| Background and objectivesEsophageal cancer(EC)is one of the most common malignant tumors and the sixth leading cause of cancer death in the world.The high incidence and mortality of EC make it one of the main causes of cancer death and burden worldwide.Saturated fatty acids are considered risk factors for many cancers.However,the evidence so far can not fully prove how it accurately promote cancer development,that still need a lot of research to prove.Early diagnosis of EC has a great significance for the survival of patients and the improvement of treatment options in different stages of esophageal cancer.Metabonomics can reveal the existence and mechanism of tumors from human metabolites,and can be used to characterize disturbed metabolic pathways and identify potential biomarkers in various cancers.Therefore,the study explored the role and mechanism of differential metabolites in the development and metastasis of esophageal cancer by identifying differential metabolites in serum and feces of esophageal cancer patients to providing research basis and data support for differential metabolites in EC as biomarkers for early diagnosis of tumors.Methods1.23 cases of esophageal cancer which diagnosed by pathology in Huai’an area and 23cases of healthy with no history of digestive system were collected,they are paired by age and sex.The serum and fecal samples were collected.The metabolites in serum and fecal were detected by high resolution tandem mass spectrometry system.2.Binary logistic regression model was used to analyze the predictive and diagnostic value of differential metabolites in fecal and serum for esophageal cancer.3.88 cases of esophageal cancer diagnosed by pathology in Huai’an area and 44 cases of healthy with no history of digestive system were collected.High resolution tandem mass spectrometry system was used to conduct targeted quantitative detection of dodecanoic acid in fecal and serum samples.4.m RNA expression levels of GPR40,GPR84 and GPR120 in esophageal cancer tissue samples and esophageal cancer cell lines were detected by q RT-PCR.5.GPR84 si RNA was used to knockdown the expression of GPR84 in esophageal cancer cell line EC109,and the knockdown efficiency of GPR84 si RNA in cells was detected by q RT-PCR and Western blot.The effects of lauric acid on the biological function of esophageal cancer cells were observed in 109 cells transfected with GPR84 si RNA.Transwell chamber was used to detect the effect of migration and invasion of esophageal cancer cells,and flow cytometry was used to detect the change of apoptosis and the distribution of cell cycle.6.The protein expression levels in the G(i/o)/PI3K/Akt pathway were detected by Western blot.ResultsI.Characteristics of fecal and serum metabolomics in esophageal cancerThe metabolites in serum and fecal were detected by HPLC-MS/MS,and it was found that the metabolites in serum and fecal of esophageal cancer were involved in the changes of multiple metabolites and metabolic pathways.And some metabolites showed different metabolic changes in serum and fecal.1.Characteristics of fecal metabolomes of esophageal cancer and healthy people182 different metabolites were screened.In esophageal cancer group,γ-linolenic acid,3,4-Dihydroxyphenylpropanoate,sphingosine,9(S)-HODE,homovanillic acid,dodecanoic acid(Lauric acid,LA)and other metabolites were downregulated,trans-Cinnamic acid,desmosterol,5-Methoxyindoleacetate were upregulated.These results indicated that in fecal metabolomics of esophageal cancer,the changes of multiple metabolites may led to the changes of multiple metabolic pathways,which may reveal the occurrence and development of esophageal cancer.3,4-Dihydroxyphenylpropanoate and dodecanoic acid was incorporated into binary logistic regression model,which can be used to predict the risk of esophageal cancer.Based on the predictive probability of predicting the risk of esophageal cancer,the area under the ROC curve was 0.941(95%CI,87.6%-100%).2.Characteristics of serum metabolomes of esophageal cancer and healthy people159 different metabolites were screened.In the esophageal cancer group,PC(18:1(9Z)/0:0)[U],glycocholic Acid,chenodeoxycholic acid,PS(18:0/18:1(9Z)),trans-cinnamic acid,5-methoxyindoleacetate and other metabolites were down-regulated.Dodecanoic acid,sphinganine,(20:5(5Z,8Z,11Z,14Z,17Z)/20:4(5Z,8Z,11Z,14Z)and(2’E,4’Z,7’Z,8E)-colnelenic acid was upregulated.Dodecanoic acid was incorporated into binary logistic regression model,which can be used to predict the risk of esophageal cancer.Based on the predictive probability of predicting the risk of esophageal cancer,the area under the ROC curve was 1.0(95%CI,100%-100%).II.Targeted detection and quantitative analysis of dodecanoic acid in human samples1.Quantitative determination of dodecanoic acid in fecal of esophageal cancer and healthy peopleThe standard curve was plotted according to the actual standard concentration and peak area,and the linear regression equation was y=540.7x+118.55,and the linear regression coefficient R~2was 0.9954.