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AIM2 Promotes Gastric Cancer Cell Proliferation via the MAPK Signaling Pathway

AIM2 Promotes Gastric Cancer Cell Proliferation via the MAPK Signaling Pathway Hindawi Journal of Healthcare Engineering Volume 2022, Article ID 8756844, 9 pages https://doi.org/10.1155/2022/8756844 Research Article AIM2 Promotes Gastric Cancer Cell Proliferation via the MAPK Signaling Pathway 1 1 2 1 3 Xiaojia Feng , Zaozhi Song , Qihui Huang , Jianguang Jia , Lingmei Zhang , 1 1 Mengqi Zhu , and Jun Qian Department of Surgical Oncology, e First Affiliated Hospital of Bengbu Medical College, Bengbu, China Department of Emergency, e ird Affiliated Hospital of Anhui Medical University, Hefei, China Department of Oncological Gynecology, e First Affiliated Hospital of Bengbu Medical College, Bengbu, China Correspondence should be addressed to Jun Qian; qianjun215036@sina.com Received 3 December 2021; Revised 28 February 2022; Accepted 10 March 2022; Published 7 April 2022 Academic Editor: Deepak Garg Copyright © 2022 Xiaojia Feng et al. .is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Gastric cancer (GC) is a highly prevalent tumor type. .e dysregulated expression of melanoma deficiency factor 2 (AIM2) has been observed in a range of tumor types. Herein, we explore the role of AIM2 in the regulation of GC progression. Methods. Gastric cancer cells BGC-823 and MGC-803 in logarithmic growth phase were divided into blank group (control), Control group (NC) and SH-AIM2 group, respectively. Control group and SH-AIM2 group were transfected with AIM2 NC and SH-AIM2, respectively. Nude mice were divided into blank group (control) and SH-AIM2 group, and the treatment methods were the same as above. Differential AIM2 expression in GC tissues was assessed via bioinformatics analyses, after which western blotting was used for analyzing the AIM2 levels in tumor and paracancerous tissues from five stomach cancer patients. In addition, qPCR and protein imprinting were used to assess AIM2 expression levels in GC cells, and AIM2 knockdown was conducted in MGC-803 and BGC-823cells, after which colony formation and EdU incorporation assay were utilized to assess cell proliferation. .e oncogenic role of AIM2 was then assessed in mice and validated through immunohistochemical analyses. Results. GC tissues and cell lines exhibited marked AIM2 overexpression. AIM2 knockdown significantly impaired GC cell proliferation and migration, as confirmed through in vitro assays. In vivo experiments showed that both the increment ability and invasion and migration ability of AIM2 knockdown group were significantly lower than that of control and NC the change of AIM2 protein level would affect the change of MAPK pathway related protein level. Conclusions. AIM2 knockdown markedly suppresses the proliferation, migration, as well as invasion of GC cells via the inhibition of MAPK signaling, thereby slowing tumor progression. Overall, these results suggest that further analyses of AIM2 may offer clinically valuable insights that can aid in the treatment of human GC. mechanistic basis for this disease has yet to be fully defined. As 1. Introduction such, it is vital that additional studies exploring the factors which control the development and regulation of GC be Gastric cancer (GC) is a substantially predominant form of conducted in order to guide patient diagnosis and treatment. cancer affecting the digestive system, and is the second Mitogen-activated protein kinase (MAPK) family pro- common cause of death related to cancer [1]. GC incidence in teins are serine/threonine kinases comprising protein 38 China is very high, with approximately 430,000 new cases and (p38), c-Jun N-terminal kinase (JNK), and extracellular 300,000 deaths per year [2]. .e primary treatments for GC signal-regulated kinase (ERK) that are able to become ac- include surgical tumor removal and adjuvant radiotherapy, but tivated with the aid of an extensive range of stimuli, where 5-year survival rates remain poor in most advanced stomach upon they control key cellular activities such as proliferation, cancer patients [3]. Many genes associated with the onset and mitosis, and migration [4]. progression of GC have been identified to date, but the 2 Journal of Healthcare Engineering Melanoma deficiency factor 2 (AIM2) is an interferon- 2.2. Human Samples. All patients (n � 5, age 41 to 78 years, inducible gene that is encoded on chromosome 1 in mean age about 59.5 years). In the First Affiliated Hospital of humans, and that is expressed at high levels in leukocytes Bengbu Medical College, the patients underwent subtotal in the peripheral blood, small intestine, and spleen [5] gastrectomy without preoperative radiotherapy and che- AIM2 orchestrates inflammatory responses and cell death motherapy, and all patients were clearly diagnosed with upon parasitic infection [6, 7], but its role in oncogenic primary gastric cancer. Pathological tissues and adjacent contexts remains to be clarified. .ere exist some facts tissues were collected during operation. .e study obtained that AIM2 can perform a dual-task in oncogenesis [8]. the consent of the patients and was approved by the Ethics Early evidence from De Young et al. [5] suggested AIM2 Committee of the First Affiliated Hospital of Bengbu to play a tumor suppressor role, and some researchers Medical College. found that AIM2 suppressed autophagy and thereby inhibited renal cancer malignancy [9]. However, in more 2.3. Cell Culture and Transfection. .e cell lines including recent research, AIM2 was shown to be frequently mu- MGG-803 [17], MKN-45 GC, SGC -7901, and BGC-823 [18] tated in colorectal neoplasms wherein It was able to in- were provided by the Shanghai Cell Bank of the Chinese hibit neoplasia development in a manner dependent upon Academy of Sciences. Control GES-1 cells were from the noninflammatory bodies [10]. AIM2 also reportedly plays Wuhan Pule Life Sciences Co. Ltd. (China). Culturing of all a role in Epstein-Barr virus (EBV)-induced