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


 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.


Introduction
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 brosus, 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 signi cant 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 Yesassociated 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 proteincoding 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 identi ed 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.
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 log 2 |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 classi cation analysis of miR-338-3p predicted target genes.

Cell proliferation assay
According to the manufacturer's manual, HNPCs (2.5×10 3 ) 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 Scienti c, Inc.).

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 pro les 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 identi ed with log 2 |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 con rmed 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 ow 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 con rmed that miR-338-3p knockdown signi cantly 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 signi cant change in luciferase activity in the mut 3′-UTR of the HIF-1α group ( Figure 3A). Results of the western blotting analysis further con rmed 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.

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 signi cantly 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 ndings demonstrated that miR-338-3p and HIF-1α regulated Hippo-YAP pathway-related protein expression in HNPCs.

Discussion
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][20][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 identi ed 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 con rmed that miR-338-3p is a degeneration-related miRNA in HNPCs that affects IDD. Our research is the rst 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 ndings 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.