MiR-135a is highly expressed and aggravates inflammatory response in sepsis by targeting MYOM1

Background : Our research attempted to explore the effect of miR-135a on lipopolysaccharide (LPS)-induced THP-1cells damage and its potential mechanism. Methods : LPS (1 μg/mL) was selected to mimic the injury of sepsis in vitro . qRT-PCR was used to detect miR-135a expression. The association between miR-135a and My- omesin 1 (MYOM1) was speculated bythe predication website and confirmed by the dual-luciferase assay. MYOM1 expression was observed by qRT-PCR and western blotting assays. The biological properties of THP-1cells were analyzed by cell counting kit-8 and flow cy- tometry assays. The concentration of TNF-α, IL-6 and IL-8 in cell supernatant was calculated by enzyme-linked immunosorbent assay. Results : A marked augmentation of miR-135a was observed in LPS-induced THP-1 cells. Moreover, depletion of miR-135a alleviated the LPS-in-duced THP-1 cells injury from the perspective of improving cell viability and reducing cell apoptosis. Important-ly, MYOM1, which was under expressed in LPS-induced THP-1 cells, was identified as a target of miR-135a and negatively regulated by miR-135a. Additionally, the mitigative impact of miR-135a inhibitor on THP-1cells damage triggered by LPS was suppressed by MYOM1 depletion. Conclusions : Our study suggested that the protective effect of miR-135a inhibitor on LPS-induced THP-1 cells injury was realized by regulation of MYOM1, which might afford a pair of novel molecules for sepsis clinical diagnosis.


INTRODUCTION
Sepsis is a severe disease, which originated from an infection and a systemic inflammatory reaction (Melvan et al., 2010). Sepsis, one of the primary reasons of death in intensive care unit (ICU) patients, is characterized by the dysfunction of an organ and systemic inflammation reaction syndrome (Zhou et al., 2016). Despite the presence of 170 different biomarkers, the complex clinical syndrome of sepsis and associated mortality have elevated morbidity (Silman, 2013). Therefore, more effective biomarkers need to be explored.
MicroRNAs (miRNAs), as non-coding RNA molecules with 17-22 nucleotides, can block mRNA expression and suppress the translation by directly modulating the specific mRNA (Mohr & Mott, 2015;Simonson & Das, 2015). Importantly, miRNAs have been reported to be implicated in multiple biological advances (Iorio & Croce, 2012;Catela Ivkovic et al., 2017;Zendjabil et al., 2017). Some reports claimed that miRNAs were tightly connected with the development of sepsis and they might serve as an important therapeutic target for sepsis (Benz et al., 2016;Kingsley & Bhat, 2017). For example, Zhou and others (Zhou et al., 2017) discovered that miR-155 suppressed the cardiac dysfunction caused by sepsis through targeting JNK. Additionally, miR-205-5b promoted the development of sepsis triggered by LPS through targeting HMGB1 (Zhou et al., 2016). Moreover, has been reported to promote the intestinal barrier dysregulation in sepsis via targeting SP-D and activating the NF-κB pathway (Du et al., 2020).
MiR-135a has been found to participate in a variety of diseases (Zhang et al., 2019). For example, exhaustion of miR-135a alleviated the injury of acute pancreatitis via targeting FAM129A (Zhang et al., 2019). Additionally, miR-135a has been noted for its pivotal role in various cancers. MiR-135a inhibited the malignant proliferation and diffusion of non-small cell lung cancer cells by reducing ROCK1 protein. Besides, miR-135a suppressed the invasion and migration of esophageal cancer stem cells via targeting Smo. Importantly, miR-135a was reported to be highly expressed in the serum of sepsis patients and aggravated the myocardial depression in sepsis . However, the effect of miR-135a on the biological behaviors of cells in sepsis and its underlying mechanism is not revealed.
In this study, we observed that miR-135a expression was increased in LPS-induced THP-1cells. Depletion of miR-135a suppressed the damage of THP-cells triggered by LPS. Moreover, we identified Myomesin 1 (MYOM1) as a target of miR-135a and down-regulated in LPS-induced THP-1cells. Importantly, we also discovered that knockdown of MYOM1 inhibited the alleviation effect of miR-135a inhibitor on THP-1 cells damage triggered by LPS. Collectively, our findings insinuated that miR-135a contributed to the progression of sepsis by targeting MYOM1, which afford the foundation for the targeted therapy of sepsis.