nasopha- cells was carried out in DMEM (Hyclone, USA) containing ryngeal cancer development [11]. .e occurrence and penicillin/streptomycin and 10% FBS (Gibco, USA) in a 5% development of gastric cancer and various signaling CO and 95% air incubator. AIM2 knockdown was achieved pathways, gene mutations and microenvironment can using shRNA constructs (AIM2: 5′-GATCCGCAAACTAT regulate and participate in the process of tumor occur- CAATCAAT CAAGAGATGTT TCAG TAGT TAGTG rence. It has been reported that AIM2 can regulate DNA- TTTTACGTGGTG-3′) and the pLKO .1 Lentivirus Particle PK-Akt, MTOR-S6K1, MAPK, and other signaling Transduction system (Gene Chemistry, Shanghai, China). pathways in colon cancer and liver cancer [10, 12, 13]. Cells in which AIM2 was stably knocked down were then Increases in reactive oxygen species (ROS) have been selected for with allopurynicin, while empty lentiviral par- detected in many cancers, and it has been reported that ticles were used as a negative control. ROS can regulate phosphorylation activation of ERK [14], promote tumor signal transduction, and enhance cell proliferation and survival. AIM2 has been proved to in- 2.4.WesternBlotting. Tissue samples from GC patients and duce ROS production by regulating mitochondrial dy- controls were lysed, and total protein extracts of gastric namic balance. in addition, By regulating the dynamic cancer cells were analyzed by BCA method. Dilution of the balance of mitochondria, AIM2 can promote the activa- samples was then carried out with 1x loading buffer, tion of ERK by extracellular growth factor, promote the denatured at 95 C, and separated via SDS-PAGE followed by phosphorylation of Dynamin related protein 1 (DRP1), their transferring to PVDF membrane. Blots were washed and be recruited to mitochondria to cause mitochondrial thrice with TBST, blocked for 15 min with a fast block division, thus leading to the proliferation of tumor cells solution, and incubated overnight with primary antibodies and drug resistance of tumor cells [15, 16] .ese reports specific for AIM2(20590-1-AP), p-ERK(ab232370), p-JN further confirm that AIM2 regulates MAPK signaling K(ab124956), ERK(ab17942), p-p38(AF4001), JNK(66210- pathway in no-small cell lung cancer(NSCLC) 1-Ig), and p38(66234-1-Ig) (1 :1000; Abcam, USA) at 4 C. .erefore, this paper aims to explore the interaction and Blots were then washed thrice for 10 minutes per wash, relationship between AIM2 and MAPK signaling pathway in succeeded by a 2 h incubation with a secondary antibody (1 : the occurrence and development of gastric cancer. 500, Abcam, USA). After three more washes, protein bands were detected with an ECL chemiluminescence kit and 2. Materials and Methods scrutinized employing the Universal Gene Gel Imaging System (Syngene, Cambridge, UK). 2.1. Bioinformatics Analyses. .e Cancer Genome Atlas (TCGA) database was queried for gene expression data pertaining to 375 GC patients (data type: HTseq-Counts). 2.5. qPCR. Extraction of total RNA from cells was carried Genes exhibiting at least a 1-fold change in expression out with Trizol, after which cDNA was synthesized and between GC tumor (n � 375) and paracancerous (n � 32) qPCR reactions were conducted using a standard two-step tissues were identified using R. Wilcoxon rank-sum tests amplification program. .e levels of gene expression were −ΔΔCt were used to assess differences in AIM2 expression be- assessed via the 2 approach. Primers were synthesized tween these samples. Median AIM2 expression levels were by Shanghai Gene Chemicals Co. Ltd. and were as follows: used to separate patients into two groups, and ROC curves AIM2-F 5′-GGCCACCATCTGTTTCTGTT-3′, AIM2-R 5′- were used to assess specificity and sensitivity values as- GCCACTAAGTCAAGCTGAAATG-3′; β-actin-F 5′-AAG sociated with this cut-off value. Analyses were conducted AGATGGCCACGGCTGCT-3′, β-actin -R 5′-TCCTTCTG after converting HTSEQ-FPKM data into the TPM for- CATCCTGTCGGC-3′. .ermocycler settings were as fol- ° ° ° mat. Any unavailable clinical data was treated as a missing lows: 95 C, 30 s; 40 cycles of 95 C for 5 s, 60 C for 30 s. Assays value. were repeated in triplicate. Journal of Healthcare Engineering 3 p=0.002 p=0.002 Normal Tumor Normal Tumor Type (a) (b) ROC curve (AUC = 0.523) T1 N1 T2 N2 T3 N3 T4 N4 T5 N5 AIM2 GAPDH 0.8 * * 0.6 0.4 0.2 0.0 N1 T1 N2 T2 N3 T3 N4 T4 N5 T5 0.0 0.2 0.4 0.6 0.8 1.0 False positive rate (c) (d) Figure 1: AIM2 is upregulated in gastric cancer. ( P< 0.05, Data obtained from three independent are represented as means and standard errors.) (a) AIM2 expression in GC tumor (n � 375) and paracancerous normal control tissues (n � 32) was assessed. (b) AIM2 expression was quantified in pairs of GC tumors and control tissues. (c) Assessment of AIM2 ROC curve sensitivity and specificity. (d) Western blotting was used to assess AIM2 protein levels in matched tumor (T) and normal (N) tissue samples. AIM2 is upregulated in GC cell lines. 2.6. Colony Formation Assay. Cells were accumulated and the bottom of each Transwell assay insert. Cells in the replated in the plates containing 6 wells (1000 cells/well). logarithmic phase of growth were then added in triplicate to Following incubation for 10 days, the fixation of cells with inserts in serum-free media. Cells were incubated for 48 h, formaldehyde (4%) was carried out followed by staining with Cells were then counted at 200x magnification. crystal violet. .e colonies containing >50 cells were counted via microscope. 2.9. Wound Healing Assays. Cells were plated in the plates containing 6 wells (5 × 105 cells/well) in triplicate until 2.7. 5-Ethynyl-2-deoxyuridine (EdU) Incorporation Assay. confluent, at which time a scratch wound was generated and GC cells were plated in the plates containing 24 wells until wells were washed thrice with PBS. Cells were then culti- 70% confluent, at which time an EdU test kit was used based vated for 24 h in serum-free DMEM, after which wound on provided directions. Briefly, cells underwent EdU la- healing was compared between 0 and 24 h employing beling, fixation, Apollo staining, DAPI staining, and imaging Photoshop. Wound Healing Rate � (1–24 h Distance/0h with a fluorescent microscope, after which cells were Range) × 100% counted using the Image-Pro software. 