The Study On The Role And Mechanism Of Vitamin D Receptor (VDR) And Its Methylation In Esophageal Squamous Cell Carcinoma

Objective:Esophageal squamous cell carcinoma(esophageal squamous cell carcinoma,ESCC)is the most common pathological type of esophageal cancer,especially in northern China,where ESCC accounts for more than 90%.In recent years,although esophageal cancer has obtained certain therapeutic effects through comprehensive treatment methods such as surgery,radiotherapy,and chemotherapy,its prognosis is still very poor.Usually,ESCC metastasis is regarded as an important factor affecting the prognosis,but the molecular mechanism of the occurrence and metastasis of ESCC is still unclear.A large number of studies have shown that dietary nutrients play a very important role in the occurrence and development of digestive tract tumors.Recent studies have confirmed that vitamin D deficiency is closely related to a variety of chronic metabolic diseases,including tumors,autoimmune diseases,diabetes,and cardiovascular diseases.Adequate vitamin D intake reduce the incidence of tumors,including colorectal cancer,prostate cancer,breast cancer,and pancreatic cancer.However,the relationship between ESCC and serum vitamin D levels is still controversial.Our previous research have shown that there was a vitamin D deficiency in patients with esophageal squamous cell carcinoma,and it was related to the single nucleotide polymorphism of VDR.At the same time,in order to reveal the molecular mechanism of the pathogenesis of esophageal squamous cell carcinoma,past studies have suggested that abnormal changes in genetic level are involved in the occurrence and development of ESCC.However,in addition to genetic changes,epigenetics is also involved in the occurrence and development of esophageal cancer.Epigenetics helps explain the role of the interaction between personal genetic susceptibility and environmental factors in the occurrence of diseases.Therefore,based on the above view,the aim of this study is to study on :1.The expression of VDR in ESCC and its relationship with clinicopathologic characteristics;2.The effect of VDR knockdown on the proliferation,apoptosis,migration,metastasis,and invasion of ESCC cells;3.The effect of VDR overexpression on the proliferation,apoptosis,migration,metastasis,invasion of ESCC cells;4.The mechanism of VDR-mediated ESCC cell proliferation,apoptosis and metastasis;5.Preliminary exploration of the relationship between VDR gene promoter DNA methylation and the occurrence and development of ESCC.Materials and Methods:1.We collected the cancer tissue and adjacent tissue(the original tumor boundary8-10cm)of 20 patients who had been diagnosed as ESCC and had undergone radical surgery for esophageal cancer from December 2018 to January 2019 in the Department of Thoracic Surgery,West China Hospital,Sichuan University.q RT-PCR was used to detect the mRNA expression level of VDR in cancer tissues and adjacent tissues of patients with ESCC,and the expression difference of VDR in ESCC was evaluated from the protein level by Western-blot.At the same time,by querying the ESCC database in the TCGA(The Cancer Genome Atlas)database,the expression of the VDR in esophageal cancer tissue and adjacent tissue was further verified;2.The differences in VDR expression in 169 cases of ESCC tissue microarray were compared by immunohistochemical staining,and the correlation between VDR expression and the patients’ age,gender,tumor differentiation,stage,metastasis,and prognosis were analyzed;3.qRT-PCR was performed on ESCC cell lines(KYSE 30,KYSE 410,TE-1,EC109,KYSE 150,KYSE 510)and normal esophageal epithelial cell lines(Het-1a),in order to detect the mRNA expression of VDR in cell lines.The normal esophageal epithelial cell line was taken as a control group.And the difference of VDR expression in ESCC cell line and normal esophageal epithelial cell line was confirmed from the protein level by WB;4.Select cells with relatively high expression of VDR(KYSE 150)and relatively low expression of VDR(KYSE 510)in cell line experiment,and transfect them with RNAi knockdown lentivirus and overexpression lentivirus of the VDR gene,respectively.KYSE 150 cell was divided into three groups: blank control group(CON:KYSE 150),negative control group(NC: negative control virus CON077)and knockdown group(KD: LV-VDR-RNAi(17379-1));KYSE 510 cell was divided into three groups : Blank control group(CON: KYSE 510),negative control group(NC:negative control virus CON238)and overexpression group(OE: LV-VDR(8718-1)).The lentivirus