Silencing lncRNA MALAT1 can promote autophagy and migration of C28/I2 chondrocytes induced by nitroprusside

doi:10.21203/rs.3.rs-3129630/v1

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Silencing lncRNA MALAT1 can promote autophagy and migration of C28/I2 chondrocytes induced by nitroprusside

https://doi.org/10.21203/rs.3.rs-3129630/v1

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Objective: To explore the effect of metastasis-associated lung adenocarcinoma transcript (MALAT1) on the autophagy, migration ability and cell survival rate of C28/I2 chondrocytes induced by sodium nitroprusside (SNP).

Methods: C28/I2 chondrocytes were treated with 0 mM, 0.5 mM, 0.75 mM, and 1 mM SNP for 12 h; si-MALAT1 was introduced into C28/I2 chondrocytes by lipofection technology followed by treatment and SNP for 12 h. The cell survival rate was measured using the CCK-8 method ,and cell migration was observed through cell scratch assays.The expression levels of autophagy-related proteins was measured using western blotting.The expression level of the MALAT1 gene was verified through qRT-PCR experiments. The effects of different concentrations of SNP on autophagy in C28/I2 chondrocytes were observed using mono-dansyl cadaverine (montane sulfonyl cadaverine, MDC) staining under a fluorescence microscope.

Results: The results showed that the expression level of Beclin-1andATG5, cell migration rate, and cell survival rate all decreased significantly,The lowest expression level was observed in cells cultured with 1 mM SNP concentration; The formation of autophagosomes was minimal when cells were cultured at 1 mM SNP concentration by fluorescence microscopy. The expression level of the MALAT1 gene was significantly increased in C28/I2 chondrocytes treated with 1 mM SNP for 12 h. Silencing MALAT1 led to up-regulation of the expression levels of two autophagy-related proteins, ATG5 and Beclin-1 in C28/I2 chondrocytes induced by SNP, resulting in increased cell migration and cell survival.

Conclusion: SNP can inhibit the autophagy, cell migration ability and survival rate of C28/I2 chondrocytes. MALAT1 plays an important role in developing osteoarthritis (OA) by affecting SNP-induced autophagy, migration ability, and cell survival rate in C28/I2 chondrocytes. Therefore, MALAT1 could be a potential target for osteoarthritis treatment.

osteoarthritis

Autophagy

Human lung adenocarcinoma metastasis-related transcript

nitropuna

Osteoarthritis (OA) is a degenerative bone disease characterized by cartilage damage, joint synovial inflammation and subchondral bone remodeling (VINA et al. 2018; ABRAMOFF et al. 2020). The current treatment options for OA are not targeted and mainly focus on anti-inflammatory, analgesic, or articular cartilage replacement to slow disease progression. However, long-term use of anti-inflammatory and analgesic drugs can lead to drug dependence and complications, while articular cartilage replacement is not a permanent solution and eventually fails. At present, the pathogenesis of the disease is still not very clear, so exploring the pathogenesis of the disease has important practical significance and theoretical value.