2.10. Immunohistochemistry (IHC). Paraffin-embedded tis- 2.8.TranswellAssay. Matrigel was diluted 1 : 8 in serum-free sue sections prepared in xenograft model experiments were DMEM, and 60 uL of this solution was used to evenly coat used for IHC analyses. probed with primary polyclonal True positive rate AIM2 expression 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 152025 30 AIM2/GAPDH 4 Journal of Healthcare Engineering 0.8 8 * * 0.6 6 AIM2 0.4 0.2 2 GAPDH 0.0 0 (a) (b) MGC803 MGC803 0.8 3 * 0.6 AIM2 0.4 0.2 0 GAPDH Control NC ShAIM2 0.0 Control NC ShAIM2 BGC823 BGC823 0.8 0.6 AIM2 0.4 Control NC ShAIM2 0.2 GAPDH 0.0 Control NC ShAIM2 (c) (d) Figure 2: Assessment and manipulation of AIM2 expression in GC cell lines ( P< 0.05, Data obtained from three independent are represented as means and standard errors.) (a, b) AIM2 levels were assessed in the indicated GC cells via Western blotting and qPCR. (c, d) An shRNA construct was used to knock down AIM2 expression in GC823 and MGC 803 cells, as confirmed via qPCR. antibodies specific for p-P38, p-JNK, or p-ERK (1 : 200, 2.12. Statistical Analysis. SPSS 21.0 was utilized for all sta- Abcam). Sections were then probed with secondary anti- tistical testing, and figures were prepared with GraphPad bodies (1 : 500, Abbkine, USA) at room temperature, washed Prism 6.0. Outcomes are given as mean± standard deviation with PBS, and color development was conducted using DAB. (SD) while Student’s t-tests or ANOVAs were employed for their comparison. .e curves of receiver operating char- acteristic (ROC) were employed for assessing the diagnostic 2.11. Tumor Xenografts. Male NOD-SCID mice (12 weeks utility of AIM2 based upon area under the curve (AUC) old) from the Cavens Laboratory (CHangzhou, China) values. P< 0.05 was the significance threshold, and all as- were randomized into control and shAIM2 groups, and sessments were executed at least three times. were subcutaneously implanted in the flank with the corresponding GC cells (0.2 mL of a 1 × 107 cell/mL 3. Results suspension in PBS). Tumor weight and diameter were measured every week for four weeks, after which mice 3.1. GC Tissues Exhibit AIM2 Upregulation. We began by were euthanized with 3% pentobarbital sodium, and for querying the TCGA database to appraise the expression of analysis, tumors were isolated. .e Animal Experimental AIM2 mRNA in 375 GC tumor specimens and 32 normal Ethics Committee of Bengbu Medical College approved gastric tissue control specimens, revealing a marked rise in this study. AIM2 expression in tumor tissues (Figures 1(a) and 1(b)). GES-1 Control Control SGC7901 NC NC MGC803 BGC823 Sh-AIM2 Sh-AIM2 MKN45 AIM2 Protein expression AIM2 Protein expression AIM2 Protein expression (Relative intensity to GAPDH) (Relative intensity to GAPDH) (Relative intensity to GAPDH) GES-1 SGC 7901 MGC803 BGC823 MKN45 Relative AIM2 mRNA expression Relative AIM2 mRNA expression Relative AIM2 mRNA expression GES-1 SGC 7901 MGC803 BGC823 MKN45 Journal of Healthcare Engineering 5 MGC803 Control NC ShAIM2 MGC803 MGC803 60 800 Hoechst EDU 400 ** Merged 0 0 BGC823 Control NC ShAIM2 Control NC ShAIM2 Control NC ShAIM2 BGC823 BGC823 60 800 Hoechst EDU 400 Merged 0 0 Control NC ShAIM2 Control NC ShAIM2 MGC803 MGC803 Control NC ShAIM2 MGC803 200 80 150 60 100 40 50 20 0 0 Control NC ShAIM2 Control NC ShAIM2 Control NC ShAIM2 BGC823 BGC823 Control NC ShAIM2 MGC803 800 50 0 0 Control NC ShAIM2 Control NC ShAIM2 Control NC ShAIM2 Figure 3: Inhibition of GC cell malignancy by the AIM2 knockdown. ( P< 0.05, Data obtained from three independent are represented as means and standard errors.) (a) BGC-823 and MGC-803 GC cells were separated into control, NC, and sh-AIM2 treatment groups and used in EdU incorporation assays to evaluate cell proliferation. (b) .e average colony counts were decreased by the AIM2 knockdown in colony formation assay. (c) A wound healing assessment was employed for appraising the influences of AIM2 knockdown on GC cell migration. (d) Transwell assessments were utilized for evaluating the impact of AIM2 knockdown on GC cell invasion. An ROC curve revealed that AIM2 exhibited an AUC value formation (Figure 3(a)). Similarly, sh-AIM2 treatment was of 52.3% (Figure 1(c)). To confirm these results, we obtained associated with the increasing reduction in EdU-positive 5 pairs of GC patient tumor and paracancerous tissue from cells number relative to control treatment (Figure 3(b)). Bengbu Medical College First Affiliated Hospital. When we Together, these data indicated that AIM2 knockdown analyzed these samples, we confirmed that AIM2 was suppresses BGC-823 and MGC 803 cell proliferation. expressed at significantly higher levels in tumor tissues relative to matched control tissues (Figure 1(d)). 3.3. AIM2 Promotes GC Cell Invasion and Migration. Next, Western blotting and qPCR assays were conducted Transwell and wound healing assays were next conducted to which revealed marked AIM2 upregulation in SGC-7901, assess the relationship between AIM2 expression and GC BGC-823, MGC803, and MKN45 in GC cells relative to cell migration. We found that AIM2 knockdown was as- control GES-1 cells (P< 0.05, Figures 2(a) and 2(b)). Sub- sociated with the decrease in the migratory activity of these sequently, the expression of AIM2 in BGC823 and MGC803 cells in an assay of wound healing at 24 h (Figure 3(c)), and cells was knocked down, as they exhibited maximal AIM2 in the invasive activity of these wells at 48 h in a Transwell expression in preliminary assays (P< 0.05, Figures 2(c) and assay relative to the control and NC groups (Figure 3(d)). 2(d)). Overall, these findings indicated that AIM2 knockdown was sufficient to suppress GC cell invasion and migration. 