| Esophageal cancer is the eighth most common cancer in the world(accounting for 3%of all cancer cases)and the sixth most common cause of cancer death(accounting for 6%of all cancer-related deaths).Incidence and mortality of esophageal cancer are on the rise,with 604,000 new cases and544,000 deaths worldwide in 2020.Those numbers are expected to increase to957,000 cases and 880,000 deaths annually by 2040.There are large variations between countries and regions,with 59.2 per cent of all cases occurring in East Asia and 53.7 per cent in China alone.Compared to individual women,male patients had a higher mortality rate of 375,000.Rates for individual men and women vary by region,with rates generally lowest in West Africa,Central America,North Africa and West Asia,and highest in East Asia,Southern and eastern Africa and northern Europe.Both have very obvious geographical pattern.The highest incidence is in East Asia,followed by southern and eastern Africa.Esophageal cancer can be divided into two main histological subtypes:adenocarcinoma(AC)and squamous cell carcinoma(ESCC).Globally,esophageal squamous cell carcinoma is the most common subtype in both male and female patients,accounting for 85%of all esophageal cancer cases,with incidence rates of 7.8 and 3.2/,respectivelyOne hundred thousand.Esophageal adenocarcinoma accounts for only14%of all esophageal cancers globally,but it is the dominant subtype in 21highly developed countries.Different subtypes of adenocarcinoma and squamous cell carcinoma have specific risk factors,and as a result,people in certain parts of the world are more susceptible to certain subtypes.For example,rates of esophageal adenocarcinoma are highest in Northern Europe and North America,while rates of esophageal squamous cell carcinoma are highest in East Asia,southern Central Asia,and South Africa.Different risk factors have been identified.In addition to tobacco and alcohol,other risk factors may contribute to the high incidence in these areas,including drinking hot beverages,opium use,low fruit and vegetable consumption,drinking water without pipes,and exposure to air pollution.Dental fluorosis and oral health are associated with an increased risk of esophageal cancer in East Africa.In most countries,the survival rate five years after diagnosis is between10%and 30%.Recent analyses in high-income countries show that,despite progress over the past two decades,five-year survival rates peak at 24 per cent.The most important prognostic factor in determining survival of esophageal cancer is the stage of diagnosis.However,most cases of esophageal cancer tend to be diagnosed at a later stage,when treatment options are limited and survival rates are poor,even in high-income countries.This is partly because esophageal squamous cell carcinoma is prone to early metastasis because the esophagus has no serous membrane.Until the diagnosis and treatment methods are improved,further elucidating the pathogenesis and risk factors is of positive significance for improving the prognosis of esophageal squamous cell carcinoma.Excessive proliferation,metastasis,and immune escape are considered hallmarks of cancer that contribute to tumor progression.Split hand and foot malformation 1(SHFM1)is highly expressed in various cancers and is reported to increase malignant behaviors.However,the biological functions of SHFM1 in esophageal squamous cell carcinomas(ESCC)progression remain unclear.SHFM1 was screened by integrated bioinformatics analysis from two GSE microarrays.SHFM1 was found to be profoundly upregulated in ESCC tissues compared to normal tissues,and SHFM1 expression was associated with clinicopathologic features.The biological effects of SHFM1 on cell growth,metastasis,and immune escape were investigated after depletion or overexpression of SHFM1 in vitro.A xenograft mouse model was established to investigate the effect of SHFM1 on ESCC progression in vivo.SHFM1overexpression promoted ESCC cell proliferation,migration,and invasion,while SHFM1 knockdown could restore the phenotype changes.SHFM1expression in xenograft models increased tumor volume and promoted tumorigenesis.In addition,SHFM1 activated NF-κB signaling by promoting P65 phosphorylation and nuclear translocation.Furthermore,SHFM1 knock-down downregulated expression of c-Myc and programmed death-ligand 1(PD-L1),key targets in cancer immunotherapy.Inhibition of SHFM1 in cancer cells enhanced NK cell-induced cell apoptosis,as well as promoted NK protease granzyme B secretion.In conclusion,these finding suggested that SHFM1 involved in ESCC progression might through activate the NF-κB pathway and enhance the resistance of ESCC cells to NK cell cytotoxicity.It might be developed as a promising target of aggressive ESCC.Part One Bioinformatic analysis and the SHFM1 exprssion level and the clinical data of esophageal squamous cell carcinomaObjective:The expression profile of esophageal squamous cell carcinoma related genes was analyzed by bioinformatics method,and SHFM1was selected as the candidate marker for esophageal squamous cell carcinoma.To investigate the expression of SHFM1 in esophageal squamous cell carcinoma and