MicroRNA-143-5p targeting eEF2 gene mediates intervertebral disc degeneration through the AMPK signaling pathway
Intervertebral disc degeneration (IDD) is a major contributor to back, neck, and radicular pain, and the treatment of IDD is costly and relatively ineffective. Dysregulation of microRNAs (miRNAs) has been reported to be involved in IDD. The purpose of our study is to illustrate the potential that miR-143-5p targeting eEF2 gene mediates IDD.
Following the establishment of the IDD rat models, expression of miR-143-5p, eEF2, Bcl-2, Bax, AMPK, mTOR, cyclinD, COL2, ACAN, and DCN was detected. The NP cells isolated from degenerative intervertebral disc (IVD) were introduced with a series of mimic, inhibitor, or AICAR to explore the functional role of miR-143-5p in IDD and to characterize the relationship between miR-143-5p and eEF2. Cell viability, cell cycle, apoptosis, and senescence were also evaluated.
A reduction in eEF2, an increase in miR-143-5p, and activation of the AMPK signaling pathway were observed in degenerative IVD. Moreover, increased senescent NP cells were observed in degenerative IVD. eEF2 was confirmed as a target gene of miR-143-5p. miR-143-5p was found to activate the AMPK signaling pathway. The restoration of miR-143-5p or the activation of AMPK signaling pathway decreased COL2, ACAN, and DCN expression, coupled with the inhibition of NP cell proliferation and differentiation, and promotion of NP apoptosis and senescence. On the contrary, the inhibition of miR-143-5p led to the reversed results.
The results demonstrated that the inhibition of miR-143-5p may act as a suppressor for the progression of IDD.
KeywordsMicroRNA-143-5p EEF2 AMPK signaling pathway Nucleus pulposus cells Differentiation Apoptosis Senescence
Analysis of variance
B cell lymphoma-2
Bovine serum albumin
Disc height index
Dual luciferase reporter
Eukaryotic elongation factor 2
Enzyme-linked immunosorbent assay
Fetal bovine serum
Human hypertrophic scar fibroblasts
Intervertebral disc degeneration
Low back pain
Magnetic resonance imaging
Mammalian target of rapamycin
Phosphate buffered saline
Rate of disc height change
Reverse transcription quantitative polymerase chain reaction
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Intervertebral disc degeneration (IDD) is a common complex disease of the spine, leading to musculoskeletal disability and poor quality of life for patients [1, 2]. IDD is often accompanied with low back pain (LBP), radiculopathy, or myelopathy, and its lifetime incidence rate is greater than 90% . Prominent changes occur during IDD, including loss of extracellular matrix, altered phenotype of normal disc cells, and the release of pro-inflammatory cytokines . It is interesting to note that nucleus pulposus (NP) was responsible for maintenance of disc function and structure . The accelerated apoptosis and senescence of NP cells were found to be a possible cause for IDD . Protection against NP cell apoptosis and senescence may be conducive for the amelioration of IDD [7, 8]. A previously conducted study demonstrated that reinsertion of activated NP cells can delay the process of disc degeneration . This prompted us to improve understanding of the biology of the intervertebral disc (IVD) healing and to identify strategies to enhance the regenerative process. In previous investigations, microRNAs (miRNAs) were reported for their deregulation in NP cells, with effect on the proliferation and apoptosis of NP cells, or their relationship with breakdown of balance between (ECM) synthesis and degradation in NP cells in IDD [10, 11, 12].
