| ObjectiveEsophageal cancer is one of the most aggressive malignant tumors of the digestive system,characterized by high mortality and poor prognosis.China is a high incidence area of esophageal cancer,and esophageal squamous cell carcinoma(ESCC)is the main histological subtype of esophageal cancer in China.Because the early symptoms are not obvious,most patients in the clinic are often diagnosed at late stage,so they missed the opportunity for surgery,and the main treatments are radiotherapy and chemotherapy.Therefore,the response of patients to chemotherapy drugs is particularly important.EphA2 is a member of Eph receptor superfamily and has been found overexpressed in a variety of cancers,including prostate cancer,breast cancer,colorectal cancer,pancreatic cancer,lung cancer,esophageal cancer,etc.It is a anticancer drug target researched widely.Targeting drugs for tyrosine kinase such as dasatinib and enzatinib also have inhibitory effects on EphA2.But recent years,with the development of research,it has been found that EphA2’s role in cancer is not consistent.There is evidence that EphA2 promotes epithelial-mesenchymal transition.After knocking down EphA2,it inhibits cancer metastasis and angiogenesis.But it also reported in the literature that EphA2 can inhibit cancer-causing Akt-mTORC1 and RAS-ERK signaling.After knocking down EphA2,it promotes the growth,migration and invasion of cancer cells.These proved that EphA2 has a dual role in cancer,it affecting the clinical application of related targeted drugs and the determination of curative effect.In 2001,EphA2 was found to be a target gene of the p53 family(p53,p63 and p73),and the p53 response element located in the EphA2 promoter was identified.This element can respond to wild-type p53 and induce apoptosis,but this element does not respond to mutant p53.At the same time,wild-type p53 can also up-regulate the expression of EphA2’s ligand ephrin-A1.Recent years,more and more evidences indicate that EphA2 inhibits the progression of cancer by ligand-dependent activation signaling.In contrast,it promotes the progression of cancer by ligand-independent signaling.These indicate that p53 may play a key role in the effect of EphA2 on tumor progression.p53 is one of the most important tumor suppressors and is involved in the regulation of various cancer-related pathways.At the same time,p53 is also the most common mutant gene in cancer.p53 mutations are usually related to the poor prognosis of cancer.p53 mutation in esophageal squamous cell carcinoma have been reported,but whether the role of EphA2 in esophageal squamous cell carcinoma is related to p53 status has not been studied yet.Therefore,this study intends to take the local high-risk ESCC with obvious regional characteristics as the research object,to investigate the role of EphA2 in p53 wild-type and p53 mutant esophageal squamous carcinoma cell lines by gene silencing technology,western blot and cell biology functional studies,respectively.To explore whether EphA2 has a dual role in esophageal squamous cell carcinoma and whether it is related to p53 status.To provide a new direction and hope for more accurate classification of tumors under the concept of precision medicine,and help patients to choose more targeted treatment options and improve drug curative effect.Methods1.Two identical esophageal squamous cell-carcinoma tissue chips were taken to detect the expression of EphA2 and p53 in esophageal squamous cell carcinoma tissue.2.Western blot was used to detect the expression of EphA2 and p53 in esophageal squamous cell carcinoma cell lines KYSE30,KYSE150,KYSE410,KYSE450,KYSE510,Eca109.Cell lines with different p53 status and high expression of EphA2 was selected for subsequent experiments.3.Knocking down EphA2 in p53 wild-type esophageal squamous carcinoma cell lines,and observe the effects on cell proliferation,migration,invasion,and EMT.(1)Establishing p53 wild-type esophageal squamous cell carcinoma stable cell lines with knocking down EphA2.KYSE30 and Eca109 cell lines with high expression of EphA2 belong to p53 wild-type esophageal squamous cell carcinoma cell lines were infected with three different shEphA2 lentiviruses,respectively,and expanded after Flow cytometry expansion.Green fluorescence of the cells were observed with a fluorescence microscope to determine the transfection efficiency of lentivirus and flow cytometry expansion efficiency.The knocking down efficiency of EphA2 in the established stable cell lines of KYSE30 and Eca109 cells was identified by Western blot.KYSE30 and Eca109 cell lins were screened for stable cell lines with the most obvious knocking down effect of EphA2.And they are used in the subsequent experiments.(2)The CCK-8 method was used to detect the proliferation capacity of KYSE30 and Eca109 cell lines after knocking down EphA2 at(0h,24h,48h,72h,96h).(3)The effect of knocking down EphA2 on cell migration ability in KYSE30 and Eca109 cell lines was detected by cell scratch test and transwell migration test.