3.2. AIM2 Promotes GC Cell Proliferation. To explore how AIM2 knockdown impacted GC cell progression, colony formation, and EdU incorporation assays were next con- 3.4. AIM2 Knockdown Inhibits Excessive MAPK Signaling In ducted using BGC-823 and MGG-803 cells in control, NC, Vitro. To explore the mechanistic basis for the above and sh-AIM2 treatment groups. AIM2 knockdown was phenotypes, we next explored the effects of AIM2 knock- found associated with an increasing reduction in colony down on the activation of proliferation-related MAPK Cell number per field Cell number per field EDU positive cell (%) EDU positive cell (%) 24 h 0 h 24 h 0 h BGC823 MGC803 Clony number Clony number The rate of migration (%) The rate of migration (%) 6 Journal of Healthcare Engineering MGC803 BGC823 1.0 0.8 0.8 0.6 p-ERK p-ERK 0.6 0.4 0.4 * ERK ERK 0.2 0.2 0.0 0.0 Control NC ShAIM2 Control NC ShAIM2 p-JNK 0.25 0.8 p-JNK 0.20 0.6 0.15 0.4 JNK 0.10 JNK ** 0.2 0.05 ** 0.00 0.0 Control NC ShAIM2 Control NC ShAIM2 p-p38 p-p38 0.8 1.0 0.8 0.6 p38 p38 0.6 0.4 ** 0.4 0.2 * GAPDH GAPDH 0.2 0.0 0.0 Control NC ShAIM2 Control NC ShAIM2 ∗ ∗∗ Figure 4: AIM2 knockdown suppresses excessive MAPK signaling activity. ( P< 0.05, P< 0.01, Data obtained from three independent are represented as means and standard errors.) (a-d) ERK, JNK, and P38 phosphorylation levels were assessed via Western blotting in BGC823 and MGC803 cells, with GAPDH as a normalization control. AIM2 facilitates GC tumor progression in vivo. ** 2500 2.0 Control 1.5 *** Sh-AIM2 1.0 ** 0.5 * 0.0 07 14 21 28 Control ShAIM2 day Control ShAIM2 (a) (b) (c) Figure 5: Continued. Control NC Sh-AIM2 p-JNK/JNK p-p38/p38 p-ERK/ERK (MGC-803 cells) (MGC-803 cells) (MGC-803 cells) Tumor weight (g) Control NC Tumor volume (mm ) Sh-AIM2 Journal of Healthcare Engineering 7 Control ShAIM2 p-ERK Control ShAIM2 p-JNK Control ShAIM2 p-p38 Control ShAIM2 (d) Figure 5: AIM2 knockdown suppresses in vivo tumor proliferation. ( P< 0.05, Data obtained from three independent are represented as means and standard errors.) (a-c) Nude mice were implanted with control or AIM2-knockdown MGC803 cells. Tumor volume and weight were then measured. (d) Phosphorylated p-ERK, p-JNK and p-p38levels in control and AIM2-knockdown tumors were assessed via immunohistochemistry. signaling molecules [19, 20] including P38, JNK, and ERK expression is highly reduced in melanoma, colorectal cancer, (Figures 4(a)–4(d)). We found that the phosphorylation of and prostate cancer, whereas it is upregulated in hepato- each of these three MAPKs was impaired following AIM2 cellular carcinoma, nasopharyngeal cancer, and oral squa- knockdown in both MGC-803 and BGC823 relative to mous cell carcinoma. As such, AIM2 functions in a tumor control cells. type-specific manner [1, 8, 23, 24]. For additional investigation of the effects of AIM2 Herein, we conducted bioinformatics analyses in order knockdown on in vivo tumor growth, we next implanted to evaluate AIM2 expression in GC, and we further con- firmed its upregulation in GC tissues and cell lines. We nude mice with GC cells that had been stably transduced with lentiviral sh-AIM2 or control constructs. We observed therefore hypothesized that AIM2 may promote the de- significant decreases in tumor growth and tumor weight velopment of GC tumors. In bioinformatics analysis, the following AIM2 knockdown (Figures 5(a)–5(c)). We also expression of AIM2 in gastric cancer tissues was significantly discovered that the p-p38, p-JNK, and p-ERK levels were increased, which was also verified in the 5 human gastric reduced in sh-AIM2 tumors relative to controls cancer tissues collected by us. .erefore, we conducted (Figure 5(d)), suggesting that AIM2 knockdown may inhibit further verification in cytology and zoology. We down- excessive MAPK signaling in GC tumors, thereby con- regulated the expression level of AIM2 in gastric cancer cells, straining their growth. and found that its proliferation ability, invasion and mi- gration ability were inhibited. In the xenotransplantation experiment, the tumor size and weight of the experimental 4. Discussion group with down-regulated AIM2 expression level were also AIM2 is a cytoplasmic member of the HIN-200 family of significantly lower than that of the blank control group. In subsequent experiments, we verified the relationship be- proteins encoded on chromosome 1q22. .e AIM2 protein is 344 amino acids in length and possesses an approximate tween AIM2 and MAPK signaling pathway, and the acti- molecular weight of 3,9487 Da [21] .e AIM2 C-terminal vation of MAPK signaling pathway in the group with low domain and N-terminal domain contain HIN-200 and pyrin EXPRESSION of AIM2 was significantly inhibited. .ere- domains, respectively, the latter of which can bind to ASC, fore, we speculated that it was highly likely that AIM2 while the former can bind to DNA and facilitate NF-kB and inhibited the activation of MAPK signaling pathway through Cystica1 activation, thereby inducing cell death [22]. 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Peng et al., “NAIF1 inhibits gastric cancer cells migration and invasion via the MAPK pathways,” Journal of Cancer Research and Clinical Oncology, vol. 141, no. 6, pp. 1037–1047, 2015. [28] K. Yoshioka, “Scaffold proteins in mammalian MAP kinase cascades,”JournalofBiochemistry, vol. 135, no. 6, pp. 657–661, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Healthcare Engineering Hindawi Publishing Corporation

AIM2 Promotes Gastric Cancer Cell Proliferation via the MAPK Signaling Pathway

