miR-199b-3p Promotes Radiation-induced Oral Mucositis via DDIT4-mediated mTOR Signal Pathway

Background: Radiation-induced oral mucositis (RIOM) is an adverse reaction in patients of head and neck cancer after radiotherapy. However, the key regulatory factors in the pathogenesis of RIOM remain largely unclear. In this article, we discover a novel role of DNA damage-inducible transcript 4 (DDIT4) in regulating RIOM pathogenesis. Methods: We established RIOM in vitro and in vivo models to mimic the biological processes of RIOM. The level of DDIT4 in RIOM was analyzed by real-time PCR and Western blot. Through the bioinformatics analysis and luciferase assay, the relationship between miR-199b-3p and DDIT4 was performed. The level of mTOR signaling were explored by Western blot. Besides, Clone Formation and EDU assay were performed to investigate the effects of miR-199b-3p/DDIT4 on cell proliferation. H&E and immunohistochemistry experiments examined the effects of miR-199b-3p/DDIT4 on RIOM in vivo. Results: We found that the level of DDIT4 was signicantly reduced during the RIOM formation, and up-regulated of DDIT4 suppressed the progression of RIOM in vitro and in vivo. Besides, we found DDIT4 was a direct target of miR-199b-3p. Ectopic expression of miR-199b-3p repressed the level of DDIT4 and activated mTOR signal conduction to promote RIOM progress, whereas the silencing of miR-199b-3p promoted the DDIT4-mediated RIOM regulation both in vitro and in vivo. Conclusion: Collectively, our studies not only identied the novel functional role of DDIT4 in modulating pathogenic processes of RIOM but also provided new directions and ideas for the future treatment of radiotherapy oral mucositis.


Introduction
Radiation-induced oral mucositis (RIOM) is the main treatment for nasopharyngeal carcinoma.
Nevertheless, radiotherapy has a direct damage on oral mucosa [1,2]. Excessive in ammation and epithelial ablation are the main features of oral mucositis. Severe oral mucositis may require feeding tubes to control severe pain or stopping radiotherapy prematurely [3]. The occurrence of RIOM is related to the effects of radiation, oxidative stress, transcription factors, pro-in ammatory cytokines and pathogenic microorganisms. The pathological process can be divided into ve phases: initiation, up-regulation, ampli cation, ulceration and healing [4]. The key regulatory factors in the pathogenesis of RIOM remain largely unknown. Therefore, we need to comprehend the pathogenesis and risk actors in order to provide basis for prevention and control of RIOM.
Ionizing irradiation leads to fatal damage in cellular DNA, principally DNA double strand breakage and the production of reactive oxygen species [5]. In RIOM, the activation of NF-κb and production of in ammatory factors cause downstream signaling pathways to be ampli ed, which ultimately lead to tissue damage and ulcer formation [6]. DNA Damage Inducible Transcript 4 (DDIT4) is a highly conserved stress-response protein, which is also regarded as REDD1 or RTP801 [7]. In previous research, DDIT4 can protect BMSC away from miR-22-mediated cell damage induced by ionizing radiation [8]. DDIT4 suppress osteoblast cells away from premature senescence induced by gamma radiation [9]. DDIT4 plays a protective role against radiotherapy in glioblastoma. In addition, DDIT4 is a suppressor of mammalian target of rapamycin (mTOR), which could regulate cell growth in response to environmental inputs [10].
The mechanism targets of mTOR are dual speci c protein kinases with phosphorylated serine/threonine and tyrosine residues [11]. Nonetheless, the function of DDIT4 in RIOM is still unclear.
In this article, we investigated the function of DDIT4 in regulating the pathogenesis of RIOM. We discovered that DDIT4 was decreased during RIOM in the mouse model and the normal NHKs after irradiation. We also discovered that silencing of DDIT4 could activate mTOR signaling and promote the progress of RIOM in vivo and vitro. We further demonstrated that DDIT4 was a direct target of miR-199b-3p. Ectopic expression of miR-199b-3p repressed DDIT4 and activated mTOR signaling to promote the formation of RIOM. Meanwhile, up-regulated of DDIT4 or down-regulation of miR-199b-3p enhanced RIOM cell model proliferation. In conclusion, our studies provide new directions and ideas for the research of RIOM.

