miR-338-3p inhibits HIF-1α to Attenuate Nucleus Pulposus Cell Proliferation and Promote Apoptosis Through the Hippo-YAP Pathway

doi:10.21203/rs.3.rs-472009/v1

Download PDF

Research Article

miR-338-3p inhibits HIF-1α to Attenuate Nucleus Pulposus Cell Proliferation and Promote Apoptosis Through the Hippo-YAP Pathway

https://doi.org/10.21203/rs.3.rs-472009/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The proliferation and apoptosis of nucleus pulposus (NP) cells (NPCs) play a crucial role in intervertebral disc degeneration (IDD). we aimed to discover the role of miRNA-induced IDD. We analyzed the miRNA expression of three NP tissues from IDD patients and three normal NP samples using the GEO2R tool, and The results revealed that miR-338-3p was upregulated in NPCs from IDD patients. miR-338-3p suppressed NPCs proliferation, and the related proteins PCNA and Ki-67 were downregulated, as demonstrated via western blotting. miR-338-3p promoted apoptosis. Furthermore, we predicted that HIF-1α was targeted by miR-338-3p, using the miRDB database, and this target was validated via dual luciferase assay. HIF-1α reversed miR-338-3p-induced NPCs proliferation and apoptosis. The Hippo-YAP pathway activation proteins YAP, CTGF, and PCNA were upregulated, unlike the inhibitory YAP phosphorylation. In conclusions, our results suggestive that miR-338-3p inhibited HIF-1α/ Hippo-YAP pathway to attenuate NPCs proliferation and apoptosis.

Molecular Biology

HIF-1α

miR-338-3p

nucleus pulposus cells

intervertebral disc degeneration

Intervertebral disc (IVD) degeneration (IDD), the primary underlying cause of low back pain, leads to chronic disability in roughly 10% of all cases [1, 2]. The current therapeutic options for IDD are mostly symptomatic treatments including pain relief and nerve block. However, there is no effective biochemical treatment for IDD and IVD functional recovery. The IVD, as the largest avascular tissue in the human body, is composed of three structures: nucleus pulposus (NP), annulus fibrosus, and endplate cartilage [3]. The NP cells (NPCs) play an important role in IVD matrix formation and metabolism. Previous studies have indicated that the accumulation of senescent NPCs may contribute to pathological changes in the IVD during aging and degeneration [4]. Notably, transplantation of NPCs into the IVD is thought to be a promising therapeutic method [5]. Therefore, further understanding of the molecular mechanisms of NPCs proliferation and apoptosis may lead to the development of better therapeutic strategies.

Hypoxia (Hx) inducible factor (HIF) is a key transcription factor in Hx stress response [6] and are greatly significant to improving tissue ischemia and Hx. Zou [8]. provided important preclinical experimental evidence concerning the HIF-1α in promoting new bone formation in a animal model. Recent study suggests that the hepatocyte growth factor regulates HIF-1α-induced NPCs proliferation [9].

The Hippo-YAP signaling pathway is a major regulator of cell proliferation and tissue growth [10]. Previous studies found that the Hippo pathway inhibits cardiomyocyte proliferation [11]. The Yes-associated protein (YAP) is an important part of the Hippo signaling pathway. Sirio [12] et al. found that mesenchymal stem cells (MSCs) accumulate YAP in their nuclei and promote osteogenic differentiation. The phosphorylation of YAP is controlled by the serine threonine kinases MST1/2 and LATS1/2 in the Hippo-YAP signaling pathway [13]. There are intricate interactions between the Hippo-YAP and HIF signaling pathways. Chen [14] et al. have shown that HIF-1α restricts the proliferation of Tca8113 cells through upregulation of the Hippo-YAP signaling pathway under Hx conditions. Zhang [15] et al. found that under Hx stress, the binding of YAP and HIF-1α promoted the glycolysis of liver cancer cells.