(S/N=2:1)The results showed that there was no significant difference in the concentration of dodecanoic acid in fecal between esophageal cancer(0.24±0.60μg/L)and healthy controls(0.30±0.60μg/L).2.Quantitative determination of dodecanoic acid in serum of esophageal cancer and healthy peopleThe concentration of dodecanoic acid in the serum of esophageal cancer(0.95±0.99μg/L)was significantly higher than that in the healthy control group(0.50±0.61μg/L).Correlation analysis of dodecanoic acid in fecal and serum showed that there was a positive correlation between the two groups(r=0.73,P<0.01).Binary logistic regression model showed that the dodecanoic acid level in serum had a high predictive specificity for esophageal cancer.The dodecanoic acid level in serum was positively correlated with the risk of esophageal cancer and was one of its risk factors(OR=1.71,95%CI:1.05-2.79,P<0.05).This indicates that the detection of dodecanoic acid in serum is of significance for the prevention of esophageal cancer.III.Expression of GPR84 in esophageal cancer cells and its association with changes in the biological function of esophageal cancer cells1.Analysis of the expression of GPR40,GPR84 and GPR120 in esophageal cancer tissuesOnline database(TCGA,https://portal.gdc.cancer.gov/genes)retrieval GPR40,GPR84and GPR120 gene expression in human esophageal tissue.Compared with paracancerous tissues,GPR84 and GPR120 genes were highly expressed in esophageal cancer tissues,1.9and 1.7 times,respectively,and the difference was statistically significant,there was no significant difference in GPR40.In our esophageal cancer tissue samples,q RT-PCR results showed that GPR84 was 2.4 times,overexpressed in the cancer tissue compared to the paracancerous tissue.The expression of GPR120 was not statistically significant.This suggests that GPR84 may be involved in the development of esophageal cancer.2.Analysis of the biological function of dodecanoic acid on esophageal cancer cells(1)Establishment of GPR84 knockdown EC109 cell lineThree si RNAs expressing GPR84 knockdown were used to transfect EC109 cell line,and the expression of GPR84 was detected by q PCR at the gene level.It was found that after si RNA transfection,the three si RNA sequences all down-regulated the level of GPR84 in the cells by 22%,14%and 8%,respectively.All the si RNA sequences can be used to establish GPR84 knockdown EC109 cell line.The cells were transfected with si RNA-3,and the expression of GPR84 was detected at the protein level by Western blot.The m RNA and protein levels of GPR84 were down-regulated.The si RNA-3 sequence can be used for subsequent verification and functional experiments.(2)Effect of LA on proliferation of esophageal cancer cells by CCK8 assayUsing dodecanoic acid(LA)with different concentration medium(0μM,10μM,50μM,100μM,200μM)to cultivate EC109 cells,respectively within 24 h,48 h,72 h.Using CCK8cell proliferation experiment kits to test LA effects on cells,and found that as the increase of concentration and the acting time,LA promote the proliferation of 109 cells,the best concentration and time was 72 h and 100μM respectively.Set up four experimental groups:NC si RNA group,NC si RNA+LA group,GPR84si RNA group,and GPR84 si RNA+LA group.Apply 100μM LA to the experimental group,and use the CCK8 experiment to detect the proliferation ability of esophageal cancer cells.It was found that GPR84 si RNA inhibited the proliferation of EC109 cells induced by 100μM LA,and GPR84 si RNA alone had no effect on the proliferation of EC109 cells.(3)Use Transwell chamber to detect the migration ability of esophageal cancer cells.The results showed that the migration number of NC si RNA group was(133.60±6.73)NC si RNA+LA group was(201.6±19.65),GPR84 si RNA group was(144.8±7.46),and GPR84si RNA+LA group was(138.8±18.61).Compared with the NC si RNA group,the difference in the NC si RNA+LA group was statistically significant.Compared with the GPR84 si RNA+LA group,the NC si RNA+LA group also has significant differences.GPR84 si RNA alone had no effect on the migration of EC109 cells.However,in the presence of LA,GPR84 si RNA inhibited the migration of EC109 cells induced by 100μM LA,indicating that LA promoted the migration of EC109 cells through GPR84.