Studies have confirmed that the accumulation of reactive oxygen species (ROS) is a significant factor in the development of OA, leading to oxidative damage in chondrocytes (BOLDUC et al. 2019; ANSARI et al. 2018). Nitroprusside (SNP), as a nitric oxide (NO) donor, can induce ROS production and activate the oxidative stress response, causing cellar damage. SNP-treated chondrocytes have been used as an in vitro OA cell model, exhibiting chondrocyte damage, reduced cell survival rate, and increased apoptosis (HUANG et al. 2021; XU et al. 2018). Autophagy, a degradation system, plays a critical role in maintaining intracellular homeostasis under conditions such as oxidative stress, hypoxia, and inflammation, which are all relevant to OA (XIA et al. 2014). Additionally, cell migration ability is associated with mitochondrial division, affected by the AMPK/Drp1 signaling pathway, thereby changing the dynamic balance of F/G-actin and impairing cell migration. Reduced cell migration can contribute to the progression of OA (TCHI et al. 2021; ZHENG et al. 2020). In this study, SNP was used as the inducer of an OA cell model to investigate its effects on autophagy, migration ability and cell survival rate of C28/I2 chondrocytes. Studies have found that lncRNA participates in the degradation of the extracellular matrix (ECM) in articular cartilage, inflammatory responses and inhibition of angiogenesis, thereby influencing the development of OA (YUDUSHKIN I. 2020; HUANG. 2020). MALAT1 is one specific LncRNA of interest, upregulated in various diseases and biological processes (FENG et al. 2019). It is highly expressed in many tumor tissues and can affect cell phenotypes such as autophagy and the cell cycle (YUE et al. 2021; SUN et al. 2019). Interestingly, studies have shown significantly upregulated expression of MALAT1 in OA tissues compared to normal cartilage tissues (WANG et al. 2021). However, the impact of MALAT1 on autophagy and migration ability of OA chondrocytes has not been reported. Therefore, this study aims to explore the effect of MALAT1 on autophagy and migration ability in SNP-induced C28/I2 chondrocytes and provide new insights and potential strategies for treating OA.

Cell culture and osteoarthritis chondrocytes induced by SNP

Human chondrocyte cell line C28/I2 was purchased from Huatuo Biotechnology Co, Ltd. (Shenzhen, China) and cultured in a high-glucose medium containing 10% fetal bovine serum, 1% streptomycin and penicillin in a 37°C, 5% CO2 cell incubator.

SNP crystals (Beyotime, Shanghai, China)were grounded into a powder, and 100 mg was weighed to prepare the SNP solution. Use complete medium for dissolving, and configure as 200 mM stock solution. The stock solution of SNP with concentrations of 0.5 mM, 0.75 mM, and 1 mM was prepared by diluting the stock solution with a complete medium. The C28/I2 chondrocytes were placed in different SNP solution concentrations and cultured at 37°C in an incubator containing 5% CO2. After 12 h, the cells were used for subsequent experiments, while untreated C28/I2 chondrocytes served as the control group.

Cell viability assay

Cell viability was measured using Cell Counting Kit-8 (NCM, Suzhou, China). C28/I2 cells were seeded into 96-well microtiter plates at a 5×103 cells/well density during the logarithmic growth period. A blank control was set up, and 100 µL of complete medium was added to each well, and complete medium containing 0.5 mM, 0.75 mM, and 1 mM SNP was added to the remaining wells, respectively. At least 3 replicate holes were made for each group. The 96-well plate was placed in an incubator containing 5% CO2 at 37°C for 12 h. After incubation, 10 µL of CCK-8 solution was added to each well and incubated for 1.5 h. The absorbance values at 450 nm and 600 nm were measured with a microplate reader. The absorbance value after 450 nm-600 nm was used as the final absorbance value.

Monodansylcadaverine (MDC) staining

MDC staining was performed for autophagy detection (Beyotime, Shanghai, China). The culture medium was discarded and the cells were washed with PBS. MDC staining solution (1 mL) was added to each well and incubated at 37°C for 30 min. Cells were washed five times with Assay Buffer. Discard Assay Buffer and add 1 mL of Assay Buffer again. Observe autophagosome formation in each group under a fluorescent microscope. Positive cells were colored in their perinuclear region, cellular autophagy was observed, and all acidic vacuoles were stained.

Western blotting detection of ATG5, Beclin-1 autophagy-related proteins

Total protein from cultured chondrocytes was extracted with RIPA Reagent (Beyotime, Shanghai, China) and analyzed by Western blotting. The concentrations of total protein were measured with BCA reagent 20 µg total protein was loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and after 120 min, proteins were transferred onto a polyvinylidene fluoride membrane, which was blocked with 5% nonfat milk for another 1 h at room temperature.Then, membranes were incubated with primary antibody against ATG5(1:1000 dilution), Beclin-1 (1:1000 dilution), β-actin (1:1000 dilution) overnight at 4°C.

