Effects and mechanism of APPL1 on proliferation of gastric cancer cells

Effects and mechanism of APPL1 on proliferation of gastric cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects and mechanism of APPL1 on proliferation of gastric cancer cells Junshan Zhai, Jianhua Zhu, Qi Wang, Mi Yang, Jing Zhang, Huaizhi Wei, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4650101/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BACKGROUND: APPL1 is highly expressed in gastric cancer tissues and it is positively correlated with TNM stage, depth of invasion, and lymph node metastasis of gastric cancer, but the specific mechanism of APPL1 on gastric cancer cell proliferation has not been defined. In our study, we prepared the gastric carcinoma cell line SGC-7901/APPL1 via plasmid transfection technique, then investigated the change of gastric carcinoma cells' proliferation ability, aimed to investigate the regulatory mechanism of APPL1 on gastric cancer cell proliferation. METHODS The gastric carcinoma cell strain SGC-7901 was transfected by APPL1 gene plasmid as the experimental group; by blank plasmid as the control group. The expression of APPL1 on the experimental group was detected by Western-blot technique. The change of the experimental group cells ' proliferation ability was detected by MTT assay, The change of the experimental group cell cycle was detected by PI staining, The change of the experimental group cell cyclin proteins were detected by Western-blot technique. RESULTS By the Western- blot technique, the expression level of APPL1 protein in the experimental group cell strain was up-regulated effectively(P<0.01). Meanwhile, the proliferation ability of the experimental group increased significantly was detected by MTT assay(P<0.05), By the PI staining, the percentage of the experimental group cell cycle in G1 phase was decreased(P<0.01), in S phase was increased(P<0.01). By the Western-bolt technique, the expression of Cyclin D1(P<0.05) was up-regulated(P<0.05) and p16, p27 was down-regulated(P0.05). CONCLUSION Through the plasmid transfection technique, the gastric carcinoma cell line that could overexpress APPL1 protein were prepared successfully and whose proliferation ability were enhanced. Gastric cancer APPL1 Proliferation Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Adaptor Protein Containing PH Domain, PTB Domain and Leucine Zipper Motif 1(APPL1) is a functional protein identified by Mitsuuchi et al. in 1999 using the yeast two-hybrid system [ 1 ] . APPL1 is involved in various cellular signaling pathways, including the insulin signaling pathway(ISP) [ 2 ] and adiponectin signaling pathway(ASP) [ 3 ] associated with the metabolism of intracellular substances, the epidermal growth factor receptor(EGFR) [ 4 ] signaling pathway associated with cell proliferation and apoptosis and the follicle stimulating hormone receptor(FSHR) [ 5 ] , et al.. Therefore, it is closely related to cell proliferation, apoptosis, the regulation of blood glucose level and the occurrence of a variety of tumors. Our research group has confirmed that APPL1 is highly expressed in gastric cancer tissues by immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR) technology, and it is positively correlated with TNM stage, depth of invasion, and lymph node metastasis of gastric cancer [ 6 ] . Liu et al. found that APPL1 can promote the migration and metastasis of gastric cancer by regulating the phosphorylation of protein kinase B(PKB) [ 7 ] , but the specific mechanism of APPL1 on gastric cancer cell proliferation has not been defined. In this study, The gastric cancer cell line overexpressed APPL1 was constructed by lentiviral vector to study the changes of gastric cancer cell proliferation capacity and the changes of related proteins in the cell proliferation cycle, aimed to investigate the regulatory mechanism of APPL1 on gastric cancer cell proliferation. 2 Materials and Methods 2.1 Materials 2.1.1 Clone The gastric cancer cell line SGC-7901 was purchased from Wuhan Prosa Life Science and Technology Co., Ltd. HEK-293T cells were a gift from Professor Wang Bo in the central laboratory of our hospital. Freezing conditions: 55% basal medium + 40% fetal bovine serum( FBS) + 5% dimethyl sulfoxide in liquid nitrogen. 2.1.2 Reagents and Equipment APPL1 antibody (rabbit source, product number: SRP06369) was purchased from Tianjin Sale Biotechnology Co., Ltd, Methylthiazol Tetrazolium(MTT) cell proliferation and cytotoxicity detection kit (C0009) was purchased from Shanghai Siger Biotechnology Co., LTD, Cell cycle and apoptosis detection kit, Propidium iodide(PI) staining method, was purchased from Shanghai Yuanmu Biotechnology Co., LTD, Cyclin D1 Antibody (# 2978), Cyclin E antibody (# 20808), p16 antibody (# 108349), p21 (# 2947) antibody, p27 antibody (# 3688), p57 antibody (# 2557) were purchased from Shanghai Yijie Biological Co., LTD, The Flow cytometer (FACSCanto II) was purchased from BD Biosciences, USA, The Western-blot Gel electrophoresis system (JY-SCZ 2+) was purchased from Beijing Junyi Dongfang Electrophoresis Equipment Co., LTD, The RIPA cell lysis solution was purchased from Changsha Abiwei Biotechnology Co., Ltd, The FluorChem® Q protein Blot imaging and quantitative analysis system was purchased from ALPHA, USA, The BCA protein quantification kit was purchased from Biyuntian Biotech Company. 2.2 Methods 2.2.1 Cell culture The gastric cancer cell line SGC-7901 frozen in liquid nitrogen tanks was revived, centrifuged and cultured on day 1 in DMEM high-glucose medium supplemented with 20% fetal bovine serum at temperature 37℃ and 5% CO2. Cultured in DMEM high glucose medium supplemented with 10% FBS on day 2. On day 3, gastric cancer cell lines were dissociated by EDTA-trypsin and passaged once, and gastric cancer cells of the third generation were selected for experimental studies. Human embryonic kidney cells(HEK-293T) used for lentiviral vector packaging were cultured in the same manner as in the gastric cancer cell line. 2.2.2 lentiviral vectors The pWPXLd plasmid vector, see http://www.addgene.org/12258/ for detailed parameters, was used to construct a lentiviral plasmid overexpressed APPL1. The upstream primer of APPL1 is 5′-GC ACGCGT ATG CCG GGG ATC GAC AAG CTG CC-3′, the downstream primer of APPL1 is 5′-GC ACTAGT TTA TGC TTC TGA TTC TCT CTT CTT TCC-3′. After digestion of APPL1 and pWPXLd plasmids by endonuclease selected by software, source from http://nc2.neb.com/NEBcutter2/ , pWPXLd plasmid and APPL1 fragment were connected, and ligation products were transformed into DH5a competent bacteria, spread on agar plate of ampicillin resistance, amplified, plasmids were extracted and identified by sequencing, recombinant plasmids and blank plasmids were packaged in HEK-293T. Plasmids containing recombinant lentviral vectors were used as experimental group, and blank plasmids as control group。 