoholic fatty liver disease (NAFLD) inflammation and its potential mechanism have not been reported. Genes and proteins expression were detected by qRT-PCR and Western blot respectively. The level of inflammatory cytokines was assessed by ELISA. HepG2 cell viability was detected by MTT assay. TG level and lipid accumulation were measured by Assay Kit and Oil red O staining, respectively. Direct binding relationship between HOTAIR and Serine/arginine splicing factor 1 (SRSF1), SRSF1 and MLX interacting protein like (MLXIPL) were confirmed by RNA-pull down and RIP assay. HOTAIR was highly expressed in free fatty acids (FFA)-treated HepG2 cells. HOTAIR knockdown alleviated FFA-induced inflammation of HepG2 cells. Then further analysis showed that HOTAIR and SRSF1 had a mutual binding relationship, and HOTAIR maintained MLXIPL mRNA stability via recruiting SRSF1 in HepG2 cells. Moreover, the inhibitory effect of HOTAIR knockdown on FFA-induced inflammation in HepG2 cells was reversed by MLXIPL overexpression. HOTAIR accelerates inflammation of FFA-induced HepG2 cells by recruiting SRSF1 to stabilize MLXIPL mRNA, which will help to find new effective strategies for NAFLD therapy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10616-023-00614-x.

Keywords: Non-alcoholic fatty liver disease, Inflammation, LncRNA HOTAIR, MLXIPL

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease characterized by triglyceride accumulation in the liver and no history of excessive drinking (Takahashi et al. 2015). NAFLD mainly includes simple sclerosis, non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma (Chen et al. 2017). In recent years, with the increasing incidence of obesity, the incidence of NAFLD is also increasing (Ganesh and Rustgi 2016). The latest research shows that liver oxidative stress and inflammation are risk factors for accelerating NAFLD progression. The specific mechanism may be that excessive free fatty acids (FFA) may lead to fat/ triglyceride (TG) accumulation, resulting in oxidative stress and insulin resistance, and finally lead to excessive production and release of inflammatory cytokines in the liver (Ding et al. 2020). Although the pathogenesis of NAFLD has been preliminarily elucidated, since NAFLD is a multi-system disease, its pathogenesis needs to be further explored.

Long non-coding RNA (LncRNA) is a cluster of transcribed RNA molecules over 200 nt in length (Hou and Bonkovsky 2013). Increasing evidence showed that lncRNA participates in the regulation of various disease progression, especially in liver diseases, and which served as a therapeutic target and potential biomarker for liver-related diseases (Huang et al. 2021). Recently, the regulation mechanism of lncRNA in NAFLD has become a research hotspot. Zhao et al. reported that lncRNA Blnc1 accelerated liver fat formation related to obesity and was closely related to the pathogenesis of NAFLD (Zhao et al. 2018a). What’s more, it was found that activated lncRNA NEAT1 accelerated liver fat accumulation in NAFLD by regulating miR-212-5p/GRIA3 axis (Hu et al. 2021). Long non-coding RNA HOX Transcript antisense intergenic RNA (LncRNA HOTAIR) with a length of about 2.2 kb, which is located on chromosome 12 (Zhao et al. 2018b). Research showed that HOTAIR widely participated in the regulation of cancer progression (Lu et al. 2012; Gao et al. 2016). Furthermore, our previous studies found that HOTAIR promoted the lipid accumulation of NAFLD (Guo, et al. 2021), but the role of HOTAIR in the inflammatory response of NAFLD remains unclear.