MicroRNAs (miRNAs), as single-stranded small endogenous RNAs, suppress the expression of protein-coding genes, and they are important biomarkers for IDD. Sherafatian [16] et al. revealed that a large number of malregulated miRNAs were involved in the IDD process. A previous study has found that upregulation of miR-199 attenuates TNF-α-induced human NPCs cells (HNPCs) apoptosis by downregulating MAP3K5 [17]. There is no research investigating whether miR-338-3p inhibits HIF-1α/Hippo-YAP pathway to attenuate NPCs proliferation and apoptosis.

In this study, we identified the effect of miR-338-3p on the cellular functions of NPCs. Mechanistically, miR-338-3p bound to HIF-1α 3′-UTR in NPCs. The roles of miR-338-3p and HIF-1α in NPCs proliferation and apoptosis were investigated. Furthermore, miR-338-3p and HIF-1α induced the expression of downstream signaling proteins of the Hippo signaling pathway. Taken together, our results demonstrated that miR-338-3p could be a promising target for IDD therapy.

HNPCs culture

HNPCs (Sciencell Research Laboratories; Carlsbad, CA, USA) maintained in NPC Medium (NPCM, Cat. #4801, ScienCell) under normoxic (Nx) culture conditions (37°C, 20% oxygen, 5% carbon dioxide, 75% nitrogen) or hypoxic (Hx) culture conditions (37°C, 1% oxygen, 5% carbon dioxide, 94% nitrogen). Hx cell culture conditions were generated in a hypoxia incubator .

qRT-PCR assay

Total RNA of HNPCs was extracted with TRIzol (Invitrogen) reagent and diluted with DEPC water (Invitrogen) and reverse transcribed into cDNA using the PrimeScript RT reagent kit (Invitrogen) according to the manufacturer's instructions. RT-qPCR was then performed using the SYBR Premix Extaq II kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) on Real-Time PCR system (7500; Applied Biosystems; Thermo Fisher Scientific, Inc.). Cycling conditions were as follows: 50°C (2min), 95 °C (10min), 72 °C (2min), followed by 40 cycles of 95 °C (15 s) and 60 °C (32s). The sequences of primers for miR-338-3p and U6 were as follows: miR-338-3p-F, 5′-ACACTCCAGCTGGGACATCAGTGATTTTGTTG-3′, miR-338-3p-R, 5′-CTCAACTGGTGTCGTGGA-3′; U6-F, 5′-CTCGCTTCGGCAGCACA-3′, U6-R, 5′-AACGCTTCACGAATTTGCGT-3′. Relative miR-338-3p expression levels were calculated using the 2^−ΔΔCt method.

Differentially expressed miRNA analysis via GEO2R

Using GEO2R (ncbi.nlm.nih.gov/geo/geo2r) for identifying the set of two or more samples differential expression of miRNAs, and with P <0.05 and log₂ |fold change| >2 for differentiation standard.

Prediction of miRNA target genes

The TargetScan7.2 database (targetscan.org/vert_72/) and the miRDB database (mirdb.org) were used to predict the miR-338-3p binding sites on the downstream target genes. Putative target genes were predicted by the two programs. Using DAVID tool (https://david.ncifcrf.gov/) to perform the functional classification analysis of miR-338-3p predicted target genes.

Dual luciferase assay

Use of Lipofectamine^® 2000 (Invitrogen) transfection HNPCs with luciferase reporter vector (psi-CHECK-2) containing a 3'-UTR fragment of HIF-1α, according to the manufacturer's instructions. co-transfected with 50 nM miR-338-3p mimics or control (mimic NC) with psi-CHECK-2 (0.5 µg) luciferase reporter vector or empty vector containing the wild-type (WT) or mutant (mut) HIF-1α 3'-UTR sequence. Then, a dual luciferase reporting detection system (Glomax; Promega Corporation, Madison, WI, USA) after 48 h of transfection. The results are normalized using Renilla/Firefly method.