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Copyright © 2022 Xiaojia Feng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Healthcare Engineering Volume 2022, Article ID 8756844, 9 pages https://doi.org/10.1155/2022/8756844 Research Article AIM2 Promotes Gastric Cancer Cell Proliferation via the MAPK Signaling Pathway 1 1 2 1 3 Xiaojia Feng , Zaozhi Song , Qihui Huang , Jianguang Jia , Lingmei Zhang , 1 1 Mengqi Zhu , and Jun Qian Department of Surgical Oncology, e First Affiliated Hospital of Bengbu Medical College, Bengbu, China Department of Emergency, e ird Affiliated Hospital of Anhui Medical University, Hefei, China Department of Oncological Gynecology, e First Affiliated Hospital of Bengbu Medical College, Bengbu, China Correspondence should be addressed to Jun Qian; qianjun215036@sina.com Received 3 December 2021; Revised 28 February 2022; Accepted 10 March 2022; Published 7 April 2022 Academic Editor: Deepak Garg Copyright © 2022 Xiaojia Feng et al. .is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Gastric cancer (GC) is a highly prevalent tumor type. .e dysregulated expression of melanoma deficiency factor 2 (AIM2) has been observed in a range of tumor types. Herein, we explore the role of AIM2 in the regulation of GC progression. Methods. Gastric cancer cells BGC-823 and MGC-803 in logarithmic growth phase were divided into blank group (control), Control group (NC) and SH-AIM2 group, respectively. Control group and SH-AIM2 group were transfected with AIM2 NC and SH-AIM2, respectively. Nude mice were divided into blank group (control) and SH-AIM2 group, and the treatment methods were the same as above. Differential AIM2 expression in GC tissues was assessed via bioinformatics analyses, after which western blotting was used for analyzing the AIM2 levels in tumor and paracancerous tissues from five stomach cancer patients. In addition, qPCR and protein imprinting were used to assess AIM2 expression levels in GC cells, and AIM2 knockdown was conducted in MGC-803 and BGC-823cells, after which colony formation and EdU incorporation assay were utilized to assess cell proliferation. .e oncogenic role of AIM2 was then assessed in mice and validated through immunohistochemical analyses. Results. GC tissues and cell lines exhibited marked AIM2 overexpression. AIM2 knockdown significantly impaired GC cell proliferation and migration, as confirmed through in vitro assays. In vivo experiments showed that both the increment ability and invasion and migration ability of AIM2 knockdown group were significantly lower than that of control and NC the change of AIM2 protein level would affect the change of MAPK pathway related protein level. Conclusions. AIM2 knockdown markedly suppresses the proliferation, migration, as well as invasion of GC cells via the inhibition of MAPK signaling, thereby slowing tumor progression. Overall, these results suggest that further analyses of AIM2 may offer clinically valuable insights that can aid in the treatment of human GC. mechanistic basis for this disease has yet to be fully defined. As 1. Introduction such, it is vital that additional studies exploring the factors which control the development and regulation of GC be Gastric cancer (GC) is a substantially predominant form of conducted in order to guide patient diagnosis and treatment. cancer affecting the digestive system, and is the second Mitogen-activated protein kinase (MAPK) family pro- common cause of death related to cancer [1]. GC incidence in teins are serine/threonine kinases comprising protein 38 China is very high, with approximately 430,000 new cases and (p38), c-Jun N-terminal kinase (JNK), and extracellular 300,000 deaths per year [2]. .e primary treatments for GC signal-regulated kinase (ERK) that are able to become ac- include surgical tumor removal and adjuvant radiotherapy, but tivated with the aid of an extensive range of stimuli, where 5-year survival rates remain poor in most advanced stomach upon they control key cellular activities such as proliferation, cancer patients [3]. Many genes associated with the onset and mitosis, and migration [4]. progression of GC have been identified to date, but the 2 Journal of Healthcare Engineering Melanoma deficiency factor 2 (AIM2) is an interferon- 2.2. Human Samples. All patients (n � 5, age 41 to 78 years, inducible gene that is encoded on chromosome 1 in mean age about 59.5 years). In the First Affiliated Hospital of humans, and that is expressed at high levels in leukocytes Bengbu Medical College, the patients underwent subtotal in the peripheral blood, small intestine, and spleen [5] gastrectomy without preoperative radiotherapy and che- AIM2 orchestrates inflammatory responses and cell death motherapy, and all patients were clearly diagnosed with upon parasitic infection [6, 7], but its role in oncogenic primary gastric cancer. Pathological tissues and adjacent contexts remains to be clarified. .ere exist some facts tissues were collected during operation. .e study obtained that AIM2 can perform a dual-task in oncogenesis [8]. the consent of the patients and was approved by the Ethics Early evidence from De Young et al. [5] suggested AIM2 Committee of the First Affiliated Hospital of Bengbu to play a tumor suppressor role, and some researchers Medical College. found that AIM2 suppressed autophagy and thereby inhibited renal cancer malignancy [9]. However, in more 2.3. Cell Culture and Transfection. .e cell lines including recent research, AIM2 was shown to be frequently mu- MGG-803 [17], MKN-45 GC, SGC -7901, and BGC-823 [18] tated in colorectal neoplasms wherein It was able to in- were provided by the Shanghai Cell Bank of the Chinese hibit neoplasia development in a manner dependent upon Academy of Sciences. Control GES-1 cells were from the noninflammatory bodies [10]. AIM2 also reportedly plays Wuhan Pule Life Sciences Co. Ltd. (China). Culturing of all a role in Epstein-Barr virus (EBV)-induced nasopha- cells was carried out in DMEM (Hyclone, USA) containing ryngeal cancer development [11]. .e occurrence and penicillin/streptomycin and 10% FBS (Gibco, USA) in a 5% development of gastric cancer and various signaling CO and 95% air incubator. AIM2 knockdown was achieved pathways, gene mutations and microenvironment can using shRNA constructs (AIM2: 5′-GATCCGCAAACTAT regulate and participate in the process of tumor occur- CAATCAAT CAAGAGATGTT TCAG TAGT TAGTG rence. It has been reported that AIM2 can regulate DNA- TTTTACGTGGTG-3′) and the pLKO .1 Lentivirus Particle PK-Akt, MTOR-S6K1, MAPK, and other signaling Transduction