Radiosensitivity in Non-Small-Cell Lung Cancer by MMP10 through the DNA Damage Repair Pathway

NSCLC (non-small-cell lung cancer) is an aggressive form of lung cancer and accompanies high morbidity and mortality. This study investigated the function and associated mechanism of MMP10 during radiotherapy of NSCLC. MMP10 expression in patients and their overall survival rate were assessed through GEPIA. Protein expression was tested by western blotting. Radioresistance was detected in vitro by apoptosis and clonogenic assay. The extent of DNA damage and repair was revealed by the comet test and γH2AX foci test. High MMP10 levels in specimens of lung adenocarcinoma were related to poor patient outcomes. Clonogenic and apoptosis assays revealed that MMP10 knockdown in A549 cells initiated radiosensitization. Furthermore, MMP10 siRNA increased damage to the DNA in NSCLC cells, while MMP10 was observed to participate in DNA damage repair post-ionizing radiation. Thus, after irradiation, MMP10 plays an essential role in NSCLC through the repair pathway of DNA damage; regulating MMP10 for NSCLC radiosensitivity might have potential treatment implications in radiotherapy of NSCLC.


Introduction
NSCLC (non-small-cell lung cancer) is an aggressive lung cancer type that accompanies increased death rates and morbidity, including squamous cell carcinoma, large cell carcinoma, and adenocarcinoma [1]. Te three methods commonly used for lung cancer treatment involve radiation, surgery, and chemotherapy [2]. However, over recent years, the development of "precision radiotherapy" is defned as stereotactic body radiation therapy, has indicated its precise, low, and noninvasive side efects, which furnishes more possibilities of treatment for lung cancer by radiotherapy [3]. While several methods for comprehensive radiotherapybased treatment are involved in NSCLC, it tolerates ionizing radiation with progressive radiotherapy, indicating that most such patients had essentially serious efects [4][5][6].
MMP10 (matrix metalloproteinase-10) is an essential member of the MMP (matrix metalloproteinase) family [7]. It is a mesenchymal lysing enzyme that can break down the core collagen IV, V, IX, X-proteins, fbronectin, laminin, elastin, gelatin, and proteoglycan [8]. Because MMP10 has roles in several pathological and physiological processes, it is essential for tissue damage repair, embryonic development, and other processes [9,10]. MMPs function in extracellular matrix (ECM) degradation and breakdown of the basement membrane tissues to facilitate tumor invasion, growth, and metastasis; besides, mediation of the ECM basement membrane is a signifcant stage for the transfer of tumors [11,12]. While several studies on MMP-2 and MMP-9 have been reported, there are only a few reports on MMP10 and tumor associations [13,14]. Recently, MMP10 was shown to play a signifcant role in pro-MMPs activation [15]; it is expressed at high levels in epithelial tumors like bladder transitional cell cancer, gastric cancer, esophageal cancer, NSCLC, and skin cancer [16][17][18][19]. Tese fndings indicate a close relationship between MMP10 and the development and occurrence of tumors.
In this research, we examined the function of MMP10 in NSCLC and observed that MMP10 conferred resistance to radiotherapy in NSCLC via the repair pathway for the damaged DNA. Te regulatory function of MMP10 on NSCLC radiosensitivity might have therapeutic possibilities in the radiotherapy of NSCLC.

Public Bioinformatics Analysis.
Diferentially genes were obtained using the limma R package from TCGA-LUAD, TCGA-LUSC, and normal lung tissues (GTEx). Here, MMP10 (NM_002425.3) expression between tumorous tissues of LUAD, LUSC, and normal surrounding tissues was analyzed using the "Expression DIY" module of GEPIA [20]. Survival analysis was performed according to the MMP10 expression status and Kaplan-Meier curves were plotted; comparison of MMP10 mutations was done according to the survival status (LIVING/DECEASED) in 514 TCGA-LUAD patients using cBioportal (https://cbioportal.org). Te Spearman method was used for the expression correlation between DDR-related genes and MMP10.

Cell
Culture and Treatment. A549, the human LUAD (lung adenocarcinoma) cell line, was procured from ATCC (USA). Tey were cultured in DMEM containing fetal bovine serum (10%) at 37°C in an incubator with a CO 2 (5%) chamber with appropriate humidity. A549 cells were radiated at dose of 8 Gy (clonogenic assay with 0 Gy, 2 Gy, 4 Gy, and 8 Gy). For apoptosis assay, the cells were detected by fow cytometry 24 h after radiation. Cellular state and density were observed during culture and fuid was changed on alternate days.