2.2.3 cell lines SGC-7901 cells were digested with EDTA/trypsin and seeded in plates, and 5×10 5 cells were seeded per plate by hemocytometer plate controlled. On day 2 of cell inoculation, SGC-7901 cells were infected with APPL1 and control plasmids at 10 multiplicity of infection(MOI). After the cells were infected for 48h, the culture medium of puromycin at 5µg/mL was selected for screening, the culture medium was replaced once every 3 days, and the culture bottle was observed after 12 to 15d. The overexpressing APPL1 and control cell lines capable of stable passage were named LV-APPL1 and LV-CON, respectively. APPL1 proteins were extracted from LV-APPL1 and LV-CON, quantified by Western-blot to compare the differences between the two groups of cell lines. 2.2.4 MTT assay SGC-7901 cells infected by lentiviral vector with APPL1 and blank plasmids were cultured into single cell suspensions and seeded in 96-well plates at a volume of 1×10 3 cells/well. At the fixed time points of days 0,1,2,3, and 4 ,5 mg/mL 10µL MTT of solution was added, and 100 µL of formazan dissolution solution was added after 4h. The OD(optical density) value was measured at 490 nm by iMark ELIASA, the growth curve of the cells with time(days) as the abscissa and OD 490 as the ordinate was drawn。 2.2.5 PI staining The LV-APPL1 and LV-CON cells in logarithmic growth phase were centrifuged at 1000rpm, and the supernatant was aspirated, the precipitated cells were moved into DMEM high glucose medium containing 10% fetal bovine serum in a 37℃ incubator. The cell suspension was collected when the cell confluence reached 60%-80%, and then according to the cell cycle detection kit instructions, PI staining, cell cycle was detected by flow cytometer, and the proportion of each phase was analyzed with the flow cytometer with software FACSCCanto Clinical. 2.2.6 Western-blot experiments The LV-APPL1 and LV-CON cells in logarithmic growth phase were lysed with RIPA cell lysis solution after 48h of culture and each group protein was extracted using BCA protein quantification kit according to the instructions. Electrophoresis was performed on SDS-polyacrylamide gel, transferred to nitrocellulose membrane, the primary antibody of corresponding cyclin (dilution 1:200) was selected and incubated overnight at 4℃. On day 2, the nitrocellulose membrane was washed 3 times with TBST(TBS + Tween) buffer, 5mins per time, and then the corresponding diluted secondary antibody (dilution 1:1000) was added. After the membrane was closed at room temperature for 1h, the nitrocellulose membrane was washed 3 times with TBST on the shaker. Finally, the protein strips were developed by the enhanced chemiluminescence(ECL) detection kit, then were quantitative analysed by FluorChem®Q protein blot imaging and quantitative analysis system. 2.3 Statistical methods Statistical analysis of the data was performed using the IBM SPSS 19.0 software. Counting data were expressed as ±s, independent sample t-test for means between two groups, one-way ANO variance for multiple groups, LSD for pairwise comparisons between groups, repeated measures ANO variance for indicators at different time points, χ 2 for measurement data, and P < 0.05 as statistical significance。 3 Results 3.1 cell lines The constructed plasmid overexpressed APPL1 and pWPXLd for blank control infected gastric cancer SGC-7901 cells in vitro respectively, after 15 days of culture, the two culture bottles were covered with round and oval cells by observation under a phase-contrast microscope(See Fig. 1 ). The APPL1 protein was extracted from two groups and quantified by Western-blot. The results showed that the expression of APPL1 protein was (1.247 ± 0.196)µg in the experimental group and (0.276 ± 0.045)µg in the control group, with a significant difference (t = 5.543, P = 0.008). See Fig. 2 Figure 2 Western-blot analysed the results of APPL1 overexpression 3.2 Proliferation capacity The OD 490 was measured at different time points in two groups, the difference was statistically significant (P < 0.05). The OD 490 measurement values gradually increased with the culture time, and the OD 490 measurements varied at different time points in each group (P < 0.05). There was an interaction on the OD 490 measurements between the two groups (P < 0.05). See Table 1 . Table 1 Comparison of cell proliferation capacity between the two groups at different times (± s) group number Day 0 Day 1 Day 2 Day 3 Day 4 experimental group 50 0.123 ± 0.032 0.221 ± 0.043 0.732 ± 0.064 1.365 ± 0.053 2.085 ± 0.086 control group 50 0.134 ± 0.013 0.173 ± 0.012 0.487 ± 0.037 0.727 ± 0.046 1.158 ± 0.043 F group/time/interactive value 3.432/2.145/1.576 P group/time/interactiv value 0.032/0.043/0.028 Note: The Mauchly method test satisfies the spherical hypothesis 3.3 Cell cycle changes The proportion of cells in G1 stage was 52.10% in the experimental group, 61.64% in the control group, the proportion of cells in G1 in the experimental group was lower than that of the control group ( χ 2 = 28.653, P = 0.003); the proportion in S phase was 33.08% in the experimental group, 25.91% in the control group, and the proportion of cells in S phase was higher in the experimental group than that of the control group ( χ 2 = 37.196, P = 0.007). See Fig. 3 . 