transfection efficiency of two groups were detected by q RT-PCR and WB respectively;5.Detect the effect of VDR knockdown and overexpression on the proliferation of ESCC cells through MTT assay and clone formation assay;6.Detect the effect of VDR knockdown and overexpression on the apoptosis of ESCC cells by Annexin V-APC single staining flow cytometry;7.Detect the effect of knockdown and overexpression of VDR on the migration of ESCC cells by Celigo scratch test;8.Detect the effect of knockdown and overexpression of VDR on the metastasis and invasion of ESCC cells by Transwell assay;9.Select the VDR knock-down group and use Tandem Mass Tags(TMT)quantitative proteomic analysis to determine the downstream functional proteins that change after VDR knocked-down.Perform the enrichment analysis of GO(Gene Ontology)function annotations and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway annotations on the downstream functional protein collections,and compare the distribution of each GO classification and KEGG pathway in the target protein set and the overall protein set through Fisher’s Exact Test.We used the Wo LF PSORT to predict and analyze the location of differential proteins.The functional domain annotation analysis of differential proteins was carried out through the Interpro database.Also,the cluster analysis on the differential protein set was performed.Finally,the differential protein set was included in the IPA(Ingenuity Pathway Analysis)analysis,and the signal pathways,functional proteins that are highly related to the results of this experiment,as well as the interaction network among the functional proteins and the regulatory network relationship with esophageal cancer were screened out;10.Perform absolute quantitative analysis through parallel reaction monitoring(PRM)on the changes of the downstream functional proteins(TP53 、 FTH1 、GCLM 、 GPX1 、 SOD2,PRDX5)that were screened out by TMT and IPA analysis,so as to verify the TMT proteomics results;11.Detect the expression level of downstream functional proteins(TP53,APOE,SOD2,JUN)screened by TMT and IPA analysis through Western-blot;12.After VDR knockdown,the downstream functional protein selected by TMT omics analysis: TP53 was confirmed being up-regulated after VDR knockdown.Then,as for phenotypic changes,functional recovery experiments was performed to observe the differential change of TP53 expression under the condition of VDR knockdown,and verify its expression by q RT-PCR and Western-blot.Meanwhile,detect the effect of knockdown of TP53 on the proliferation of KYSE 150 VDR knockdown cells;13.Nude mouse tumor formation assay: select negative control virus-infected KYSE 150(inoculation amount: 2E+06 cells/head)and knockdown lentivirusinfected KYSE 150(inoculation amount: 2E+06 cells/head),and inoculate through tail vein of BALB/c nude mice(5 weeks,female),each group was inoculated with 10 nude mice.In vivo imaging of mice in each group was performed 42 days after inoculation,and Total Radiant Efficiency [p/s] / [μW/cm2](the amount of trace light expression in the area)was used as a reference standard to indirectly reflect the number of cells carrying fluorescent markers or the size of the tumor.At the same time,starting from the day of inoculation for each group,the weight data of each group was collected once a week,and the mice were sacrificed 43 days after the inoculation to evaluate the tumor metastasis;14.Select the cancer tissues and adjacent tissues of 9 ESCC patients who had undergone radical surgery from December 2018 to January 2019 at the Department of Thoracic Surgery of West China Hospital of Sichuan University and 5 cases of ESCC patients who underwent radical surgery at the Peace Hospital Affiliated to Changzhi Medical College from January 2018 to January 2019.Through conventional methylation-specific PCR(Methylation-specific PCR,MSP),and bisulfite amplicon sequencing(BSAS),explore whether there is a difference in VDR methylation between ESCC and adjacent tissues preliminarily.Results:1.VDR mRNA expression in ESCC tissues are significantly higher when compared with adjacent tissues.At the same time,Western-blot indicated that the protein bands of ESCC tissues were deeper than that of adjacent tissues,which was consistent with the results of q RT-PCR.Inquiring the TCGA-ESCA database,the mRNA expression of VDR mRNA in 184 cases of ESCC tissues was significantly higher than that of 11 cases of normal tissues.2.After immunohistochemical staining of ESCC tissue microarray,the expression of VDR in ESCC tissue was significantly