Carbohydrate response element (ChoRE) binding protein (ChREBP) and its binding chaperone Max-like protein X (MLX) are important transcription factors that regulate glucose metabolism, storage and redistribution. They can sense glucose levels in the microenvironment, dynamically coordinate their target genes to participate in many metabolic processes of glycolysis, lipogenesis and glucose transport, and maintain metabolic homeostasis (Wang et al. 2015). MLX interacting protein like (MLXIPL) is a glucose-responsive transcription factor, which was originally reported to be involved in glucose metabolism, lipogenesis and tumor progression (Zhan et al. 2020). Also, MLXIPL is an important liver transcription factor and plays an important role in lipid metabolism (Wang et al. 2020). Research showed that MLXIPL transcriptionally activated lipid metabolism genes including FASN, SCD1, ACACA and MGAT1, and its overexpression will lead to lipid accumulation (Stoeckman et al. 2004). It has been reported that knockdown of MLXIPL impeded HCC cell growth, invasion, migration, and lipid formation (Chang et al. 2023). Furthermore, studies have shown that MLXIPL mediated the inflammatory response and apoptosis of mesangial cells in diabetic nephropathy (Park et al. 2014). Interestingly, our previous study found that knocking down HOTAIR significantly down-regulated MLXIPL. However, it is unclear whether HOTAIR affects NAFLD by regulating MLXIPL.

In summary, we hypothesized that HOTAIR accelerated the inflammatory response of NAFLD by regulating MLXIPL. Our study explored the role and underlying mechanism of HOTAIR in FFA-induced lipid accumulation and cellular inflammation in HepG2 cells. HOTAIR may be used as a novel target for NAFLD clinical treatment.

Materials and methods

Cell lines and culture

Human HepG2 cells was provided by American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, California, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured in a humidified incubator at 37 °C and 5% CO2. The cell culture medium was changed every 48 to 72 h. HepG2 cells were treated with 1 mmol/L free fatty acid (FFA) (oleic acid: palmitic acid = 2:1) for 24 h to construct NAFLD cell model in vitro. Oleic acid and palmitic acid were purchased from Sigma-Aldrich (MO, USA).

Cell transfection

For cell transfection, HepG2 cells were added into 96-well plate for 12 h, then Lipofectamine™ 3000 Transfection Reagent (Invitrogen, CA, USA) was used to transfect corresponding plasmids into cells. Following 48 h transfection, HepG2 cells were used in subsequent experiments. All vectors used in our study were synthesized by Sangon Biotech (Shanghai, China), including sh-HOTAIR, sh-SRSF1, oe-MLXIPL, oe-HOTAIR and their negative controls (sh-NC and pcDNA3.1).

MTT assay

HepG2 cells were inoculated on a 96-well cell culture plate with a density of 5 × 103 cells for 24 h. Then 20 μL MTT solution (Beyotime, Shanghai, China, C0009M, 1 mg/mL) was added to each well plate and incubated under aseptic condition for 2 h. The absorbance of each hole at 490 nm was detected by a miniature tablet reader, which reflects the HepG2 cell viability.

Triglyceride (TG) level detection

HepG2 cells in the 6-well cell culture plate were washed twice with PBS and collected by trypsin digestion. TG was extracted by chloroform/methanol (2:1 v/w) method and dried in a chemical cover. Subsequently, 40 μL 1% Triton X-100 was added into HepG2 cells. Triton X-100 is a non-ionic surfactant used to facilitate the dissolution of lipids and proteins. Then a TG reagent kit (Solarbio, Beijing, China, BC0625) was used to measure the concentration of TG.

Oil red O staining

HepG2 cells were washed twice with PBS and fixed with 10% formaldehyde for 30 min. An Oil red O staining kit (Jiancheng Biotech, Nanjing, China, #D027) was used to perform Oil red O staining, according to previous report (Wang et al. 2019).

ELISA

1 × 106 HepG2 cells were added into 6-well cell culture plate for 12 h, after treatment, cell supernatant was collected. The ELISA kits (Boster, Wuhan, China) were applied to detect TNF-α, IL-6 and IL-1β levels, according to previous report (Li et al. 2021).

qRT-PCR

TRIzol reagent was used to extract total RNA from HepG2 cells. Then the RNA samples were reverse transcribed into cDNA using a reverse transcription first-strand cDNA synthesis kit (Thermo Fisher Scientific, MA, USA). One-Step SYBR Prime Script PLUS RT-PCR kit (TakaRa, Osaka, Japan) was applied to detect the RNA levels. GAPDH was used as the endogenous control in data analysis. Fold changes were calculated using the 2−ΔΔCt method. The whole process was repeated three times. The primers were shown in Table 1.

