| Introduction:Gastric cancer（GC）is a common malignant tumor and the third leading cause of cancer-related deaths worldwide.In 2018,more than one million new cases of GC and 78,000 deaths were reported worldwide.Owing to improved awareness and diagnostic technologies,an increasing number of cases of GC are being diagnosed at an early stage.However,many cases with hidden onset and poor prognosis are still diagnosed at a late stage,and the five-year survival rate of patients with GC is only 36.9%.Therefore,early GC detection is crucial for reducing its mortality rate,and prognostic biomarkers need to be urgently identified.Primary cilia are censors that play an important role in the regulation of several signaling pathways.They receive signals from the outside of the cell and transmit them to the inside of the cell triggering a cascade reaction that regulates cell proliferation,differentiation,transcription,migration,polarity,and histological morphology.The ability to form primary cilia is lost in several cancers,such as breast,pancreatic,ovarian,and renal cancers.Additionally,defective cilia were shown to play a role in tumorigenesis.Transmembrane protein 17（TMEM17）is a newly identified protein consisting of 198amino acid residues and is encoded by a gene on human chromosome 2.It is a component of the Meckel syndrome（MKS）complex located in the transition zone（TZ）of cilia and promotes the formation of cilia.Zhang et al.demonstrated that non-small cell lung cancer（NSCLC）cells express low levels of TMEM17 and that TMEM17 inhibits the invasion and migration of NSCLC cells by inactivating the ERK-p90RSK-Snail signaling pathway.Conversely,Zhao et al.showed that TMEM17 was highly expressed in breast cancer cells and promoted the proliferation,invasion,and migration of these cells by activating the Akt/GSK3βpathway.Therefore,TMEM17 plays different roles in different tumor types.However,its role in gastric tumorigenesis is still unknown.The present study aimed to investigate the role,underlying mechanisms,and diagnostic and prognostic utility of TMEM17 in stomach adenocarcinoma（STAD）.The expression and subcellular localization of TMEM17 in gastric cancer（GC）cells and tissues collected from 75 patients undergoing surgery were determined using immunohistochemical analysis.TMEM17 level was determined in GC cell lines using Western Blot.TMEM17 was overexpressed by plasmid transfection and knocked down using small interfering RNAs in GC cells.The effect of TMEM17 on the proliferation of these transfected cells was determined using CCK-8 and colony formation assays,and its role in the cell cycle and apoptosis was confirmed using flow cytometry.Western Blot and rescue experiments were performed to confirm the underlying mechanisms of TMEM17.Materials and Methods:1.Date acquisition and pan-cancer analysis of TMEM17To determine the expression level of TMEM17 in normal healthy tissues and tumor samples,TPM-normalized expression data for TMEM17 was obtained from The Cancer Genome Atlas（TCGA）,which included 33 types of tumors and relevant normal tissues.We performed the Wilcoxon rank-sum test to compare TMEM17 expression among 27types of cancer and paired adjacent normal tissue samples.2.Survival analysisTo assess the prognostic value of TMEM17 protein expression,the differences in overall survival（OS）rates between the high and low TMEM17 expression groups were determined using Kaplan-Meier survival analysis in conjunction with the Wilcoxon log-rank test.3.Differential expression analysis and functional analysis enrichmentTCGA STAD samples were divided into high-and-low expression groups based on median TMEM17 expression levels,and differential expression analysis was conducted between these groups using the Limma package,and volcano maps and heat maps were generated for high and low