miR-19b-3p relieves intervertebral disc degeneration through modulating PTEN/PI3K/Akt/mTOR signaling pathway

Emerging studies have revealed that non-coding RNAs contribute to regulating intervertebral disc degeneration (IVDD). Here, we intended to probe into the function of miR-19b-3p in IVDD evolvement. The miR-19b-3p level in the intervertebral disc (IVD) tissues of IVDD patients and IL-1β/TNF-α/hydrogen peroxide-treated human nucleus pulposus cells (HNPCs) was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Also, qRT-PCR was conducted to examine the profiles of MMP-3, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5. The PTEN/PI3K/Akt/mTOR pathway was examined by Western blot (WB). The miR-19b-3p overexpression assay was carried out, and HNPC proliferation and apoptosis were compared by the cell counting kit-8 (CCK-8) assay and flow cytometry (FCM). In addition, the mechanism of action of miR-19b-3p was clarified using the PTEN inhibitor (VO-Ohpic triphosphate) or the mTOR inhibitor (Rapamycin) on the basis of IL-1β intervention and miR-19b-3p mimics transfection. Our results testified that miR-19b-3p expression was curbed in IVD tissues of the IVDD patients (vs. normal IVD tissues) and IL-1β-, TNF-α, or hydrogen peroxide-treated HNPCs. Up-regulating miR-19b-3p enhanced HNPC proliferation and hampered its apoptosis. Moreover, miR-19b-3p dampened the PTEN profile and activated the PI3K/Akt/mTOR pathway. Interestingly, attenuating PTEN reduced IL-1β-, TNF-α-, or hydrogen peroxide-mediated HNPC apoptosis and up-regulated PI3K/Akt/mTOR, while inhibiting the mTOR pathway offset the protective function of miR-19b-3p. Further mechanism studies illustrated that miR-19b-3p targeted the 3’untranslated region (UTR) of PTEN and abated the PTEN level. This research confirmed that miR-19b-3p suppressed HNPC apoptosis in the in-vitro model of IVDD by regulating PTEN/PI3K/Akt/mTOR pathway.


INTRODUCTION
The intervertebral disc (IVD) is a moderately moving joint that provides load transfer and flexibility to the spine [1]. On the other hand, intervertebral disc degeneration (IVDD) is a chronic progressive process accompanied by loss of living cells in IVD, especially in the internal region of the nucleus pulposus (NP), and the imbalance of extracellular matrix (ECM) decomposition and catabolism [2,3]. IVDD often causes low back pain and radiating leg pain in patients. Discogenic low back pain (DLBP) is the main risk factor of disability, and inflammation is considered to be the main driver of IVDD [4,5]. Disappointingly, the treatment of IVDD does not fully restore the biological function of IVD, so there is still a need to explore the relevant mechanisms to bring novel theoretical guidelines for the clinical treatment of IVDD.
miR-19b-3p is a non-coding RNA that has been exhibited to contribute to cell inflammation and cell growth. Studies have revealed that pathologic circulatory stretch enhances vascular smooth muscle cell (VSMC) proliferation by curbing the miR-19b-3p/connective tissue growth factor (CTGF) pathway, thus inducing vascular remodeling [6]. On the other hand, several reports have confirmed the value of miR-19b-3p in orthopedic diseases. For example, miR-19b-AGING 3p boosts the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by interacting with lncRNA H19, thereby hindering postmenopausal osteoporosis (OP) [7]. Some reports have stated that miR-19b-3p attenuates OA evolvement by targeting EZH2 [8]. In another study, miR-19b-3p impedes GRK6 expression in a targeted manner, thereby attenuating IL-1β-induced cartilage extracellular matrix degradation and inflammation [9]. Overall, miR-19b-3p exerts an essential function in regulating cell inflammation, while its role in IVDD remains elusive.
In recent years, the PTEN/PI3K/Akt signaling pathway has attracted wide attention on account of its regulation on cells. Meanwhile, multiple reports have proved that PTEN/PI3K/Akt advances the growth of various cancers, such as osteosarcoma [10], invasive gallbladder adenocarcinoma [11] and non-small cell lung cancer [12]. In contrast, in the non-tumor field, some researchers have found that arctiin chokes high glucosemediated proliferation of human retinal capillary endothelial cells (HRCECs) by activating ROCK1 and PTEN and inactivating PI3K and Akt [13]. Moreover, miR-140-3p heightens C2C12 cell proliferation and differentiation and hampers apoptosis by targeting PTEN and choking PTEN/PI3K/Akt in OP [14]. Notably, PTEN has been validated to regulate the behaviors of NP cells by directly targeting PI3K/AKT [15]. As a classical signaling pathway, PTEN/ PI3K/Akt's interaction with miR-19b-3p in IVDD remains largely unknown.
The mTOR pathway boosts metabolism, contributes to cell apoptosis and autophagy, and exerts a considerable role in diversified diseases. For example, Zheng RH et al. have stated that liraglutide chokes myocardial fibrosis and dysfunction by abating the mTOR/p70S6K signaling and enhancing autophagy [16]. In addition, several researchers have found through in vitro experiments in rats that hUMSCs dampen the autophagy of interstitial cells (ICs) by reducing oxidative stress and regulating the AMPK/mTOR pathway, thereby decreasing the apoptosis of ICs [17]. It is worth noting that miR-21 facilitates the catabolism of type II collagen (Col II) and aggrecan by repressing the PTEN/Akt/mTOR signal autophagy in HNPCs [18].
Overall, this research focuses on probing the regulatory role and mechanism of miR-19b-3p on IVDD. We set up in-vitro IVDD models by treating HNPCs with IL-1β, TNF-α, or hydrogen peroxide. Our results manifested miR-19b-3p expression was impeded in IVDD tissues and HNPCs treated with IL-1β, TNF-α, or hydrogen peroxide separately. Further investigations suggested that overexpressing miR-19b-3p attenuated the IL-1β-mediated apoptosis of HNPCs and motivated the PI3K/Akt/mTOR pathway. These data hinted that miR-19b-3p and the PI3K/Akt/mTOR pathway are essential therapeutic targets for IVDD.