MiRNAs can downregulate the gene expression by targeting mRNAs for translational repression and/or cleavage, which enables them critical roles in cellular processes such as proliferation, invasion, apoptosis, and senescence . A previous study has confirmed that miR-143-5p plays a significant role in epithelial-mesenchymal transition (EMT) and metastasis of gallbladder cancer . Besides, it has also been proven to involve in the progression and prognosis of cervical cancer . Furthermore, recent evidence has revealed that miR-143 is upregulated in degenerative disc tissues . A study conducted by Mu et al. found that miR-143 affects proliferation and apoptosis of human hypertrophic scar fibroblasts (HSFs) and inhibits ECM production-associated protein through suppression of the Akt/mTOR pathway . As was previously reported, eukaryotic elongation factor 2 (eEF2) is a target of mTOR , and thus it was speculated that miR-143 could modulate eEF2. Furthermore, eEF2 has been predicted to be a target of miR-143-5p by using the online prediction program. The eEF2 gene has been proven to be a common calcium/calmodulin (Ca/CaM)-dependent Ser/Thr-kinase that is activated by mitotic agents involved in cell proliferation and apoptosis . eEF2 kinase (eEF2K), an enzyme that inactivates eEF2, is activated by AMP-kinase (AMPK) and contributes to cell survival [20, 21]. Tumor cells exploit this pathway to adapt to nutrient deprivation via reactivating the AMPK-eEF2K signaling pathway . Recently, Wang et al. have provided evidence showing that resveratrol has the ability to promote NP cell autophagy via activation of AMPK signaling pathway, thus acting as a novel preventive role in IDD . Based on the aforementioned literature, a hypothesis can be drawn that miR-143-5p is involved in IDD via the AMPK signaling pathway by regulating eEF2. In this present study, the functions of miR-143-5p on NP cells of IDD rats were explored, as was the underlying mechanism involving the AMPK signaling pathway.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize suffering of the animals. The experimental procedures were approved by the Animal Ethics Committee of Taihe Hospital.
Fifteen male Lewis rats, weighing 297 to 323 g and aged 12 to 14 weeks, were purchased from the Laboratory Animal Research Center of Southern Medical University (Guangzhou, China). A week before treatment, the rats were housed with free access to water and food, under a 12-h light/dark cycle with the environment at 22~24 °C with the humidity of 50~60%.
Establishment of IDD model
IDD models were developed as previously reported . In brief, 15 male Lewis rats aged 12~14 weeks were selected for the experiments. Rat coccygeal vertebrae (Co6/Co7 and Co7/Co8) were punctured using the 18-G needle, in order to establish the model of IDD. Besides, the rats (Co8/Co9) without puncture were regarded as the control group. The central NP (nucleus pulposus), 5 mm distant from the skin, was the object of puncture. The pinpoint was rotated for 360° and kept for 30 s. Images of X-ray and magnetic resonance imaging (MRI) were obtained of every rat coccygeal vertebra before the puncture and in the second week and the fourth week after the puncture. The disc height index (DHI) (%) and rate of disc height change (RDHC) were calculated . The degeneration degree of caudal IVD of rats was graded as follows: I was normal, II was slight IDD, III was moderate, and IV was serious , and the rate of IDD was calculated as previously described . All IDD grades and imaging analysis were conducted by three independent surgeons, twice respectively so as to evaluate the reliability of the rating system. The rats with typical IDD, which was confirmed through imaging, were all euthanatized by CO2 inhalation 4 weeks later. IDD specimen, obtained after dissecting the rats, was used for hematoxylin-eosin (HE) staining and immunohistochemistry.
Parts of degenerative IVD tissues (Co6/Co7 or Co7/Co8) and normal IVD tissues (Co8/Co9) were extracted for the HE staining. The target IVD tissues (Co7-Co8 and Co8-Co9) and adjacent caudal vertebrae were excised, then fixed with 3% neutral formaldehyde, embedded with paraffin, and cut into 5-μm-thick sections. Following the cutting period, the tissue sections were dewaxed with xylene twice (each time for 5 min), then rehydrated with 100%, 95%, 80%, and 75% gradient ethanol respectively for 1 min and rinsed for 2 min under running water. Sections were then stained with hematoxylin for 2 min and rinsed with running water for 10 s, followed by color separation with 1% hydrochloric acid ethanol for 10 s. Then sections were then rinsed by distilled water for 1 min and stained with eosin for 1 min. Following the eosin staining periods, sections were dehydrated by 95% and 100% ethanol for two times (each time 1 min) after being washed by distilled water for 10 s. Finally, sections were permeabilized by xylene and then mounted by neutral balsam.
Safranin O staining
The tissues were fixed with 4% paraformaldehyde, dehydrated, permeabilized, and then paraffin embedded. The paraffin-embedded tissues were sliced into 5-μm-thick sections. After this, the tissue sections were dewaxed and stained conventionally using hematoxylin and eosin. Following that, the sections were stained with 0.5% Safranin O for 5 min. After washing under distilled water, the tissue sections were dehydrated twice using 95% and 100% gradient ethanol (2 min each time). After that, the sections were permeabilized using xylene and sealed with neutral balsam.