Table 1.

Primer sequences

Primer name Primer sequences
F- HOTAIR 5′-AAGGCCCCAAAGAGTCTGAT-3′
R- HOTAIR 5′-GCTTGGGTGTAATTGCTGGT‐3′
F- SRSF1 5′-CCGCAGGGAACAACGATTG -3′
R- SRSF1 5′-GCCGTATTTGTAGAACACGTCCT‐3′
F-TNF-α 5′-CCCCAGGGACCTCTCTCTAA-3′
R-TNF-α 5′-TGAGGTACAGGCCCTCTGAT‐3′
F-IL-6 5′-TACCCCCAGGAGAAGATTCC-3′
R- IL-6 5′-TTTTCTGCCAGTGCCTCTTT‐3′
F-IL-1β 5′-CGATGCACCTGTACGATCAC-3′
R-IL-1β 5′-TCTTTCAACACGCAGGACAG‐3′
F-MLXIPL 5′-GTCATCCACAGCGGTCACTTC-3′
R-MLXIPL 5′-GTCTCTGCAGAGCAGCTTGAG‐3′
F-GAPDH 5′-CCAGGTGGTCTCCTCTGA-3′
R-GAPDH 5′-GCTGTAGCCAAATCGTTGT‐3′
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Western blot

The total cell was prepared with RIPA cleavage buffer (Beyotime, Nanjing, China). The protein concentration was determined with the BCA Assay Kit (Thermo-Fisher Scientific, MA, USA). 10% SDS-PAGE gel was used to separate the protein and then transferred to the PVDF membrane. 5% skimmed milk powder in TBST were used to seal the PVDF membrane. Then, the PVDF membrane was incubated overnight with specific antibodies, including SRSF1 (Invitrogen, CA, USA) and MLXIPL (Abcam, Cambridge, UK) at 4 ℃. GAPDH (Abcam, Cambridge, UK) was used as the endogenous control. On the second day, the membrane was incubated with specific secondary antibodies at room temperature for 1 h, and the signal was detected by ECL kit (ab133406, Abcam).

Actinomycin D assay

To block transcription, actinomycin D (2 mg/mL) was used to treat HepG2 cells for 0 h, 3 h, 6 h, 9 h and 12 h (Sigma-Aldrich, MO, USA). To assess RNA stability, qRT-PCR was used to assess the remaining RNAs (MLXIPL) extracted from treated cells.

RNA pull-down

RNA pull-down kit (BersinBio, Guangzhou, China) was used to detect the proteins that bind to HOTAIR and MLXIPL. Biotin labeled probes specifically targeting HOTAIR or MLXIPL were designed and synthesized by GenePharma (Shanghai, China), and scramble and NC were used as the control. RNA–protein complexes were formed by incubating specific probes with cell lysates. The complex was then separated using streptavidin-coupled magnetic beads. After separation by 10% polyacrylamide gel electrophoresis (PAGE), they were stained with silver, followed by Western blotting.

RIP assay

RIP detection was performed using BersinBio RIP Detection Kit (BersinBio, Guangzhou, China). Cell lysates were incubated with magnetic beads and anti-SRSF1 (Proteintech, CHI, USA) or anti-IgG (ABclonal, Wuhan, China). qRT-PCR was used to analyze the enriched RNA.

Statistical analysis

The mean ± standard deviation (SD) represents data from three independent experiments. Statistical analysis of all data was performed by using GraphPad Prism 7.0 Software (GraphPad Software, Inc.). Comparison between two groups was performed by two-tailed Student t test. One-way analysis of variance (ANOVA) was used for pairwise comparison among multiple groups. When P < 0.05, the difference is statistically significant.