miR-19b-3p was notably down-regulated in IVD tissues of IVDD patients
First, we performed qRT-PCR to monitor the miR-19b-3p and PTEN profiles in the IVD tissues of IVDD patients. According to the results, by contrast with that in normal IVD tissues, the miR-19b-3p level was attenuated (P<0.05, Figure 1A), while PTEN was upregulated in the IVD tissues of IVDD patients (P<0.05, Figure 1B). Furthermore, Pearson analysis confirmed that the expression of miR-19b-3p and PTEN was reversely associated (R 2 =0.4495, P<0.01, Figure 1C). These outcomes manifested that miR-19b-3p influenced IVDD by regulating PTEN.

The influence of overexpressing miR-19b-3p on PTEN/PI3K/Akt/mTOR pathway
WB was implemented to clarify the impact of miR-19b-3p on PTEN/PI3K/Akt/mTOR pathway. The findings uncovered that the PTEN level was strengthened and the PI3K/Akt/mTOR signal was inactivated after IL-1β, TNF-α, or hydrogen peroxide treatment. In contrast, the PTEN level was elevated and the PI3K/Akt/mTOR axis was inactivated after cell transfection compared with that of the IL-1β/TNF-α/hydrogen peroxide group (P<0.05, Figure 4A, 4B).

DISCUSSION
IVD is the largest avascular organ in the human body [19], and the incidence of IVD disease increases proportionally with the aging of the population [20]. AGING However, the current conservative treatment and surgical treatment have limited effects, which can only relieve symptoms and cannot restore the structural and biological function of the IVD [21]. Here, we discovered that overexpressing miR-19b-3p attenuates IL-1β-, TNF-α-, hydrogen peroxidemediated HNPC apoptosis. Further studies displayed that miR-19b-3p targeted the 3'UTR of PTEN and choked PTEN expression, thus activating PI3K/Akt/mTOR pathway.
Multiple pieces of research have confirmed that miRNAs contribute to regulating IVDD. It is reported that MIR660 knockout exerts a protective role in the apoptosis of NPs by up-regulating SAA1mRNA [22]. Other scholars have claimed that miR-499a-5p AGING reduces TNF-α-induced HNPC apoptosis and the imbalance between anabolism and catabolism of extracellular matrix by down-regulating SOX4 [23]. On the other hand, miR-19b-3p studied in this paper is a miRNA involved in regulating cell inflammation and metabolic-related diseases. For example, H19 overexpression dampens BMSC proliferation and differentiation by hampering miR-19b-3p expression in postmenopausal OP [7]. In addition, Xu H et al. discovered that the miR-19b-3p level in sepsis patients' serum is notably reduced and is reversely linked to the IL-6 and TNF-α contents. More importantly, miR-19b-3p facilitation mitigates sepsis-induced inflammation [24]. Here, it was revealed that miR-19b-3p is downregulated in the tissues of IVDD patients and HNPCs treated with IL-1β, TNF-α, or hydrogen peroxide. Interestingly, overexpressing miR-19b-3p attenuates the anti-proliferative and pro-apoptotic effects-mediated by IL-1β, TNF-α, and hydrogen peroxide on HNPCs, suggesting that miR-19b-3p serves as a potential therapeutic target against IVDD.
PTEN, a homolog of phosphatase and tension protein missing from chromosome 10, is an effective tumor suppressor that regulates cell growth and survival [25,26]. For instance, miR-221 has been reported to curb PTEN expression and activate phosphatidylinositol 3 kinase (PI3K)/AKT, thus repressing the apoptosis of cardiomyocytes (CMs) [27]. In addition, Per2 facilitates NHAC-kn inflammation in osteoarthritis by heightening the PTEN level and dampening the PI3K/Akt expression [28]. Of special note, PTEN is overexpressed in denatured NP. Moreover, VO-OHpic (VO), a PTEN inhibitor, can prevent NP degradation by diminishing oxidative stress and increasing cell proliferation through the PTEN/Akt pathway [29]. In our study, qRT-PCR and WB confirmed that PTEN is up-regulated in the tissues of IVDD patients and HNPCs treated with IL-1β, TNF-α, or hydrogen peroxide. Furthermore, the functional assays confirmed that PTEN inhibition relieved HNPCs injury, and miR-19b-3p exert protective effects on HNPCs via targeting PTEN.