TMEM17-expression groups.A P-value of 0.05 and log2（1.5）were set as the threshold for significantly differential expression.Next,based on the lists of up-regulated and down-regulated genes,functional module enrichment analysis was performed selectively.Finally,the Cluster Profiler package was also used to determine which functional modules were significantly enriched.The gene set collections from the Molecular Signatures Database（MSig DB,version 3.0）were used for differential gene set enrichment analysis.The Kyoto Encyclopedia of Genes and Genomes（KEGG）（https://www.kegg.jp/）and Gene Ontology（GO）signaling pathways were selected to demonstrate the primary biological actions of major potential m RNA.Enrichment was considered significant at a false discovery rate（FDR）of<0.25,an adjusted P<0.05,and Normalized Enrichment Score（NES）>1.4.Sample collectionPrimary tumor samples were collected from 75 patients（54 males and 21 females）with STAD who underwent surgery at the First Affiliated Hospital of China Medical University between January and September 2010.The study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University（AF-SOP-07-1.1-01）.All the participants provided written informed consent.Patients diagnosed with GC but without other serious diseases were enrolled in this study.During surgery,75 samples of the tumor,peritumoral（within 3 cm of the tumor edge）were collected from the 75 patients and stored at-80°C for future use.The inclusion criteria were as follows:（1）patients pathologically confirmed with GC,（2）patients who underwent surgery,and（3）patients aged 18–80 years.The exclusion criteria were as follows:（1）receiving neoadjuvant chemotherapy or radiotherapy,（2）remnant GC,and（3）postoperative death within 3 months.Pathological diagnoses and classifications were performed according to the American Joint Committee on Cancer Staging Manual（7th edition）.The samples were classified into histopathologic subtypes:papillary（n=4,5.3%）,tubular（n=18,24%）,poorly differentiated（n=28,37.3%）,signet ring（n=16,21.3%）,and mucinous subtypes（n=9,12%）.5.Immunohistochemistry（IHC）To determine the differences between TMEM17 protein expression levels in tumor and adjacent non-tumor tissues and whether TMEM17 protein expression correlates with other clinicopathological characteristics,IHC was performed on the validation cohort of tumor and adjacent non-tumor tissue specimens.IHC was performed according to standard methods as previously described using anti-TMEM17（1:500;Santa Cruz Biotechnology Inc.,Santa Cruz,CA,USA）,a goat anti-rabbit secondary antibody（1:200）（G1213;monoclonal;Servicebio Inc.,Wuhan,China）,and phosphate-buffered saline was used as a blank control.Staining intensity was scored as 0（negative）,1（weak）,2（moderate）,or 3（strong）.The percentage of stained cells was scored as 1（1–25%）,2（26–50%）,3（51–75%）,or 4（76–100%）.A final combined score of 0–12 was obtained by multiplying the intensity and percentage scores.Specimens with scores≥4 were considered TMEM17-positive.Patients were classified into high-or low-TMEM17 protein expression groups based on the median scores.The t-test（two-tailed）was used to compare TMEM17 protein expression between tumor and adjacent non-tumor tissue specimens.The Pearsonχ2 test was used to compare clinicopathological characteristics between the high-and low-TMEM17 expression groups.6.Cell cultureThe human gastric epithelial cell line（GES-1）and three human gastric cancer cell lines（AGS,HGC-27,and MGC-803）were purchased from China National Infrastructure of Cell Line Resource（Beijing,China）.Culture media were:Dulbecco’s Modified Eagle’s Medium（DMEM;Hy Clone Inc.,Logan,Utah,USA）for GES-1 and MGC-803,F12medium（Hy Clone Inc.,Logan,Utah,USA）for AGS,and Roswell Park Memorial Institute（RPMI）-1640 medium（Hy Clone Inc.,Logan,Utah,USA）for HGC-27.All the media were