system (Gene Chemistry, Shanghai, China). pathways in colon cancer and liver cancer [10, 12, 13]. Cells in which AIM2 was stably knocked down were then Increases in reactive oxygen species (ROS) have been selected for with allopurynicin, while empty lentiviral par- detected in many cancers, and it has been reported that ticles were used as a negative control. ROS can regulate phosphorylation activation of ERK [14], promote tumor signal transduction, and enhance cell proliferation and survival. AIM2 has been proved to in- 2.4.WesternBlotting. Tissue samples from GC patients and duce ROS production by regulating mitochondrial dy- controls were lysed, and total protein extracts of gastric namic balance. in addition, By regulating the dynamic cancer cells were analyzed by BCA method. Dilution of the balance of mitochondria, AIM2 can promote the activa- samples was then carried out with 1x loading buffer, tion of ERK by extracellular growth factor, promote the denatured at 95 C, and separated via SDS-PAGE followed by phosphorylation of Dynamin related protein 1 (DRP1), their transferring to PVDF membrane. Blots were washed and be recruited to mitochondria to cause mitochondrial thrice with TBST, blocked for 15 min with a fast block division, thus leading to the proliferation of tumor cells solution, and incubated overnight with primary antibodies and drug resistance of tumor cells [15, 16] .ese reports specific for AIM2(20590-1-AP), p-ERK(ab232370), p-JN further confirm that AIM2 regulates MAPK signaling K(ab124956), ERK(ab17942), p-p38(AF4001), JNK(66210- pathway in no-small cell lung cancer(NSCLC) 1-Ig), and p38(66234-1-Ig) (1 :1000; Abcam, USA) at 4 C. .erefore, this paper aims to explore the interaction and Blots were then washed thrice for 10 minutes per wash, relationship between AIM2 and MAPK signaling pathway in succeeded by a 2 h incubation with a secondary antibody (1 : the occurrence and development of gastric cancer. 500, Abcam, USA). After three more washes, protein bands were detected with an ECL chemiluminescence kit and 2. Materials and Methods scrutinized employing the Universal Gene Gel Imaging System (Syngene, Cambridge, UK). 2.1. Bioinformatics Analyses. .e Cancer Genome Atlas (TCGA) database was queried for gene expression data pertaining to 375 GC patients (data type: HTseq-Counts). 2.5. qPCR. Extraction of total RNA from cells was carried Genes exhibiting at least a 1-fold change in expression out with Trizol, after which cDNA was synthesized and between GC tumor (n � 375) and paracancerous (n � 32) qPCR reactions were conducted using a standard two-step tissues were identified using R. Wilcoxon rank-sum tests amplification program. .e levels of gene expression were −ΔΔCt were used to assess differences in AIM2 expression be- assessed via the 2 approach. Primers were synthesized tween these samples. Median AIM2 expression levels were by Shanghai Gene Chemicals Co. Ltd. and were as follows: used to separate patients into two groups, and ROC curves AIM2-F 5′-GGCCACCATCTGTTTCTGTT-3′, AIM2-R 5′- were used to assess specificity and sensitivity values as- GCCACTAAGTCAAGCTGAAATG-3′; β-actin-F 5′-AAG sociated with this cut-off value. Analyses were conducted AGATGGCCACGGCTGCT-3′, β-actin -R 5′-TCCTTCTG after converting HTSEQ-FPKM data into the TPM for- CATCCTGTCGGC-3′. .ermocycler settings were as fol- ° ° ° mat. Any unavailable clinical data was treated as a missing lows: 95 C, 30 s; 40 cycles of 95 C for 5 s, 60 C for 30 s. Assays value. were repeated in triplicate. Journal of Healthcare Engineering 3 p=0.002 p=0.002 Normal Tumor Normal Tumor Type (a) (b) ROC curve (AUC = 0.523) T1 N1 T2 N2 T3 N3 T4 N4 T5 N5 AIM2 GAPDH 0.8 * * 0.6 0.4 0.2 0.0 N1 T1 N2 T2 N3 T3 N4 T4 N5 T5 0.0 0.2 0.4 0.6 0.8 1.0 False positive rate (c) (d) Figure 1: AIM2 is upregulated in gastric cancer. ( P< 0.05, Data obtained from three independent are represented as means and standard errors.) (a) AIM2 expression in GC tumor (n � 375) and paracancerous normal control tissues (n � 32) was assessed. (b) AIM2 expression was quantified in pairs of GC tumors and control tissues. (c) Assessment of AIM2 ROC curve sensitivity and specificity. (d) Western blotting was used to assess AIM2 protein levels in matched tumor (T) and normal (N) tissue samples. AIM2 is upregulated in GC cell lines. 2.6. Colony Formation Assay. Cells were accumulated and the bottom of each Transwell assay insert. Cells in the replated in the plates containing 6 wells (1000 cells/well). logarithmic phase of growth were then added in triplicate to Following incubation for 10 days, the fixation of cells with inserts in serum-free media. Cells were incubated for 48 h, formaldehyde (4%) was carried out followed by staining with Cells were then counted at 200x magnification. crystal violet. .e colonies containing >50 cells were counted via microscope. 2.9. Wound Healing Assays. Cells were plated in the plates containing 6 wells (5 × 105 cells/well) in triplicate until 2.7. 5-Ethynyl-2-deoxyuridine (EdU) Incorporation Assay. confluent, at which time a scratch wound was generated and GC cells were plated in the plates containing 24 wells until wells were washed thrice with PBS. Cells were then culti- 70% confluent, at which time an EdU test kit was used based vated for 24 h in serum-free DMEM, after which wound on provided directions. Briefly, cells underwent EdU la- healing was compared between 0 and 24 h employing beling, fixation, Apollo staining, DAPI staining, and imaging Photoshop. Wound Healing Rate � (1–24 h Distance/0h with a fluorescent microscope, after which cells were Range) × 100% counted using the Image-Pro software. 