miR-4731-5p Enhances Apoptosis and Alleviates Epithelial-Mesenchymal Transition through Targeting RPLP0 in Non-Small-Cell Lung Cancer

Background/Aim. MircoRNA-4731-5p (miR-4731-5p) is a new miRNA involved in different human cancers, but its function has not been clarified in non-small-cell lung cancer (NSCLC). The present study attended to resolve the role of miR-4731-5p in NSCLC. Materials and Methods. The expression level of miR-4731-5p or ribosomal protein large P0 (RPLP0) and NSCLC clinicopathologic characteristics were analyzed. The binding between miR-4731-5p and RPLP0 was confirmed by TargetScan prediction and luciferase reporter experiment. Also, the probable role of miR-4731-5p in NSCLC via RPLP0 was elaborated by the MTT, western blotting, immunofluorescence, transwell, flow cytometry, and TUNEL assays. Moreover, in vivo verification was conducted in xenografted nude mice. Results. The level of miR-4731-5p was notably declined in vivo and in vitro, which was involved in the prognosis of lung cancer patients. The miR-4731-5p mimic could remarkably restrain cell viability, invasion, and the translational expression level of vimentin and e-cadherin, with promoted cell apoptosis in NSCLC, which were notably reversed by RPLP0 overexpression. Conclusion. miR-4731-5p/RPLP0 axis might be an underlying therapeutic target for NSCLC.


Introduction
Lung cancer has become the dominating cause of death from all kinds of cancers worldwide and is currently the most frequently diagnosed malignancy [1]. It is reported that about 80% of lung cancer is authenticated as non-small-cell lung cancer (NSCLC), including cell carcinoma and adenocarcinoma [2]. e molecularly targeted systemic therapies of NSCLC have significantly improved the outcomes for patients, but the disease control rate and increased overall 5year survival rate remain poor [3]. erefore, expounding the pathogenesis of lung cancer is quite necessary and also can contribute to the development of an effective treatment.
A growing body of researches has exhibited differential expression of miRNAs in the development of NSCLC [4][5][6]. miR-4731-5p is a novel miRNA that has rarely been studied, which is associated with tumor suppression [7], and was able to distinguish tumor stage with high specificity and sensitivity [8], indicating miR-4731 may have a tumor-suppressive activity [7]. Some evidences show that miR-4731-5p suppresses glioma development [9]. Additionally, miR-4731-5p has been exhibited to be down-expressed in several cancers, such as oral lichen planus [10] and glioblastoma [9,11]. Nevertheless, the effect of miR-4731-5p on NSCLC still needs further studies. e human ribosomal P complex, which contains RPLP0, RPLP1, and RPLP2, accelerates protein synthesis through recruiting translational factors. RPLP0 is a member of the RPLP family and a crucial modulator in the progress of many diseases, including cancers. e overexpression of RPLP0 mRNA is seen in human colorectal and hepatocellular carcinomas [12]. RPLP0 contributes to the onset and development of gastric cancer [13] and gynecologic tumors [14]. Although the previous study reports that RPLP0 always acts as a reference gene for gene expression studies on NSCLC [15], more and more literature confirm that RPLP0 is differently expressed in lung cancer. Ali et al. [16] show that RPLP0 exhibits low expression stability in the NSCLC cell lines NCI-H A549, NCI-H446, and NCI-H460. Moreover, the level of RPLP0 is demonstrated to be downregulated in samples from patients with squamous-cell carcinoma compared to that in normal tissues based on the RNA sequencing (RNA-Seq) analysis [17]. Furthermore, our bioinformatics analysis, which predicted that RPLP0 and miR-4731-5p were each other's targets, led us to hypothesize that RPLP0 was silenced by miR-4731-5p.
us, the underlying mechanism of miR-4731-5p in NSCLC was illuminated in the present study. e results demonstrated that the level of miR-4731-5p was dramatically diminished in NSCLC tissues. e upregulation of miR-4731-5p may inhibit NSCLC development by directly decreasing the expression of RPLP0. 35 pairs of NSCLC tissues and relevant normal tissue were acquired from the Tianjin Chest Hospital. All samples were resected during the operation and instantly preserved in liquid nitrogen for the following study.

Sample Collection.
e experiment was in agreement with the ethics committee of the Tianjin Chest Hospital and all participants enrolled in the study offered signed informed consent.