Animal experiment
All animal experimental procedures were approved by the Animal Trials of the A liated Hospital of ZunYi Medical University. We anesthetized C57/BL6 mice (18-20g; 6-8 weeks) and exposed them to irradiation on the head and neck at a dose of 10 Gy for 3 times. After 7 days of irradiation, the mice were euthanized by exsanguination prior after intraperitoneal anesthesia. The tongue tissues were immediately cut out and xed in 4% formaldehyde solution, embedded in para n and sectioned [12].
The miR-199b-3p inhibitors, DDIT4 OE and DDIT4 KD (Ribobio, Guangzhou, China) were employed to assess the impacts of miR-199b-3p or DDIT4 on the model of RIOM. In brief, miR-199b-3p inhibitors (5'-TAACCAATGTGCAGACTACTGT-3') (10 nmol) in 50 µL PBS were administered into RIOM mouse caudal veins once for 3 days to conduce miR-21 knockdown. In order to achieve up-regulated or down-regulated of DDIT4 in RIOM mice, Adeno-associated viral vectors (DDIT4 OE) or lentivirus-loaded short hairpin RNA (DDIT4 KD) were injected into RIOM mice by caudal vein. The sequence of the DDIT4 OE was consistent with the coding sequence of mouse DDIT4. The sequence of the DDIT4 KD was designed and synthesized by Ribobio (China).

Hematoxylin-Eosin (H&E) Staining
Tongue tissues were xed in 4% paraformaldehyde for 24 h. Then, the samples were embedded in para n, and sectioned. The slices were immersed in 0.5% hematoxylin for 5 minutes and eosin solution for 3 minutes. Images of stained sections were observed under a light microscope (Nikon, Japan).

Immunohistochemistry (IHC) assay
Tongue tissues were xed with 4% paraformaldehyde, sliced after para n embedding. The sections were incubated with antibody CD45 (Abcame, ab281586) or PCNA(Abcame, ab92552) at 4°C overnight treatment. The sections were washed with PBS and then incubated with the secondary antibody of goat anti-rabbit HRP conjugate (CST) at room temperature for 1 h, after which haematoxylin was used for the counterstain. Images of each group were viewed under a light microscope (Nikon, Japan).

Clone Formation Assay
NHKs Cells were seeded into 6-well plates with 500 cells/well and incubated with 37°C, 5% CO 2 for 14 days. Subsequently, cells were xed with 10% formaldehyde, treated with 0.1% crystal violet. At last, the number of clonies were taken by a light microscope (Olympus, Japan).

EdU assay
Cellular proliferation rate was measured by EdU assay. NHKs cells (5 × 10 4 /well) were cultured in 24-well plates and transfected for 48 h. Then NHKs cells were xed with 4% paraformaldehyde, Triton X-100 was used to permeabilize the nuclear membrane, and NHKs cells were blocked with goat serum for 1 h. Further, NHKs cells were stained according to the manufacturer's suggestions.

Immuno uorescence analyses
The NHKs cells were incubated on the coverslip for 24 hours. Washing by PBS for 3 times, the NHKs cells were xed in 4% paraformaldehyde for 15 minutes, and then xed in 0.2% TritonX-100 for 10 minutes at 20°C. After blocking with 3% BSA for 30 minutes, incubated the DDIT4 antibody at -4°C overnight. The Alexa Fluor 488 conjugated second antibody was incubated at 20°C for 1 h. Afterwards, NHKs Cells were stained by rhodamine phalloidin for 25 mins. The nuclei of NHKs were stained with SlowFade® Gold Antifade Mountant (Thermo Fisher, S36942) for 30 mins. Finally, the samples added Anti-fade solution to prevent quenching. Imaging was performed by confocal microscopes (Leica TCS SP8).