Degenerative IVD and normal IVD tissues were respectively fixed with 10% formalin, embedded into paraffin, and cut into 4-μm sections. The tissue sections were then baked at 60 °C for 20 min; dewaxed with xylene I, xylene II, and xylene III (10 min for each); immersed in absolute ethanol I, absolute ethanol II, and 90% water-ethanol (2 min for each); treated with 3% H2O2 for 15 min; and then washed by distilled water three times (each time for 2 min). The tissue sections then underwent microwave antigen retrieval. Following the antigen revival period, the sections were immersed in the 0.01 M citrate buffer solution (pH 6.0) and heated in a microwave oven until boiling. After 5 min, sections were further heated until boiling. After another 5-min period, sections were cooled down to room temperature. Then, the sections were blocked with 10% bovine serum albumin at 37 °C for 30 min. After the surplus serum was discarded, primary antibody to eEF2 (1:2000, ab75748, Abcam, Cambridge, MA, USA) was added into sections until they were completely covered, and incubated at 4 °C overnight. After that, sections were incubated at 37 °C for 1 h and then washed by 0.02 M phosphate buffered saline (PBS) three times (each time for 5 min). After being washed via PBS, sections were added with secondary antibody and goat anti-rabbit immunoglobulin G (IgG) (DF7852, Shanghai Yao Yun Biotechnology Co., Ltd., Shanghai, China) for incubation at 37 °C for 30 min, and washed with 0.02 M PBS three times (each time for 5 min). Sections were added with a horseradish peroxidase-labeled streptomycin avidin reagent, incubated at 37 °C for 30 min, and then washed with 0.02 PBS three times (each time for 5 min). Following that, sections were developed by diaminobenzidine (DAB) under conditions void of light, which was controlled under the microscope for 5 min, and then fully washed by distilled water. After being washed by distilled water, sections were counterstained with hematoxylin for 1 min, conventionally dehydrated by ethanol and permeabilized, and sealed with neutral gum. The staining was observed under a microscope, with the brownish yellow particles regarded as positive. Five high-power visual fields (× 400) were randomly chosen from each section with 100 cells counted in each field. The mean values of percentage of positive cells in total cells were then calculated. The experiment was performed three times.
Following a successful model establishment for 4 weeks, 4 rats were given lethal doses of CO2, with the spine separated to expose normal and IDD spine under an aseptic condition. The fibrous ring was cut with a scalpel to separate gelatinous nucleus, which then was rinsed in sterile D-hanks three times to remove bloodstain. NP tissues were cut into blocks at a size of 1 × 1 × 1 mm and placed into a centrifuge tube. After that, the tube was added with twofold volume of 0.1% type II collagenase, placed in a water bath at 37 °C for digestion, and then shaken every 10 min. After the digestion period, the tissues were centrifuged at 179×g for 5 min with the supernatant removed, and detached with 10 U/mL hyaluronidase in a water bath for 2 h. Tissues were centrifuged at 179×g for 5 min and then washed by Dulbecco’s modified Eagle’s medium (DMEM)-F12 three times. Following the counting period, cells were inoculated into 25-cm culture flasks at 1 × 106, added with DMEM-F12 containing 100 U/mL streptomycin and 15% fetal bovine serum (FBS), and cultured in a cell incubator with 5% CO2 at 37 °C. The medium was changed after a week and then changed every 3 days. Following a 20-day period, when the cells were detached from the wall, a fibroblast region with long spindle and polygon-shaped cells was scrapped with cell scraper and then suspended in culture medium. Meanwhile, the circular and short shuttle-shaped NP cells were retained under a phase-contrast microscope. Afterwards, the flask was rinsed with culture medium two times and continually cultured. Until the circular and short shuttle-shaped NP cells turned into the clone population with single morphology, the cells were treated with 0.25% trypsin for resuspension, and then inoculated into another culture flask for further culture.