Western blotting

Transfected NPCs were cleaved using RIPA lysis buffer (Beyotime, Shanghai, China) on ice using BCA protein assay kit (Boster Biological Technology co.ltd; California, USA) pyrolysis lysate protein concentration calculation. Equivalent of denatured proteins were resolved using 10% SDS-polyacrylamide gel electrophoresis (Solarbio), and protein bands were transferred to a polyvinylidene fluoride membrane (Solarbio). Using TBST (Beyotime) wash membranes twice and blocked(Solarbio), incubated overnight with the primary antibodies at 4 °C, rinsed, and then incubated with secondary antibody (goat anti-rabbit; 2 mg/ml; ab205718; Abcam, Cambridge, UK) for 2 h at 23 ± 2 °C. Protein bands were visualized by the addition of ELC-enhanced chemiluminescence reagent (Thermo Fisher Scientific) in conjunction with an imaging system (FluorChem R; ProteinSimple co., Ltd., Silicon Valley, California, USA). An anti-GAPDH antibody (1:10,000) was used as the loading control. Anti-HIF-1α antibody (1:1,000; ab179483; Abcam), Anti-PCNA antibody (1:1,000; ab92552; Abcam), Anti-Ki-67 antibody (1:5,000; ab92742; Abcam), Anti-Caspase-3 antibody (1:2,000; ab184787; Abcam), Anti-caspase-9 antibody (1:2,000; ab202068; Abcam), Anti-YAP antibody (1:1,000; ab205270; Abcam), Anti-p-YAP antibody (2.5 µg/ml; ab76252; Abcam) and Anti-CTGF antibody (1:1,000; ab209780; Abcam) were used.

Cell proliferation assay

According to the manufacturer's manual, HNPCs (2.5×10³) was incubated for the indicated time periods (12, 24, 48, and 72 h) in an incubator, and 10 μL of CCK8 solution (Beyotime) was added and incubate for 1 hour. The absorbance at 490 nm was measured with the enzyme-labeled instrument (Thermo Fisher Scientific, Inc.).

Cell apoptosis assay

Annexin V-FITC (5 μL; BD Biosciences, CA, USA) and propidium iodide (10 μL; BD Biosciences) were incubated with HNPCs (1×10⁶ cells/ml) in the dark (15 min, 23±2°C), and rinsed twice with phosphate-buffered saline (Gibco). Apoptosis was assessed by flow cytometry (FCM; BD Biosciences).

Statistical analysis

All experiments were triplicate and the data are expressed as the mean ± standard deviation (SD). Using SPSS version 19.0 statistical analysis package (SPSS Inc., Chicago, IL, USA) for more than two groups of data. Student’s t-test was used to analyses independent two-group (unpaired).

Screening of differentially expressed miRNAs in HNPCs of IDD patients

To investigate the involvement of miRNAs in HNPCs degeneration in IDD patients, we analyzed the expression profiles using a miRNA microarray of three HNPCs from IDD patients (GSM3259528, GSM3259529, and GSM3259530) and three normal HNPCs from fresh traumatic lumbar fracture patients (GSM3259525, GSM3259526, and GSM3259527) using the NCBI GEO2R online database of GSE116726. The results of hierarchically clustered heatmaps and a volcano plot demonstrated that 52 differentially expressed miRNAs were identified with log₂|fold change| >2 and P <0.05. There were 44 upregulated miRNAs and 8 downregulated miRNAs in HNPCs from IDD patients, compared to controls (Figure 1A, and B).

miR-338-3p was a candidate degeneration-related miRNA in HNPCs

It is well known that Hx promotes HNPCs proliferation and inhibits apoptosis and that normoxia (Nx) has the opposite effect [18]. We examined the proliferation and apoptosis of HNPCs under hypoxic (Hx) or Nx conditions, and our results were consistent with previous studies (Figure 1C, and D). Additionally, we confirmed that miR-338-3p was substantially upregulated in the Nx treatment group, compared to the Hx treatment group (Figure 1E). The results indicated that further experimentation could highlight miR-338-3p as a key regulator of HNPCs proliferation and apoptosis that affect IDD.

