Knockout of miR-17-92 Cluster Protect Against Intervertebral Disc Degeneration by Inhibiting Apoptosis of Nucleus Pulposus Cells in Mice via the Activation of PI3K/Akt Pathway

Background: microRNA(miR)-17-92 cluster is involved in a variety of physiological and pathological processes, and the purpose of this study is to preliminarily explore the role of miR-17-92 cluster in disc degeneration and the corresponding mechanisms. Methods: Hematoxylin and Eosin (HE) and Safranin O Staining were used to evaluate the degeneration of intervertebral disc. qRT-PCR was applied to evaluate the mRNA level of miR-17-92 cluster and functional genes of nucleus pulposus (NP) tissues, whose protein level was evaluated with Western-blot. Terminal-Deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) was used to evaluate the apoptotic level of nucleus pulposus cell (NPC). Results: The expression levels of members of the miR-17-92 cluster were signicantly increased in the NP tissues from patients with intervertebral disc degeneration (IVDD). Furthermore, in the 3-months and 24-months miR-17-92-ccKO mice, the degree of IVDD was signicantly lower than that of the control group. At the same time, we also detected the expression levels of related functional genes in the NP tissues of mice in two groups. The results showed that the mRNA and protein levels of Bax and Caspase-3 in the knockout group were signicantly lower than those in the control group, and the mRNA and protein levels of Bcl-2 were signicantly higher. The TUNEL results showed that the apoptosis level of NPCs in the 3-month knockout mice was signicantly lower than that in the control group. Finally, the assessment of pathway-related protein levels showed that p-Ser473-Akt expression ratio in the nucleus pulposus of mice in the knockout group were signicantly increased, suggesting that the PI3K/Akt pathway was activated after miR-17-92 cluster knockout. Conclusion: To sum up, miR-17-92 cluster does play an important regulating role in IVDD, and the results showed that miR-17-92 cluster could inhibiting NPCs apoptosis by activating PI3K/Akt pathway, eventually producing protective effect against IVDD.


Introduction
Intervertebral disc degenerative (IVDD) is a common disease in spinal surgery. The clinical symptoms caused by IVDD, including neck and shoulder pain, limb weakness, and paresthesia, will seriously affect the quality of life of patients. At present, the clinical treatment of IVDD is mainly achieved through decompression surgery, which adds a heavy economic burden to the patient's family and even the whole society [1,2]. Therefore, the development of a non-surgical treatment is imperative and urgent. Generally, studies on IVDD are divided into three levels: (1) studies on the mechanism of IVDD (2) Studies on the non-surgical treatment of IVDD; (3) Studies on drug delivery methods of intervertebral disc (IVD), while the most important part is to clarify the pathological basis of IVDD. In this regard, a large number of articles have discussed and analyzed the relevant mechanisms, and the conclusions obtained have gradually improved the construction of "IVDD signal network". MicroRNA (miR) mechanisms account for a large proportion in the whole network. miRs are a class of non-coding single-stranded RNA molecules encoded by endogenous genes between 19-25 nucleotides, which inhibit expression by complementing and binding to the 3'UTR region of the target gene. They are involved in various physiological and pathological processes, and play a key regulatory role in many cases. MIR17HG gene is a nucleotide sequence with a length of about 7000bp located on human chromosome 13. After further transcription and shear processing, 7 mature miRs will eventually be formed, including miR-17-3p, miR-17-5p, miR-18a-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p and miR-92a-3p, collectively known as the miR-17-92 family cluster. At present, a large number of studies have con rmed that miR-17-92 clusters are involved in the development and progression of many diseases [3][4][5].
There have been abundant studies on IVDD and miRs. For example, miR-21-5p can regulate the process of IVDD by promoting the proliferation of nucleus pulposus cell (NPC) and the degradation of extracellular matrix (ECM)[6-8]. miR-140-5p can regulate IDD by inhibiting in ammatory response and protecting NPCs [9,10]. In our previous work, we found that miR-17-92 clusters are involved in cartilage development and also play an important regulatory role in bone healing. Considering that the nucleus pulposus (NP), annulus brosus (AF), and endplate of the IVDs are all cartilaginous tissues, we considered whether miR-17-92 could also regulate the degeneration process of the IVDs. Therefore, the purpose of this study is to verify the above hypothesis [11].
Materials And Methods