Results

HOTAIR knockdown alleviated FFA-induced inflammation in HepG2 cells

To explore the effect of HOTAIR on NAFLD, HepG2 cells were treated with FFA for 24 h to construct NAFLD cell model in vitro. qRT-PCR analysis indicated that HOTAIR was significantly up-regulated in FFA-treated HepG2 cells (Fig. 1A). Then, sh-HOTAIR vector was transfected into HepG2 cells for HOTAIR knockdown (Fig. 1B). As shown in Fig. 1C, HOTAIR was significantly up-regulated in FFA-treated HepG2 cells, but HOTAIR knockdown reversed the upward trend. Besides, MTT assay analysis showed that FFA treatment inhibited HepG2 cell viability, which was alleviated by HOTAIR knockdown (Fig. 1D). Additionally, the high level of TG induced by FFA was down-regulated by HOTAIR knockdown (Fig. 1E). Oil red O staining analysis indicated that lipid accumulation in FFA induced HepG2 cells was decreased by HOTAIR inhibition (Fig. 1F). As shown in 1G and 1H, the high expression of TNF-α, IL-6 and IL-1β in FFA induced HepG2 cells was abolished by HOTAIR knockdown. What’s more, we examined the effect of HOTAIR knockdown on the expression of multiple genes involved in lipid synthesis and storage (such as GPAM, MOGAT, FASN, MLXIPL, ACACA, APOC3, SREBP, and PLIN2). Results showed that knocking down HOTAIR has the most obvious effect on MLXIPL (Supplementary Fig. 1). MLXIPL is an important liver transcription factor and plays an important role in lipid metabolism (Wang et al. 2020). In general, HOTAIR knockdown alleviated FFA-induced inflammation in HepG2 cells, and this effect may be related to the regulation of MLXIPL.

Fig. 1.

Fig. 1

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HOTAIR knockdown alleviated FFA-induced inflammation in HepG2 cells. A-C, the level of HOTAIR was detected by qRT-PCR. D, MTT assay was used to detect cell proliferation after HepG2 cells induced by FFA and transfected with sh-HOTAIR. E, TG level was assessed by using CheKine™ Triglyceride (TG) Colorimetric Assay Kit. F, lipid accumulation was evaluated by Oil red O staining. Scale bars = 100 μM. G-H, the levels of TNF-α, IL-6 and IL-1β were detected by ELISA and qRT-PCR, respectively. The data were expressed as the mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001

HOTAIR bound to SRSF1 and SRSF1 bound to MLXIPL in HepG2 cells

Next, we continued to explore the underlying mechanism by which HOTAIR regulates FFA-induced inflammation. A large number of studies have found that lncRNA can affect the stability of downstream mRNA by regulating RNA binding proteins (RBPs) (Lv et al. 2023; Ni et al. 2019). Through Starbase database prediction, 38 RBPs with the possibility of combining with HOTAIR and MLXIPL were found (Supplementary Fig. 2A). Among which, we selected 8 RBPs, which were reported to be expressed in hepatocytes. Through RIP detection, we further found that only SRSF1 and HOTAIR have a mutual association relationship (Supplementary Fig. 2B). Therefore, we hypothesized that HOTAIR might regulate MLXIPL levels in hepatocytes by recruiting RNA binding protein SRSF1. And Supplementary Fig. 2C showed the possible combination of HOTAIR and SRSF1 as predicted by Starbase database. Subsequently, Fig. 2A and 2B found that SRSF1 was significantly increased in FFA-treated HepG2 cells. RNA-pull down assay further found that SRSF1 was enriched by biotin-labeled HOTAIR, but no such enrichment was observed in negative or blank control group (Fig. 2C). What's more, results showed that there were target binding relationship between SRSF1 and MLXIPL, and which was verified by RNA-pull down and RIP assay (Fig. 2D and 2E). Collectively, our findings indicated that SRSF1 bound to both HOTAIR and MLXIPL in HepG2 cells.

Fig. 2.