supplemented with 10%fetal bovine serum（FBS;Hy Clone Inc.,Logan,Utah,USA）.And the cells was incubated at 37℃in a humid incubator containing 5%CO2.7.Western BlotWestern Blot was performed according to standard methods as previously described using anti-TMEM17（1:500;Santa Cruz Biotechnology Inc.）,anti-cyclin D1,anti-P21,anti-P27,anti-cyclin B1,anti-Bax,anti-Bcl-2,anti-P-Akt,anti-Akt,anti-P-Foxo3a,and anti-Foxo3a（1:500;Sigma-Aldrich）.anti-GAPDH（1:5000;Sigma-Aldrich,St.Louis,MO,USA）was as a loading control.TMEM17 expression levels were compared among groups using a t-test（two-tailed）.8.Real-time PCRTotal RNA,which was extracted from GC cultured cells using TRIzol reagent（Invitrogen,Carlsbad,CA,USA）following the manufacturer’s instructions,was reverse transcribed and subjected to real-time PCR as previously described.Expression data were normalized to the geometric mean of the housekeeping gene GAPDH to control the variability in expression levels and calculated as 2-[（CT of indicated genes）-（CT of GAPDH）],where CT represents the threshold cycle for each transcript.The primers used were:TMEM17,forward:5’-GGTGGGTGAGCAGCATTATG-3’;TMEM17 reverse:3’-GATACAACCGGATGGCTTCA AT-5’.Three independent experiments were performed.9.Plasmid transfections and small interfering RNA treatment The plasmids CMV-MCS-poly A-EF1A-zs Green-sv40-puromycin were purchased from Genechem Co.,Ltd.（Shanghai,China）.TMEM17-si RNA（sc-94962）and Con-si RNA（sc-37007）were purchased from Santa Cruz Biotechnology Inc.,and Lipo8000 reagent（C0533;Beyotime Biotechnology,Shanghai,China）was used for transfection,according to the manufacturer’s instructions.10.CCK-8 assayCell proliferation was evaluated using a CCK-8 assay（C0041;Beyotime Biotechnology,Shanghai,China）according to the manufacturer’s instructions.The cells（after transfected24h）were seeded in 96-well plates at a density of 2000 cells per well in triplicate and incubated for 24h in a medium supplemented with 5%FBS at 37°C with 5%CO2.At 24,48,and 72h,each well was incubated with 10μL/well of CCK-8 solution.Optical density was measured at 450nm using a microplate reader.Three independent experiments were performed.11.Colony formation assayAGS and HGC-27 cells were transfected with overexpression plasmids or si RNA targeting TMEM17 for 24h.The cells were seeded in 6cm plates（1000 cells/well）and incubated for14d at 37°C and 5%CO2.Colonies were fixed in 100%ice-cold ethanol and were visualized by hematoxylin staining.Colonies（>50 cells）were counted,and the average number of colonies from three separate experiments was calculated.12.Flow cytometryTransfected cells were seeded into 6-well plates（2×105 cells per well）in triplicate,incubated at 37°C and 5%CO2 for 24h,and were then collected and fixed with 75%chilled ethanol overnight at 4°C.Cell cycle was assessed by staining with 500μL of a binding buffer containing 5μL RNase A（5 mg/ml;Sigma-Aldrich）for 30min at 37°C.Then,5μL PI（5 mg/m L;BD Biosciences,San Jose,CA,USA）was added and incubated for 30min on ice,in the dark.Red fluorescence intensity was detected at an excitation wavelength of488nm using flow cytometry to analyze the cell cycle.Cell cycle distribution was assessed using the Mod Fit software（BD Biosciences）.To assess apoptosis,the cells were suspended in 500μL binding buffer and stained with 5μL APC-linked Annexin V（BD Biosciences）and 5μL PI（5μg/m L;BD Biosciences）for 30min,in the dark.BD LSRFortessa（BD Biosciences）was used for the analysis,and 10,000 cells were gated.13.Statistical analysisAll statistical analyses were performed using SPSS v23.0.Group pairs were compared using Student’s t-test.Comparisons of more than two groups were performed using a one-way analysis of variance（ANOVA）.Statistical significance was set at P<0.05.Data are presented as the mean±SEM.Results:1.TMEM17 is highly expressed in