Irradiation.
For cell radiation treatment, we used 60 Co c-rays (Radiation Center, Faculty of Naval Medicine of the Second Military Medical University, Shanghai, China). A specifc dose was given to the cells at a rate of 1 Gy/min. All irradiations were performed at room temperature.

siRNA and Cellular
Transfections. MMP10 siRNA was obtained from Termo Fisher (Cat. # AM16708). MMP10 siRNA was transfected along with lipofectamine 3,000 from Invitrogen as per the provided instructions. Cells transfected with the empty vector were used as negative control (NC), along with untransformed cells (parental). A549 cells were cultured for at least 24 h after transfection and then exposed to radiation. Cells that were successfully transfected were used for assays at specifed time points.

Clonogenic
Assay. A549 cell survival was examined by clonogenic assay. Te cells were trypsinized, counted, and seeded in 60-mm culture dishes in two sets of three for each dose of radiation; the number of cells seeded was according to the dose of radiation (0 Gy-200 cells, 2 Gy-400 cells, 4 Gy-800 cells, and 8 Gy-1600 cells), followed by irradiation with 0, 2, 4, and 8 Gy after 24 h. After ten days, cells were fxed using paraformaldehyde and methylene blue (1%) stain. Tirty minutes later, dishes were washed using phosphate-bufered saline (PBS) and dried naturally. Ten, the clone formation was counted.

Apoptosis Assay.
To stain the irradiated A549 cells, Annexin V-fuorescein isothiocyanate (AV) and propidium iodide (PI) in the kit for apoptosis detection from Invitrogen (California, USA) were utilized. Te cells were plated in sixwell plates at a density of 10 5 cells per well and allowed to attach for 24 h. 24 h after 8 Gy radiation, the cells were harvested by trypsin digestion, washed with precooled phosphate-bufered saline (PBS) twice, and resuspended. Ten, the cells were stained with AV and PI at room temperature for 15 min in a dark room. Flow cytometry (Beckman CytoFLEX) was conducted for analyses as per the instructions of the manufacturer.

Neutral Comet Assay.
Te extent of damage to DNA of A549 cells was examined by the neutral comet assay using a kit from Trevigen Inc. (Gaithersburg, MD) that was used at 4 h and 8 h post-irradiation as per the protocol provided by the manufacturer. First, slides were immersed in a 1% NMA and dry thoroughly. Next, the single cell suspension prepared (2 × 10 4 cells/ml) was immersed in LMA under a 40°C water bath. Tird, cell suspension was mixed and rapidly pipetted onto the surface of the precoated slide. Te slides were then incubated at 4°C for 25 min at 25 V in TBE. Ten, the gel was stained with PI (10 μg/ml) for 20 min and then rinsed gently with ddH2O. Finally, all slices were examined by an Olympus BX60 fuorescence microscope. Total 100 images in each slide were analyzed using CASP 1.2.3b2 software (CASPlab, Poland).

Immunofuorescence
Staining. For this, cH2AX foci, a marker for DNA double-strand breaks, was detected via immunofuorescence assay. Post-2 Gy radiation, transfection of A549 cells with siRNA against MMP10 was conducted. At specifed times, cells were fxed with chilled methanol/acetone (1 : 1); then, BSA (3%; in PBS) was used for blocking at room temperature for 60 min. Ten, cells allowed to bind to a cH2AX primary antibody (1 : 300; Abcam, US) were reacted with the secondary antibody (1 : 1000). Ten, confocal and conventional microscopy was used to monitor immunofuorescence; each group recorded the number of cH2AX foci in 30 cells and took the average.

Statistical Analysis.
Data were acquired after conducting a minimum of three experiments conducted independently and were presented as the mean ± standard deviation. Te statistical signifcance limit was considered to be P < 0.05. Per treatment, for all the experimental groups, mean and standard error (SEM) was calculated. For all pairwise comparison procedures, Student's t-test was used, including calculating P values.