the correlation between SHFM1 and the clinicopathologic features and prognosis of esophageal squamous cell carcinoma.Methods:1.Esophageal squamous cell carcinoma gene expression profile chips were selected from GEO database,GEO2R was used to screen differentially expressed genes of esophageal squamous cell carcinoma and adjacent tissues,and overlapping up-regulated genes were obtained by Veen-style interaction analysis.The PPI network was constructed and the Hub gene was obtained by GO enrichment analysis and KEGG pathway enrichment analysis.SHFM1was selected as a biological marker in esophageal squamous cell carcinoma according to the survival analysis data of TCGA.2.The expression of SHFM1 in 58 cases of esophageal squamous cell carcinoma was detected by IHC technology,and the clinicopathological features of patients were statistically analyzed to predict the correlation between SHM1 expression and clinicopathological features.Results:1.Bioinformatic analysis of genes associated with esophageal squamous cell carcinomaEsophageal squamous cell carcinoma GSE17351 and GSE33810 were studied in GEO database.To|log2FC|>2,P<0.5 for screening criteria,select3185 genetic variations,including GSE17351 contains 262 raised genes and728 down-regulated genes;GSE33810 contained 1215 up-regulated genes and980 down-regulated genes.Online Veen mapping software was used to make intersection of up-regulated genes in two data sets,and 75 DGEs were obtained.GO enrichment analysis and KEGG pathway enrichment analysis were performed on 75 DEGs using DAVID.GO analysis showed that 75 up-regulated genes were significantly enriched in extracellular matrix tissues and collagen catabolism of biological processes(BP).The changes of molecular function(MF)mainly focused on the effects of metallopeptidase activity and extracellular matrix structure on the molecular function.The changes in CC of DEGs were mainly concentrated in extracellular matrix containing collagen and collagen trimers.KEGG pathway enrichment analysis showed that 75up-regulated genes were mainly concentrated in interleukin-17 signaling pathway and tumor transcription error regulation.75 up-regulated genes were imported into STRING database to construct PPI network.PPI network showed that the three most important node genes were COL1A1,SPP1 and SHFM1.Further survival analysis was performed to assess the prognostic value of these important genes.Survival analysis data of TCGA showed that the expression of SHFM1 was negatively correlated with the overall survival rate,while the expression of COL1A1 and SPP1 was not significantly correlated with the survival rate of ESCC patients.According to the prognostic value,SHFM1 was identified as a candidate biomarker,and the expression of SHFM1 in different cancers was analyzed in the TCGA database.Analysis of data from the TCGA database showed that SHFM1 was highly expressed in various cancer types,including esophageal squamous cell carcinoma.2.Expression and clinicopathologic features of SHFM1 in esophageal squamous cell carcinoma and adjacent tissues.The expression of SHFM1 in patients with esophageal squamous cell carcinoma was positively correlated with lymph node metastasis and pathological stage,but not with histological grade.To further confirm the expression of SHFM1 in the progression of esophageal squamous cell carcinoma,we collected 58 esophageal squamous cell carcinoma tissues to examine the correlation between SHFM1 and clinicopathologic features.Interestingly,high expression of SHFM1 was significantly correlated with TNM staging(P=0.048)and lymph node metastasis(P=0.006),which was consistent with the database results.IHC staining showed that the expression of SHFM1 was significantly increased in cancer tissues compared to adjacent normal tissues.Summary:SHFM1 was highly expressed in esophageal squamous cell carcinoma tissues,and the high expression of SHFM1 was significantly correlated with lymph node metastasis of ESCC and TNM staging.SHFM1may be involved in ESCC development.Part Two The effect of SHFM1 on biological characteristics of esopha-geal squamous cell carcinomaObjective:The effects of SHFM1 on the growth,proliferation,migration and invasion of esophageal squamous cell cells were studied by WB experiment,CCK-8 experiment,Transwells experiment and other methods,and the effects of SHFM1 on the biological function of esophageal squamous cell cells were evaluated.The effect of SHFM1 on the tumor formation of esophageal squamous cell cells in nude mice was explored by constructing subcutaneous transplantation tumor model in nude mice.Methods:1.Cell culture,transfection and detectionHuman esophageal squamous cell line KYSE-410 was cultured in RPMI-1640 medium containing 10%fetal bovine serum,and TE-1 cells were cultured in RPMI-1640 medium containing 10%fetal bovine serum.The cells were cultured in an incubator containing 5%CO2 at 37℃.SHFM1 expression was silenced using Lipofectamine 3000(Invitrogen,USA)and two small interfering Rnas(Si Rnas)for SHFM1(si SHFM1-1 and si SHFM1-2)or non-specific control Si Rnas(si Control).The encoding sequence of SHFM1was constructed into pc DNA3.1 vector.pc