(4)Use Transwell chamber to detect the invasion ability of esophageal cancer cells.It was found that the number of invasions in the NC si RNA group was(64.0±4.30),the NC si RNA+LA group was(85.4±2.41),the GPR84 si RNA group was(65.2±2.68),and the GPR84 si RNA+LA group was(65.0±4.53).Compared with the NC si RNA group,the difference in the NC si RNA+LA group was statistically significant.Compared with the GPR84 si RNA+LA group,the NC si RNA+LA group also has significant differences.GPR84si RNA alone had no effect on the invasion of EC109 cells.However,in the presence of LA,GPR84 si RNA inhibited the invasion of EC109 cells induced by 100μM LA,indicating that LA promoted the invasion of EC109 cells through GPR84.(5)Use flow cytometry to detect apoptosis of esophageal cancer cells.The results showed that the apoptosis rate in the NC si RNA group was(8.79±0.11)%,the NC si RNA+LA group was(4.40±0.08)%,the GPR84 si RNA group was(8.43±0.30)%,and the GPR84 si RNA+LA group was(8.56±0.18)%.After GPR84 si RNA transfection,LA treatment had no effect on the apoptosis rate of transfected EC109 cells,and the difference was not statistically significant.However,in the presence of GPR84,LA treatment inhibited EC109 cell apoptosis.It shows that dodecanoic acid(LA)inhibits the apoptosis of esophageal cancer 109 cells through GPR84.(6)Use flow cytometry to detect cell cycle changes of esophageal cancer cells.The cycle distribution of NC si RNA group was G1(57.26±1.07)%,S phase(30.33±0.83)%,G2 phase(12.41±0.54)%,NC si RNA+LA group was G1 phase(56.44±0.84)%,S phase(31.87±0.89)%,G2 phase(11.69±0.24)%,GPR84 si RNA group cycle distribution is G1 phase(60.77±6.17)%,S phase(26.72±3.14)%,G2 phase(12.51±3.05)%,GPR84 si RNA+LA group was(56.64±1.57)%in G1 phase,(29.51±3.05)%in S phase,and(13.86±1.51)%in G2 phase.Statistical analysis found that the difference in cycle distribution between groups was not statistically significant(P>0.05),it can be considered that neither GPR84 knockdown nor LA has an effect on the cell cycle.IV.Mechanism of lauric acid regulating the biological function of esophageal cancer cells through GPR84(1)Use WB to detect the expression levels of PI3K-Akt pathway related proteins,including the levels of Akt and p-Akt proteins.It was found that the phosphorylation level of Akt was significantly increased in the NC si RNA+LA group,and the PI3K-Akt pathway was activated,which subsequently caused functional changes of downstream target genes,such as cell migration and invasion.(2)WB was used to detect the expression level of MMP-9 in PI3K-Akt pathway.The results showed that the expression level of MMP-9 protein was significantly increased in the NC si RNA+LA group,indicating that LA activates PI3K-Akt through GPR84 receptor to activate the collateralization,and activated Akt activates its target protein MMP-9 through phosphorylation,degrades the basement membrane and extracellular matrix,and destroys the histologic barrier of cancer cell migration and invasion to promote the migration and invasion of esophageal cancer cells.(3)Use WB to detect the expression level of Caspase9.The results showed that the expression level of Caspase9 protein was significantly decreased in the NC si RNA+LA group,indicating that LA activates PI3K-Akt pathway through GPR84 receptor,and then inactivates Caspase9 protein,inhibiting the apoptosis of esophageal cancer cells.(4)Use WB to detect the expression levels of target proteins Bcl-2 and Bax in PI3K/Akt/Bcl-2/Bax pathway.The results showed that in the NC si RNA+LA group,the expression level of Bcl-2 protein was significantly increased,and the expression of Bax was significantly decreased.Activated Akt activated the expression of Bcl-2,and the heterodimer of Bcl-2 and Bax was increased,and the trend of apoptosis was decreased,inhibiting the apoptosis of esophageal cancer cells.Conclusion1.Fatty acid metabolism is the main metabolic disorder during the progression of esophageal cancer.Dodecanoic acid,the medium-chain fatty acids,has significant changes in serum and fecal,which may be related to the occurrence and development of esophageal cancer.2.The concentration of dodecanoic acid in serum of esophageal cancer patients was significantly higher than that of healthy people,and was positively correlated with the concentration of dodecanoic acid in feces.3.GPR84,a specific receptor for medium-chain fatty acids,is specifically and highly expressed in esophageal cancer tissues and cells.Dodecanoic acid may regulate the biological function changes of esophageal cancer cells through GPR84.4.Dodecanoic acid promotes the proliferation,migration and invasion of esophageal cancer cells through GPR84,and inhibits cell apoptosis.5.Dodecanoic acid can activate G(i/o)/PI3K/Akt and acting on its downstream target protein MMP-9 to promote migration and invasion of esophageal cancer cells,and acting on Caspase9,Bcl-2 and Bax proteins to play an inhibitory role in cells apoptosis. |
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