STADTMEM17 expression was significantly high in most of The Cancer Genome Atlas dataset tumor subtypes,including STAD（P<0.001）.The expression of TMEM17 protein as seen through Western Blot was significantly up-regulated in the three GC cell lines（AGS,P=0.0005;HGC-27,P=0.0004;and MGC-803,P=0.0026）relative to the GES-1 cell line.Furthermore,real-time PCR analysis verified that TMEM17 m RNA expression was indeed upregulated（AGS,P=0.002;HGC-27,P=0.023;MGC-803,P=0.0072）relative to the GES-1.2.TMEM17 protein expression was correlated with clinicopathologic characteristics The clinical characteristics of the patients and TMEM17 transcript levels for the 75 primary tumors（at the First Affiliated Hospital of China Medical University）.Samples without corresponding clinical information were excluded from the subsequent statistical analysis.Both STAD tissue specimens and adjacent non-tumor tissue specimens exhibited cytoplasmic TMEM17.However,TMEM17 staining was significantly more intense in STAD tissue specimens（score>4 in 76%（57/75））than in adjacent non-tumor tissue specimens（score>4 in 48%（36/75））（Figure 2Aa-b）（P=0.0026）.The patients were divided into two groups with relatively low（n=18）and high（n=57）TMEM17 expression.The patients（males,n=54,72%and females,n=21,28%）included in the analysis were aged between 36 and 79 years（≥60 years,n=38,50.7%and<60,n=37,49.3%）.Patients with clinical stages I（n=7,9.3%）,II（n=37,49.3%）,III（n=28,37.4%）,and IV（n=3,4%）were included.Moreover,high TMEM17 protein expression correlated positively with clinical stage（P=0.006）,T stage（P=0.025）,and N stage（P=0.008）.3.The survival analysis of TMEM17The high TMEM17 expression group exhibited significantly poorer OS than the low TMEM17 expression group（HR=1.56[1.12-2.18];P=0.008）.Furthermore,in different T classification subgroups,patients with high TMEM17 expression also had a significantly poor prognosis（HR=1.54[1.10-2.15];P=0.012）.The high TMEM17 expression group exhibited poorer OS than the low TMEM17 expression group)（P=0.015）.Finally,multivariate Cox regression was used to determine clinical staging（HR=1.966;95%CI[1.042-3.711];P=0.037）and TMEM17 protein expression levels（HR=2.415;95%CI[1.175-4.962];P=0.016）as an independent prognostic factor for OS.4.Differentially expressed genes in correlation with TMEM17 and KEGG/GO analysis in STADThe differential gene expression analysis identified 550 significantly differentially expressed genes（DEGs）（459 up-regulated and 91 down-regulated）.Most of these DEGs were dominated by upregulated genes.Functional enrichment analysis KEGG and GO pathway classification analyses were performed for upregulated and downregulated DEGs selectively.The GO analysis shows a highly significant enrichment of processes related to muscle development and muscle contraction.The up-regulation of KEGG pathways was associated mainly with malignancy-related pathways such as PI3k-Akt,Wnt,and c GMP signaling pathways.PI3k-Akt pathways were the three most significantly enriched pathways in KEGG pathway analysis,suggesting that TMEM17 may regulate PI3K-Akt pathway activity in GC cells.5.TMEM17 enhances the proliferation of GC cellsTo determine the effect of TMEM17 on the proliferation of GC cells,TMEM17 was overexpressed and knocked down in AGS and HGC-27 cell lines,and cell viability was assessed using CCK-8 assays.TMEM17 overexpression significantly promoted AGS and HGC-27 cell proliferation（at 72h,P=0.013 and P=0.009,respectively）,whereas TMEM17knockdown significantly inhibited their proliferation（at 72h,AGS cells,P=0.001 and HGC-27 cells,P=0.005）.Additionally,cell survival was assessed using colony formation assays.Following TMEM17 overexpression,the number of AGS（P=0.0052）and HGC-27（P=0.0039）colonies significantly increased.In contrast,the number of AGS（P=0.0027）and HGC-27（P=0.0019）cells