Cell transfection and grouping
Mimic or inhibitor sequence for cell transfection
Dual luciferase reporter gene assay
The target genes of miR-143-5p were analyzed by biology prediction website Targetscan (http://www.targetscan.org). HEK-293T cells (AT-1592, ATCC, Manassas, VA, USA) were plated into a 24-well plate and cultured for 24 h. The total RNA of cells was extracted and reversely transcribed into cDNA. The full-length sequence of eEF2 3′-UTR was obtained by polymerase chain reaction (PCR) amplification with cDNA as the template. According to the sequence of eEF2, the primers were designed (forward primer: 5′-ATGAGGGCAAGATGAAGCTG-3′, and reverse primer: 5′-ATGAAGGACGGGATGTTCAC-3′) and amplified with genome extracted from HEK-293T cells as a template. Following the amplification period, the primers were digested by enzyme and cloned into the downstream of pmiRRB carrier luciferase coding gene to obtain eEF2 dual luciferase reporter (DLR) vector, namely pmiRRB-eEF2-3′UTR, which was separately co-transfected with miR-143-5p vector, siRNA-miR-143-5p, or negative control into HEK-293T cells. After transfection for 48 h, the culture medium was aspirated, and cells were rinsed twice with PBS. Then cells were then collected and lysed. The luciferase activity was measured with Dual-Luciferase® Reporter Assay System (E1910, Promega, Madison, WI, USA). A total of 50 μL of firefly luciferase working fluid was added into every 10-μL aliquot of cells to examine firefly luciferase activity. Then a 50-μL aliquot of renilla luciferase working liquid was added to examine renilla luciferase activity. The ratio of firefly luciferase activity to renilla luciferase activity represented the relative luciferase activity. The experiment was repeated three times.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Primer sequence for RT-qPCR
Western blot analysis
Total protein was extracted using a total protein extract kit (R0010, Beijing Solarbio Technology Co. Ltd., Beijing, China). The transfected cells were washed using the precooling PBS three times. The protein lysate (60% radio immunoprecipitation assay [RIPA] lysis buffer + 39% sodium dodecyl sulfate [SDS] + 1% protease inhibitors) was added into every cell bottle, and the mixture was lysed on ice for 30 min in the EP tube. Then cells were centrifuged at 32,608×g in the high-speed freezing centrifuge for 30 min at 4 °C, and the supernatant was collected and placed on the icebox to measure protein concentration using bicinchoninic acid (BCA) method. Subsequently, 10% separation gel and 5% spacer gel were prepared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) kit, and the protein was separated using electrophoresis on polyacrylamide gel. Following that, protein was transferred onto a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h and incubated with primary antibodies of rabbit anti eEF2 (1:10000, ab75748), p-AMPK (1:2000, ab133448), Bax (1:1000, ab32503), p-mTOR (1:5000, ab137133), cyclinD (1:10000, ab134175), Bcl-2 (1:1000, ab196495), t-AMPK (1:2000, ab32047), t-mTOR (1:2000, ab2732), and GAPDH (1:1000, ab9485). All the above antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). The following day, the membrane was incubated with the secondary antibody, goat anti-rabbit IgG (1:2000, ab205718, Abcam Inc., Cambridge, MA, USA) at 4 °C for 1 h. The proteins were visualized with a developing agent and imaged using a Bio-rad imaging system (MG8600, Beijing Thmorgan Biotechnology Co., Ltd., Beijing, China). Quantitative analysis was conducted using IPP7.0 software (Media Cybernetics, Silver Springs, MD, USA). The ratio of gray value of eEF2, p-AMPK, p-mTOR, Bcl-2, cyclinD, Bax, t-AMPK, and t-mTOR protein bands to that of the internal reference GAPDH protein band represented the protein levels of genes respectively. The procedures were also applicable for cell experiments.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The transfected cells in each group were incubated in RPMI 1640 culture medium containing 10% FBS based on the predetermined concentration with 5% CO2 at 37 °C for 48 h. Cells in the logarithmic growth phase were collected for the following experiment. The cell suspension was transferred into a centrifuge tube and then triturated evenly using a sterile straw. Single-cell suspension was stained by trypan blue staining solution. The number of living cells was counted. The living cells were seeded into a 96-well plate (180 μL of cells per well) at a density of 1 × 104 cells/well. Afterwards, cells were cultured at 37 °C with 5% CO2 for 16 to 48 h. Each well was incubated with 20 μL of 5% MTT under conditions void of light. The 96-well plate was cultured in a 5% CO2 incubator at 37 °C under conditions void of light for 4 h. After that, the cells were centrifuged at 179×g for 10 min with the supernatant removed. Then, 100 μL dimethyl sulfoxide (DMSO) solution was used to dissolve the crystal fully under conditions void of light for 10 min. Fifteen minutes later, optical density (OD) value was detected at 570 nm using enzyme-linked immunosorbent assay (ELISA) reader (SAF-680T, Multiskan GO, Thermo, Waltham, MA, USA). Cell growth curve was plotted with time as the abscissa and OD value as the ordinate.