Fig. 2

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HOTAIR bound to SRSF1 and SRSF1 bound to MLXIPL in HepG2 cells. A-B, the level of SRSF1 was measured by qRT-PCR and Western blot. C, the binding relationship between HOTAIR and SRSF1 was verified by RNA-pull down assay. D-E, the binding relationship between SRSF1 and MLXIPL was verified by RNA-pull down and RIP assay. The data were expressed as the mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001

HOTAIR maintained MLXIPL mRNA stability via recruiting SRSF1 in HepG2 cells

Then we further investigated the underlying mechanism of HOTAIR on regulating MLXIPL. Firstly, sh-SRSF1 vector was transfected into HepG2 cells for SRSF1 knockdown (Fig. 3A and B). It could be seen that after treating with actinomycin D, MLXIPL mRNA stability in HepG2 cells was decreased by SRSF1 knockdown (Fig. 3C). As shown in 3D and 3E, the level of MLXIPL was significantly decreased in SRSF1 knockdown-HepG2 cells. Similarly, HOTAIR knockdown decreased MLXIPL mRNA stability and down-regulated MLXIPL level in HepG2 cells (Fig. 3F–H). Furthermore, oe-HOTAIR was transfected into HepG2 cells for HOTAIR overexpression. qRT-PCR analysis indicated that HOTAIR expression was significantly up-regulated in HepG2 cells after transfected with oe-HOTAIR (Fig. 3I). Our results discovered that after treating with actinomycin D, MLXIPL mRNA stability in HepG2 cells was enhanced by HOTAIR overexpression, while the effect of HOTAIR overexpression on MLXIPL mRNA stability was reversed by SRSF1 knockdown (Fig. 3J). Taken together, the results showed that HOTAIR affected MLXIPL through SRSF1..

Fig. 3.

Fig. 3

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HOTAIR maintained MLXIPL mRNA stability via recruiting SRSF1 in HepG2 cells. A-B, the level of SRSF1 was measured by qRT-PCR and Western blot. C, the remaining of MLXIPL mRNA was detected by qRT-PCR. D-E, the level of MLXIPL was detected by qRT-PCR and Western blot. F, the remaining of MLXIPL mRNA was detected by qRT-PCR. G-H, the level of MLXIPL was detected by qRT-PCR and Western blot. I, qRT-PCR was performed to detect HOTAIR level. J, the remaining of MLXIPL mRNA was detected by qRT-PCR. The data were expressed as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

The inhibition of HOTAIR knockdown on FFA-induced inflammation in HepG2 cells was reversed by MLXIPL overexpression

To further explore the underlying molecular mechanism of HOTAIR regulating FFA-induced inflammation in HepG2 cells, pcDNA3.1 MLXIPL vector was transfected into HepG2 cells for MLXIPL overexpression (Fig. 4A and B). Then HepG2 cells were transfected with sh-HOTAIR and pcDNA3.1 MLXIPL vectors for HOTAIR knockdown and MLXIPL overexpression, simultaneously. Findings indicated that the high expression of MLXIPL in FFA induced HepG2 cells was antagonized by HOTAIR down-regulation, which was reversed by MLXIPL overexpression (Fig. 4C and D). As shown in Fig. 4E–G, MLXIPL overexpression largely neutralized HOTAIR knockdown-mediated increasing of HepG2 cell vability, decreasing of TG level and lipid accumulation in FFA-induced HepG2 cells. Moreover, the decreased level of TNF-α, IL-6 and IL-1β caused by HOTAIR knockdown was hindered by MLXIPL overexpression in FFA-treated cells (Fig. 4H and I). Taken together, our findings suggested that the inhibitory effect of HOTAIR knockdown on FFA-induced inflammation in HepG2 cells was reversed by MLXIPL overexpression. HOTAIR accelerated FFA-induced inflammatory responses in HepG2 cells by regulating MLXIPL.

Fig. 4.

