The Mechanism Of APOC1 Promote Esophageal Squamous Cell Carcinoma Growth And Metastasis Via MAPK/ERK Pathway

Esophageal squamous cell carcinoma(ESCC)is a characteristic malignancy in China.First of all,esophageal cancer is one of the four most common tumors in China,with a high incidence[1].Secondly,one of the most important reasons why esophageal cancer in China is different from Western countries is that squamous cell carcinoma is the most common histological type in China.Most esophageal cancer in China was dignosed with metastatic lymph node or distant metastasis at the initial,and the prognosis is poor,with a 5-year survival rate of only 10%and a 5-year survival rate after surgery of only 15-40%[2].The development process of ESCC is a long process,from mild,moderate and severe dysplasia to carcinoma in situ,to invasive carcinoma,and then to metastatic carcinoma is a multifactorial,multi-signal joint regulatory process.Therefore,it is particularly important to screen key genes that affect the development of ESCC at the molecular biological level,we could find out the key gene for ESCC development from the GEO database(Gene Expression Omnibus database)and may provide potential targets for early screening and subsequent therapeutic intervention for ESCC in the future.Previous study from our research group showed apolipoprotein C1(APOC1)was one of the key up regulating gene btween ESCC and the normal esophageal tissue through the tissue transcriptome sequencing and the bioinformatics analysis[3].In order to validate this finding,we use the same screening method to find out the key up regulating gene btween ESCC and the normal esophageal tissue by GEO database(Gene Expression Omnibus database)and find out APOC1 was really up reglation in ESCC.APOC1 is a member of the apolipoprotein family,a small molecule protein,mainly expressed in the liver,the main physiological role is to participate in the metabolism of very low-density lipoprotein and high-density lipoprotein[4].APOC1 plays an important role in the development of various malignancies.Retrospective studies suggest that elevated APOC1 predicts a poor prognosis in gastric cancer,cervical cancer,colorectal cancer,and thyroid cancers[5-10].The expression of APOC1 in colorectal and renal cancer is higher than that in adjacent tissues[7,8].In studies in which APOC1 affects the proliferation of colorectal cancer,sequencing suggests a possible mechanism is associated with the MAPK/P38 pathway[7].In addition,in breast and kidney cancer,APOC1 can promote the process of EMT in breast and kidney cancer,thereby promoting the occurrence of metastasis[8,10].On the other hand,renal cancer cells can also promote the migration and invasion ability of vascular endothelial cells through APOC1 in exosomes,thereby promoting the metastasis of kidney cancer[8].However,so far,there has been a lack of in-depth systematic studies on the expression,prognosis and related mechanisms of APOC1 in esophageal squamous cells.In this study,the relevant research of APOC1 in ESCC was carried out from the following three aspects:1.To explore the expression differences of APOC1 in ESCC cancer tissues and adjacent tissues and its correlation with clinicopathological factors and survival prognosis;2.To explore the effects of APOC1 on ESCC proliferation and metastasis.3.Explore the specific mechanism of APOC1 affecting the proliferation and metastasis of ESCC,and provide potential intervention targets for the treatment of ESCC in the future.Part I Expression of APOC1 in esophageal squamous cell carcinoma and its relationship with clinical staging,prognosis,and other indicatorsObjective:1.Validate whether the APOC1 was the key differential expressed gene between ESCC and normal tissue or not through GEO database.2.Explore the mRNA expression level of the candidate gene APOC1 in pancarcinoma and esophageal cancer and its correlation with survival prognosis from the gene level;3.To explore the correlation between the candidate gene APOC1 and clinicopathological factors and prognostic survival of esophageal squamous cell