After transfection of 24 h, the cells were collected, washed with cold PBS three times, centrifuged with the supernatant removed, and then resuspended with PBS to adjust cell density to 1 × 105/mL. Cells were then fixed by 1 mL of 75% ice ethanol (precooled at − 20 °C) at 4 °C for 1 h. After that, they were centrifuged and washed with PBS two times. Cells were incubated with 100 μL RNase A under conditions void of light, water-bathed at 37 °C for 30 min, and stained with 400 μL of propidium iodide (PI) (D0820, Sigma, San Francisco, CA, USA). They were incubated at 4 °C under conditions void of light for 30 min. After that, the red fluorescence at the wavelength of 488 nm was recorded by flow cytometry (Gallios, Beckman Coulter, S.Kraemer Boulevard Brea, CA, USA) to detect cell cycle. After transfection for 48 h, cells were treated with ethylenediamine tetraacetic acid (EDTA)-free trypsin and centrifuged to discard the supernatant. Cells were rinsed by cold PBS three times and centrifuged with the supernatant discarded. In accordance with the instructions of Annexin-V-FITC kit (4030ES20, Sigma, SF, CA, USA), the Annexin-V-FITC/PI staining solution was prepared with Annexin-V-FITC, PI, (2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in the proportion of 1:2:50. The 1 × 106 cells were resuspended in every 100-μL aliquot of staining solution. Cells were then incubated at room temperature for 15 min and then added with 1 mL of HEPES. The cells were excited at 488 nm, the FITC fluorescence was measured at 525 nm, and PI fluorescence was measured at 620 nm with a band-pass filter.
Senescence-associated β-galactosidase (SA-β-gal) staining
The senescence of NP cells was assessed by SA-β-gal staining. NP cells in the control group and experimental groups were collected and seeded into six-well plates at a density of 3 × 105 cells/well. Upon reaching 70% confluence, cells were washed using PBS and fixed by 1 mL SA-β-gal staining solution for 15 min at room temperature. The cells were washed three times by PBS (3 min per wash). The cells were stained with 1 mL SA-β-gal staining solution and then sealed with a preservative film at 37 °C overnight. The SA-β-gal-stained cells (the cytoplasm stained in dark blue) and total cells were counted under an optical microscope. The SA-β-gal-positive rate of NP cells = the number of SA-β-gal-stained NP cells/the number of total cells. The cytoplasm of SA-β-gal-positive cells presented dark blue staining.
Statistical analysis was conducted using SPSS 17 (SPSS, Chicago, IL, USA), and data were expressed by mean ± standard deviation. Comparison of data between two groups was conducted using a t-test and that of data among multiple groups was performed by one-way analysis of variance (ANOVA). Disc height index and rate of disc height change were analyzed by one-way ANOVA. The grade of IDD was analyzed by kappa. If the kappa value was 0–0.2, it means the consistency deviation was comparatively large and the result was very poor. If the kappa value was 0.21–04, it means the result was poor. If the kappa value was 0.41–0.60, it means the result was ordinary. If the kappa value was 0.61–0.8, it means the consistency of the result was comparatively good. Besides, if the kappa value was over 0.81, the result was basically the same and the result was reliable. p < 0.05 was considered statistically significant.