High Levels of MMP10 in Lung Adenocarcinoma
Correlates with Poor Patient Outcomes. We analysed the expression of MMP10 in both LUAD and LUSC specimens by Rlanguage according to TCGA and GTEx databases. Te expression of MMP10 in the two types of NSCLC was higher than that in normal lung tissue, but only the expression of MMP10 in LUSC was statistically signifcant (Figure 1(a)). Te MMP10 level in 486 primary LUAD specimens (Figure 1(b)) was remarkably higher than 338 healthy tissues. We then explored the relationship between MMP10 levels and LUAD/LUSC patient lifespan through R-language. Overall survival and disease-free survival were signifcantly lower for patients with MMP10 high LUAD (Figures 1(c) and 1(d)) relative to those with MMP10 low tumors (P < 0.05). Ten, we analyzed the clinical data combined with MMP10 gene mutation data in TCGA--LUAD via cBioPortal (https://cbioportal.org) online tools. Te results show that a total of 11 mutation sites (including 9 Missense, 1 Truncating, and 1 Splice) were found between 0 and 476 amino acids of MMP10 and 9 mutations in the domain. Tese mutations were all concentrated in the previous LIVING group, and none of the 186 cases in the DECEASED group had mutations, which illuminated the prognosis of LUAD patients with MMP10 mutation that shows better survival level (Figure 1(e)).

Impact of MMP10 siRNA on A549 Cell Survival and
Apoptosis Post-Irradiation. To reveal the efects of MMP10 in radio treatment, we frst used MMP10 siRNA for inhibiting the expression of MMP10 in A549 cells (Figure 2(a)). Ten, using these cellular models for clonogenic assay, MMP10 knockdown rendered these cells signifcantly sensitive to IR (Figure 2(b)). Furthermore, we explored the efect of siMMP10 on the apoptosis of A549 cell after irradiation using fow cytometry. As we could see in Figures 2(c) and 2(d), although there were little diferences in early apoptotic rate (fourth quadrant) and late apoptotic rate (frst quadrant) between group parental and group NC (negative control) due to little diference in detection time, the total number of apoptosis (frst and fourth quadrant) detected in the siMMP10 group was signifcantly less than that in groups parental and NC, which meant MMP10 knockdown signifcantly promotes A549 cell apoptosis post-IR.

Increase in NSCLC Cell DNA Damage Due to MMP10
siRNA Post-IR. Ten, comet assay was conducted to reveal the activity of MMP10 in NSCLC post-IR and examine the extent of DNA damage using MMP10 siRNA. Indeed, MMP10 expression knockdown enhanced the damage to DNA post-IR (Figures 3(a)-3(c)), suggesting a possible, important function of MMP10 in the repair pathway for DNA damage post-IR.

Involvement of MMP10 in the Pathway for DNA Damage
Repair. For confrming our inference, we examined the pathway for DNA damage repair by using western blot and immunofuorescence post-IR and treatment with MMP10 siRNA. Te cH2AX foci assay revealed a much higher foci number at 8 h after IR in the siMMP10 + IR group than in the IR group (Figures 4(a)-4(d)), suggesting signifcant

BARD1
BLM BRCA1  BRCA2  BRIP1  EME1  GEN1  MRE11A  MUS81  NBN  PALB2  RAD50  RAD51  RAD52  RBBP8  SHFM1  TOP3A  TP53BP1  XRCC2  XRCC3   LIG4  NHEJ1  POLL  POLM  PRKDC  XRCC4  XRCC5   Journal of Oncology impairment of DNA repair as a result of MMP10 knockdown in response to IR. DNA damage repair in body after radiation is mainly carried out through NHEJ (nonhomologous end-joining) and HR (homologous recombination) pathways. Among them, HR is a completely correct repair pathway, because it requires homologous sister chromatids as templates, so the repair process only occurs during the S and G2 phases of DNA replication. In contrast, the NHEJ pathway plays roles throughout the cell cycle because it can directly rejoin broken DNA without the need for homologous sequences, and thus is often the primary repair modality for DSBs [21]. Related studies have shown that the DNA damage repair mechanism in tumor cells is extremely active, and a series of DNA damage repairrelated proteins (ATM, DNA-PKcs, and Rad51) are involved in the regulation of tumor radiation resistance [22]. In order to further explore the relationship between MMP10 and DNA damage repair pathway, we tested the correlation between core genes of DDR pathway (HR and NHEJ) and expression of MMP10 in LUAD, which we found partial core genes of the DDR pathway were positively correlated with MMP10 expression, especially the genes in the HR pathway ( Figure 5(a)). Further examination showed the inhibition of phosphorylation of proteins involved in DNA damage repair post-MMP10 siRNA treatment ( Figure 5(b)), indicating the involvement of MMP10 in the pathway for DNA damage repair post-IR.