higher than that in adjacent tissues.The expression of VDR was related to gender(P=0.026),T stage(P=0.002)and TNM stage(P=0.044).At the same time,according to logistic regression analysis,it was also confirmed that males were the independent protect facot related to the expression of VDR in ESCC(P=0.033;OR=0.459,95%CI: 0.224,0.491),and T stage(P=0.003);OR=2.652,95%CI: 1.382,5.088)and TNM stage(P=0.041;OR=1.671,95%CI: 1.021,2.735)have also been confirmed as independent risk factors related to the expression of VDR in ESCC.Kaplan-Meier survival analysis suggests that patients with higher VDR expression in ESCC have a poorer prognosis(P<0.001).COX multivariate regression analysis showed the expression of VDR in ESCC was the independent prognostic facotr related to ESCC patients.3.Compared with normal esophageal epithelium cell—Het-1a,VDR is highly expressed in KYSE 410,TE-1,EC109,KYSE 150,and KYSE 510,but is poorly expressed in KYSE 30.Among them,KYSE 150 got the highest VDR expression,followed by KYSE 410,and the expression is relatively low in TE-1,EC109 and KYSE 510.4.The MTT assay indicated that after VDR knockdown lentivirus infects KYSE150 cells,the number of viable cells in the knockdown group was significantly reduced when compared with the negative control group,that is,the cell proliferation of the knockdown group was significantly decreased(P=0.00016).After VDR overexpression lentivirus infected KYSE 510 cells,compared with the negative control group,the number of viable cells in the overexpression group increased significantly,that is,the cell proliferation of the overexpression group increased significantly(P=0.00875).5.Annexin V-APC single-staining flow cytometry showed that: when VDR was knocked down,the apoptosis rate of knockdown group was significantly increased(P<0.001).When VDR was overexpressed,compared with the negative control group,there was no significant change in the number of apoptosis cells in the overexpression group(P<0.001 but the apoptosis rate was less than 5%,it was judged that no obvious cell apoptosis occurred).6.Clone formation assay suggests: when VDR was knocked down,compared with the negative control group,the number of cell clones in the knockdown group was significantly reduced(P=0.00106).When VDR was overexpressed,the number of cell clones in the overexpression group increased significantly when compared with the negative control group(P=0.00112).7.Celigo scratch assay showed: when VDR was knocked down,compared with the negative control group,the 24 hour cell migration rate of the knockdown group was significantly reduced(P<0.001).When VDR was overexpressed,the cell migration rate of cells in the overexpression group increased significantly during 56hours(P=0.00083).8.Transwell assay indicated: when VDR was knocked down,compared with the negative control group,the cell metastasis rate(P<0.001)and invasion rate(P<0.001)of the knockdown group were significantly reduced.When VDR was overexpressed,the cell metastasis rate(P<0.001)and invasion rate(P=0.001629)of the knockdown group increased significantly.9.TMT quantitative proteomics showed: VDR knockdown induced a total of 984 downstream protein expression changed,of which,639 proteins were up-regulated and 345 proteins were down-regulated.GO analysis results showed that after VDR knockdown,the differential protein changes were mainly concentrated in platelet degranulation,type I interferon signaling,receptor-mediated endocytosis,response to calcium ions,gamma interferon-mediated signaling and regulation of complement activation,etc.Knockdown of VDR significantly enriches heparin binding,oxygen binding,fibronectin binding integrin binding,growth factor activity,cholesterol transfer activity,activity of serine endopeptidase and serine endopeptidase inhibitor.The changes of differential proteins caused by VDR knockdown are mainly concentrated in the extracellular space,cell surface and endoplasmic reticulum cavity.KEGG pathway analysis showed that the differential proteins after VDR knockdown were mainly enriched in: complement and coagulation cascade,ferroptosis,colorectal cancer,cell adhesion molecules,p53 signaling pathway,and cancer pathways.After VDR knockdown,most of the differential proteins located in the nucleus(29.0%)and cytoplasm(24.8%),followed by the plasma membrane(16.6%),extracellular(14.4%)and mitochondria(11.3%).IPA analysis showed that after VDR knockdown,the LXR/RXR Activation pathway was significantly activated,and the Z-score was 3.638.At the same time,p53 Signaling was also activated in the