Regulation of miR-338-3p in HNPCs

To determine the functions of miR-338-3p in HNPCs, we transfected the cells with an miRNA inhibitor and a negative control inhibitor under Nx conditions. The miR-338-3p expression was decreased in the miRNA inhibitor group compared to the negative control inhibitor group and normal HNPCs (Figure 2A). The CCK8 assay results showed that HNPCs transfected with an miRNA inhibitor proliferated faster than HPNCs transfected with a negative control inhibitor and normal HNPCs (Figure 2B). The flow cytometry (FCM) results revealed that miRNA inhibitor treatment reduced apoptosis, and miR-338-3p induced rapid apoptosis in normal cells within 48 h of transduction (Figure 2C). Moreover, DAVID tool result revealed that predicted target genes IGF1, AKT3, CAMK2G, CAMK2A, and MAPK1 were enriched in the HIF-1 signaling pathway, and the predicted target genes PPP1CB, CDH1, DVL3, WNT2B, and DLG2 were enriched in the Hippo-YAP signaling pathway (Figure 1D). Then, the western blotting analysis confirmed that miR-338-3p knockdown significantly upregulated the pro-proliferation proteins PCNA and Ki-67 and downregulated the pro-apoptosis proteins caspase-3 and caspase-9 (Figure 2E). In general, our data suggest that miR-338-3p is an important biomarker for IDD that also regulates HNPCs proliferation and apoptosis.

Dual-luciferase analysis of miR-338-3p- HIF-1α binding

Via miRDB database, we found that HIF-1α, a critical regulator of the response to Hx, is a potential target of miR-338-3p. Then, a luciferase reporter vector with the WT or mut HIF-1α 3′-UTR including the putative miR-338-3p target site were constructed. The results of luciferase assay revealed that overexpression of miR-338-3p inhibited the cell luciferase activity of the WT 3′-UTR of HIF-1α group, compared to the mimic NC group, whereas no significant change in luciferase activity in the mut 3′-UTR of the HIF-1α group (Figure 3A). Results of the western blotting analysis further confirmed the luciferase reporter gene assay results. Contrary to miRNA, HIF-1α was downregulated in the Nx group compared to the Hx group. Overexpression of miR-338-3p inhibited the HIF-1α protein level in HNPCs (Figure 3B). These results revealed that miR-338-3p targets the HIF-1α 3′-UTR.

HIF-1α reversed miR-338-3p-induced HNPCs proliferation and apoptosis

To investigate whether HIF-1α reversed miR-338-3p-induced HNPCs proliferation and apoptosis, we transfected cells with negative control and si-HIF-1α. As evident from the western blot in Figure 4A, the expression of HIF-1α was significantly decreased in HNPCs transfected with si-HIF-1α+ miR-338-3p inhibitor compared to HPNCs transfected with si-NC+ miR-338-3p inhibitor. Moreover, CCK8 results showed that HNPCs transfected with si-HIF-1α and miR-338-3p inhibitor proliferated slower than HPNCs transfected with si-negative control and miR-338-3p inhibitor. These results indicate that HIF-1α can promote proliferation and is capable of reversing proliferation downregulated by miR-338-3p (Figure 4B). Moreover, FCM analysis results revealed that HNPCs transfected with si-HIF-1α and miR-338-3p inhibitor enhanced miR-338-3p-induced apoptosis within 48 h of transduction. These results indicate HIF-1α could reduce apoptosis and reverse miR-338-3p-induced apoptosis upregulation (Figure 4C). Taken together, our data suggested that HIF-1α reversed miR-338-3p-induced HNPCs proliferation and apoptosis.

miR-338-3p- HIF-1α regulated Hippo-YAP pathway related proteins in HNPCs

We performed the western blotting assay to validate the effect of miR-338-3p or HIF-1α on the Hippo-YAP pathway. Contrary to Hippo-YAP pathway inhibition, knockdown of the phosphorylation of YAP and miR-338-3p significantly increased the expression of the Hippo-YAP pathway activator proteins YAP, CTGF, and PCNA. HIF-1α knockdown reduced the expression of YAP, CTGF, and PCNA, contrary to YAP phosphorylation (Figure 5). In general, these findings demonstrated that miR-338-3p and HIF-1α regulated Hippo-YAP pathway-related protein expression in HNPCs.