IDD models are successfully established
Degeneration of rat caudal IVD at different time points
Total degenerative IVD
At the beginning
100 ± 0
The 2nd week
3.35 ± 0.30*
86.44 ± 3.00*
0.75 ± 0.03*
The 4th week
5.07 ± 0.41*
69.00 ± 2.37*
0.70 ± 0.02*
eEF2 protein is lowly expressed in degenerative IVD tissues
IDD is associated with increased NP cell senescence
miR-143-5p and AMPK are upregulated while eEF2 is downregulated and cartilage differentiation is inhibited in degenerative IVD tissues
The protein levels of cartilage differentiation-related genes in normal IVD and degenerative IVD were detected using Western blot analysis (Fig. 4d). When compared to the normal IVD, degenerative IVD showed significantly decreased levels of COL2, ACAN, and DCN proteins (p < 0.05).
Therefore, these results demonstrated that miR-143-5p highly expressed and AMPK signaling pathway activation was induced in degenerative IVD, while eEF2 lowly expressed and cartilage differentiation was inhibited.
eEF2 is a target gene of miR-143-5p
miR-143-5p-mediated eEF2 inhibition activates AMPK signaling pathway in NP cells
Inhibition of miR-143-5p promotes NP cell differentiation by inactivating the AMPK signaling pathway
Inhibition of miR-143-5p induces NP cell proliferation via the inactivation of the AMPK signaling pathway
Flow cytometry analysis was further carried out to confirm the cell cycle, the results of which in Fig. 8 b, c suggested that, when compared to the control group, the blank, NC, miR-143-5p mimic, AICAR, miR-143-5p inhibitor, and miR-143-5p inhibitor + AICAR groups exhibited prolonged G0/G1 phase (increased cell proportion at G0/G1 phase), but shortened S phase (decreased cell proportion at G0/G1 phase) (p < 0.05). When compared to that of the blank and NC groups, G0/G1 phase shortened (cell proportion decreased) and S phase lengthened (cell proportion increased) in the miR-143-5p inhibitor group, while G0/G1 phase lengthened (cell proportion increased). At the same time, the S phase was shortened (cell proportion decreased) in the miR-143-5p mimic and AICAR groups (p < 0.05), and no significant difference was found in the miR-143-5p inhibitor + AICAR group (p > 0.05). There was no significant difference in cell proportion in the G2 phase among all groups (p > 0.05). Based on these results, it was concluded that miR-143-5p depletion promoted NP cell proliferation and cell cycle entry by inhibiting the AMPK signaling pathway.
Inhibition of miR-143-5p suppresses NP cell apoptosis and senescence by inactivating the AMPK signaling pathway
SA-β-gal staining was carried out to observe NP cell senescence. From the results of SA-β-gal staining, the cytoplasm of NP cells from normal IVD presented dark blue without any obvious vacuoles, but no obvious staining in nucleus was observed under the optical microscope (Fig. 9c, d). Positive rate in NP cells from degenerative IVD was significantly higher than that in the NP cells from normal IVD (p < 0.05). When compared to that of the control group, cell senescence significantly increased in the remaining groups (p < 0.05). In contrast to the blank and NC groups, cell senescence was diminished in the miR-143-5p inhibitor group (p < 0.05), while significantly increased in the miR-143-5p mimic and AICAR groups (p < 0.05). No significant difference was observed in the cell senescence among the blank, NC, and miR-143-5p inhibitor + AICAR groups (p > 0.05). Taken together, miR-143-5p inhibition impeded NP cell apoptosis and senescence by inhibiting the AMPK signaling pathway.
Human IDD is associated with dysfunction of NP cells, including differentiation, migration, proliferation, and apoptosis, and epigenetic processes and genetic background also are risk factors of IDD . Furthermore, abnormal expression of miRNAs has been linked with various musculoskeletal disorders and also has a vital role to play in the pathogenesis of IDD . However, the detailed mechanism of miR-143-5p in IDD remains poorly understood. The purpose of this study was to illustrate that miR-143-5p targeting the eEF2 gene could mediate IDD. Collectively, the data of this study revealed that miR-143-5p was highly expressed in rats with IDD while the inhibition of miR-143-5p was observed to inhibit NP cell senescence and apoptosis and promote cell proliferation and differentiation via repressing the activity of the AMPK signaling pathway.