Cell Transfection.
Cells were plated into 6-well plates with an inoculation density of 3 × 10 6 cells/well and then maintained for 24 h at 37°C with 5% CO 2 . When the cells reached 70-80% confluence, 100 nmol/L miR-4731-5p mimic was transfected into the cells through Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) based on the operating manual. For the up-regulation of RPLP0 expression, pcDNA3.1 containing the full-length complementary DNA (cDNA) of RPLP0 (5 μg) was used in the NSCLC cells. e cells were gathered for subsequent detection after 48 h of transfection.

MTT Assay.
Cells were plated into 96-well plates with an inoculation density of 3 × 10 3 cells/well and hatched with 10 μl MTT solution (Sigma) at the indicated time for 4 h. Subsequently, the supernatant fluid was abandoned, and each well was appended with 100 μl DMSO to dissolve the crystals. e enzyme-labelling measuring instrument was applied to determine the absorbance at 570 nm, and each individual experiment was repeated for 3 times at least.

Electronic Microscope Observation.
e cells were maintained in 6-well plates with an inoculation density of 1 × 10 5 cells/ml and then maintained for 24 h to promote the total attachment to the plates' surface. Subsequently, the cells were diverted with the miR-4731-5p mimic or mimic-NC. e cells were imaged by an inverted light microscope (Nikon Corporation, Tokyo, Japan).

Immunofluorescence
Staining. Cells were administrated with vehicle for 24 h. After blocking in phosphate buffer saline (PBS) containing 0.05% BSA, the cells were hatched with primary antibodies overnight at 4°C. en, the cells were maintained with anti-rabbit immunoglobulin G (IgG) coupled to Alexa-Fluor-630 or -488 (2 μg/mL) at room temperature for 60 min. e nuclear component was counterstained with DAPI (Vector Laboratories, Burlingame, CA, USA). e results were imaged under a fluorescence microscope (Carl Zeiss, ornwood, NY, USA).

Transwell Assays.
Transwell inserts were used to measure the invasion abilities with matrigel. e transwell chamber was loaded with 200 μl of cell suspension with 5 × 10 5 /ml cells. en, the lower transwell chamber was loaded with DMEM added with FBS (500 μl). e cells were rinsed by PBS buffer after being maintained at 37°C with 5% CO 2 for 36 h. Next, the cells were immobilized with 100% ethanol and stained with 0.1% crystal violet solution. For microscope-based observations, five random fields were chosen to determine the cell numbers.
2.9. Apoptosis Analysis. Cell apoptosis was assessed with the flow cytometry experiment. Briefly, the cells were gathered and rinsed by PBS twice after transfection. Subsequently, 5 μl Annexin V/FITC and propidium iodide (PI) were applied to stain the cells for quarter at room temperature following resuspension by 0.5 ml of bind buffer. e apoptosis of the cells was measured on a FACScan flow cytometer via CellQuest software (BD Biosciences).

TUNEL.
e in situ cell death detection kit (Roche, Budapest, Hungary) was utilized to evaluate the TUNEL assay based on the operating manual. e tissue sample was immobilized in 10% formaldehyde solution, and then embedded with paraffin. 5 μm sections were cut and dehydrated in graded concentrations of ethanol, cleared in xylene. en, the slides were hatched with proteinase K for 20 min at 37°C, blocked with 3% H 2 O 2 for 10 min, and fixed with 4% paraformaldehyde. Next, the sections were hatched with the TUNEL reaction mixture for 1 h at 37°C. For the detection of fluorescein-labeled DNA, horseradish peroxidase (HRP)conjugated antibody was added. e number of TUNELpositive cells among the total number of cells was counted.
e miR-4731-5p-mimics or specified luciferase reporter vectors were transfected into the cells. Luciferase activities were assessed by a dual-luciferase reporter assay system (Promega) after 48 h of transfection.