IDD treatment options are limited and mostly symptomatic, including pain relief and nerve block. NPCs are one of the most important components of IDD. NPCs proliferation inhibition and apoptosis directly induce IDD [19–21]; thus, the molecular mechanism of NPCs proliferation and apoptosis should be closely related to IDD pathogenesis. In this study, 52 differentially expressed miRNAs were identified with |fold change| >2 and P < 0.05. Additionally, 44 miRNAs were upregulated and 8 miRNAs were downregulated in HNPCs from IDD patients, compared to controls, as compared via the NCBI GEO2R online database. miR-338-3p was upregulated in the Nx group, compared to the Hx treatment group. Furthermore, HNPCs transfected with an miRNA inhibitor proliferated faster than HPNCs transfected with a negative control inhibitor. HNPCs treated with an miRNA inhibitor showed reduced apoptosis. Moreover, miR-338-3p knockdown upregulated the expression of the pro-proliferation proteins PCNA and Ki-67 and downregulated the pro-apoptosis proteins caspase-3 and caspase-9. In brief, our results confirmed that miR-338-3p is a degeneration-related miRNA in HNPCs that affects IDD. Our research is the first study to report that miR-338-3p attenuates NPCs proliferation and promotes apoptosis.

HIF-1α is the key molecular regulator of the initiation and development of IDD. Previous studies demonstrated that expression of prolyl hydroxylases is selectively controlled by the HIF-1 and HIF-2 proteins in NPCs of the IVD [22]. In particular, our findings supported that HIF-1α promoted NPCs proliferation and inhibited apoptosis. Therefore, HIF-1α could be a promising therapeutic target in IDD.

The Hippo-YAP signaling pathway is an essential intracellular signaling pathway, which is involved in many physiological processes such as cell growth and apoptosis. Heallen et al. [11] found that the Hippo pathway inhibits the proliferation of cardiomyocytes by inhibiting the Wnt signaling pathway. Previous study demonstrated that the Hippo pathway acts via various miRNAs to control cellular functions and pro-apoptotic gene expression during tissue growth [23]. In our study, NPCs proliferation promotion and apoptosis inhibition activated the Hippo-YAP signaling pathway, and vice versa. Furthermore, The levels of the major Hippo-YAP pathway-related proteins determined via western blotting were assessed as key indicators for the degree of pathway activation. After Hippo-YAP pathway activation, the levels of the activation proteins YAP, CTGF, and PCNA were elevated, whereas the levels of the inhibitory YAP phosphorylation were decreased. Notably, all these results indicate that the Hippo-YAP signaling pathway proteins YAP, CTGF, and PCNA are IDD biomarkers.

miR-338-3p was the central regulator of IDD in our study. To understand the manner by which miR-338-3p regulated IDD, we conducted experiments and found that it attenuates NPCs proliferation and promotes apoptosis. In addition, miR-338-3p repressed HIF-1α expression. Moreover, miR-338-3p regulated the Hippo-YAP pathway-related proteins in HNPCs. All these results indicated that miR-338-3p inhibited HIF-1α to attenuate NPCs proliferation and promoted apoptosis through the Hippo-YAP pathway.

In conclusion, we believe that systematic miR-338-3p molecular experiments and bioinformatics analysis may contribute to our understanding of the molecular mechanisms underlying the regulation of NPCs proliferation and apoptosis. Finally, it is important to note that targeting the miR-338-3p/HIF-1α/Hippo-YAP pathway axis provides a novel therapeutic opportunities for treating IDD.