The mammalian target of rapamycin (mTOR) is a vital mediator of the PTEN/PI3K/AKT signaling [30]. mTOR signals control basic biological functions, including proliferation, growth, metabolism, autophagy, and aging, and its excessive activation leads to various human diseases [31]. For example, the downregulation of miR-181 dampens the PTEN level and induces the expression of PI3K, P-Akt, and p-mTOR, confirming that knockdown of miR-181 strengthens the proliferation and migration of A549/DDP cells [32]. On the other hand, resveratrol (RSV) boosts cell viability and impedes apoptosis by motivating the miR-17-regulated PTEN/PI3K/AKT and mTOR path-ways and hampers inflammatory damage of HaCaT cells by reducing the production of IL-6/IL-8/TNF-α [33]. Above all, osteoblasts protein 1 (OP-1) dampens the apoptosis of rat NPs by activating PI3K/ Akt/mTOR in hypertonic cultures [34]. In contrast, another study revealed that the PI3K/Akt phosphorylation protects IDD, ascribing to increased ECM concentrations, apoptosis prevention, cell proliferation elevation, induction or repression of autophagy, reduction of oxidative damage and adaptive hypoxic microenvironment [35]. Here, we discovered that inhibition of mTOR offsets the protection of miR-19b-3p on HNPCs. Overall, through a series of experiments, it was discovered that miR-19b-3p is down-regulated in the tissues of IVDD patients and HNPCs. Meanwhile, miR-19b-3p overexpression motivates PI3K/Akt/mTOR by hampering PTEN, thus heightening HNPC proliferation and choking its apoptosis, which provides new insights and theoretical references for the treatment and intervention of IVDD patients clinically.

Collection and treatment of clinical specimens
The IVD tissues from 20 IVDD patients who received surgical treatment in Qilu Hospital of Shandong University (Qingdao), and the normal IVD tissues removed from 10 patients due to trauma from January 2019 to May 2019 were harvested. All patients signed the informed consent, and the experiment was authorized by the Ethics Committee of Qilu Hospital of Shandong University (Qingdao) and met the ethical requirements of international and national regulatory authorities for biomedical research. All the patients were diagnosed with IVDD by two senior pathologists, and the tissues were placed in liquid nitrogen at -196° C for future use.

Cell culture
Human NP cells (HNPCs) were isolated from the normal IVD tissues. In short, the tissues were cut into sections of about 1 mm 3 and treated for 0.5 hours with 0.25% trypsin (Gibco, Life Technologies, Paisley, UK), and then digested for 3 hours with 0.2% type II collagenase (Invitrogen, Carlsbad, CA, USA) at 37° C. After filtration and washing with PBS, the suspension was centrifuged, and the cells were cultured in the Dulbecco modified Eagle medium comprising F12 nutrient mixtures (Gibco, Grand Island, NY, USA), 15% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37° C with 5% CO2. After fusion, HNPCs were digested and sub-cultured.

AGING
The second-generation cells were applied for subsequent experiments.