3.4 Western-blot The expression of Cyclin D1 protein was higher than that of the control group, and the expression of p16 and p27 were lower than those of the control group, all the differences were statistically significant (P 0.05). See Fig. 4 , and Table 2 . Table 2 Comparison of cyclin expression the between experimental and control groups (± s, µ g) group number Cyclin D1 p16 p27 Cyclin E p21 p57 experimental group 50 1.895 ± 0.053 0.318 ± 0.064 0.595 ± 0.083 2.768 ± 0.321 1.834 ± 0.331 3.219 ± 0.876 Control group 50 1.354 ± 0.241 0.687 ± 0.057 1.028 ± 0.039 2.347 ± 0.485 1.967 ± 0.454 3.564 ± 0.542 t value 3.357 2.485 2.012 0.567 1.313 0.817 P value 0.023 0.031 0.043 0.763 0.126 0.474 4 Discussion APPL1 gene contains multiple domains and expresses various active proteins, which has been demonstrated in lung cancer [ 8 ] and colorectal cancer [ 9 ] , which can promote tumor proliferation and inhibit tumor apoptosis. Broussard et al. confirmed the intrinsic mechanism of APPL1 as a protein regulator during cell migration [ 10 ] . The previous study of our research group found that APPL1 is closely related to the biological behavior and clinical characteristics of gastric cancer patients. Recently, the studies found that APPL 1 is also a prognostic predictor for kidney cancer and prostate cancer [ 11 – 12 ] . The incidence and mortality rate of gastric cancer have increased year by year, especially in East Asian countries, becoming the main fatal malignant tumor [ 13 ] . The mechanism of gastric cancer proliferation is complex, and the specific signaling pathways is important for the diagnosis and treatment of gastric cancer [ 14 ] . The previous study of our research group showed that the protein expression and mRNA level of APPL1 were higher in gastric cancer tissues compared with those of adjacent tissues, and are closely related to the tumor biological behavior of gastric cancer and the clinical characteristics of gastric cancer patients [ 6 ] . In this study, the plasmid carrying the APPL1 gene was further constructed by a lentiviral vector and successfully transfected with a gastric cancer cell line. MTT proliferation assay found the proliferative capacity of the gastric cancer cells overexpressed APPL1 was significantly enhanced. The flow cytometry found that gastric cancer cell lines overexpressed APPL1 had an higher proportion of cells in the S phase during the cell cycle, but decreased in the G1 phase. The Western-blot analysis found that overexpression of APPL1 promoted the expression of Cyclin D1 and inhibited the expression of p16 and p27, with no obvious effect on the expression of Cyclin E, p21, and p57. The results of this study suggest that the proportion of S/G1 of the gastric cancer overexpressed APPL1 in the cell cycle increased. CyclinD1 is one of the key proteins in the regulation of cell proliferation, which regulates cell proliferation mainly by regulating the G1/S conversion rate of the cell cycle [ 15 ] . In cell mitosis, Cyclin D1 is able to phosphorylate Rb by binding to a cyclin-dependent kinase, which in turn regulates cells from G1 phase to S phase [ 16 – 17 ] . Both p16 and p27 can block the formation of a complex with Cyclin D1 by binding to cyclin-dependent kinase 4, thus arresting cell division and proliferation [ 18 – 19 ] . The results of this study found that APPL1 promotes gastric cancer cell proliferation by regulating cyclin. Due to the upregulation expression of Cyclin D1 and downregulation expression of p16 and p27, the proportion of gastric cancer cells in S/G1 phase increased, and the proliferation ability of cells was significantly strengthened. Unlike the mechanism that APPL1 promotes proliferation in human hepatocellular carcinoma and triple-positive breast cancer. Ding et al. found that APPL1 stimulated cancer cell proliferation mainly by binding to the receptor of leptin after phosphorylation [ 20 ] in the above two cancer tissues, which suggests that APPL1 promotes cancer cell proliferation in different manner in different cancer tissues. It has been shown that the same gene or protein in different cancer tissues exerts the biological effects through different mechanisms of action [ 21 – 22 ] , This study shows that APPL1 also has this property. 5 Conclusion Our study revealed that the cell cycle and cyclin changes of SGC-7901 gastric cancer cell line overexpressed APPL1, but the specific mechanism or link between gene changes and protein expression differences is not clear, and further research is needed Declarations Ethics approval and consent to participate All procedures conducted in this study were in compliance with the principles outlined in the Declaration of Helsinki and were approved by the Ethics Committee of China PLA General Hospital (Approval number: 2023018). Additionally, this study obtained written informed consent from all patients. Consent for publication Consent for publication was obtained from the participants. Conflict of interest The authors have no conflicts of interest related to this research. Funding acquisition: KW Author Contribution JHZ did the work of Interpretation and analysis of data. QW and MY did the work of Experiment. JSZ wrote the manuscript.Revision for important intellectual content: JZ and HZW. Funding acquisition: KWSupervision: all authors All authors have read and approved this manuscript Data Availability The data that support the findings of this study are available from the corresponding author, Kai Wu, upon reasonable request. References Mitsuuchi Y, Johnson SW, Sonoda G et al. Identification of a chromosome 3p14.3-21.1 gene,APPL,encoding an adaptor molecule that interacts with the oncoprotein-serine/threonine kinase AKT2[J].Oncogene,1999,18(35):4891–8. Fan Y, Chen H, Peng H, et al. Molecularmechanisms of curcuminrenoprotection in experimental acute renal injury[J]. Front Pharmacol. 2017;8:912. Artimani T. Najafi R.APPL1 as an important regulator of insulin and adiponectin-signaling pathways in the PCOS:a narrative review[J]. Cell Biol Int 2020,44(8):1577–87. York HM, Patil A, Moorthi U, K, et al. Rapid whole cell imaging reveals a calcium-APPL1-dynein nexus that regulates cohort trafficking of stimulated EGF receptors[J]. Commun Biol. 2021;4(1):224. Zhou N, Wang N, Qin X, et al. Expression of follicle-stimulating hormone receptor(FSHR),protein kinase B-2(AKT2) and adapter protein with PH domain,PTBdomain,andleucine zipper(APPL1) in pig ovaries[J]. Pol J Vet Sci. 2017;20(4):661–7. Zhai JS, Song JG, Zhu CH et al. Expression of APPL1 is correlated with clinicopathologic characteristics and poor prognosis in patients with gastric cancer[J]. Curr Oncol 2016,23(2):e95–101. Liu Y, Zhang C, Zhao L, et al. APPL1 promotes the migration of gastric cancer cells by regulating Akt2 phosphorylation[J]. Int J Oncol. 2017;51(4):1343–51. Lakoduk AM, Roudot P, Mettlen M, et al. 