RNA extraction and qRT-PCR
Total RNA was extracted and reverse-transcribed to cDNA by Trizol reagent and PrimeScript™RT kit (Takara, Japan). The cDNA reactions were ampli ed using SYBR Premix Ex TaqII (Takara, USA) by uorescent quantitative PCR 7500 (ABI, USA). All target gene transcripts were normalized to U6 or β-actin using the 2 −ΔΔCT method. The sequence was shown in Supplementary table 1.

Western blotting analysis
Tissues and cells were fully lysed with 500 µL protein lysate of RIPA 2.10 miRNA prediction and dual-luciferase reporter assay The candidate miRNA of DDIT4 was predicted with miRDB, miRwalk and TargetScan, and mmu-miR-199b-3p was chosen as a target miRNA. The wild type and mutant DDIT4 3'-UTR dual-luciferase reporter vectors were constructed. 80 ng luciferase reporter vectors and miR-199b-3p mimic or inhibitor using the lipofectamine 2000 (Invitrogen, CA, USA) were transfected into NHKs. Luciferase activity was measured after 24 h.

Data analysis
All Experimental results were repeated three times and the data were presented mean ± standard deviation (SD) and analyzed by GraphPad Prism 8. The student's t-test or one-way ANOVA with Turkey's test was used to measure statistical signi cance of differences between two groups or multiple groups, respectively. A P value < 0.05 was considered statistically signi cant.

Effects of DDIT4 overexpression on RIOM
In order to investigate the effect of DDIT4 in RIOM, western blot analysis was performed to assess DDIT4 expression pro le in the tongue tissue of RIOM mouse. The results showed that DDIT4 level in RIOM mouse tongue tissues was signi cantly decreased compared with non-irradiated ( Figure.1a).
Subsequently, H&E experiments were performed and showed that there was no change of the tongue tissues in the DDIT4 overexpressed mice under non-irradiation treatment. However, the ulcer areas were signi cantly reduced and the strati ed squamous keratinized epithelium complete depletion in the group of DDIT4 overexpressed mice under 10 Gy*3 ( Figure.1b-c). CD45 is a cell surface tyrosine phosphatase which could regulate T cell and B cell activation and maturation to facilitate radiation damage repair. [13]. Proliferating cell nuclear antigen (PCNA) is a key factor that initiates recombination-associated DNA synthesis after irradiated injury [14]. Immunohistochemical experiments indicated that up-regulated DDIT4 in RIOM in vivo could decrease in ltration of in ammatory cells at the sub-epithelial connective tissue and promote cell proliferation ( Figure.1d).
3.2 DDIT4 modulates proliferation in NHKs exposed to irradiation To con rm whether DDIT4 was also down-regulated by irradiation in vitro, NHKs were exposed to 10 Gy irradiation three times. qRT-PCR analyzed the mRNA level of DDIT4 was no difference at irradiation. Western blot analyzed the protein level of DDIT4 was down-regulation. In addition, immuno uorescence staining showed that the uorescence intensity of DDIT4 in 10 Gy*3 group was signi cantly weaker than Non-irradiation ( Figure.2a). NHKs were transduced with lentivirus expressing DDIT4, the result of EDU experiment showed that up-regulated of DDIT4 increased the survival fraction of NHKs subjected to irradiation ( Figure.2b). Colony-forming assay showed that up-regulated of DDIT4 suppressed the sensitivity of NHKs when exposed to irradiation ( Figure.2c).
3.2 DDIT4 suppressed the formation of RIOM by regulating the mTOR pathway DDIT4 was reported to promote cell survival under radiation via suppressing mTOR pathway, therefor we inferred the involvement of DDIT4 mediated mTOR pathway during the formation of RIOM [11,15]. DDIT4 as an inhibitor of mTOR can be actived the tuberous sclerosis 1/2 (TSC1/2) complex to interfere glioblastoma cell death induction by radiotherapy. Western blot detected the expression of TSC1/2, mTOR and mTOR downstream protein, including P-mTOR, mTOR, P-P70S6K, P70S6K, P-4EBP1 and 4EBP1 in the model of RIOM [16]. The results showed that TSC1/2 was down-regulated, mTOR and its downstream protein were up-regulated in the model of RIOM (Figure.