carcinoma from the mRNA level;4.Determine the expression of the candidate gene APOC1 in ESCC and its correlation with clinicopathological factors and prognostic survival from the protein level;Methods:Firstly,we searched the ESCC related data,including ESCC and the corresponding normal tissue,from GEO database,then we pick up the GPL 570 platform as the training cohort and the GPL 571 platform as the viladation cohort to screen the key differential expressed gene between ESCC and the normal esophageal tissue.We extracted the expression levels of APOC1mRNA in the high-throughput sequencing data in The Cancer Genome Atlas(TCGA)database and the corresponding normal tissues in Genotype-Tissue Expression(GTEx).We obtained the expression level of APOC1 protein in ESCC postoperative samples by immunohistochemical staining and retrospectively extracted the clinicopathological factors and survival data of corresponding patients.Firstly,the expression of APOC1 in TCGA cancer tissues and corresponding GTEx was compared by t-test.Secondly,the effect of APOC1 on the survival prognosis of pan-carcinoma,esophageal cancer and esophageal squamous cell carcinoma was explored by Kaplan-Meier method and Cox survival regression analysis.The influence of ESCC clinicopathological factors on APOC1 expression in TCGA database was again explored by Wilcoxon rank sum test(continuous variable)and logistic regression analysis(categorical variable).Finally,in the postoperative ESCC specimens,the Wilcoxon rank sum test(continuous variable)and chi-square test(categorical variable)were used to analyze the difference of APOC1 expression in ESCC cancer tissue and adjacent tissue,and its relationship with clinicopathological factors.Results:1.APOC1 is one of the key differential expressed gene between ESCC and the normal esophageal tissue from the GEO database.2.APOC1 is highly expressed in various malignant tumors such as esophageal cancer,colon cancer,and renal cell carcinoma,and its high expression is significantly correlated with poorer overall survival(OS)in esophageal cancer,renal clear cell carcinoma,and hepatocellular carcinoma.3.Compared with normal tissues,APOC1 is significantly overexpressed in esophageal cancer(p<0.05).APOC1 mRNA expression is significantly increased in stage II and stage III esophageal cancer compared to stage I(p=0.006 and p=0.0245,respectively).Compared to the low-expression group of APOC1,the high-expression group is associated with shorter progression-free survival(PFS)(hazard ratio=1.632,95%confidence interval 1.031-2.582,p=0.03)and OS(hazard ratio=1.768,95%C11.069-2.923,p=0.026).4.Further verification using TCGA database of ESCC subgroup and postoperative specimens confirmed that APOC1 is highly expressed in ESCC tissues,and its expression is higher in advanced stages,and it is an independent risk factor for PFS and OS.Conclusions:APOC1 is one of the key differential expressed gene between ESCC and the normal esophageal tissue from the GEO database.APOC1 is highly expressed in a variety of malignant tissues,including esophageal cancer.High APOC1 expression was significantly associated with poor OS in a variety of malignancies,including esophageal cancer.In ESCC,APOC1 was significantly expressed in cancer tissues,and its high expression was significantly correlated with later clinical stage,and significantly correlated with poor PFS and OS.Part Ⅱ APOC1 can promote the proliferation and metastasis of esophageal squamous cell carcinomaObjective:Explore the effect of APOC1 on the malignant biological behavior of ESCC,including its effect on ESCC proliferation,migration,and invasion,as well as its possible mechanisms affecting ESCC proliferation,migration,and invasion.Methods:The expression of APOC1 in 5 ESCC cell lines(Kyse 30,Kyse 150,Kyse 180,Kyse 450,Kyse 510)was detected by real-time quantitative PCR and Western blot.Kyse 30 and Kyse 450 were selected and screened by plasmid transfection and corresponding resistant antibiotic,and stable transfected APOC1 overexpression and knockdown cell lines were screened.The effect of APOC1 