Cell culture and transfection
The THP-1 cells were obtained from American Type Culture Collection (ATCC, Manassas, UAS). The cells were cultured in DMEM medium including 10% fetal bovine serum (FBS) and antibiotic in a humidified atmosphere with 5% CO 2 , at a 37°C incubator. The model was established by continuous stimulation with lipopolysaccharide (LPS) for 12 h.

Western blotting
The treated cells were lysed in lysis buffer. The total protein concentration was measured by the bicinchoninic acid (BCA) method. Afterwards, the same amount of protein (20 μg) was isolated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with non-fat milk and incubated overnight at 4°C against the following specific primary antibodies: MYOM1 (Abcam, Ab201228, 1:1000) and GAPDH (Abcam, Ab181602, 1:5000). This was followed by incubation with secondary antibodies. Finally, the blots were detected by enhanced chemiluminescence and analyzed by Image J software.

Determination of cell viability
Cell counting kit-8 (CCK-8) agent was implemented to assess cell viability. First, 1×10 3 cells were seeded into a 96-well plate and incubated overnight. At per time point (0 h, 24 h, 48 h, 72 h), 15 μL CCK-8 (Beyotime, Shanghai, China) reagent was put into per well and cultured for another 1.5 h at 37°C. Absorbance at 450 nm was detected by a microplate reader.

Enzyme-linked immunosorbent assay (ELISA)
The amount of TNF-α, IL-6 and IL-8 secreted into the culture supernatants was detected by using ELISA kit (Beyotime, Shanghai, China) based on the supplier's guidance.

Cell apoptosis detection
Flow cytometry was adopted to measure cell apoptosis with the support of an Annexin V/Propidium Iodide (PI) Apoptosis Detection Kit (Bytotime, Shanghai, China). First, THP-1 cells were collected, washed, resuspended in Annexin V binding buffer, and then stained with Annexin V and PI reagent. After gently vortex and cultivation away from the light at 37°C for 12 min, the cell apoptosis was detected by flow cytometry.

Plasmid Construction
pGL3-Basic vectors were obtained from Promega. Mutations of the miR-135a binding site of wild-type pGL3-MYOM1 plasmids were carried out using a Mut Express II Fast Mutagenesis kit following the manufacturer's instructions (Vazyme Biotech, Nanjing, China, C214-01). All constructs were confirmed by sequencing.

Dual-luciferase reporter assay
The association between miR-135a and MYOM1 was verified by dual-luciferase reporter assay. First, the wild type (wt) or mutant type (mut) of MYOM1was cloned into the pGL3 vector. Then, plasmid DNA (wt-MYOM1 or mut-MYOM1) and miR-135a inhibitor or negative control (NC) were co-transfected into cells. The luciferase activity was measured by using a double-luciferase reporter assay kit (Promega, Madison, USA) based on the supplier's instruction, which was normalized to firefly luciferase activity.

Statistical analysis
All experiment values were presented as mean ± standard deviation (S.D.) of at least three independent experiments. All statistical analyses were implemented by SPSS 22.0 and GraphPad Prism 5.0. The student's t-test was used to perform the difference between the two groups. Comparisons between two groups were implemented by one-way ANOVA followed by Dunnett's post hoc test. p<0.05 was deemed as statistically significant.