Fig. 4

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The inhibition of HOTAIR knockdown on FFA-induced inflammation in HepG2 cells was reversed by MLXIPL overexpression. A-D, the level of MLXIPL was detected by qRT-PCR and Western blot. E, HepG2 cell proliferation was detected by MTT assay. F, TG level was assessed by using CheKine™ Triglyceride (TG) Colorimetric Assay Kit. G, Lipid accumulation was evaluated by Oil red O staining. Scale bars = 100 μM. H-I, the levels of TNF-α, IL-6 and IL-1β were detected by ELISA and qRT-PCR. The data were expressed as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

NAFLD is a spectrum of liver disease defined as hardening of more than 5 percent of liver cells with little or no alcohol consumption. There are three important processes in the progression of NAFLD, namely hardening, lipid accumulation and inflammatory response (Cobbina and Akhlaghi 2017). Here, we focused on the mechanism of HOTAIR in FFA-induced lipid accumulation and inflammatory response in NAFLD progression. Our findings illustrated that HOTAIR accelerated lipid accumulation and inflammation of NAFLD by stabilizing MLXIPL mRNA.

LncRNA is an important class of non-coding RNA that lack the ability to encode proteins (Li et al. 2020). Di Mauro et al. reported that lncRNA could be served as a biomarker of NAFLD and the severity of fibrosis (Mauro et al. 2019). Excessive lipid deposition is a risk factor for NAFLD, and lncRNA Gm15622 has been proved to be a vital factor in promoting liver fat accumulation in NAFLD progression (Ma et al. 2020). Therefore, functional lncRNA is an effective target for NAFLD treatment. Here, HOTAIR is closely associated with inflammatory disease progression. HOTAIR has also been reported to regulate the rheumatoid arthritis progression by targeting miR-138 and regulating NF- κ B pathway (Zhang et al. 2017). Furthermore, it was reported that HOTAIR promoted synovitis by activating Wnt/β-catenin signal pathway (Mao et al. 2019). Our findings elucidated that HOTAIR was significantly increased in FFA-induced NAFLD model, and HOTAIR knockdown alleviated FFA-induced inflammation in HepG2 cells. Our study firstly reported the promoting effect of HOTAIR on NAFLD inflammation.

MLXIPL, initially identified as a glucose activator, is strongly associated with the pathogenesis of metabolic diseases and tumors (Iizuka et al. 2020). Wang et al. showed that lncRNA H19 induced liver cirrhosis by activating MLXIPL in hepatocytes (Wang et al. 2020). MLXIPL regulates the accumulation of lipids in lipid metabolism (Iizuka 2013). Observingly, as a glucose-sensitive transcription factor in hepatocytes, high level of MLXIPL accelerated NAFLD progression (Lei et al. 2020). Our results revealed that HOTAIR maintained MLXIPL mRNA stability via recruiting SRSF1 in HepG2 cells, and the inhibitory effects of HOTAIR knockdown on FFA-induced inflammation could be offset via MLXIPL upregulation.

The serine/arginine-rich (SR) proteins are the splicing regulators needed to form the pre-splicing and alternative splicing of mRNA (Xie et al. 2017). Serine/arginine splicing factor 1 (SRSF1) is one of the more conserved members of the SR protein family, which plays an irreplaceable regulatory role in mRNA metabolism (Zhou et al. 2019). The multi-function of SRSF1 is mainly reflected in the binding potential of RNA, nuclear-cytoplasmic shuttling and the interaction with different proteins (Das et al. 2012). SRSF1 has a vital effect on maintaining genomic stability, cell bioactivity (Anczuków et al. 2015; Sun and Hu 2020). In addition, SRSF1 is involved in regulating inflammatory responses. It was found that nuclear output of inflammatory mRNAs (including Cxcl1, TNF and Cxcl2) could be regulated by SRSF1 in LPS treated Hela cells (Zhou, et al. 2017). What's more, SRSF1 promoted LPS-induced inflammation in acute lung injury by stabilizing thymic stromal lymphopoietin mRNA (Fu et al. 2021). Here, we found that SRSF1 was the RBP that had a combination relationship with both HOTAIR and MLXIPL. Also, we confirmed for the first time that SRSF1 acted as a molecular scaffold to mediate the promoting effect of HOTAIR on MLXIPL and on FFA-induced inflammatory response in HepG2 cells.