significantly decreased after TMEM17 knockdown.These results indicate that TMEM17 may enhance the proliferation of GC cells.6.TMEM17 promotes G1/S phase transition of the cell cycleTo determine the role of TMEM17 in the cell cycle,we analyzed the cell cycle using flow cytometry and western blot analysis in TMEM17-overexpressed and TMEM17-knockdown AGS and HGC-27 cells.Compared to that normal cells,TMEM17-overexpressed AGS and HGC-27 cells showed a low percentage of cells in the G0/G1phase and a high percentage of cells in the S phase（AGS,P=0.0098 and HGC-27,P=0.0051）.Conversely,TMEM17-knockdown AGS and HGC-27 cells showed an increase in the percentage of cells in the G0/G1 phase and a decrease in the percentage of cells in the S phase（AGS,P=0.0066 and HGC-27,P=0.0049）.Additionally,Western Blot showed that TMEM17 overexpression in AGS and HGC-27 cell lines upregulated the expression of the cell cycle promoter cyclin D1 and downregulated that of the cell cycle inhibitors P21 and P27.Conversely,TMEM17 knockdown downregulated cyclin D1 and upregulated P21 and P27 expression.However,TMEM17 overexpression did not affect the cell cycle promoter cyclin B1.These results suggest that TMEM17 promotes the cell cycle G1/S transition in GC cells.7.TMEM17 inhibits apoptosis in GC CellsApoptosis analysis using flow cytometry was used to investigate whether TMEM17inhibits apoptosis.TMEM17 overexpression in AGS and HGC-27 cells reduced the percentage of apoptotic cells in the early stage of apoptosis（AGS,P=0.0004 and HGC-27,P=0.0002）,whereas TMEM17 knockdown increased the percentage of apoptotic cells in the early stages of apoptosis in these cells（AGS,P=0.0045 and HGC-27,P=0.0105）.Additionally,Western Blot showed that TMEM17 overexpression in AGS and HGC-27cells upregulated the expression of the apoptotic inhibitor Bcl-2 and downregulated that of the apoptotic promoters Bax.Conversely,TMEM17 knockdown downregulated Bcl-2 and upregulated Bax expression.These results suggest that TMEM17 inhibits apoptosis in GC cells.8.TMEM17 regulates proliferation,G1/S phase transition,and apoptosis via the Akt/Foxo3a signaling pathwayTo confirm these results,we evaluated the effects of TMEM17 on the expression of p-Akt and total Akt in GC cells,as well as the downstream proteins p-Foxo3a and total Foxo3a using Western Blot.The results showed that TMEM17 did not affect the expression of total Akt and total Foxo3a in AGS and HGC-27 cells.However,TMEM17 overexpression upregulated p-Akt and p-Foxo3a expression in GC cells.Additionally,we treated TMEM17-transfected AGS cells with the Akt inhibitor AZD5363and evaluated its effects using Western Blot.AZD5363 treatment reduced the levels of p-Akt and p-Foxo3a and restored the expression of P21/P27,Bax,and BAL-2.Additionally,CCK-8 and colony formation analyses showed that the increase in cell proliferation was reversed by AZD5363 treatment（at 72 h,T17+DMSO group vs.T17+AZD5363 group,P=0.012,Figure 7B;T17+DMSO group vs.T17+AZD5363 group,P=0.001）.Cell cycle analysis using flow cytometry showed that the TMEM17-induced G1/S phase transition was reversed by AZD5363 treatment in GC cells（T17+DMSO group vs.T17+AZD5363group,P=0.03）.Apoptosis analysis using flow cytometry showed that the TMEM17-induced inhibition of apoptosis in GC cells was also reversed by AZD5363 treatment（T17+DMSO group vs.T17+AZD5363 group,P=0.0028）.Conclusions:1.Our results indicated that TMEM17 is overexpressed in GC tissues and cells and is significantly correlated with high TNM stage and poor prognosis.It can be used as an independent risk factor to predict the OS of patients with GC.2.TMEM17 promotes proliferation,Cell cycle progression and inhibits apoptosis by promoting the phosphorylation of the Akt/Foxo3a signaling pathway in GC cells. |
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