Discussion
Tis study revealed the involvement of MMP10 in NSCLC radiosensitivity through the pathway for DNA damage repair. First, R-language was used to reveal a signifcantly high expression of MMP10 in LUAD samples as per TCGA and GTEx samples, relative to that in normal tissue. Next, analysis of the correlation between the expression of MMP10 with LUAD patient lifespan revealed signifcantly lower rates of overall survival for patients with MMP10 high LUAD than those having tumors with MMP10 low (P < 0.05). We also found that MMP10 gene mutation data in TCGA-LUAD showed a better survival level which meant MMP10 might play a role in promoting tumor progression indirectly. IR can induce double-stranded DNA breaks and the subsequent apoptosis of corresponding cells [23]. Based on the bioinformatics analysis results, next, we used siMMP10 on A549 cells which rendered radioresistance for a better cell survival rate and lower apoptosis rate compared with negative control. Besides, we found MMP10 was closely associated with DNA damage through neutral comet assay. Tis brought us great interest so that we did series of experiments to detect the relationship between MMP10 and DNA damage repair after IR. Ten, we illuminated that the knockdown of MMP10 increased the damage to DNA post-IR through the inhibition of the pathway for DNA damage repair, which we deem the primary reason for resistance to radiotherapy in NSCLC. Radio treatment is an important approach to the treatment of tumors in lung cancer, although the outcome is not so satisfactory [24]. Studies on radiosensitization involve the following aspects: tolerance of tumor cells to hypoxia, repair of damaged DNA, apoptosis, angiogenesis, and disorders of the cell cycle [25][26][27]. While research on radiosensitization has progressed, it is still in the preliminary stage. With the resistance to radiotherapy of lung cancer cells, several uncertainties still exist in the treatment [28,29].
MMP10, as an essential component of the MMP family, is active in various pathological and physiological processes and is essential for the repair of the damaged tissue, development of the embryo, and other processes [30]. Recently, MMP10 was found to be essential for pro-MMP activation [31]; a high expression of MMP10 was observed in tumors epithelial cells, such as transitional cell cancer of the bladder, gastric cancer, skin cancer esophageal cancer, and NSCLC [17,[32][33][34]. In this study, we observed that MMP10 knockdown signifcantly inhibited A549 cell survival and facilitated apoptosis post-IR. Furthermore, MMP10 knockdown could enhance the extent of DNA damage post-IR. Studies have shown that abnormally active DNA damage repair ability is the core mechanism of tumor cell to resist IR, which is also the main reason why tumor cells have a better survival rate and lower apoptosis rate [35]. DNA strand breaks are severe damages caused by IR, which can be divided into DNA single-strand breaks, DNA double-strand breaks, DNA base damage, and DNA crosslinks. Among them, DNA double-strand breaks (DSBs) are the most important form of damage caused by IR, and it is also recognized as the most serious form of damage [36]. In response to DSBs, cells establish complex signaling networks for the activation of DNA damage checkpoints. Once the cell detects damage, a host of DNA repair factors localize to the site of chromatin damage and initiate the DNA repair machinery by recruiting other repair proteins. In eukaryotic cells, DSBs are mainly repaired by the NHEJ and HR pathways [37]. Terefore, we made an assessment of the repair pathway for DNA damage by western blotting and immunofuorescence suggested a crucial function of MMP10 in NSCLC post-radiation through the pathway for DNA damage repair and the regulatory role of MMP10 on NSCLC radiosensitivity may confer therapeutic indications to radiotherapy. In addition, as both the NHEJ and HR pathways proteins (DNA-PKcs, ATM, and Rad51) phosphorylation were reduced in our result; MMP10 might afect the upstream proteins of the NHEJ and HR pathways, which meant that MMP10 might be involved in the core regulation of DNA damage repair; this is our next research direction. Although radiotherapy is currently the mainstay of NSCLC treatment, tumor radioresistance has greatly limited the efcacy of radiotherapy. As DNA strand breaks are the main reason for cell death caused by IR, screening and discovering the key molecules involved in DNA radiation damage repair and elucidating their mechanism are the core basic issues in the feld of radiotherapy. But so far, there are very few genes that could be clinically targeted for radiosensitization. Our research results suggest that MMP10 may play an important role in tumor radioresistance. In the next step, we will continue to study how MMP10 regulates DNA damage repair pathway and carry out clinical transformation.
To conclude, this is the frst report to show that knockdown MMP10 can signifcantly radiosensitize NSCLC. We also fnd that MMP10 regulates tumor radiosensitivity through the DNA damage repair pathway. Tese novel fndings would possibly aid in discovering new mechanism to enhance radiosensitivity to NSCLC.

Data Availability
Te datasets are available under reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.