(4)Transwell invision test was used to detected the the invasion ability of KYSE30 and Eca109 cell lines after knocking down EphA2.(5)After knocking down EphA2 in KYSE30 and Eca109 cell lines,western blot was used to detect the changes of epithelial markers include E-cadherin and Claudin-1,mesenchymal markers N-cadherin,Vimentin and Snail were also detected.To verify the effect of knocking down EphA2 on EMT process in KYSE30 and Eca109 cell lines.4.Knocking down EphA2 in p53 mutant esophageal squamous cell carcinoma cell line,and explore the effects of koncking down EphA2 on cell proliferation,migration,invasion and EMT.(1)Two different siRNA sequences was used to infect KYSE150 cell line belongs to p53 mutant esophageal squamous cell carcinoma cell lins and with high expression of EphA2,respectively.72 hours later,Western blot was used to detect the knocking down efficiency of EphA2 in KYSE150 cell line.The siEphA2 sequence with the most obvious knocking down effect was selected for subsequent experiments.(2)The CCK-8 method was used to detect the proliferation capacity of KYSE150 cell line after knocking down EphA2 at(0h,24h,48h,72h,96h).(3)The migration ability of KYSE 150 cell line was detected by cell scratch test and transwell migration test after knocking down EphA2.(4)The effect of knocking down EphA2 on the migration ability of KYSE 150 cell line was detected by transwell invision test.(5)Western blot was used to detect the changes of epithelial markers E-cadherin and Claudin-1,and the mesenchymal markers Vimentin and Snail was also detected after knocking down EphA2 in KYSE150 cell line.To verify the effect of knocking down EphA2 on EMT process in KYSE150 cell line.Result1.In esophageal squamous cell carcinoma tissues,there are cases that EphA2 overexpresses with p53 mutation and EphA2 overexpresses with wild-type p53.2.In different esophageal cancer cell lines,the states of TP53 are different,and the expression of p53 and EphA2 are also different.(1)Western blot results showed that p53 was low expressed in KYSE30,KYSE510,and Eca109,and high expressed in KYSE150,KYSE410,and KYSE450.(2)In p53 wild-type esophageal squamous cell carcinoma cell lines,the expression of EphA2 in KYSE30 and Eca109 cell lines are higher.In p53 mutant esophageal squamous cell carcinoma cell lines,the expression of EphA2 in KYSE150 cells is higher;so choose KYSE30,Eca109,KYSE150 cell lines to perform the subsequent experiments.3.In p53 wild-type esophageal squamous carcinoma cell lines,the effect of knocking down EphA2 on cell proliferation,migration,invasion,and EMT.(1)The results of CCK-8 experiments showed that knocking down EphA2 significantly promote the proliferation of KYSE30 and Eca109 cells.(2)The results of cell scratch test and transwell migration test showed that knocking down EphA2 promote the migration ability of KYSE30 and Eca109 cell lines.(3)The results of transwell invision test showed that the knocking down EphA2 promote the invasion ability of KYSE30 and Eca109 cell lines.(4)Western blot results showed that after knocking down EphA2,the expression of KYSE30’ epithelial marker E-cadherin was decreased,but the expression of Eca109’ epithelial marker E-cadherin was increased.And the expression of KYSE30’ and Eca109’ epithelial marker Claudin-1 was decreased,the expressions of mesenchymal markers N-cadherin,Vimentin and Snail were all increased.Indicating that knocking down EphA2 promote the EMT process of KYSE30 and Eca109 cell lines.4.Effects of knockdown of EphA2 on cell proliferation,migration,invasion,and EMT in p53 mutant esophageal squamous carcinoma cell line.(1)The results of CCK-8 experiment showed that knocking down EphA2 significantly inhibite the proliferation of KYSE150 cell line.(2)The results of cell scratch and transwell migration test showed that the knocking down EphA2 inhibite the migration ability of KYSE150 cell line.(3)Transwell invision test showed that the invasion ability of KYSE150 cell line was inhibited after knocking down the expression of EphA2.(4)Western blot results showed that the expression of epithelial markers E-cadherin and claudin-1 were increased,and the expression of mesenchymal markers Vimentin and Snail were decreased after knocking down EphA2.Indicating that knocking down EphA2 inhibite the EMT process of KYSE150 cell line.Conclusions1.High expression of EphA2 in p53 wild type esophageal squamous cell carcinoma cell lines inhibited cell proliferation,migration and invasion ability;high expression of EphA2 in p53 mutant esophageal squamous cell carcinoma cell line promoted cell proliferation,migration and invasion ability.2.High expression of EphA2 in p53 wild-type esophageal squamous cell carcinoma cell lines inhibited the EMT process.High expression of EphA2 in p53 mutant esophageal squamous cell carcinoma cell line promoted the EMT process.3.EphA2 has a dual role in esophageal squamous cell carcinoma,and the different effects are related to different status of p53. |
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