β-actin was used as an internal loading control. After being incubated with horseradish peroxidase-conjugated secondary antibody(1:5000 dilution) at room temperature for 1 h, the membranes were visualized using enhanced chemiluminescence assay on a chemiluminescence instrument. The quantification analysis was conducted using Image J 1.8.0.

RNA isolation and quantitative real-time PCR

The total RNA of chondrocytes was extracted using TRIzol reagent (CW BIO, Jiangsu, China).RNA quality and quantity were assessed using a Nano Drop 2000 spectrophotometer. Reverse transcription was done to reverse RNA into cDNA using a reverse transcription kit (TOTOBO, Shanghai, China). cDNA amplification was performed on an Applied Biosystems 7500 Real-Time Fluorescent Polymerase Chain Reaction System using the Premix Ex Taq™ II Kit (TaKaRa, Dalian, China). The reaction system (20 µL) included 10 µL of SYBR TB Green Premix Ex Taq II, 1.6 µL of Forward and Reverse Primer Mix (10 µM), 2 µL of cDNA (1 µg), 0.4 µL of ROX Reference Dye II and 6 µL of sterilized water. The real-time quantitative polymerase chain reaction program is: react at 95°C for 1min. React at 95°C for 5 s, 60°C for 30 s, 72°C for 30 s, and experience 40 cycles. React at 4°C for 10 minutes. The relative expression of MALAT1 was calculated using the 2 − ΔΔCT method, and the U6 gene was used as the reference gene. Primers were as follows: MALAT1: 5 '-GCCTGGAAGCTGAAAAACGG-3 ' (forward)and 5'- TGGAAAACGCCTCAATCCCA-3 ' (Reserve); U6: 5 '- GCTTCGGCAGCACATATACTAA-3 ' (forward)and 5 '- CGAATTTGCGTGTCATCCTT-3 ' (Reserve);

Transient transfection

HANBIO (China) provided small interfering RNAs (siRNAs) against MALAT1 and control siRNAs. Sequences were as follows: si-MALAT1, 5'-GAGGUGUAAAGGGAUUUAUTT-3'. Using LipoFiter 3.0 (HANBIO,Shanghai,China), cell transfection was performed and results analyzed 48 h later .

Cell migration experiment

The cell migration ability was tested with a cell scratch experiment. As soon as the cells reached the logarithmic growth phase, the cell density was adjusted to 5×104 cells/mL and the cells were seeded into 6-well plates and cultured for 12 h. After the cells were treated differently according to the needs of the experiment, the culture medium was discarded, and a 200 µL gun tip was used to scratch the bottom of the plate against the ruler to form cell scratches. We observed the migration of cells by using an inverted microscope at 0 and 12 h after scratching the cells. In Image-Pro Plus 6.0, the gap area was quantified and the migration percent was calculated.

Statistical analysis

A median and a standard deviation (SD) are used to express all experimental data. GraphPad Prism 8.3.0 was used to perform the statistical analysis. The t-test was used for comparisons between two groups, while the one-way ANOVA was used to compare multigroups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

SNP can affect autophagy, migration and cell survival of C28/I2 chondrocytes

In this study, we aimed to establish in vitro OA cell model by inducing injury to C28/I2 chondrocytes by administering a specific concentration of SNP. Our found that compared with the control group, the cell migration rate was significantly reduced (P < 0.01) (Fig. 1A). Furthermore, cell viability analysis using the CCK-8 assay showed a significant reduction in cell survival rate after 12 h of cell culture with 1 mM SNP, the cell survival rate reached about 50% (P < 0.01) (Fig. 1B). To investigate the effect of SNP on autophagy, the expression levels of autophagy-related proteins ATG5 and Beclin-1 were measured by western blot analysis. The results demonstrated a gradual decrease in the expression levels of ATG5 and Beclin-1 with the increasing concentrations of SNP, with the lowest expression observed in cells cultured with 1 mM SNP concentration (Fig. 1C). Additionally, the formation of autophagosomes was evaluated by MDC staining. The results showed a decrease in the number of autophagosomes with the increase in drug concentration, indicating that the phenomenon of autophagy gradually weakened, which was consistent with western Blot findings (Fig. 1D). Based on these results, it is evident that a specific concentration of SNP induces damage to C28/I2 chondrocytes, leading to reduced cell migration ability and impaired autophagy. To construct a stable in vitro OA cell model, we selected a drug concentration of 1 mM SNP for subsequent experiments.