A marked augmentation of miR-135a was presented in LPS-induced THP-1 cells
To detect the impact of miR-135a in sepsis, an in vitro model was established. By analysing the viability of THP-1cells with different concentration of LPS stimulation, 1 μg/mL LPS was selected to mimic the injury of sepsis in vitro (Fig. 1A). Next, the data from qRT-PCR showed that the expression level of miR-135a is dose-dependently upregulated in response to LPS in THP-1 cells (Fig. 1B). To further investigate the impact of miR-135a in THP-1cells, we used the miR-135a inhibitor to reduce its expression. The data from Fig. 1C-D illustrated that miR-135a expression was significantly reduced after miR-135a inhibitor transfection, which provided a foundation for the following experiments.

Knockdown of miR-135a alleviated the damage of THP-1 cells induced by LPS
Thereafter, a series of biological experiments were adopted to evaluate the effect of miR-135a on LPS-induced THP-1cells. First, we observed that the reduced THP-1 cells viability caused by LPS stimulation was suppressed by miR-135a treatment ( Fig. 2A). Moreo-MiR-135a in sepsis by targeting MYOM1 ver, the strengthened apoptosis ability of THP-1cells caused by LPS was impaired through miR-135a inhibitor stimulation (Fig. 2B-C). Furthermore, the observation from ELISA kit showed that the increasing concentration of TNF-α, IL-6 and IL-8 in LPS-induced THP-1cells was reduced after miR-135a inhibitor treatment (Fig. 2D-F). All these findings suggested that depletion of miR-135a alleviated the damage of THP-1cells induced by LPS.

MYOM1 was confirmed as a target of miR-135a and down-regulated in LPS-induced THP-1 cells
To further explore the mechanism of miR-135a on LPS-induced THP-1 cells injury, the downstream target of miR-135a was excavated. By bioinformatics analysis, MYOM1 was predicted as a target of miR-135a. The targeting sites were exhibited (Fig. 3A). Then, the dual -uciferase assay exhibited that miR-135a inhibitor treatment elevated the luciferase activity of the wt MYOM1 group. But the change of luciferase activity in mut MYOM1 group was no statistical significance (Fig. 3B). In addition, we observed that MYOM1 was under expressed in THP-1 cells triggered by LPS. Furthermore, MYOM1 expression was obviously increased in LPS-induced THP-1 cells after miR-135a inhibitor stimulation (Fig. 3C-E). The whole findings indicated that a negative association existed between miR-135a and MYOM1.

The alleviation effect of miR-135a inhibitor on THP-1 cells injury can be inhibited by MYOM1 depletion
Based on the above observations, we performed biological experiments to investigate the function of miR-135a and MYOM1 on LPS-induced THP-1 cells damage. Firstly, we performed a qRT-PCR assay and western blot assay to investigate the expression of MYOM1 after being transfected with si-MYOM1. The data showed that the expression level of MYOM1 is significantly downregulated both in mRNA level and protein level after being transfected with si-MYOM1 ( Fig. 4A and 4B). We discovered that the elevated viability of THP-1 cells caused by miR-135a inhibitor in LPS condition was suppressed by MYOM1 knock-   down (Fig. 4C). Then, the reduced apoptotic ability of THP-1cells created by miR-135a inhibitor was inhibited by MYOM1 depletion in LPS treatment (Fig. 4D-E). Additionally, we also observed that the decreased trend of TNF-α, IL-6 and IL-8 concentration in THP-1cells caused by miR-135a inhibitor was reversed by MYOM1 exhaustion (Fig. 4F-H). These results suggested that the function of miR-135a on LPS-induced THP-1cells was realized by targeting MYOM1.