In Vivo Assay.
ymus-free nude mice (nu/nu; 8-weekold males) were bought from the Experimental Animal Center of Tianjin Chest Hospital. e mice were housed individually and fed in a temperature-controlled animal room with 12 hours/12 hours light-dark cycle. Animal assays were ratified by the Tianjin Chest Hospital (SYXK Jin 2019-0001).
e lentiviral vector with OE-miR-4731-5p or its negative control (OE-NC) acquired from GeneChem (Shanghai, China) was injected into the backs of nude mice subcutaneously. en, the tumor volume was supervised every 5 d by an electronic vernier caliper when they were visible. e mice were sacrificed with an intraperitoneal injection of sodium pentobarbital (200 mg/kg) after the introduction of tumor cells for 28 days, and also the tumors samples were removed and weighed. e tumor size was quantified based on the formula: volume � 1/2 × length × width 2 .
2.13. Immunohistochemistry. Immunohistochemistry was performed using the e-cadherin (Abcam) and vimentin antibodies (Abcam). Slides were repaired with sodium citrate buffer (10 mM, pH 6.0) at 94°C for 25 min, and then got back to room temperature. After rinsing, 1% bovine serum albumin (BSA) was utilized to seal the sections for 30 min. en, the sections were hatched with biotinylated secondary antibody. Restaining with hematoxylin was performed after the slices were washed with PBS for 3 × 10 min.
2.14. Statistical Analysis. All statistical data were analyzed through the SPSS 20.0 software (IBM, Armonk, New York, USA). e results were shown as mean ± standard deviation (SD). Statistical differences between the two groups were determined by Student's t-test, whereas differences among multiple groups were tested by one-way analysis of variance (ANOVA) followed by the post hoc Bonferroni test. e survival curve was established by the Kaplan-Meier method and the difference was evaluated with the log-rank test. p < 0.05 represents significant difference.

miR-4731-5p Level Was Reduced in NSCLC Cell Lines and Tissues and Related in the Prognosis of NSCLC.
e expressions of miR-4731-5p in the NSCLC tissues and five NSCLC cell lines were first examined via qRT-PCR. In comparison with the control group, the relative level of miR-4731-5p was markedly declined in the tissues and cells of NSCLC (Figures 1(a) and 1(b)). Since the expression level of miR-4731-5p in A549 and NCI-H1299 NSCLC cells was obviously diminished relative to that in the other three cell lines, A549 and NCI-H1299 NSCLC cells were selected for the following evaluation. Moreover, the transfection of the miR-4731-5p mimic markedly elevated the level of miR-4731-5p compared with the mimic-NC group in A549 and NCI-H1299 cells (Figures 1(c) and 1(d)). Besides, the interaction between the level of miR-4731-5p and NSCLC clinicopathologic characteristics was examined (Table 1). e expression level of miR-4731-5p was observably related in lymph node metastasis, distance metastasis, and TNM stage, though no statistical difference was indicated between the level of miR-4731-5p and age, gender, smoking, and tumor size. Furthermore, patients with lower expression levels of miR-4731-5p generally showed lymph node metastasis, distance metastasis, and III/IV TNM stage. Altogether, the results clarified that miR-4731-5p was declined in NSCLC tissues and cell lines, which was tightly relevant in the prognosis of NSCLC.

e Upregulation of miR-4731-5p Reduced Cell Viability, Invasion, and EMT with Elevated Apoptosis in both A549 and NCI-H1299 Cells.
To identify the role of miR-4731-5p in the Journal of Oncology NSCLC cells, the miR-4731-5p mimic and mimic-NC were diverted into A549 and NCI-H1299 cells separately. e upregulation of miR-4731-5p notably dampened the cell viability of A549 and NCI-H1299 cells relative to the mimic-NC group (Figure 2(a)). Morphological alterations were also discovered. As indicated in Figure 2(b), both A549 and NCI-H1299 cells treated with the miR-4731-5p mimic became sparser with an obvious spindle shape change compared with those in the mimic-NC group. To illustrate the role of miR-4731-5p in EMT, the translational expressions of e-cadherin and vimentin were analyzed via western blot. e miR-4731-5p mimic prominently elevated the expression level of e-cadherin protein with declined translational levels of vimentin both in A549 and NCI-H1299 cells relative to those in the mimic-NC group (Figures 2(c)-2(e)). Moreover, immunofluorescence results verified that the miR-4731-5p mimic memorably attenuated the level of vimentin both in A549 and NCI-H1299 cells (Figure 2(f )). Besides, the invasion ability of A549 and NCI-H1299 cells transfected with the miR-4731-5p-mimic was notably declined relative to that in the mimic-NC group (Figure 2(g)). However, the transfection of the miR-4731-5p-mimic signally promoted the apoptosis rate of both the cells, as shown by an increase in the apoptosis rate (Figure 2    Journal of Oncology upregulated level of miR-4731-5p repressed the cell viability, invasion, and EMT with increased apoptosis in both A549 and NCI-H1299 cells.