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Novelpiperazine based benzamide derivatives as potential anti-glioblastoma agents inhibiting cell proliferation and cell cycle progression[J]. Eur J Med Chem. 2022;227:113908. DING, Y,CAO Y,WANG, B et al. APPL1-mediating leptin signaling contributes to proliferation and migration of cancer cells[J]. PLoS One 2016,11(11):e0166172. LIU, L,MICHOWSKI W,KOLODZIEJCZYK, A et al. The cell cycle in stem cell proliferation,pluripotency and differentiation[J]. Nat Cell Biol 2019,21(9):1060–7. LAVOIE, H,GAGNON J,THERRIEN. M.ERK signaling:a master regulator of cell behaviour,life and fate[J]. Nat Rev Mol Cell Biol 2020,21(10):607–32. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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4","display":"","copyAsset":false,"role":"figure","size":246834,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of cyclin expression between the experimental and control groups\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4650101/v1/8cf417b7b0ffd52a3d1d6b72.jpg"},{"id":68451982,"identity":"fa955576-54a7-40b5-a420-3c828c2e9e16","added_by":"auto","created_at":"2024-11-07 12:02:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1817764,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4650101/v1/5bbe9edd-0b26-4599-b0d3-b2665193207f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects and mechanism of APPL1 on proliferation of gastric cancer cells","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eAdaptor Protein Containing PH Domain, PTB Domain and Leucine Zipper Motif 1(APPL1) is a functional protein identified by Mitsuuchi et al. in 1999 using the yeast two-hybrid system\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. APPL1 is involved in various cellular signaling pathways, including the insulin signaling pathway(ISP)\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e and adiponectin signaling pathway(ASP)\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e associated with the metabolism of intracellular substances, the epidermal growth factor receptor(EGFR)\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e signaling pathway associated with cell proliferation and apoptosis and the follicle stimulating hormone receptor(FSHR)\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, et al.. Therefore, it is closely related to cell proliferation, apoptosis, the regulation of blood glucose level and the occurrence of a variety of tumors. Our research group has confirmed that APPL1 is highly expressed in gastric cancer tissues by immunohistochemistry and reverse transcription-polymerase chain reaction(RT-PCR) technology, and it is positively correlated with TNM stage, depth of invasion, and lymph node metastasis of gastric cancer\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Liu et al. found that APPL1 can promote the migration and metastasis of gastric cancer by regulating the phosphorylation of protein kinase B(PKB)\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, but the specific mechanism of APPL1 on gastric cancer cell proliferation has not been defined. In this study, The gastric cancer cell line overexpressed APPL1 was constructed by lentiviral vector to study the changes of gastric cancer cell proliferation capacity and the changes of related proteins in the cell proliferation cycle, aimed to investigate the regulatory mechanism of APPL1 on gastric cancer cell proliferation.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 \u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/h2\u003e\u003cspan\u003e\n \u003cp\u003e2.1.1 \u003cstrong\u003eClone\u003c/strong\u003e The gastric cancer cell line SGC-7901 was purchased from Wuhan Prosa Life Science and Technology Co., Ltd. HEK-293T cells were a gift from Professor Wang Bo in the central laboratory of our hospital. Freezing conditions: 55% basal medium\u0026thinsp;+\u0026thinsp;40% fetal bovine serum( FBS)\u0026thinsp;+\u0026thinsp;5% dimethyl sulfoxide in liquid nitrogen.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.1.2 \u003cstrong\u003eReagents and Equipment\u003c/strong\u003e APPL1 antibody (rabbit source, product number: SRP06369) was purchased from Tianjin Sale Biotechnology Co., Ltd, Methylthiazol Tetrazolium(MTT) cell proliferation and cytotoxicity detection kit (C0009) was purchased from Shanghai Siger Biotechnology Co., LTD, Cell cycle and apoptosis detection kit, Propidium iodide(PI) staining method, was purchased from Shanghai Yuanmu Biotechnology Co., LTD, Cyclin D1 Antibody (# 2978), Cyclin E antibody (# 20808), p16 antibody (# 108349), p21 (# 2947) antibody, p27 antibody (# 3688), p57 antibody (# 2557) were purchased from Shanghai Yijie Biological Co., LTD, The Flow cytometer (FACSCanto II) was purchased from BD Biosciences, USA, The Western-blot Gel electrophoresis system (JY-SCZ 2+) was purchased from Beijing Junyi Dongfang Electrophoresis Equipment Co., LTD, The RIPA cell lysis solution was purchased from Changsha Abiwei Biotechnology Co., Ltd, The FluorChem\u0026reg; Q protein Blot imaging and quantitative analysis system was purchased from ALPHA, USA, The BCA protein quantification kit was purchased from Biyuntian Biotech Company.\u003c/p\u003e\n \u003c/span\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 \u003cstrong\u003eMethods\u003c/strong\u003e\u003c/h2\u003e\u003cspan\u003e\n \u003cp\u003e2.2.1 \u003cstrong\u003eCell culture\u003c/strong\u003e The gastric cancer cell line SGC-7901 frozen in liquid nitrogen tanks was revived, centrifuged and cultured on day 1 in DMEM high-glucose medium supplemented with 20% fetal bovine serum at temperature 37℃ and 5% CO2. Cultured in DMEM high glucose medium supplemented with 10% FBS on day 2. On day 3, gastric cancer cell lines were dissociated by EDTA-trypsin and passaged once, and gastric cancer cells of the third generation were selected for experimental studies. Human embryonic kidney cells(HEK-293T) used for lentiviral vector packaging were cultured in the same manner as in the gastric cancer cell line.