DNA3.1 empty vector was used as control.SHFM1 expression plasmid or empty vector was combined with Lipofectamine 3000 for transfection of TE-1 and KYSE-410 cells.The transfection efficiency was detected by qRT-PCR.2.Western blot was used to detected the expressions of SHFM1 andβ-actin.3.Counting Kit-8(CCK-8)was used to detected ty the effects of SHFM1 on the proliferation of esophageal squamous cell cancer cells.4.Flow cytometry was used to analyze the cycle distribution of esophageal squamous cell.5.ELISA was used to detecte the level of granulozyme B.6.Transwell assay was used to detecte the effect of SHFM1 silencing and overexpression on the invasion ability of TE-1 and KYSE-410 cells.7.Twenty-four nude mice aged 4-5 weeks were housed under standard conditions.The mice were randomly divided into four groups(n=6/group):the sh Control group,the sh SHFM1 group,the Vector group,and the SHFM1group.From the 8th day,KYSE-410/vector cells or SHFM1-overexpression KYSE-410 cells(1×10~6)were subcutaneously injected into the right flank of mice.In addition,a total of 1×106 negative or SHFM1-silenced KYSE-410cells were injected into mice.Tumor volumes(L×W~2)/2 were examined every4 days.After 28 days,animals were euthanized and tumor tissues were dissected and fixed in 4%paraformaldehyde for further analysis.8.The expressions of SHFM1 and Ki-67 were detected by immunohis-tochemical staining.IHC staining was performed on patients with esophageal squamous cell carcinoma and xenograft tissues using antibodies against SHFM1 or Ki-67.Cultured with goat anti-rabbit HRP-conjugated secondary antibodies,the slices were prepared with DAB and stained with hematoxylin.9.The expression levels of MMP-2 and MMP-9 were detected by Western blot.Results:1.Validation of the silencing and overexpression efficiency of SHFM1.In this study,KYSE-410 and TE-1 cells were used to construct SHFM1silencing and overexpression model,and qRT-PCR test results indicated successful silencing and overexpression,P<0.01.2.In vitro cell experiments showed that SHFM1 overexpression promoted the proliferation,migration and invasion of esophageal squamous cell cells,and SHFM1 knockout could restore phenotypic changes.3.In vivo experiments showed that overexpression of SHFM1 promoted the growth of esophageal squamous cell carcinoma xenografts,and promoted cell proliferation,migration and invasion.Summary:In vitro and in vivo experiments confirmed the carcinogenic role of SHFM1 in the progression of esophageal squamous cell carcinoma.The silencing of SHFM1 inhibited the proliferation,migration and invasion of esophageal squamous cell carcinoma,and the overexpression of SHFM1promoted the progression of esophageal squamous cell carcinoma.Part Three The mechanism of SHFM1 deletion suppresses the progres-sion of esophageal squamous cell carcinoma by mediating NF-κB signaling pathway and natural killer cell immune responseObjective:To investigate the mechanism of SHFM1 deletion suppresses the progression of esophageal squamous cell carcinoma.Methods:1.Western blotting was used to detect the expression levels of P65,p-P65,c-myc and PD-L1 proteins in esophageal squamous cell carcinoma cells silenced or overexpressed by SHFM1.2.The position of P65 in cells was analyzed by immunofluorescence staining.3.qRT-PCR was used to detect the expression levels of c-Myc and PD-L1 m RNA in esophageal squamous cell cells silenced or overexpressed by SHFM1.4.The cycle distribution of esophageal squamous cell was analyzed by flow cytometry.5.ELISA assay was used to detect the level of granzyme B.Results:1.Western blotting assay and immunofluorescence assay suggested that SHFM1 may play a role in promoting the activation of NF-κB signaling pathway.2.SHFM1 silencing decreased c-Myc and PD-L1 expression levels in esophageal squamous cell carcinoma cells.3.The silencing of SHFM1 enhanced the ability of NK cell-mediated immune cell killing.4.ELISA experiments suggested that silencing SHFM1 could significantly increase the expression level of granulosa B,and the expression of SHFM1 partially blocked the immune response mediated by NK cells.Summary:The deletion of SHFM1 can inhibit the expression of NF-κBp-P65 and nuclear translocation of P65.Conclusions:1.SHFM1 was highly expressed in esophageal squamous cell carcinoma tissues,and the high expression of SHFM1 was significantly correlated with lymph node metastasis of ESCC and TNM staging.SHFM1 may be involved in ESCC development.2.In vitro and in vivo experiments confirmed the carcinogenic role of SHFM1 in the progression of esophageal squamous cell carcinoma.The silencing of SHFM1 inhibited the proliferation,migration and invasion of esophageal squamous cell carcinoma,and the overexpression of SHFM1promoted the progression of esophageal squamous cell carcinoma.3.SHFM1 may play a role in promoting the activation of NF-κB signaling pathway.After SHFM1 silencing,the NK cell-mediated immune cell killing ability is enhanced,and the expression level of granulosa B is significantly increased.The expression of SHFM1 partially blocks the NK cell-mediated immune response. |
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