MALAT1 is upregulated in SNP-induced chondrocytes

The results found that compared with the NC group, the expression level of MALAT1 in the SNP group was significantly higher than that in the NC group. The expression level was significantly upregulated (P < 0.01), indicating that the SNP can promote the expression of MALAT1 in C28/I2 chondrocytes. Moreover, after transfection of si-MALAT1 and negative control (si-NC) into C28/I2 chondrocytes and observed a significant down-regulation of MALAT1 expression in the SNP-treated cells transfected with si-MALAT1 compared with the untransfected group, and the difference was statistically significant (P < 0.01) (Fig. 2).

MALAT1 knockout promotes SNP-induced autophagy in C28/I2 chondrocytes

The expression of autophagy-related proteins (ATG5, Beclin-1) in C28/I2 chondrocytes induced by SNP was inhibited.Silencing MALAT1 could significantly attenuate SNP-induced autophagy inhibition (Fig. 3A)

Knockdown of MALAT1 promotes SNP-induced migration and cell survival of C28/I2 chondrocytes

After the C28/I2 chondrocytes were induced by SNP, the cell migration ability and survival rate were reduced, and the above phenomenon could be reversed after knocking out the MALAT1 gene (Fig. 4A B).

OA is a severe joint disease that can cause pain, swelling, stiffness, and limited mobility, significantly affecting the quality of life of patients with OA (GOGUET-RUBIO et al. 2017). Previous studies have shown that nitric oxide (NO) donors can produce ROS and activate oxidative stress responses leading to cellular damage in chondrocytes, the cells responsible for maintaining cartilage. This chondrocyte cellular damage reduces cell survival and promotes cell apoptosis (HUANG et al. 2021; XU et al. 2018). In this study, the different concentrations of sodium nitroprusside (SNP) reduced the cell survival rate to about 50% after 12 h of administration. The 1 Mm concentration showed a significant decrease in cell survival, consistent with previous reports.

Furthermore, SNP treatment decreased the expression of autophagy-related proteins, specifically Beclin-1 and ATG5, indicating a reduction in autophagy initiation. IL-1β and LPS are standard drugs for constructing OA cell models in vitro, which can damage C28/I2 chondrocytes by reducing autophagy and inducing apoptosis (XU et al. 2022; JIA et al. 2022). However, whether SNP-mediated cellular oxidative damage can affect autophagy in C28/I2 chondrocytes has not been reported. In our study, we found that different concentrations of SNP could induce the decrease of the expression of autophagy-related proteins Beclin-1 and ATG5 in C28/I2 chondrocytes. Beclin-1 is the first autophagy-related gene discovered, which can directly participate in the formation of autophagy lysosomes, is a critical factor in the regulation of autophagy, and is generally considered a marker of autophagy initiation (VAN et al. 2007). Studies have found that Bcl-2 can bind to the BH3 domain of Beclin-1 so that Bcl-2 can be dissociated from Bax to induce apoptosis. At the same time, the complex formed by Beclin-1 and bcl-2 inhibits the formation of autophagy precursors, thereby reducing the occurrence of autophagy (LIANG et al. 1998). The literature has reported that NO can regulate the formation and degradation of autophagy by interfering with the activity of its C-Jun-N-terminal kinase 1. C-Jun-N-terminal kinase 1 can induce Bcl-2 phosphorylation by affecting the Bcl-2/Beclin − 1, and the formation and dissociation of the Bcl-2/Bax complex affect apoptosis and autophagy. When Bcl-2 is phosphorylated and dissociated from Beclin-1, autophagy is promoted, but Bcl-2 persists. Phosphorylation will inhibit the occurrence of autophagy (WEI et al. 2008). Studies have shown that SNP can promote the expression of the pro-apoptotic protein Bax in chondrocytes and inhibit the expression of Bcl-2 to promote the apoptosis of chondrocytes (LIN et al. 2018). Therefore, as an NO donor, SNP may promote the dissociation of the bcl-2/Beclin-1 complex by regulating the activity of C-Jun-N-terminal kinase 1, thereby promoting the apoptosis of C28/I2 chondrocytes and reducing autophagy. ATG5 is a key protein in the autophagy pathway; it combines with ATG12 to form a protein complex and autophagic vacuoles.