Initially, based on the findings of this study, miR-143-5p was found to be expressed at a high level in degenerative IVD. Recent studies have confirmed that miR-143 is a muscle-enriched miRNA capable of mediating the myoblast differentiation and muscle aging . Another previous study noted that the overexpression of miR-143-5p accelerates cell migration, proliferation, and melanogenesis in alpaca melanocyte in melanoma . In addition, silencing miR-143-5p contributes to the differentiation dental pulp stem cells into odontoblasts by enhancing Runx2 expression via the OPG/RANKL signaling pathway . Another important finding of this present study was that miR-143-5p suppressed NP cell proliferation and differentiation but promoted cell apoptosis and senescence in IDD. Consistently, a prior study also revealed upregulated miR-143 expression in degenerative disc tissues and restored miR-143 expression could promote IDD via negatively regulating Bcl-2 . The results from the dual luciferase reporter gene assay confirmed that miR-143-5p targeted and negatively regulated eEF2. This study also found that eEF2 was expressed at a low level in degenerative IVD. eEF2, belonging to the GTP-binding translation elongation factor family, played a significant role in protein synthesis . Recent evidence has indicated that the downregulation of eEF2 induced by IL-6 can cause impairment on myogenic differentiation during muscle homeostasis . Furthermore, overexpression of eEF2 contributes to the inhibition of cardiomyocyte apoptosis during myocardial ischemia reperfusion via upregulating Bcl-2 expression .
In the following experiments, it was found that NP cells displayed a decreased expression ratio of p-mTOR/t-mTOR and an increased expression ratio of p-AMPK/t-AMPK following transfection with miR-143-5p mimic. The phosphorylation of the AMPK substrates was shown to be associated with upregulation of miR-143/145 in regulating the angiotensin-converting enzyme . EEF2K is the enzyme that inactivates eEF2, which is activated by AMPK [20, 21]. It should be noted that mTOR is a protein kinase that participates in translation control and long-lasting synaptic plasticity , and it also played a significant role in cell senescence . Furthermore, a previous study has suggested that AMPK signaling pathway is capable of inhibiting mTOR activity . When the AMPK signaling pathway was activated by AICAR, NP cell proliferation and differentiation were inhibited but NP cell apoptosis and senescence were promoted. As a significant regulator of cell apoptosis , Bcl-2 has been confirmed to block apoptosis in degenerative IVD . Bax is a member of Bcl-2 family . Moreover, the stabilizing of the Bax/Bcl-2 ratio induces cell death and cell senescence . It was also found that the cartilage differentiation-related genes COL2, ACAN, and DCN were suppressed when miR-143-5p was increased or the AMPK signaling pathway was activated. A recent study suggests that the COL2 protein level was increased when AMPK was poorly expressed . It has also confirmed that DCN promotes nephron progenitor differentiation in the embryonic kidney . The loss-of-function experiments further revealed that the inhibition of miR-143-5p blocked the AMPK signaling pathway and reversed the effects caused by the AMPK signaling pathway activation. Altogether, the inhibition of miR-143-5p promoted NP cell proliferation and differentiation yet restrained cell apoptosis and senescence by disrupting the AMPK signaling pathway.
In conclusion, this study indicated that miR-143-5p was overexpressed in IDD. Furthermore, it has been shown that miR-143-5p promotes cell senescence and inhibits cell proliferation and differentiation in NP cells. Notably, eEF2 is a target gene of miR-143-5p, and miR-143-5p activates the AMPK signaling pathway in the regulation of IDD. It was speculated that miR-143-5p is a promising therapeutic target for IDD treatment. However, further studies are still required with more detailed methods to investigate more specific mechanism of miR-143-5p in IDD.
We acknowledge and appreciate our colleagues for their valuable efforts and comments on this paper.
Availability of data and materials
The datasets generated/analyzed during the current study are available.
QY and XPG carried out the molecular genetic studies, participated in the sequence alignment, and drafted the manuscript. QY carried out the immunoassays. YLC participated in the sequence alignment. YW participated in the design of the study and performed the statistical analysis. XPG, YLC, and YW conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize suffering of the animals. The experimental procedures were approved by the Animal Ethics Committee of Taihe Hospital.
Consent for publication
The authors declare that they have no competing interests.
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