Patient Materials
Non-degenerated IVD samples and degenerative IVD samples were abtained from idiopathic scoliosis patients and patients with IVDD undergoing disectomy, respectively. All patients performed routine magnetic resonance imaging (MRI) of spine before surgery. This study had been approved by the Ethics Committee of West China Hospital, Sichuan University and the Ethics Approvals have been provided.
Informed consent was obtained from all subjects and all methods were carried out in accordance with relevant guidelines and regulations.

Clinical Evaluation of Disc Degeneration with Magnetic Resonance Imaging (MRI)
The disc degeneration degree was analyzed according to T2-weighted images by using the modi ed P rrmann classi cation [12].

Animals and Experimental Groups
The miR-17-92 ox/ ox mouse strain used in this study was supported by The Jackson Laboratory and the collagen II (Col2a1)-Cre mouse strain was supported by The Maine Medical Research Center in The United States. By hybridization of miR-17-92 ox/ ox with Col2a1-Cre mouse strain, miR-17-92-ccKO mice were obtained with speci c knockout of miR-17-92 in chondrocytes, including NPCs. The mice of the control group in this study were C57BL/6 mice (WT), purchased from Beijing HFK Bioscience CO., LTD. All experimental protocols of the animal part of this study had been approved by the Animal Ethics Committee of West China Hospital, Sichuan University and the Ethics Approvals have been provided. All methods were carried out in accordance with relevant guidelines and regulations.

Histopathologic Analysis
Parts of mouse IVD tissues (Co6-Co8) were extracted for the histopathologic analysis. The target IVDs were excised, then xed with 4% paraformaldehyde and decalci ed with Ethylene Diamine Tetraacetic Acid (EDTA) solution, embedded with para n, and cut into 5um-thick section. The disc specimens were staind with hematoxylin-eosin (HE) and Safranin O staining.

Histological Evaluation of Intervertebral Disc Degeneration
Histological assessment of the degree of IVDD was based on Tam's report [13]. The whole scoring system is divided into three parts to evaluate the histological appearance of NP, AF and NP/AF. The speci c scoring system can be referred to Table-1.

Terminal-Deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL)
Apoptosis levels of NP tissues in each group were measured utilizing a TUNEL kit (Takara Bio Tech CO., LTD, Beijing, China) following the manufacturer's instructions. Green uroescence (FITC) indicated DNA fragments and the blue uroescence (DAPI) indicated cell nucleus. The percentage cells undergoing apoptosis to the total number of NP cells was calculated and considered the apoptotic index.

Western Blot Analysis
Total protein of each group of NP tissue was extracted with RIPA buffer, and the BCA kit (EpiZyme, Shanghai, China.) was used to detect the protein concentration. Protein samples were separated by electrophoresis using a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to a polyvinylidene uoride membrane (Millipore, Billerica, MA, USA). After processing in a 5% defatted milk in TBST, the blots were incubated with anti-β-actin, anti-GAPDH, anti-Akt, anti-p-Ser473-Akt, anti-Bcl-2, anti-Bax, anti-Caspase-3, anti-Cleaved-Caspase-3 (ProteinTech, Wuhan, China.), diuted from 1:500 to 1:1000, at 4℃, for over 12 hours. β-actin and GAPDH served as the internal reference. After washing with TBS containing Tween 20, the appropriate secondary antibody was incubated with the blots at 25℃ for 60min, treated with Chemiluminescence Kit (EpiZyme, Shanghai, China.) for 30s, and placed in a dark room for observation. Gray values were analyzed with Image J software v1.46.