DISCUSSION
Sepsis is a fearful clinical disease due to the host's inflammatory response to the infection (Xu et al., 2020). Herein, we reported that miR-135a expression was obviously increased in LPS-induced THP-1cells. Exhausting miR-135a increased the proliferative ability of THP-1 cells, as well as alleviated the apoptosis ability and inflammatory response. Moreover, the downstream effector of miR-135a was identified as MYOM1, which presented a lower expression in LPSinduced THP-1 cells. The alleviation effect of miR-135a inhibitor on THP-1 cells damage triggered by LPS was suppressed by depletion of MYOM1. The observations suggested that the function of miR-135a on THP-1 cells was achieved by targeting MYOM1.
MiR-135a has been illustrated to be abnormally upregulated and therefore could be a meritorious prognostic biomarker in numerous human diseases such as glioma, primary nasopharyngeal carcinoma and Parkinson's disease (Tang et al., 2014;Liu et al., 2016;Cheng et al., 2017). Additionally, a marked augmentation of miR-135a was presented in the serum of a patient with sepsis . Interestingly, we also discovered that miR-135a expression was obviously increased in THP-1 cells with LPS stimulation. Furthermore, we illustrated that knockdown of miR-135a attenuated the injury of THP-1 cells triggered by LPS from the perspective of augmentation of cells proliferation, as well as reduction of cells apoptosis and inflammatory response. Moreover, we identified the downstream target of miR-135a in this model. Therefore, we not only revealed a marked augmentation of miR-135a presented in the in vitro model of sepsis, but also discovered the influence of miR-135a on the biological behaviors of THP-1 cells and the potential downstream mechanism.
It is well documented that miRNAs exert functions in the development and progression of various cells through modulating the expression of specific targets (Vangoor et al., 2019). Thus, we investigated the underlying target gene, which mediates the role of miR-135a in LPS-induced THP-1 cells. Dual-luciferase assay illustrated that miR-135a inhibitor elevated the luciferase activity of cells cloned with wt MYOM1 3'UTR. MYOM1 encodes myomesin, a major binding partner of another sarcomeric protein Titin (Shamseldin et al., 2015). MYOM1, which was discovered to be down-regulated in various cancer, was related to the worse clinical outcomes in tumors . In addition, MYOM1 has been identified as one of the lethal genes in human embryos (Shamseldin et al., 2015). MYOM2, as an important paralog of MYOM1, has been reported to function in localized aggressive periodontitis by regulation of inflammatory processes (Sørensen et al., 2008). However, little research has been done on the role of MYOM1 in inflammation. In our study, we creatively discovered that MYOM1 was lower expressed in THP-1 cells triggered by LPS. Moreover, knockdown of MYOM1 suppressed the alleviation impact of miR-135a inhibitor on THP-1 cells injury triggered by LPS. The whole observation underscored the important role of MYOM1 in the in vitro model of sepsis.
The original model of sepsis was the immune response to endotoxin, an LSP discovered in the cell walls of Gram-negative bacteria (Ulevitch & Tobias, 1999). The occurrence of sepsis stimulated macrophages to produce TNF and ILs (Faix, 2013). These pro-inflammatory cytokines produced a systemic inflammatory response which was characteristic of early sepsis (Faix, 2013). Additionally, several reports have suggested that levels of IL-6 and IL-8 were all higher in patients who died from sepsis (Andaluz-Ojeda et al., 2012;Gouel-Chéron et al., 2012). In our study, we observed that the levels of TNF-α, IL-6 and IL-8 were raised in THP-1 cells with LPS treatment, which was in step with the previous report (Faix, 2013). However, depletion of miR-135a suppressed the levels of TNF-α, IL-6 and IL-8 in THP-1 cells. While knockdown of MYOM1 reversed the above results triggered by the miR-135a inhibitor, which indicates that function of miR-135a/MYOM1 on THP-1 cells was achieved by regulating the inflammatory response.
Despite several findings that have been made in our study, limitations in this research need to be revealed. Firstly, we only found these results in LPS-induced THP-1 cells, more results should be verified in vivo. Second, we just found the downstream target of miR-135a, the upstream regulator of miR-135a should be explored in future.

CONCLUSIONS
Depletion of miR-135a alleviated the LPS-induced THP-1 cells injury from the perspective of improving cell viability and reducing cell apoptosis. The mitigative impact of miR-135a inhibitor on THP-1cells damage triggered by LPS was suppressed by MYOM1 depletion. In conclusion, we proposed that the therapeutic exploitation of miR-135a might afford intriguing perspectives to prevent the progression of sepsis.