2.10. Immunohistochemistry (IHC). Paraffin-embedded tis- 2.8.TranswellAssay. Matrigel was diluted 1 : 8 in serum-free sue sections prepared in xenograft model experiments were DMEM, and 60 uL of this solution was used to evenly coat used for IHC analyses. probed with primary polyclonal True positive rate AIM2 expression 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 152025 30 AIM2/GAPDH 4 Journal of Healthcare Engineering 0.8 8 * * 0.6 6 AIM2 0.4 0.2 2 GAPDH 0.0 0 (a) (b) MGC803 MGC803 0.8 3 * 0.6 AIM2 0.4 0.2 0 GAPDH Control NC ShAIM2 0.0 Control NC ShAIM2 BGC823 BGC823 0.8 0.6 AIM2 0.4 Control NC ShAIM2 0.2 GAPDH 0.0 Control NC ShAIM2 (c) (d) Figure 2: Assessment and manipulation of AIM2 expression in GC cell lines ( P< 0.05, Data obtained from three independent are represented as means and standard errors.) (a, b) AIM2 levels were assessed in the indicated GC cells via Western blotting and qPCR. (c, d) An shRNA construct was used to knock down AIM2 expression in GC823 and MGC 803 cells, as confirmed via qPCR. antibodies specific for p-P38, p-JNK, or p-ERK (1 : 200, 2.12. Statistical Analysis. SPSS 21.0 was utilized for all sta- Abcam). Sections were then probed with secondary anti- tistical testing, and figures were prepared with GraphPad bodies (1 : 500, Abbkine, USA) at room temperature, washed Prism 6.0. Outcomes are given as mean± standard deviation with PBS, and color development was conducted using DAB. (SD) while Student’s t-tests or ANOVAs were employed for their comparison. .e curves of receiver operating char- acteristic (ROC) were employed for assessing the diagnostic 2.11. Tumor Xenografts. Male NOD-SCID mice (12 weeks utility of AIM2 based upon area under the curve (AUC) old) from the Cavens Laboratory (CHangzhou, China) values. P< 0.05 was the significance threshold, and all as- were randomized into control and shAIM2 groups, and sessments were executed at least three times. were subcutaneously implanted in the flank with the corresponding GC cells (0.2 mL of a 1 × 107 cell/mL 3. Results suspension in PBS). Tumor weight and diameter were measured every week for four weeks, after which mice 3.1. GC Tissues Exhibit AIM2 Upregulation. We began by were euthanized with 3% pentobarbital sodium, and for querying the TCGA database to appraise the expression of analysis, tumors were isolated. .e Animal Experimental AIM2 mRNA in 375 GC tumor specimens and 32 normal Ethics Committee of Bengbu Medical College approved gastric tissue control specimens, revealing a marked rise in this study. AIM2 expression in tumor tissues (Figures 1(a) and 1(b)). GES-1 Control Control SGC7901 NC NC MGC803 BGC823 Sh-AIM2 Sh-AIM2 MKN45 AIM2 Protein expression AIM2 Protein expression AIM2 Protein expression (Relative intensity to GAPDH) (Relative intensity to GAPDH) (Relative intensity to GAPDH) GES-1 SGC 7901 MGC803 BGC823 MKN45 Relative AIM2 mRNA expression Relative AIM2 mRNA expression Relative AIM2 mRNA expression GES-1 SGC 7901 MGC803 BGC823 MKN45 Journal of Healthcare Engineering 5 MGC803 Control NC ShAIM2 MGC803 MGC803 60 800 Hoechst EDU 400 ** Merged 0 0 BGC823 Control NC ShAIM2 Control NC ShAIM2 Control NC ShAIM2 BGC823 BGC823 60 800 Hoechst EDU 400 Merged 0 0 Control NC ShAIM2 Control NC ShAIM2 MGC803 MGC803 Control NC ShAIM2 MGC803 200 80 150 60 100 40 50 20 0 0 Control NC ShAIM2 Control NC ShAIM2 Control NC ShAIM2 BGC823 BGC823 Control NC ShAIM2 MGC803 800 50 0 0 Control NC ShAIM2 Control NC ShAIM2 Control NC ShAIM2 Figure 3: Inhibition of GC cell malignancy by the AIM2 knockdown. ( P< 0.05, Data obtained from three independent are represented as means and standard errors.) (a) BGC-823 and MGC-803 GC cells were separated into control, NC, and sh-AIM2 treatment groups and used in EdU incorporation assays to evaluate cell proliferation. (b) .e average colony counts were decreased by the AIM2 knockdown in colony formation assay. (c) A wound healing assessment was employed for appraising the influences of AIM2 knockdown on GC cell migration. (d) Transwell assessments were utilized for evaluating the impact of AIM2 knockdown on GC cell invasion. An ROC curve revealed that AIM2 exhibited an AUC value formation (Figure 3(a)). Similarly, sh-AIM2 treatment was of 52.3% (Figure 1(c)). To confirm these results, we obtained associated with the increasing reduction in EdU-positive 5 pairs of GC patient tumor and paracancerous tissue from cells number relative to control treatment (Figure 3(b)). Bengbu Medical College First Affiliated Hospital. When we Together, these data indicated that AIM2 knockdown analyzed these samples, we confirmed that AIM2 was suppresses BGC-823 and MGC 803 cell proliferation. expressed at significantly higher levels in tumor tissues relative to matched control tissues (Figure 1(d)). 3.3. AIM2 Promotes GC Cell Invasion and Migration. Next, Western blotting and qPCR assays were conducted Transwell and wound healing assays were next conducted to which revealed marked AIM2 upregulation in SGC-7901, assess the relationship between AIM2 expression and GC BGC-823, MGC803, and MKN45 in GC cells relative to cell migration. We found that AIM2 knockdown was as- control GES-1 cells (P< 0.05, Figures 2(a) and 2(b)). Sub- sociated with the decrease in the migratory activity of these sequently, the expression of AIM2 in BGC823 and MGC803 cells in an assay of wound healing at 24 h (Figure 3(c)), and cells was knocked down, as they exhibited maximal AIM2 in the invasive activity of these wells at 48 h in a Transwell expression in preliminary assays (P< 0.05, Figures 2(c) and assay relative to the control and NC groups (Figure 3(d)). 2(d)). Overall, these findings indicated that AIM2 knockdown was sufficient to suppress GC cell invasion and migration. 