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.2.2 \u003cstrong\u003elentiviral vectors\u003c/strong\u003e The pWPXLd plasmid vector, see \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.addgene.org/12258/\u003c/span\u003e\u003c/span\u003e for detailed parameters, was used to construct a lentiviral plasmid overexpressed APPL1. The upstream primer of APPL1 is 5\u0026prime;-GC ACGCGT ATG CCG GGG ATC GAC AAG CTG CC-3\u0026prime;, the downstream primer of APPL1 is 5\u0026prime;-GC ACTAGT TTA TGC TTC TGA TTC TCT CTT CTT TCC-3\u0026prime;. After digestion of APPL1 and pWPXLd plasmids by endonuclease selected by software, source from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://nc2.neb.com/NEBcutter2/\u003c/span\u003e\u003c/span\u003e, pWPXLd plasmid and APPL1 fragment were connected, and ligation products were transformed into DH5a competent bacteria, spread on agar plate of ampicillin resistance, amplified, plasmids were extracted and identified by sequencing, recombinant plasmids and blank plasmids were packaged in HEK-293T. Plasmids containing recombinant lentviral vectors were used as experimental group, and blank plasmids as control group。\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.2.3 \u003cstrong\u003ecell lines\u003c/strong\u003e SGC-7901 cells were digested with EDTA/trypsin and seeded in plates, and 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were seeded per plate by hemocytometer plate controlled. On day 2 of cell inoculation, SGC-7901 cells were infected with APPL1 and control plasmids at 10 multiplicity of infection(MOI). After the cells were infected for 48h, the culture medium of puromycin at 5\u0026micro;g/mL was selected for screening, the culture medium was replaced once every 3 days, and the culture bottle was observed after 12 to 15d. The overexpressing APPL1 and control cell lines capable of stable passage were named LV-APPL1 and LV-CON, respectively. APPL1 proteins were extracted from LV-APPL1 and LV-CON, quantified by Western-blot to compare the differences between the two groups of cell lines.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.2.4 \u003cstrong\u003eMTT assay\u003c/strong\u003e SGC-7901 cells infected by lentiviral vector with APPL1 and blank plasmids were cultured into single cell suspensions and seeded in 96-well plates at a volume of 1\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well. At the fixed time points of days 0,1,2,3, and 4 ,5 mg/mL 10\u0026micro;L MTT of solution was added, and 100 \u0026micro;L of formazan dissolution solution was added after 4h. The OD(optical density) value was measured at 490 nm by iMark ELIASA, the growth curve of the cells with time(days) as the abscissa and OD 490 as the ordinate was drawn。\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.2.5 \u003cstrong\u003ePI staining\u003c/strong\u003e The LV-APPL1 and LV-CON cells in logarithmic growth phase were centrifuged at 1000rpm, and the supernatant was aspirated, the precipitated cells were moved into DMEM high glucose medium containing 10% fetal bovine serum in a 37℃ incubator. The cell suspension was collected when the cell confluence reached 60%-80%, and then according to the cell cycle detection kit instructions, PI staining, cell cycle was detected by flow cytometer, and the proportion of each phase was analyzed with the flow cytometer with software FACSCCanto Clinical.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.2.6 \u003cstrong\u003eWestern-blot experiments\u003c/strong\u003e The LV-APPL1 and LV-CON cells in logarithmic growth phase were lysed with RIPA cell lysis solution after 48h of culture and each group protein was extracted using BCA protein quantification kit according to the instructions. Electrophoresis was performed on SDS-polyacrylamide gel, transferred to nitrocellulose membrane, the primary antibody of corresponding cyclin (dilution 1:200) was selected and incubated overnight at 4℃. On day 2, the nitrocellulose membrane was washed 3 times with TBST(TBS\u0026thinsp;+\u0026thinsp;Tween) buffer, 5mins per time, and then the corresponding diluted secondary antibody (dilution 1:1000) was added. After the membrane was closed at room temperature for 1h, the nitrocellulose membrane was washed 3 times with TBST on the shaker. Finally, the protein strips were developed by the enhanced chemiluminescence(ECL) detection kit, then were quantitative analysed by FluorChem\u0026reg;Q protein blot imaging and quantitative analysis system.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2.3 \u003cstrong\u003eStatistical methods\u003c/strong\u003e Statistical analysis of the data was performed using the IBM SPSS 19.0 software. Counting data were expressed as \u0026plusmn;s, independent sample t-test for means between two groups, one-way ANO variance for multiple groups, LSD for pairwise comparisons between groups, repeated measures ANO variance for indicators at different time points, \u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e for measurement data, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as statistical significance。\u003c/p\u003e\n \u003c/span\u003e\n\u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e3.1 \u003cstrong\u003ecell lines\u003c/strong\u003e The constructed plasmid overexpressed APPL1 and pWPXLd for blank control infected gastric cancer SGC-7901 cells in vitro respectively, after 15 days of culture, the two culture bottles were covered with round and oval cells by observation under a phase-contrast microscope(See Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The APPL1 protein was extracted from two groups and quantified by Western-blot. The results showed that the expression of APPL1 protein was (1.247\u0026thinsp;\u0026plusmn;\u0026thinsp;0.196)\u0026micro;g in the experimental group and (0.276\u0026thinsp;\u0026plusmn;\u0026thinsp;0.045)\u0026micro;g in the control group, with a significant difference (t\u0026thinsp;=\u0026thinsp;5.543, P\u0026thinsp;=\u0026thinsp;0.008). See Fig. 2\u003c/p\u003e\n\u003cp\u003eFigure 2 Western-blot analysed the results of APPL1 overexpression\u003c/p\u003e\n\u003cp\u003e3.2 \u003cstrong\u003eProliferation capacity\u003c/strong\u003e The OD 490 was measured at different time points in two groups, the difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The OD 490 measurement values gradually increased with the culture time, and the OD 490 measurements varied at different time points in each group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was an interaction on the OD 490 measurements between the two groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). See Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of cell proliferation capacity between the two groups at different times (\u0026plusmn;\u0026thinsp;s)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003egroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003enumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 0\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 2\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 3\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDay 4\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eexperimental group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.123\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.221\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.732\u0026thinsp;\u0026plusmn;\u0026thinsp;0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.365\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.085\u0026thinsp;\u0026plusmn;\u0026thinsp;0.086\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003econtrol group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.134\u0026thinsp;\u0026plusmn;\u0026thinsp;0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.173\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.487\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.727\u0026thinsp;\u0026plusmn;\u0026thinsp;0.046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.158\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003csub\u003egroup/time/interactive\u003c/sub\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e3.432/2.145/1.576\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003csub\u003egroup/time/interactiv\u003c/sub\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003e0.032/0.043/0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eNote: The Mauchly method test satisfies the spherical hypothesis\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e3.3 \u003cstrong\u003eCell cycle changes\u003c/strong\u003e The proportion of cells in G1 stage was 52.10% in the experimental group, 61.64% in the control group, the proportion of cells in G1 in the experimental group was lower than that of the control group (\u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;28.653, P\u0026thinsp;=\u0026thinsp;0.003); the proportion in S phase was 33.08% in the experimental group, 25.91% in the control group, and the proportion of cells in S phase was higher in the experimental group than that of the control group (\u003cem\u003e\u0026chi;\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;37.196, P\u0026thinsp;=\u0026thinsp;0.007). See Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003e3.4 \u003cstrong\u003eWestern-blot\u003c/strong\u003e The expression of Cyclin D1 protein was higher than that of the control group, and the expression of p16 and p27 were lower than those of the control group, all the differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The expression of Cyclin E, p21 and p57 in the control group were not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). See Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, and Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of cyclin expression the between experimental and control groups (\u0026plusmn;\u0026thinsp;s, \u0026micro; g)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003egroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003enumber\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCyclin D1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep16\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep27\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCyclin E\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep21\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ep57\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eexperimental group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.895\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.318\u0026thinsp;\u0026plusmn;\u0026thinsp;0.064\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.595\u0026thinsp;\u0026plusmn;\u0026thinsp;0.083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.768\u0026thinsp;\u0026plusmn;\u0026thinsp;0.321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.834\u0026thinsp;\u0026plusmn;\u0026thinsp;0.331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.219\u0026thinsp;\u0026plusmn;\u0026thinsp;0.876\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.354\u0026thinsp;\u0026plusmn;\u0026thinsp;0.241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.687\u0026thinsp;\u0026plusmn;\u0026thinsp;0.057\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.028\u0026thinsp;\u0026plusmn;\u0026thinsp;0.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.347\u0026thinsp;\u0026plusmn;\u0026thinsp;0.485\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.967\u0026thinsp;\u0026plusmn;\u0026thinsp;0.454\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.564\u0026thinsp;\u0026plusmn;\u0026thinsp;0.542\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003et\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.357\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.485\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.012\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.313\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.817\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.043\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.763\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.474\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eAPPL1 gene contains multiple domains and expresses various active proteins, which has been demonstrated in lung cancer\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e and colorectal cancer\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, which can promote tumor proliferation and inhibit tumor apoptosis. Broussard et al. confirmed the intrinsic mechanism of APPL1 as a protein regulator during cell migration\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The previous study of our research group found that APPL1 is closely related to the biological behavior and clinical characteristics of gastric cancer patients. Recently, the studies found that APPL 1 is also a prognostic predictor for kidney cancer and prostate cancer\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe incidence and mortality rate of gastric cancer have increased year by year, especially in East Asian countries, becoming the main fatal malignant tumor\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The mechanism of gastric cancer proliferation is complex, and the specific signaling pathways is important for the diagnosis and treatment of gastric cancer\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe previous study of our research group showed that the protein expression and mRNA level of APPL1 were higher in gastric cancer tissues compared with those of adjacent tissues, and are closely related to the tumor biological behavior of gastric cancer and the clinical characteristics of gastric cancer patients\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In this study, the plasmid carrying the APPL1 gene was further constructed by a lentiviral vector and successfully transfected with a gastric cancer cell line. MTT proliferation assay found the proliferative capacity of the gastric cancer cells overexpressed APPL1 was significantly enhanced. The flow cytometry found that gastric cancer cell lines overexpressed APPL1 had an higher proportion of cells in the S phase during the cell cycle, but decreased in the G1 phase. The Western-blot analysis found that overexpression of APPL1 promoted the expression of Cyclin D1 and inhibited the expression of p16 and p27, with no obvious effect on the expression of Cyclin E, p21, and p57.\u003c/p\u003e \u003cp\u003eThe results of this study suggest that the proportion of S/G1 of the gastric cancer overexpressed APPL1 in the cell cycle increased. CyclinD1 is one of the key proteins in the regulation of cell proliferation, which regulates cell proliferation mainly by regulating the G1/S conversion rate of the cell cycle\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In cell mitosis, Cyclin D1 is able to phosphorylate Rb by binding to a cyclin-dependent kinase, which in turn regulates cells from G1 phase to S phase\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Both p16 and p27 can block the formation of a complex with Cyclin D1 by binding to cyclin-dependent kinase 4, thus arresting cell division and proliferation\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results of this study found that APPL1 promotes gastric cancer cell proliferation by regulating cyclin. Due to the upregulation expression of Cyclin D1 and downregulation expression of p16 and p27, the proportion of gastric cancer cells in S/G1 phase increased, and the proliferation ability of cells was significantly strengthened. Unlike the mechanism that APPL1 promotes proliferation in human hepatocellular carcinoma and triple-positive breast cancer. Ding et al. found that APPL1 stimulated cancer cell proliferation mainly by binding to the receptor of leptin after phosphorylation\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e in the above two cancer tissues, which suggests that APPL1 promotes cancer cell proliferation in different manner in different cancer tissues. It has been shown that the same gene or protein in different cancer tissues exerts the biological effects through different mechanisms of action\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, This study shows that APPL1 also has this property.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eOur study revealed that the cell cycle and cyclin changes of SGC-7901 gastric cancer cell line overexpressed APPL1, but the specific mechanism or link between gene changes and protein expression differences is not clear, and further research is needed\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eAll procedures conducted in this study were in compliance with the principles outlined in the Declaration of Helsinki and were approved by the Ethics Committee of China PLA General Hospital (Approval number: 2023018). Additionally, this study obtained written informed consent from all patients.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eConsent for publication was obtained from the participants.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConflict of interest\u003c/strong\u003e \u003cp\u003eThe authors have no conflicts of interest related to this research.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eacquisition: KW\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJHZ did the work of Interpretation and analysis of data. QW and MY did the work of Experiment. JSZ wrote the manuscript.Revision for important intellectual content: JZ and HZW. Funding acquisition: KWSupervision: all authors All authors have read and approved this manuscript\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author, Kai Wu, upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMitsuuchi Y, Johnson SW, Sonoda G et al. Identification of a chromosome 3p14.3-21.1 gene,APPL,encoding an adaptor molecule that interacts with the oncoprotein-serine/threonine kinase AKT2[J].Oncogene,1999,18(35):4891\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan Y, Chen H, Peng H, et al. Molecularmechanisms of curcuminrenoprotection in experimental acute renal injury[J]. Front Pharmacol. 2017;8:912.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArtimani T. Najafi R.APPL1 as an important regulator of insulin and adiponectin-signaling pathways in the PCOS:a narrative review[J]. Cell Biol Int 2020,44(8):1577\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYork HM, Patil A, Moorthi U, K, et al. Rapid whole cell imaging reveals a calcium-APPL1-dynein nexus that regulates cohort trafficking of stimulated EGF receptors[J]. Commun Biol. 2021;4(1):224.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou N, Wang N, Qin X, et al. Expression of follicle-stimulating hormone receptor(FSHR),protein kinase B-2(AKT2) and adapter protein with PH domain,PTBdomain,andleucine zipper(APPL1) in pig ovaries[J]. Pol J Vet Sci. 2017;20(4):661\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhai JS, Song JG, Zhu CH et al. Expression of APPL1 is correlated with clinicopathologic characteristics and poor prognosis in patients with gastric cancer[J]. Curr Oncol 2016,23(2):e95\u0026ndash;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Y, Zhang C, Zhao L, et al. APPL1 promotes the migration of gastric cancer cells by regulating Akt2 phosphorylation[J]. Int J Oncol. 