DDIT4 is a direct target of miR-199b-3p in RIOM
The mRNA level of DDIT4 had no change signi cantly under 10 Gy irradiation and combined with the changes of DDIT4 protein in Fig. 2a, we speculated that there was a post-transcriptional regulation mechanism to regulate the expression of DDIT4 in RIOM. It was reported that miRNA could regulate posttranscriptionally by mRNA cleavage or translational repression. We performed three online databases (miRDB, Targetscan, miRWalk) to predict miRNAs, and a total of 15 intersecting miRNAs were found ( Figure.4a). qRT-PCR analysis indicated that the expression of miR-199p signi cantly increased in RIOM in vivo and vitro ( Figure.4b). In addition, the predicted miR-199b-3p binding sites in the 3'UTR of DDIT4 were shown in Figure.4c. Moreover, luciferase reporter was performed to con rm the interaction between DDIT4 and miR-199b-3p. The results indicated that miR-199b-3p mimic suppressed the luciferase activity of wild-type DDIT4, and miR-199b-3p inhibitor promoted the luciferase activity of wild-type DDIT4. Meanwhile, miR-199b-3p mimic and inhibitor had no effects on the mutant group ( Figure.4d). Western blot and qRT-PCR further showed that miR-199b-3p negatively regulated the expression of DDIT4 (Figure.4e). These data indicated that DDIT4 was a target of miR-199b-3p.

MiR-199a accelerated RIOM through targeting DDIT4 in vivo
To evaluate the therapeutic potential of miR-199b-3p in vivo, mice were divided into eight groups: Nontreat group, NC inhibitor group, miR-199b-3p inhibitor group, miR-199b-3p inhibitor +DDIT4 KD group, Non-treat+10 Gy*3 group, NC inhibitor group+10 Gy*3 group, miR-199b-3p inhibitor group+10 Gy*3, miR-199b-3p inhibitor +DDIT4 KD+10 Gy*3 group. As similar to the results of the in vitro study, H&E indicated that down-regulation of miR-199b-3p with no change in Non-irradiated group. However, down-regulation of miR-199b-3p caused the in ammatory cells and ulcer areas were signi cantly reduced, the strati ed squamous keratinized epithelium complete depletion in the group of 10 Gy*3. In addition, downregulation of DDIT4 could restore the RIOM inhibition of miR-199b-3p inhibitor on RIOM mouse models ( Figure.6a-b). CD45 and PCNA were detected by immunohistochemical assay and found that downregulation of miR-199b-3p in RIOM in vivo could decrease in ltration of in ammatory cells at the subepithelial connective tissue and promote cell proliferation and up-regulation of DDIT4 could decrease the function of down-regulated miR-199b-3p in RIOM in vivo. Western blot analyzed TSC1/2, mTOR and its downstream protein and discovered that down-regulation of miR-199b-3p in RIOM in vivo could HYPERLINK "javascript:;" accelerate TSC1/2 and suppress mTOR and its downstream protein. Downregulation of DDIT4 could inhibit the suppression of mTOR and its downstream protein by miR-199b-3p inhibitor (Figure.6d). These results suggested that miR-199b-3p regulated RIOM through DDIT4/TSC1/2/mTOR pathway, and then participated in regulating the development of RIOM in vivo.