3.2. AIM2 Promotes GC Cell Proliferation. To explore how AIM2 knockdown impacted GC cell progression, colony formation, and EdU incorporation assays were next con- 3.4. AIM2 Knockdown Inhibits Excessive MAPK Signaling In ducted using BGC-823 and MGG-803 cells in control, NC, Vitro. To explore the mechanistic basis for the above and sh-AIM2 treatment groups. AIM2 knockdown was phenotypes, we next explored the effects of AIM2 knock- found associated with an increasing reduction in colony down on the activation of proliferation-related MAPK Cell number per field Cell number per field EDU positive cell (%) EDU positive cell (%) 24 h 0 h 24 h 0 h BGC823 MGC803 Clony number Clony number The rate of migration (%) The rate of migration (%) 6 Journal of Healthcare Engineering MGC803 BGC823 1.0 0.8 0.8 0.6 p-ERK p-ERK 0.6 0.4 0.4 * ERK ERK 0.2 0.2 0.0 0.0 Control NC ShAIM2 Control NC ShAIM2 p-JNK 0.25 0.8 p-JNK 0.20 0.6 0.15 0.4 JNK 0.10 JNK ** 0.2 0.05 ** 0.00 0.0 Control NC ShAIM2 Control NC ShAIM2 p-p38 p-p38 0.8 1.0 0.8 0.6 p38 p38 0.6 0.4 ** 0.4 0.2 * GAPDH GAPDH 0.2 0.0 0.0 Control NC ShAIM2 Control NC ShAIM2 ∗ ∗∗ Figure 4: AIM2 knockdown suppresses excessive MAPK signaling activity. ( P< 0.05, P< 0.01, Data obtained from three independent are represented as means and standard errors.) (a-d) ERK, JNK, and P38 phosphorylation levels were assessed via Western blotting in BGC823 and MGC803 cells, with GAPDH as a normalization control. AIM2 facilitates GC tumor progression in vivo. ** 2500 2.0 Control 1.5 *** Sh-AIM2 1.0 ** 0.5 * 0.0 07 14 21 28 Control ShAIM2 day Control ShAIM2 (a) (b) (c) Figure 5: Continued. Control NC Sh-AIM2 p-JNK/JNK p-p38/p38 p-ERK/ERK (MGC-803 cells) (MGC-803 cells) (MGC-803 cells) Tumor weight (g) Control NC Tumor volume (mm ) Sh-AIM2 Journal of Healthcare Engineering 7 Control ShAIM2 p-ERK Control ShAIM2 p-JNK Control ShAIM2 p-p38 Control ShAIM2 (d) Figure 5: AIM2 knockdown suppresses in vivo tumor proliferation. ( P< 0.05, Data obtained from three independent are represented as means and standard errors.) (a-c) Nude mice were implanted with control or AIM2-knockdown MGC803 cells. Tumor volume and weight were then measured. (d) Phosphorylated p-ERK, p-JNK and p-p38levels in control and AIM2-knockdown tumors were assessed via immunohistochemistry. signaling molecules [19, 20] including P38, JNK, and ERK expression is highly reduced in melanoma, colorectal cancer, (Figures 4(a)–4(d)). We found that the phosphorylation of and prostate cancer, whereas it is upregulated in hepato- each of these three MAPKs was impaired following AIM2 cellular carcinoma, nasopharyngeal cancer, and oral squa- knockdown in both MGC-803 and BGC823 relative to mous cell carcinoma. As such, AIM2 functions in a tumor control cells. type-specific manner [1, 8, 23, 24]. For additional investigation of the effects of AIM2 Herein, we conducted bioinformatics analyses in order knockdown on in vivo tumor growth, we next implanted to evaluate AIM2 expression in GC, and we further con- firmed its upregulation in GC tissues and cell lines. We nude mice with GC cells that had been stably transduced with lentiviral sh-AIM2 or control constructs. We observed therefore hypothesized that AIM2 may promote the de- significant decreases in tumor growth and tumor weight velopment of GC tumors. In bioinformatics analysis, the following AIM2 knockdown (Figures 5(a)–5(c)). We also expression of AIM2 in gastric cancer tissues was significantly discovered that the p-p38, p-JNK, and p-ERK levels were increased, which was also verified in the 5 human gastric reduced in sh-AIM2 tumors relative to controls cancer tissues collected by us. .erefore, we conducted (Figure 5(d)), suggesting that AIM2 knockdown may inhibit further verification in cytology and zoology. We down- excessive MAPK signaling in GC tumors, thereby con- regulated the expression level of AIM2 in gastric cancer cells, straining their growth. and found that its proliferation ability, invasion and mi- gration ability were inhibited. In the xenotransplantation experiment, the tumor size and weight of the experimental 4. Discussion group with down-regulated AIM2 expression level were also AIM2 is a cytoplasmic member of the HIN-200 family of significantly lower than that of the blank control group. In subsequent experiments, we verified the relationship be- proteins encoded on chromosome 1q22. .e AIM2 protein is 344 amino acids in length and possesses an approximate tween AIM2 and MAPK signaling pathway, and the acti- molecular weight of 3,9487 Da [21] .e AIM2 C-terminal vation of MAPK signaling pathway in the group with low domain and N-terminal domain contain HIN-200 and pyrin EXPRESSION of AIM2 was significantly inhibited. .ere- domains, respectively, the latter of which can bind to ASC, fore, we speculated that it was highly likely that AIM2 while the former can bind to DNA and facilitate NF-kB and inhibited the activation of MAPK signaling pathway through Cystica1 activation, thereby inducing cell death [22]. AIM2 some mechanism. is involved not only in innate immune responses, but also in Several recent studies have clarified the role of the oncogenesis and tumor progression [8]. .e AIM2 MAPK pathway as a key regulator of GC devlopment Positive cells rate Positive cells rate Positive cells rate 8 Journal of Healthcare Engineering [2] M. C. Mendoza, E. E. Er, and J. Blenis, “.e Ras-ERK and [25–27]. MAPK proteins are kinases that are phosphorylated PI3K-mTOR pathways: cross-talk and compensation,” Trends in a series of steps whereupon these proteins can control key in Biochemical Sciences, vol. 36, no. 6, pp. 320–328, 2011. biological functions within tumor cells [28]. We found that [3] P. Zhao, D. Chen, and H. 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Dunn et al., “HIN-200 proteins marker and potential therapeutic target in GC. regulate caspase activation in response to foreign cytoplasmic DNA,” Science, vol. 323, no. 5917, pp. 1057–1060, 2009. Data Availability [8] D. Choubey, “Absent in melanoma 2 proteins in the devel- opment of cancer,” Cellular and Molecular Life Sciences, .e data used to support the findings of this study are vol. 73, no. 23, pp. 4383–4395, 2016. available from the corresponding author upon request. [9] D. Chai, H. Shan, G. Wang et al., “AIM2 is a potential therapeutic target in human renal carcinoma and suppresses its invasion and metastasis via enhancing autophagy induc- Ethical Approval tion,” ExperimentalCell Research, vol. 370, no. 2, pp. 561–570, .is study was granted ethical approval by the Institutional [10] J. E. Wilson, A. S. Petrucelli, L. 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Published: Apr 7, 2022

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