Statistical Analysis
All statistical analyses were performed using SPSS 17.0 (SPSS, Chicago, Illinois). The results are presented as mean ± standard deviation. The differences between various groups were analyzed using Student T test. P 0.05 was considered to indicate statistical signi cance.

Differential Expression of miR-17-92 Cluster in Patients' NP with Intervertebral Disc Degeneration
The expression of miR-17-92 cluster was evaluated by qRT-PCR in patients with or without disc degeneration, and the results were shown in Figure-1. As we can see, the members of miR-17-92 cluster differentially expressed in two groups, and the expression levels were relatively lower in patients without disc degeneration, which at least suggests that miR-17-92 cluster is correlated with disc degeneration.

Knockout of miR-17-92 Cluster Relieve the Degree of Intervertebral Disc Degeneration in Mice
To further prove the correlation between miR-17-92 cluster and disc degeneration, we constructed genetically-engineered mice with the knockout of miR-17-92 cluster, and the knockout veri cation of miR-17-92 cluster has been shown in Figure- Figure-3E), we found that, in whatever age group, the disc morphology of the miR-17-92-ccKO group was signi cantly better than that of the control group, which was also con rmed by the histological scores ( Figure-3F to Figure-3M).

Knockout of miR-17-92 Cluster Protects Intervertebral Disc from Degeneration by Inhibiting Apoptosis of NPCs
We do not yet know with which mechanism miR-17-92 cluster affects intervertebral disc degeneration, so we examined the expression levels of a range of functional genes associated with nucleus pulposus cells. As shown in Figure-4A to Figure-4D, there were no signi cant differences in mRNA expression levels between two groups in Col2a1, Aggrecan, Proliferating Cell Nuclear Antigen (PCNA), Ki67, reminding us that the knockout of miR-17-92 cluster would not affect the matrix metabolism and proliferation of NPCs. As shown in Figure-4E to Figure-4H, the mRNA expression levels of Bax and Caspase-3 were signi cantly lower than that of the control group, while the mRNA expression level of Bcl-2 and the ratio of Bax/Bcl-2 in miR-17-92-ccKO group was signi cantly higher. Further, the Western Blot also showed the same results, as shown in Figure-4I to Figure-4M. To obtain a more intuitive result, we performed a direct histological evaluation of apoptosis in the caudal intervertebral discs with TUNEL ( Figure-4N

Discussion
The basic research of IVDD has become the clinical hotspot, and as mentioned above, the ultimate goal of the related research is to nd the non-surgical methods that can effectively relieve IVDD. Although not every study can be translated into clinical practice, the more complete the mechanism network of IVDD, the more abundant the treatment that can eventually be transformed. Therefore, studies on the mechanism of IVDD provide more possibilities [14].
The construction of non-coding RNA system has provided new ideas for the pathogenesis research of many diseases, including IVDD. Up to now, there have been more than hundreds of studies focusing on the correlation between non-coding RNA and IVDD, among which miR has been well studied. The results of this research provide new materials and possibilities for the construction and improvement of this system.
In previous work, we found that miR-17-92 cluster was involved in cartilage development and also play an important regulatory role in bone healing. Accordingly, we attempted to verify the effect of miR-17-92 cluster on IVDD. As described above, we rstly determined that the expression levels of miR-17-92 cluster in the NP of patients with IVDD were signi cantly higher than those in the control group. Next, we build the miR-17-92-ccKO mice, and evaluate the degenerative levels of IVDs. We found that in 3 months and 24 months of mice, the degenerative leves of IVDs in miR-17-92-ccKO mice were much lower than that of the control group, further con rming the regulatory role of miR-17-92 cluster in IVDD. In subsequent studies, we attempt to preliminary explore the regulating mechanism of miR-17-92 cluster in the IVDD. There're no signi cant difference in the expression levels of Col2a1, Aggrecan, PCNA and Ki67 mRNA in the NP tissues of the two groups of mice, suggesting that miR-17-92 cluster knockout did not affect the ECM synthesis and proliferation functions of NPCs. The mRNA levels of Bax and Caspase-3 in the nucleus pulposus of the knockout group were signi cantly lower than those of the control group, while the mRNA levels of Bcl-2 and the ratio of Bcl-2/Bax were signi cantly higher than those of the control group, and the protein results were also consistent. Therefore, we believe that miR-17-92 cluster knockout protect IVDs from inhibiting the apoptosis of NPCs. Furthermore, we demonstrate the above conclusions at the histological level with the TUNEL test. Finally, we screened the signaling pathways related to nucleus pulposus apoptosis with the help of existing literature reports, and the results indicated that the activity of PI3K/Akt pathway was signi cantly increased after miR-17-92 cluster deletion. Therefore, we believe that miR-17-92 cluster knockout can inhibit the apoptosis of nucleus pulposus cells by activating the PI3K/Akt pathway, and nally produce a protective effect on the IVDs against degeneration.
The main shortcomings of this paper which need further study in the future are as follows: (1) The elaboration of the relevant mechanism is not intact, which needs to be further analyzed with the mRNA high-throughput sequencing technology; (2) This study did not explore the possibility of clinical transformation.