Discussion
Radiation-induced oral mucositis (RIOM) is a common toxicity in patients receiving radiotherapy for head and neck cancer [17]. The clinical manifestations in the acute stage range from mild erythema to deep mucosal ulceration [18]. RIOM not only signi cantly affects the oral function of patients, but also affects the short-term and long-term quality of life. In severe cases, it can reduce the tumor control rate and thus impact the survival of patients [1,19]. The key regulatory factors in the pathogenesis of RIOM remain largely unknown. Therefore, preventing or reducing radiation injury of the oral cavity is very necessary.
Previous studies have shown that the level of DDIT4 was related to the sensitivity of human cancer cells to chemotherapy and radiotherapy [9,10,20,21]. In addition, DDIT4 could protect BMSC from damage induced by ionizing radiation [8]. We discovered direct evidence that DDIT4 serves as a key regulator of the pathogenesis of RIOM for the rst time.
Our data revealed that DDIT4 was signi cantly decreased in RIOM mouse and cells model. Furthermore, the level of DDIT4 related to the formation of RIOM. Functionally, up-regulated of DDIT4 accelerated cell proliferation of RIOM in vitro. DDIT4 was a negative regulator of mTOR signal pathway in cellular response to stress. It has been con rm that irradiation generates excessive amounts of reactive oxygen species (ROS) resulting in oxidative stress which is the reason for the formation of RIOM [22]. Oxidative stress-related critical protein kinase mTOR which could regulate cell growth, cell death, cancer and metabolism [23][24][25]. Previous study has shown that inhibition of mTOR can protect normal tissues via suppression of radiation-induced senescence [26]. DDIT4 as an inhibitor of mTOR which can be ctivedactivated by the tuberous sclerosis 1/2 (TSC1/2) complex to interfere glioblastoma cell death induction by radiotherapy [10]. Ribosomal protein S6 kinases (P70S6K) and eukaryotic initiation factor 4E (eIF4E)-binding protein (4EBP1) are the characteristic downstream effectors of mTOR. P70S6K and 4EBP1 play particularly important roles in the mTOR signaling pathway growth acceleration function [27].
In this work, we speculated that DDIT4 inhibited the formation of RIOM by activating TSC1/2 to inhibit mTOR and downstream proteins (P70S6K). Western blot detected that the down-regulated of TSC1/2, upregulated of mTOR and its downstream protein in RIOM mouse and cell model. However we transfected DDIT4 OE could up-regulated TSC1/2, down-regulated mTOR and its downstream proteins at the model of RIOM in vitro and in vivo. The results con rmed our conjecture that DDIT4 suppresses the formation of RIOM by activating TSC1/2 to inhibit the mTOR signaling pathway related to oxidative stress.
MicroRNAs (miRNAs) are small non-coding RNA molecules, which only contain 18-25 nucleotides in length [28][29][30]. MiRNAs participate in many physiological and pathological processes through completely or incompletely complementary between seed region and 3'-UTR of target genes [31]. For instance, downregulated of miR-200c reduced radiation-induced ROS generation and DNA damage in the initiation stage of RIOM [32,33]. In this article, we detected that the mRNA level of DDIT4 without signi cantly changes in RIOM. However, the results of western blot indicated the protein level of DDIT4 was downregulated. Therefore, we speculated that the post-transcriptional regulation of DDIT4 was regulated by miRNA.
Therefore, we used TargetScan, miRDB, miRwalk software to predict, and found 15 common miRNAs which could combine with DDIT4-3'UTR. qRT-PCR analysis indicated only miR-199b-3p was up-regulated in RIOM. Bioinformatics and luciferase reporter assays suggested that miR-199b-3p speci cally bound to DDIT4. miR-199b-3p is a member of the miR-199 family, which could inhibit head and neck squamous carcinoma cell migration and invasion [34]. In addition, miR-199b-3p can suppress the apoptosis of cerebral microvascular endothelial cells in ischemic stroke [34]. In this research, we found that miR-199b-3p negatively regulates the expression of DDIT4 protein. Soon afterwards, we further explored the role of miR-199b-3p in RIOM. We proved that miR-199b-3p promoted the formatiom of RIOM by suppressing DDIT4 and activating mTOR pathway.

Conclusion
In summary, our data provided the evidence that DDIT4 modulates the pathogenesis of RIOM. We identi ed the miR-199b-3p promotes RIOM trough DDIT4/TSC1/2/mTOR pathway. This may provide a new perspective for the treatment and prognosis of RIOM.

Declarations
Funding This work was supported by National Natural Science Foundation of China(No. 81960202).

Con icts of interest
The authors declared that there are no con icts of interest.

Availability of data and material
The data used to support the ndings of this study are available from the corresponding author upon request.