Intervertebral disc (IVD) degeneration (IDD); nucleus pulposus (NP) cells (NPCs); human NPCs (HNPCs); hypoxia-inducible factors (HIFs); hypoxia (Hx); normoxia (Nx); Yes-associated protein (YAP); mesenchymal stem cells (MSCs)

Acknowledgements

Not applicable.

Funding

This work was supported by the Natural Science Foundation of Hainan Province under Grant number [819QN350].

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The authors declare that the microarray data (GSE116726) referenced in the study are available in a public repository from the GEO website (ncbi.nlm.nih.gov/geo) and the other data were available from the corresponding authors upon reasonable request.

Code availability

Not applicable

Author’s Contribution

DZ, JL, WC and GW conceived and designed the study and developed the methodology. JC, BW and WC performed the experiments and collected the data. DZ, JL, ML, JC, YF, YL and WC analyzed and interpreted the data. DZ and JL drafted the manuscript. All authors endorse and approved this manuscript.

Ethics approval

Not applicable

Consent to participate

Not applicable

Consent for publication

Not applicable

Andersson GB (1999) Epidemiological features of chronic low-back pain. Lancet 354(9178):581–585
Xi Y, Jiang T, Wang W, Yu J, Wang Y, Wu X, He Y (2017) Long non-coding HCG18 promotes intervertebral disc degeneration by sponging miR-146a-5p and regulating TRAF6 expression. Sci Rep 7(1):13234
Zhang Z, Lin J, Nisar M, Chen T, Xu T, Zheng G, Wang C, Jin H, Chen J, Gao W, Tian N, Wang X, Zhang X (2019) The Sirt1/P53 Axis in Diabetic Intervertebral Disc Degeneration Pathogenesis and Therapeutics. Oxid Med Cell Longev 2019 7959573
Huang H, Cheng S, Zheng T, Ye Y, Ye A, Zhu S, Lin X (2019) Vitamin D retards intervertebral disc degeneration through inactivation of the NF-kappaB pathway in mice. Am J Transl Res 11(4):2496–2506
Wang W, Deng G, Qiu Y, Huang X, Xi Y, Yu J, Yang X, Ye X (2018) Transplantation of allogenic nucleus pulposus cells attenuates intervertebral disc degeneration by inhibiting apoptosis and increasing migration. Int J Mol Med 41(5):2553–2564
Kawaguchi SI, Gonda Y, Yamamoto T, Sato Y, Shinohara H, Kobiki Y, Ichimura A, Dan T, Sonoda M, Miyata T, Ogawa A, Tsujita T (2018) Furan- and Thiophene-2-Carbonyl Amino Acid Derivatives Activate Hypoxia-Inducible Factor via Inhibition of Factor Inhibiting Hypoxia-Inducible Factor-1. Molecules 23 (4)
Kong KH, Oh HJ, Lim BJ, Kim M, Han KH, Choi YH, Kwon K, Nam BY, Park KS, Park JT, Han SH, Yoo TH, Lee S, Kim SJ, Kang DH, Choi KB, Eremina V, Quaggin SE, Ryu DR, Kang SW (2017) Selective tubular activation of hypoxia-inducible factor-2alpha has dual effects on renal fibrosis. Sci Rep 7(1):11351
Zou D, He J, Zhang K, Dai J, Zhang W, Wang S, Zhou J, Huang Y, Zhang Z, Jiang X (2012) The bone-forming effects of HIF-1alpha-transduced BMSCs promote osseointegration with dental implant in canine mandible. PLoS One 7(3):e32355
Itsuji T, Tonomura H, Ishibashi H, Mikami Y, Nagae M, Takatori R, Tanida T, Matsuda KI, Tanaka M, Kubo T (2020) Hepatocyte growth factor regulates HIF-1alpha-induced nucleus pulposus cell proliferation through MAPK-, PI3K/Akt-, and STAT3-mediated signaling. J Orthop Res