Cell transfection
HNPCs at the logarithmic growth stage were seeded in 6-well plates at 5×10 6 /well after digestion and subculture. After stable cell growth, HNPCs were transfected with miR-19b-3p mimics and corresponding negative control group (miR-NC), PTEN overexpression plasmids and Vectors, according to the specification of the FuGENE®HD Transfection Reagent (Roche, Shanghai, China). At last, they were maintained at 37° C with 5% CO2 for 24 hours.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNAs were separated from IVD tissues or HNPCs with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After the RNA concentration and purity were tested by utilizing Thermo NanoDrop 2000, the miRNA and mRNA were reversely transcribed into cDNA by adopting the One Step PrimeScript miRNA cDNA synthesis kit (Bao Biological Engineering Co., Ltd., Dalian, China) and RevertAid First Strand cDNA short Kit (Thermo Fisher Scientific, Waltham, MA, USA) respectively. The cDNA synthesis was performed at 37° C for 40 min and 85° C for 5 s. PCR amplification was implemented with the SYBRGreen method and cDNA as template, while miR-19b-3p, MMP-3, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5 served as specific primers (Sangon Biotech, Shanghai, China). PCR was performed with 40 cycles of 95° C for 30 s, 95° C for 5 s, 60° C for 30 s and 73° C for 10s. U6 was the endogenous control of miR-19b-3p, while GAPDH was that of MMP-3, MMP-9, MMP-13, ADAMTS-4 and ADAMTS-5. The relative expression was measured with the 2 -Δ Δ CT method, and the primer sequences were exhibited in Table 1.

Cell counting kit-8 (CCK-8) experiment
HNPCs were digested and collected, then the single cell suspension was made (2×10 3 /mL). Then, they were dispersed in 96-well plates with 100 μL cell suspension per well, and each group had three repetitive wells. Next, the plates were placed in an incubator. After 24 hours, 10 μL CCK-8 solution (Hubei Biossci Biotechnology Co., Ltd, China) was added to each well for incubation for another 1 hour. After culture, the plates were put in a microplate reader to observe the optical density (OD) of each well at 450 nm. Afterward, the OD of each cell was measured at the 24 th , 48 th and 72 ed hour, respectively.

TdT-mediated dUTP nick end labeling (TUNEL) assay
HNPCs treated with the different factors were seeded into 24-well plates (1 × 10 5 per well) and further maintained for 24 hours. Cells were fastened with 4% paraformaldehyde for 30 min and cleaned with PBS. 0.2% DE Triton X-100 was added and incubated with the cells at RT for 15 min. Next, cell apoptosis was examined by employing the TUNEL apoptosis detection kit (Shanghai Xinyu Biotech. Co., Ltd, Shanghai, China). Briefly, 50 μL of TUNEL reaction solution was AGING added and incubated at RT away from light for 1 hour. Then, DAPI (Shanghai Biotime Biotechnology Co., Ltd, Shanghai, China.) was applied for re-staining for 1 min. Cells were immersed in PBS and sealed with the antifade mounting medium. Five randomly chosen nonoverlapping fields of view were reviewed with a microscope, and the TUNEL-positive cells were counted.

Dual-luciferase reporter gene assay
The bioinformatics predicted that miR-19b-3p targeted 3'-UTR of PTEN. All luciferase reporter vectors (PTEN-WT and PTEN-MUT) were constructed by Promega (Promega, Madison, WI, USA). 293T cells (4.5×10 4 ) were dispersed in 48-well plates and cultured to reach a 70% confluence rate. Then, PTEN-WT and PTEN-MUT were co-transfected with miR-19b-3p mimics or negative controls, respectively, using liposome 2000. After the transfection for 48 hours, the luciferase activity was determined as requested by the manufacture. All tests were made in triplicate.

Cellular immunofluorescence
HNPCs were inoculated into 24-well plates (1 × 10 5 cells/well) and transfected with miR-19b-3p and miR-NC, respectively, for 48 hours. Cells were immobilized with 4% paraformaldehyde at RT for 20 min and endogenous peroxidase was inactivated by the addition of 3% H2O2 for 15 min. Cells were subjected to blocking with 5% goat serum for 1 hour and incubation with the primary PTEN antibody (1:80, ab170941, Abcam) overnight at 4° C. The following morning, a secondary antibody, Goat Anti-Rabbit (1:100, ab6721, Abcam), was added dropwise and maintained for one hour at RT. DAPI staining solution was utilized for cell dyeing for 5 min at RT away from light. After mounting with the anti-fade mounting medium, the cells were reviewed and photographed under a fluorescence microscope.

Statistical analysis
Experimental data were processed with the SPSS18.0 statistical software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8 (GraphPad Software, USA). The differences between the two groups were ascertained by the t test, while one-way ANOVA was employed to analyze differences between multiple groups. P <0.05 indicated statistical significance. Pearson correlation coefficient R was adopted to measure the correlation between miR-19b-3p and PTEN in the IVD tissues of IVDD patients. Quantitative data were presented as mean values and SD values of independent experiments that were repeated at least three times.

Ethics statement
Our study was approved by the Ethics Review Board of Qilu Hospital of Shandong University (Qingdao).

Data availability
The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.