To sum up, our research illustrated the role of HOTAIR in inflammation of FFA-induced HepG2 cells and the potential molecular mechanism. This study indicated that HOTAIR/SRSF1/MLXIPL axis participate in the regulation of inflammation in NAFLD. These findings may provide a new strategy for NAFLD treatment.

The supplementary materials-WB raw data

The supplementary materials-WB raw data were shown in URL: https://osf.io/7mrek/. 10.17605/OSF.IO/7MREK.

Supplementary Information

Below is the link to the electronic supplementary material.

10616_2023_614_MOESM1_ESM.tif (2.6MB, tif)

Supplementary Figure 1 HOTAIR knockdown has the most obvious effect on MLXIPL.The levels of GPAM, MOGAT, FASN, MLXIPL, ACACA, APOC3, SREBP and PLIN2 were detected by qRT-PCR after transfected with sh-HOTAIR. The data were expressed as the mean ±SD of at least three independent experiments. *P <0.05, ***P< 0.001.

Supplementary file1 (TIF 2685 KB)

10616_2023_614_MOESM2_ESM.tif (6.9MB, tif)

Supplementary Figure 2 RBPs with the possibility of combination with both HOTAIR and MLXIPLHOTAIR bound to SRSF1..A, Venn diagram showed the RBPs that had the potential to combine with both HOTAIR and MLXIPL. 8 kinds of RBPs which was reported to be expressed in hepatocytes. B, RIP assay was performed to detect the binding relationship between 8 different RBPs (IGF2BPs, SRSF1, KHSRP, PTBP1, FUS, SOX2, UPF1, WDR4) and R. HOTAIR. ***P< 0.001.

Supplementary file2 (TIF 7067 KB)

Abbreviations

NAFLD

Non-alcoholic fatty liver disease

LncRNA HOTAIR

Long non-coding RNA HOX Transcript antisense intergenic RNA

SRSF1

Serine/arginine splicing factor 1

MLXIPL

MLX interacting protein like

TG

Triglyceride

qRT-PCR

Quantitative real-time PCR

FFA

Free fatty acids

Author contributions

Bo Guo: Conceptualization; Methodology; Validation; Formal analysis; Writing - Original Draft; Shengzhe Yan: Investigation; Resources; Lei Zhai: Data Curation; Visualization; Supervision; Yanzhen Cheng: Writing - Review & Editing; Project administration; Funding acquisition

Funding

No.

Data availability statement

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

Declarations

Competing interest

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

10616_2023_614_MOESM1_ESM.tif (2.6MB, tif)

Supplementary Figure 1 HOTAIR knockdown has the most obvious effect on MLXIPL.The levels of GPAM, MOGAT, FASN, MLXIPL, ACACA, APOC3, SREBP and PLIN2 were detected by qRT-PCR after transfected with sh-HOTAIR. The data were expressed as the mean ±SD of at least three independent experiments. *P <0.05, ***P< 0.001.

Supplementary file1 (TIF 2685 KB)

10616_2023_614_MOESM2_ESM.tif (6.9MB, tif)

Supplementary Figure 2 RBPs with the possibility of combination with both HOTAIR and MLXIPLHOTAIR bound to SRSF1..A, Venn diagram showed the RBPs that had the potential to combine with both HOTAIR and MLXIPL. 8 kinds of RBPs which was reported to be expressed in hepatocytes. B, RIP assay was performed to detect the binding relationship between 8 different RBPs (IGF2BPs, SRSF1, KHSRP, PTBP1, FUS, SOX2, UPF1, WDR4) and R. HOTAIR. ***P< 0.001.

Supplementary file2 (TIF 7067 KB)

Data Availability Statement

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

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