Heidary Arash E, Song KM, Song S, Shiban A, Attisano L (2014) Arhgef7 promotes activation of the Hippo pathway core kinase Lats. EMBO J 33(24):2997–3011
Heallen T, Zhang M, Wang J, Bonilla-Claudio M, Klysik E, Johnson RL, Martin JF (2011) Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size. Science 332(6028):458–461
Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore N, Piccolo S (2011) Role of YAP/TAZ in mechanotransduction. Nature 474(7350):179–183
Clattenburg L, Wigerius M, Qi J, Rainey JK, Rourke JL, Muruganandan S, Sinal CJ, Fawcett JP (2015) NOS1AP Functionally Associates with YAP To Regulate Hippo Signaling. Mol Cell Biol 35(13):2265–2277
Chen JY, Zhang YG, Du JD (2018) HIF-1alpha restricts proliferation and apoptosis of Tca8113 cells through up regulation of Hippo signaling pathway under hypoxic conditions. Eur Rev Med Pharmacol Sci 22(20):6832–6837
Zhang X, Li Y, Ma Y, Yang L, Wang T, Meng X, Zong Z, Sun X, Hua X, Li H (2018) Yes-associated protein (YAP) binds to HIF-1alpha and sustains HIF-1alpha protein stability to promote hepatocellular carcinoma cell glycolysis under hypoxic stress. J Exp Clin Cancer Res 37(1):216
Sherafatian M, Abdollahpour HR, Ghaffarpasand F, Yaghmaei S, Azadegan M, Heidari M (2019) MicroRNA Expression Profiles, Target Genes, and Pathways in Intervertebral Disk Degeneration: A Meta-Analysis of 3 Microarray Studies. World Neurosurg 126:389–397
Wang W, Guo Z, Yang S, Wang H, Ding W (2018) Upregulation of miR-199 attenuates TNF-alpha-induced Human nucleus pulposus cell apoptosis by downregulating MAP3K5. Biochem Biophys Res Commun 505(3):917–924
Choi H, Merceron C, Mangiavini L, Seifert EL, Schipani E, Shapiro IM, Risbud MV (2016) Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling. Autophagy 12(9):1631–1646
Sakai D, Grad S (2015) Advancing the cellular and molecular therapy for intervertebral disc disease. Adv Drug Deliv Rev 84:159–171
Li ZY, Xiong SH, Hu M, Zhang CS (2011) Knockdown Epithelial Membrane Protein 1 Suppresses Human Degenerative Intervertebral Disc-Derived Nucleus Pulposus Cell Proliferation. Cartilage 2(3):300–306
Chen D, Xia D, Pan Z, Xu D, Zhou Y, Wu Y, Cai N, Tang Q, Wang C, Yan M, Zhang JJ, Zhou K, Wang Q, Feng Y, Wang X, Xu H, Zhang X, Tian N (2016) Metformin protects against apoptosis and senescence in nucleus pulposus cells and ameliorates disc degeneration in vivo. Cell Death Dis 7(10):e2441
Fujita N, Markova D, Anderson DG, Chiba K, Toyama Y, Shapiro IM, Risbud MV (2012) Expression of prolyl hydroxylases (PHDs) is selectively controlled by HIF-1 and HIF-2 proteins in nucleus pulposus cells of the intervertebral disc: distinct roles of PHD2 and PHD3 proteins in controlling HIF-1alpha activity in hypoxia. J Biol Chem 287(20):16975–16986
Zhang W, Cohen SM (2013) The Hippo pathway acts via p53 and microRNAs to control proliferation and proapoptotic gene expression during tissue growth. Biol Open 2(8):822–828

Download PDF

Version 1

posted

You are reading this latest preprint version

miR-338-3p inhibits HIF-1α to Attenuate Nucleus Pulposus Cell Proliferation and Promote Apoptosis Through the Hippo-YAP Pathway

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Abbreviations

Declarations

References

Status:

Version 1