on ESCC growth was evaluated by clonal formation experiments,growth curves,apoptosis flow cytometry,cell cycle flow cytometry and NOD-scid mouse subcutaneous transplantation tumor model.The effect of APOC1 on ESCC metastasis ability was evaluated by Transwell with and without matrix gel and tail vein nude mouse transplantation model.The west blot was used to explore the effects of APOC1 in regulating ESCC proliferation and metastasis of key proteins such as cyclin and EMT transformation of key proteins.Results:1.APOC1 expression levels in ESCC cell lines were ordered from low to high:mRNA Kyse 150,Kyse 30,Kyse 450,Kyse 510,Kyse 180;Protein levels:Kyse 30,Kyse 510,Kyse 150,Kyse 450,Kyse 180.We selected Kyse 30 with the lowest APOC1 expression and Kyse 450 with higher levels to establish stable overexpressed or knocked cell lines for subsequent experiments.2.In vitro experiments have shown that APOC1 can promote the formation and proliferation of ESCC clones,promote the cell cycle transition from G0/G1 to S phase to promote cell proliferation,and promote cell migration and invasion,on the contrary,APOC1 knockdown inhibits ESCC clone formation and proliferation ability,can inhibit cell cycle conversion from G0/G1 to S phase to inhibit cell proliferation,and can inhibit cell migration and invasion.APOC1 had no significant effect on apoptosis of ESCC.3.In vivo experiments confirmed that APOC1 overexpression can promote the tumorigenic ability of Kyse 450 and the growth of subcutaneous transplanted tumors;The tail vein injection metastasis model showed that overexpression of APOC1 promoted the formation of ESCC liver metastases,whereas knocking out APOC1 inhibited the formation of ESCC liver metastases.4.West blot experiment showed that overexpression of APOC1 can upregulate the expression of transcription factors c-Myc,slug and snail,thereby upregulating the expression of Cyclin D1,Vimentin,N-cadherin and Claudin-1,upregulating the expression of E-cadherin,conversely,knocking down APOC1 can downregulate the expression of transcription factors c-Myc and slug,thereby upregulating the expression of E-cadherin.Down-regulation of downstream Cyclin D1,Vimentin,N-cadherin and Claudin-1 expressions.Conclusions:APOC1 plays an important role in the ESCC development process.APOC1 can promote the proliferation and metastasis of ESCC,which provides new ideas for the subsequent treatment of ESCC.Part III APOC1 promotes proliferation and metastasis of esophageal squamous cell carcinoma through MAPK/ERK pathwayObjective:The specific mechanism of APOC1 promoting ESCC proliferation and metastasis was further clarified;Downstream pathway key proteins were identified and intervened to reverse the proliferation and metastatic capacity of APOC1 to ESCCs.Methods:First,Kyse 30 and Kyse 30 control cells overexpressing APOC1 were sequenced for transcriptome at the cellular level to search for differential genes and enriched into key cell signaling pathways to find downstream key signaling pathways.Secondly,the common signaling pathway MAPK/ERK affected by APOC1 was explored in vivo and in vitro.Then,ERK inhibitors,clonal formation experiments and Transwell experiments were used to verify that APOC1 is regulated by MAPK/ERK to regulate ESCC proliferation and metastasis.Results:1.Transcriptome sequencing of APOC1 overexpression Kyse 30 and Kyse 30 NC cells looked for differential genes overexpressing APOC1,and enriched KEGG pathway including the MAPK pathway.APOC1 upregulates the expression of c-raf.2.West blot and IHC stain showed that overexpression of APOC1 and knockdown of APOC1 could cause changes in the expression of key proteins of the downstream MAPKKK(C-RAF)/MAPKK(p-MEK1/2)/MAPK(p-ERK1/2)pathway in vovo and in vitro.3.The application of ERK inhibitors can reverse the effect of APOC1 overexpression on the proliferation and metastasis of ESCCs.Conclusions:APOC1 regulates ESCC proliferation and metastasis by upregulating the expression of C-raf,activating the MAPKKK(C-RAF)/MAPKK(p-MEK1/2)/MAPK(p-ERK1/2)pathway,and upregulating Cyclin D1 and EMT formation.