RPLP0 Directly Targeted to miR-4731-5p.
To deeply assess the molecular role of miR-4731-5p in regulating NSCLC, the relative level of RPLP0 in the NSCLC tissues was examined. An increased level of RPLP0 in tissues of NSCLC was observed relative to that in normal tissues (Figure 3(a)). Additionally, in accordance with the results of miR-4731-5p, the level of RPLP0 was also notably related in lymph node metastasis, distance metastasis, and TNM stage, but no statistical difference was found between the expression of RPLP0 and age, gender, smoking, or tumor size. On the contrary, patients with higher expression of RPLP0 usually exhibited distance metastasis, lymph node metastasis, and III/IV TNM stage (Table 2). Besides, the expression level of RPLP0 was negatively involved in the level of miR-4731-5p in NSCLC tissues (Figure 3(b)). e expression of RPLP0 was measured in the five NSCLC cells. e results exhibited that the level of RPLP0 was signally overexpressed in the five NSCLC cell lines relative to that in normal lung cancer cells, among which the level of RPLP0 was higher in A549 and NCI-H1299 cells relative to that in the other three cell lines (Figure 3(c)). e possible binding between miR-4731-5p and RPLP0 was analyzed using the TargetScan (Figure 3(d)).

miR-4731-5p Modulated Cell Viability, Invasion, Apoptosis, and EMTin A549 Cells via Targeting RPLP0.
To deeply explore whether miR-4731-5p modulated the development of NSCLC by targeting RPLP0, a cotransfection assay was executed. e repressive role of the miR-4731-5p mimic in the translational level of RPLP0 was significantly antagonized by the cotransfection of the miR-4731-5p mimic and OE-RPLP0 in A549 cells (Figure 4(a)). e MTTassay results indicated that the miR-4731-5p mimic inhibited the cell viability, which could be reversed by OE-RPLP0 (Figure 4(b)). Phase contrast microscopic evaluation further elucidated that A549 cells treated with OE-RPLP0 suppressed the changes caused by the miR-4731-5p mimic (Figure 4(c)). Cotransfection of the miR-4731-5p mimic and OE-RPLP0 markedly diminished the translational level of e-cadherin in A549 cells relative to that in the miR-4731-5pmimic group. e opposite effect on vimentin was also observed (Figures 4(d) and 4(e)). Immunofluorescence detection displayed that the expression level of vimentin was observably reduced in A549 cells treated with the miR-4731-5p mimic and OE-RPLP0 relative to that in the miR-4731-5p-mimic group (Figure 4(f )). Additionally, the transwell and apoptosis assays revealed decreased cell invasion and increased apoptosis following cotransfection, which was different from the results from the only transfection with the miR-4731-5p mimic (Figures 4(g) and 4(h)). Increased counts of TUNEL-positive cells resulted by the miR-4731-5p mimic were memorably inverted by the cotransfection of the miR-4731-5p mimic and OE-RPLP0 in A549 cells (Figure 4(i)). Besides, after A549 cells were cotreated with the miR-4731-5p mimic and OE-RPLP0, the protein level of Bax was markedly decreased with notably enhanced translational expression of Bcl-2, Wnt1, and Nuc-β-catenin vimentin relative to that in the miR-4731-5p-mimic group (Figures 4(j) and 4(k)). In brief, these results clarified that miR-4731-5p attenuated NSCLC cell growth and invasion with enhanced apoptosis by targeting RPLP0.