It has been reported that SNP can delay the wound healing of rabbit gastric epithelial cells by inhibiting cell migration and proliferation. These effects may be related to superoxide anion or peroxynitrite, not Cgmp-mediated (KIVILUOTO et al. 2001). Alfke H et al. (ALFKE et al. 2000) found that SNP inhibited primary fibroblast growth factor-stimulated migration of bovine vascular smooth muscle cells in vitro and excluded the side effects of superoxide-derived oxidation products. Zhan R et al. (ZHAN et al. 2015) found that SNP promoted the migration ability of human keratinocyte cell lines through the cGMP signaling pathway and promoted cytoskeleton reorganization by up-regulating the expression of Rho-GTPase. We found that after stimulating C28/I2 chondrocytes with different concentrations of SNP for 12 h, the cell migration ability was reduced, and the cGMP signaling pathway may play a significant role. Still, the exact molecular mechanism needs further research to confirm.

Studies have found that lncRNA participates in the degradation of articular cartilage ECM, inflammation, and inhibition of angiogenesis by regulating autophagy, migration, proliferation, and apoptosis of articular chondrocytes or synoviocytes, thereby affecting the occurrence and development of OA (YUDUSHKIN I. 2020; HUANG. 2020). With the maturity of high-throughput sequencing technology, many lncRNAs differentially expressed in lesioned cartilage tissue have been screened out. However, we have only elucidated a small part of the roles of these differentially expressed lncRNAs, and we need a deeper understanding.

lncRNA MALAT1 was initially discovered in the metastasis-related genes of human non-small cell lung cancer and is involved in various diseases and biological processes. The expression of lncRNA MALAT1 is usually up-regulated in multiple cancers, and up-regulation can promote cell proliferation and inhibit cell apoptosis. In contrast, down-regulation can promote cell apoptosis and inhibit cell proliferation (ZHANG et al. 2019). Studies have reported that the expression of lncRNA MALAT1 is upregulated in glioma stem cells, which enhances the activity and proliferation of cells and promotes the occurrence of glioma tumors (XIONG et al. 2018). In recent years, it has been found that lncRNA MALAT1 can regulate cell autophagy and migration; for example, it is upregulated in breast cancer and promotes autophagy and migration of breast cancer cells (SHIH et al. 2021; XU et al. 2022). Yaxian Song et al. (SONG et al. 2019) studied the expression of lncRNA MALAT1 and found it was upregulated in high glucose-treated endothelial cells. Further studies found that lncRNA MALAT1 affects the expression of NLRP3 by competitively binding miR-22 and promotes elevated glucose-induced endothelial cell apoptosis. However, the biological role of lncRNA MALAT1 in OA is still unclear. It has been reported in the literature that the expression of MALAT1 is up-regulated in OA patient cartilage tissue and SNP-induced chondrocytes, inhibits the viability of chondrocytes, and promotes the degradation of the extracellular matrix of cartilage. When the MALAT1 gene is knocked out, the viability of chondrocytes can be improved (LIU et al. 2019). However, the literature has also reported that the knockout of the MALAT1 gene can inhibit the viability and proliferation of chondrocytes through the PI3K/Akt pathway (LIANG et al. 2018). Our study found SNP-induced upregulation of MALAT1 expression in chondrocytes, consistent with previous reports. However, the difference is that when the MALAT1 gene is knocked out, the survival rate of chondrocytes is improved.