Conclusion
To sum up, miR-17-92 cluster in IVDD does play an important regulating role, and preliminary results showed that miR-17-92 cluster could activate the PI3K/Akt pathway, which in turn, inhibit the apoptosis of NPCs, eventually producing protective effect on IVDs against degeneration. In the future to further explore the speci c mechanism, we will try to turn this conclusion for clinical transformation and application. The contents of the clinical specimen part of this study had been approved by the Ethics Committee of West China Hospital, Sichuan University and the Ethics Approvals have been provided. Informed consent was obtained from all subjects and all methods were carried out in accordance with relevant guidelines and regulations.
Animal Ethics approval: All experimental protocols of the animal part of this study had been approved by the Animal Ethics Committee of West China Hospital, Sichuan University and the Ethics Approvals have been provided. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication:
Not applicable.

ARRIVE guidelines:
The study was carried out in compliance with ARRIVE guidelines.
Availability of data and material: Data will not be shared. These are the preliminary results of a long-term study. Many of the image content is only a selective display. We don't want to damage the originality of this study by sharing the results too much. But, the data is available from the corresponding author on reasonable request.         Figure-4D, there's no signi cant difference in the mRNA expression level of PCNA in NP tissues between WT and miR-17-92-ccKO group of mice (P=0.5570). Figure-4E, the mRNA expression level of Bcl-2 in NP tissues is signi cantly higher in miR-17-92-ccKO than that of WT group of mice (P 0.0001). Figure-4F, the mRNA expression level of Bax in NP tissues is signi cantly higher in WT than that of miR-17-92-ccKO group of mice (P=0.0065). Figure-4G, the mRNA expression level ratio of Bcl-2/Bax in NP tissues is signi cantly higher in miR-17-92-ccKO than that of WT group of mice (P 0.0001). Figure-4H, the mRNA expression level of Caspase-3 in NP tissues is signi cantly higher in WT than that of miR-17-92-ccKO group of mice (P=0.0011). Figure-4I, Western-blot results of apoptosis-related genes in NP tissues of WT and miR-17-92-ccKO group of mice (This grouping of blots cropped from different gels). Figure-4J, the protein expression level ratio of Bcl-2/β-Actin in NP tissues is signi cantly higher in miR-17-92-ccKO than that of WT group of mice (P=0.0214). Figure-4K, the protein expression level ratio of Bax/β-Actin in NP tissues is signi cantly higher in miR-17-92-ccKO than that of WT group of mice (P=0.0074). Figure-4L, the protein expression level ratio of Caspase-3/β-Actin in NP tissues is signi cantly higher in WT than that of miR-17-92-ccKO group of mice (P=0.0023). Figure