2017;51(4):1343\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLakoduk AM, Roudot P, Mettlen M, et al. Mutant p53 amplifies a dynamin-1/APPL1 endosome feedback loop that regulates recycling and migration[J]. J Cell Biol. 2019;218(6):1928\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Moura NA, Caetano BF, R,de Moraes LN et al. Enhancement of colon carcinogenesis by the combination of indole-3 carbinol and synbiotics in hemin-fed rats[J].FoodChem Toxicol,2018,112:11\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBroussard JA, Lin WH, Majumdar. D,Theendosomal adaptor protein APPL1 impairs the turnover of leading edge adhesions to regulate cell migration[J].MolBiol Cell,2012,23(8):1486\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYANG, M,GONG C,SONG, K. et al.APPL1 is a prognostic biomarker and correlated with treg cell infiltration via oxygen-consuming metabolism in renal clear cell carcinoma[J]. Oxid Med Cell Longev,2023:5885203.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKIEŁB, P,KOWALCZYK K,GURWIN, A et al. Novel histopathological biomarkers in prostate cancer:implications and perspectives[J].Biomedicines,2023,11(6):1552.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSEKIGUCHI, M,ODA I,MATSUDA, T et al. Epidemiological trends and future perspectives of gastric cancer in eastern. Asia[J] Digestion 2022,103(1):22\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR\u0026Ouml;CKEN C. Predictive biomarkers in gastric cancer[J]. J Cancer Res Clin Oncol 2023,149(1):467\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu H, Miao ZF, Wang ZN et al. HCRP1 downregulation confers poor prognosis and induces chemoresistance through regulation of EGFR-AKT pathway in human gastric cancer[J]. Virchows Arch 2017,471(6):743\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKitamura S, Yanagi T, Maeda T et al. Cyclin-dependent kinase 4/6 inhibitors suppress tumor growth in extramammary Paget's disease[J]. Cancer Sci 2022,113(2):802\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Q, Zhu H, Cui Z, et al. The HPV16E7 Affibody as a Novel Potential Therapeutic Agent for Treating Cervical Cancer Is Likely Internalized through Dynamin and Caveolin-1 Dependent Endocytosis[J]. Biomolecules. 2022;12(8):1114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoulonval K, Vercruysse V, Paternot S, et al. Monoclonal antibodies to activated CDK4:use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27[J]. Cell Cycle. 2022;21(1):12\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLU, Y,FENG Y,LI, Z, et al. Novelpiperazine based benzamide derivatives as potential anti-glioblastoma agents inhibiting cell proliferation and cell cycle progression[J]. Eur J Med Chem. 2022;227:113908.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDING, Y,CAO Y,WANG, B et al. APPL1-mediating leptin signaling contributes to proliferation and migration of cancer cells[J]. PLoS One 2016,11(11):e0166172.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLIU, L,MICHOWSKI W,KOLODZIEJCZYK, A et al. The cell cycle in stem cell proliferation,pluripotency and differentiation[J]. Nat Cell Biol 2019,21(9):1060\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLAVOIE, H,GAGNON J,THERRIEN. M.ERK signaling:a master regulator of cell behaviour,life and fate[J]. Nat Rev Mol Cell Biol 2020,21(10):607\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gastric cancer, APPL1, Proliferation","lastPublishedDoi":"10.21203/rs.3.rs-4650101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4650101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND: \u003c/strong\u003eAPPL1 is highly expressed in gastric cancer tissues and it is positively correlated with TNM stage, depth of invasion, and lymph node metastasis of gastric cancer, but the specific mechanism of APPL1 on gastric cancer cell proliferation has not been defined. In our study, we prepared the gastric carcinoma cell line SGC-7901/APPL1 via plasmid transfection technique, then investigated the change of gastric carcinoma cells' proliferation ability, aimed to investigate the regulatory mechanism of APPL1 on gastric cancer cell proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS \u003c/strong\u003eThe gastric carcinoma cell strain SGC-7901 was transfected by APPL1 gene plasmid as the experimental group; by blank plasmid as the control group. The expression of APPL1 on the experimental group was detected by Western-blot technique. The change of the experimental group cells ' proliferation ability was detected by MTT assay, The change of the experimental group cell cycle was detected by PI staining, The change of the experimental group cell cyclin proteins were detected by Western-blot technique.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS \u003c/strong\u003eBy the Western- blot technique, the expression level of APPL1 protein in the experimental group cell strain was up-regulated effectively(P\u0026lt;0.01). Meanwhile, the proliferation ability of the experimental group increased significantly was detected by MTT assay(P\u0026lt;0.05), By the PI staining, the percentage of the experimental group cell cycle in G1 phase was decreased(P\u0026lt;0.01), in S phase was increased(P\u0026lt;0.01). By the Western-bolt technique, the expression of Cyclin D1(P\u0026lt;0.05) was up-regulated(P\u0026lt;0.05) and p16, p27 was down-regulated(P\u0026lt;0.05), Cyclin E、p21、p57 were not changed(P\u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION \u003c/strong\u003eThrough the plasmid transfection technique, the gastric carcinoma cell line that could overexpress APPL1 protein were prepared successfully and whose proliferation ability were enhanced.\u003c/p\u003e","manuscriptTitle":"Effects and mechanism of APPL1 on proliferation of gastric cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-02 20:48:05","doi":"10.21203/rs.3.rs-4650101/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a8928fd-af78-45c0-a88b-cce406f07afd","owner":[],"postedDate":"August 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-07T11:54:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-02 20:48:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4650101","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4650101","identity":"rs-4650101","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}