miR-4731-5p Expression Restrained Tumor Growth In
Vivo. To investigate whether miR-4731-5p played an analogous antitumor role was determined in vivo. e backs of nude mice received lentiviral vector with OE-miR-4731-5p, and a negative control was established as well. As presented in Figures 5(a) and 5(b), the elevation of miR-4731-5p prominently reduced the tumor volume of mice. Also, the upregulation of miR-4731-5p observably reduced the tumor weight of mice ( Figure 5(c)); however, no significant effect was found on body weight ( Figure 5(d)). e Kaplan-Meier analysis also exhibited that mice with a high level of miR-4731-5p in tumors had longer recurrence-free survival time than the control group miR-4731-5p mimic :    ( Figure 5(e)). An increased level of miR-4731-5p and a decreased level of RPLP0 were indicated in NSCLC tissues relative to those in the adjacent tissues (Figures 5(f) and 5(g)). e TUNEL assay data further revealed that apoptosis was notably elevated in the OE-miR-4731-5p group relative to the control group ( Figure 5(h)). e IHC data presented that the level of e-cadherin in NSCLC tissues overexpressing miR-4731-5p was memorably increased relative to that in the control group with an opposite pattern for vimentin ( Figure 5(i)), which was consistent with the results of western blotting (Figures 5(j)-5(l)). e translational expression of Bax was significantly elevated following OE-miR-4731-5p treatment in NSCLC tissues with reduced protein levels of Bcl-2, Wnt1, and Nucβ-catenin ( Figures 5(j), 5(k), and 5(m)). us, the findings demonstrated that the upregulated level of miR-4731-5p restrained NSCLC tumor growth by suppressing RPLP0 at the mRNA level.