classic pathway analysis of IPA,with a Z-score of 0.378.Cancer-related functions include activated functions:Apoptosis of carcinoma cell lines(0.761),and inhibited functions: Metastasis of tumor cell lines(-0.926),significantly Inhibited Oxidative stress(-2.175)and Cell proliferation of carcinoma cell lines(-2.026),etc.Including the functional genes in the LXR/RXR pathway and p53 pathway: APOE and TP53 simultaneously,the regulation effect with the highest correlation coefficient(Consistency Score)is Oxidative stress(Consistency Score: 14.23).Finally,after integrating the results of IPA analysis,it can be obtained that the genes: ABCG2,APOE,FTH1,GCLM,GPX1,HMOX1,JUN,PRDX5,SOD2,TP53,regulated by ADRB,APP,CAPN3,EGF,EIF4 E,F2,FOXO3,IL1,IL1 A,Jnk,MEF2 D,NFE2L2,NFKBIA,have an activation effect on Apoptosis through genes and have an inhibitory effect on Oxidative stress,Metastasis of cells and Proliferation of cells regulator Among them,the diseases and functions related to APOE interaction network was: cell cycle.The diseases and functions related to the TP53 interaction network was: Gastrointestinal Disease,both of which were involved in the occurrence and development of VDR-mediated esophageal cancer.10.TMT omics combined with IPA analysis results verification—PRM analysis results: this analysis based on the functional protein selected in the p53 signaling pathway: TP53 was confirmed to be up-regulated after VDR knockdown,and at the same time,among the functional proteins(ABCG2,APOE,FTH1,GCLM,GPX1,HMOX1,JUN,PRDX5,SOD2,TP53)obtained from the final IPA analysis,FTH1,GCLM,GPX1,SOD2 were comfirmed to be up-regulated after VDR knockdown as well.Although PRDX5 has been confirmed to be up-regulated,the up-regulation effect is not significant.11.TMT omics combined with IPA analysis result verification—antibody-based verification(WB verification)results: In the KYSE150 cell line,the expression of TP53 and APOE was up-regulated compared with the negative control group after VDR knockdown.At the same time,other differential proteins in the regulation of oxidative stress: SOD2 and JUN were all confirmed to be up-regulated through Western-blot.12.TMT omics combined with IPA analysis results verification—functional recovery experimental verification results: When TP53 knocked down followed by VDR knockdown,compared with the negative control group,the cell proliferation of the TP53 knockdown group was significantly increased(P=0.0107),and the number of cell clones in the TP53 knockdown group was significantly increased(P=0.0426).13.Results of nude mouse tumor formation assay: the nude mouse tumor formation model was successfully constructed.Live imaging of mice in each group was carried out 42 days after inoculation,suggesting that compared with the negative control group,the tumorigenesis of mice in the knockdown group was reduced(P=0.01725).Tumor metastasis in mice—lung metastasis: compared with the negative control group,the number of lung metastases in the knockdown group was reduced(P=0.00088).Liver metastasis: there were no obvious liver metastase both in the negative control and knockdown group.Other metastases: compared with the negative control group,the weight of the neck and skin axillary solid tumors in the knockdown group did not change(P=0.204719).14.VDR methylation test results: the methylation level of VDR in esophageal cancer tissues is lower than that of adjacent tissues.Of course,due to the small number of cases included in this experiment,the difference of VDR methylation in cancer and was not obvious,and the sample size needs to be expanded later to confirm the results of this experiment further.Conclusion:1.The expression of VDR in ESCC is significantly higher than that of adjacent tissues,and is related to the poor prognosis of patients with ESCC;2.VDR can promote the proliferation,migration and invasion of ESCC cells and inhibit the apoptosis of ESCC cells;3.VDR may activate the oxidative stress effect of ESCC cells by inhibiting LXR/RXR Activation and p53 signaling pathways,thereby promoting tumor angiogenesis and metastasis,and inhibiting tumor cell apoptosis.And the regulation of TP53 by VDR has been preliminarily confirmed,and VDR can down-regulate the expression of TP53.4.Knockout of VDR in ESCC cells inhibit the formation and growth of metastatic tumors in nude mice.5.It is preliminarily shown that the VDR methylation level of ESCC tissue is lower than the methylation level of adjacent tissues,but the difference is not obvious.It still needs to be further confirmed by enlarging test samples.