The factors that regulate autophagy in chondrocytes are still not very clear. We have shown that SNPs inhibit autophagy in C28/I2 chondrocytes, and silencing of MALAT1 reverses this trend. The results indicated that MALAT1 was involved in SNP-mediated autophagy in C28/I2 chondrocytes. Studies have found that MALAT1 is also highly expressed in hippocampal neurons of epileptic rats, and the downregulation of MALAT1 activates the PI3K/Akt signaling pathway to protect hippocampal neurons of epileptic rats from autophagy and apoptosis (Wu et al. 2018). Hu H et al. (HU et al. 2019) simulated cardiomyocyte injury with hypoxic injury stimulation and found that MALAT1 expression was increased and cardiomyocyte apoptosis was enhanced by inhibiting autophagy by regulating TSC2-mTOR signaling. It has been reported that the PI3K/Akt/mTOR signaling pathway is closely related to its regulatory effect on OA, and MALAT1 may target the PI3K/Akt/mTOR signaling pathway to regulate SNP-mediated autophagy in C28/I2 chondrocytes.

The literature has reported that when using NO donors to construct oxidatively damaged cells, the concentration needs to reach 0.5–1 mM (KRONCKE et al. 1997). This study found that the cell survival rate reached about 50% when the drug concentration was 1 mM, combined with ATG5 and Beclin-1, two autophagy-related proteins, and cell migration. Finally, it used a 1 mM drug concentration to construct an in vitro OA cell model for subsequent experiments. Many lncRNAs have been shown to play a key role in OA, and we confirmed for the first time that lncRNA MALAT1 plays an essential role in the process of OA by affecting cell autophagy and migration. Our findings provide a basis for studying the exact molecular mechanism of the role of lncRNA MALAT1 in OA and developing new strategies for treating this disease. The multiple effects of lncRNA MALAT1 on chondrocytes indicate that the regulatory network of OA is highly complex, and this gene is expected to be an effective target for the treatment of OA. Finally, animal experiments need to verify further the effect of lncRNA MALAT1 on chondrocytes in vivo.

SNP can inhibit autophagy and reduce the cell migration ability of C28/I2 chondrocytes.lncRNA MALAT1 plays an important role in the process of OA by affecting the autophagy and migration abilities of C28/I2 chondrocytes.

Funding

This work was supported by [Natural Science Foundation of Heilongjiang Province] and [Basic Scientific Research operating expenses of Heilongjiang provincial universities] and [Mudanjiang Medical College graduate tutor research program] (Grant numbers [LH2022H100] and [2022-KYYWF-0689] and [YJSZX2022077]).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Kai Chen], [Junzi Cong] and [Li Tang]. The first draft of the manuscript was written by [Hong Jin] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Silencing lncRNA MALAT1 can promote autophagy and migration of C28/I2 chondrocytes induced by nitroprusside

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Abstract

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Introduction

Materials and methods

Cell culture and osteoarthritis chondrocytes induced by SNP

Cell viability assay

Monodansylcadaverine (MDC) staining

Western blotting detection of ATG5, Beclin-1 autophagy-related proteins

RNA isolation and quantitative real-time PCR

Transient transfection

Cell migration experiment

Statistical analysis

Results

SNP can affect autophagy, migration and cell survival of C28/I2 chondrocytes

MALAT1 is upregulated in SNP-induced chondrocytes

MALAT1 knockout promotes SNP-induced autophagy in C28/I2 chondrocytes

Knockdown of MALAT1 promotes SNP-induced migration and cell survival of C28/I2 chondrocytes

Discussion

Conclusions

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References

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