Discussion
Lung cancer is a kind of heterogeneous disease derived from the abnormal cells of the respiratory epithelium. MiRNAs play a core role in initiating cancer and its development in different types of malignancies [18]. A large number of evidences have suggested the dysregulation of miRNAs related to a variety of diseases, including lung cancer [19,20]. Extensive miRNAs are dysregulated in NSCLC reported by the high-throughput analysis [21,22]. erein, miR-4731-5p has been confirmed to be a tumor-suppressed miRNA in melanoma [7] and glioma development [9]. e results of the current study suggested that NSCLC was associated with low expression level of miR-4731-5p, thereby indicating the altered level of miR-4731-5p might be the usual characteristic of NSCLC.
Plenty of studies have shown that miR-4731-5p targets and suppresses the level of oncogenes involved in cancer development, such as cellular proliferation, migration, and apoptosis [23]. e upregulation of miR-4731-5p suppressed the proliferation, migration, and invasion via targeting FOXM1 in breast cancer [24]. miR-4731-5p/E2F2 axis also regulated the progresses of glioma cells [9]. Interference of miR-4731-5p promoted the growth, migration, and invasion of choriocarcinoma by targeting HIF3A as well [25]. In the present study, the level of miR-4731-5p was consistently downregulated both in NSCLC tissues and cells. Plenty of studies have reported that miRNAs exhibited a satisfactory prognostic value in NSCLC [26,27]. Consistent with these findings, patients with a lower expression of miR-4731-5p usually exhibited distance metastasis, lymph node metastasis, and III/ IV TNM stage, which indicated that the level of miR-4731-5p was also correlative with the prognosis of lung cancer patients. Moreover, in subsequent transfection assays, A549 and NCI-H1299 treated with the miR-4731-5p mimic exhibited markedly decreased cell viability, invasion, and EMT, as well as strengthened apoptosis as relative to that in the controls. erefore, these data offered direct evidence of a tumorsuppressive activity of miR-4731-5p against the NSCLC cells, which was tightly correlative in the prognosis of lung cancer.
Previous studies have shown the importance of RPLP protein inhibition, as their downregulation may be critical for the therapy in cancer [28]. Growing evidence has indicated that RPLP proteins are highly regulated in endometrial carcinoma, ovarian cancer, colon carcinoma [14], and other numerous types of cancers [29,30]. Except for the endometrial carcinoma, ovarian cancer, and colon carcinoma [14], RPLP0, as a vital member of the RPLP family, is also upregulated in acute myeloid leukemia [31], clear cell renal cell carcinoma [32], and breast cancer [33]. Moreover, it has been demonstrated that patients with high expressions of RPLP0 are prominently associated with poor prognosis in clear cell renal cell carcinoma [32] and breast cancer [33]. In line with these findings, RPLP0 was also found markedly upregulated in NSCLC cell lines and tissues in our study, and the level of RPLP0 was also related in the prognosis of lung cancer patients. Furthermore, bioinformatics analysis predicted a reciprocity between miR-4731-5p and RPLP0, and the findings displayed that miR-4731-5p directly restrained RPLP0 by interacting with its 3'UTR. e results illustrated that miR-335-5p directly targets RPLP0.
Additionally, previous work has demonstrated that the down-expression of RPLP proteins affected cell growth and cell cycle progression [12]. RPLP0 has been found to regulate cell apoptosis and cycle arrest of cervical tumor cells [34]. e down-regulation of RPLP0 led to G1 arrest of gastric cancer cells [13]. Natalie et al. reported that RPLP0 was stably expressed in melanoma cells [35]. RPLP0 also modulates a variety of cellular functions in neurodegenerative diseases [36]. Overall, these results indicate that RPLP0 made vital contributions to the cell activity in various diseases. Here, we discovered that the over-regulation of miR-4731-5p resulted in the low expression of RPLP0, leading to the retardation of NSCLC cell viability and invasion, and consequently inhibited tumor growth. Epithelial cells can obtain the mesenchymal features during the EMT process [37]. In tumor, EMT is contacted with the progress of tumor beginning, invasion, and metastasis [38][39][40]. Furthermore, a growing body of research has indicated EMT is correlative with the progress and metastasis of NSCLC [41,42]. In the present study, we discovered that the EMT restraint which was induced by the miR-4731-5p mimic was significantly reversed by OE-RPLP0 in A549 cells. However, there is no relevant reports that describe the antitumor effect of RPLP0 dysregulation modulated by other miRNAs; thus, we discovered a novel target axis that might contribute to the NSCLC treatment in the current study. Taken together, further mechanistic experimentation revealed that RPLP0 contributed to the regulation of cell viability, invasion, EMT, and apoptosis by miR-4731-5p in NSCLC.
It has been shown that animal experiment models are a crucial method for NSCLC studies. Much evidence in vivo has demonstrated that the dysregulation of miRNAs plays a role in NSCLC development. For example, miR-367 promotes NSCLC progression in vivo [43], overexpression of miR-103 is capable of inhibiting NSCLC growth in vivo and promoting mouse survival [44], miR-146a-5p is overexpressed in the NSCLC cell line, and the repressive role of miR-146a-5p in the angiogenesis and tumorigenesis in a tumor model is also found [45]. In the final step, based on our findings, all animal experiments were implemented and finished successfully. In vivo tumor growth was suppressed by increasing miR-4731-5p. Besides, the overexpression of miR-4731-5p promoted apoptosis and inhibited EMT in vivo. ese in vitro effects were confirmed by adopting the nude mice model under in vivo conditions.
In short, we discovered that miR-4731-5p and RPLP0 dysregulated NSCLC tissues and cell lines, which are involved in lymph node metastasis, distance metastasis, and III/IV TNM stage, with no relation to age, gender, smoking, or tumor size.
us, the level of miR-4731-5p and RPLP0 was related in the prognosis of lung cancer patients. Besides, our findings elucidated the mechanistic interaction between miR-4731-5p and RPLP0 in NSCLC. miR-4731-5p-mediated modulation pathway via targeting RPLP0 provides new insights into the therapeutic strategies for NSCLC. Nonetheless, several limitations of the study should be addressed: [1] although NSCLC is greatly sensitive to chemotherapy, it quickly obtains resistance. Only two NSCLC cell lines are utilized in this study, and thus validation in other chemoresistance NSCLC cell lines and clinical specimens is needed in subsequent studies; [2] the results of miR-4731-5P influence on other RPLP family members have been rarely reported. e exact mechanism of RPLP0 regulation of cell viability, invasion, apoptosis, and EMT of NSCLC is still unclear and needs more studies.
Data Availability e data analyzed and used during the present study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
All authors approved the final manuscript. Chang was responsible for conceptualization, methodology, software, and data curation, prepared the original draft, and visualized, investigated, and supervised the study. Chang also wrote the manuscript. Meilin Xu validated, reviewed, and edited the manuscript.

Supplementary Materials
Chang et al. showed that the expression of miR-4731-5p was notably declined in NSCLC tissues and cell lines, which was related to the prognosis of lung cancer patients. Mechanically, miR-4731-5p promotes cell apoptosis and alleviates epithelial-mesenchymal transition of non-small-cell lung cancer via targeting RPLP0. (Supplementary Materials)