| ObjectiveEsophageal cancer is one of the common malignant tumors in the digestive system,the main pathological type is esophageal squamous cell carcinoma.In recent years,surgery and radiotherapy and chemotherapy have got some progress in the treatment of esophageal cancer,but patients with advanced esophageal cancer should not be treated with surgery,and some patients are resistant to radiotherapy and chemotherapy,molecular targeted therapy and immunotherapy are new approaches to treat tumors,and in-depth elucidation of the molecular mechanism of esophageal cancer is helpful for targeted treatment of esophageal cancer.Long non-coding RNA(LncRNA)is involved in the progression of many cancers.The LncRNA-miRNA-mRNA regulatory network plays an important role in the occurrence and development of esophageal cancer.Long non-coding RNA HOXA remote transcript(lncRNA HOTTIP)plays an oncogene role in a variety of tumors.HOTTIP affects cell proliferation and migration in esophageal cancer,but its effect on esophageal cancer cell apoptosis,metastasis,and tumor immune escape and its mechanism of action in the progression of esophageal cancer are still not completely clear.Therefore,this study intends to study the role of lncRNA HOTTIP in the development of esophageal cancer.Bioinformaties analysis revealed that lncRNA HOTTIP may target the regulation of miR-516b-5p expression,and further predictions show that the target gene of miR-516b-5p may be SPIN1,and the role of miR-516b-5p and SPIN1 in esophageal cancer and its mechanism have not yet been fully determined.It is also unknown whether HOTTIP affects the occurrence and development of esophageal cancer through the miR-516b-5p/SPIN1 axis.To further reveal the molecular mechanism of esophageal cancer apoptosis,metastasis and tumor immune escape,this study intends to analyze from the following four aspects:Chapter 1:The effect of lncRNA HOTTIP on the apoptosis,metastasis,and immune escape of esophageal cancer;Chapter 2:Effect of lncRNA HOTTIP on the process of esophageal cancer by regulating miR-516b-5p;Chapter 3:Effect of miR-516b-5p on the process of esophageal cancer by regulating SPIN1;Chapter 4:lncRNA HOTTIP regulates the expression of SPIN1 through sponge miR-516b-5p and affects the PI3K/AKT/mTOR signaling pathway.Chapter 1:Effects of HOTTIP on apoptosis,metastasis and immune escape of esophageal cancerBackground:Esophageal cancer is a common malignant tumor of the digestive tract in China,and its incidence is high because its early symptoms are not obvious and it is difficult to find,so many patients are often in the middle and late stages when they are diagnosed,and the best treatment opportunity is lost,resulting in a poor prognosis,And the case fatality rate is high.With the research of molecular biology and precision medicine,molecular targeted therapy has become an important research direction of tumor therapy.Studying the specific mechanisms of esophageal cancer development and the mechanisms affecting tumor-related immune escape can provide new ideas for the prevention and treatment of esophageal cancer.Method and reference basis.It has been reported that lncRNA also affects the progress of esophageal cancer.Some lncRNA mediates the occurrence,development and metastasis of esophageal cancer,and can be used as a new target for the diagnosis and treatment of esophageal cancer.Aim:To explore the relationship between HOTTIP and apoptosis,metastasis and immune escape of esophageal cancer.Methods:A total of 73 patients with esophageal cancer treated in our hospital from January 2012 to December 2013 were selected as the research objects,and the clinical and pathological data of the patients were collected.Normal esophageal epithelial cells Het-1A and esophageal cancer cells KYSE410 and ECA109 were cultured in vitro.Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of HOTTIP in esophageal cancer tissues and esophageal cancer cell lines.Based on the median of the relative expression of HOTTIP,the esophageal cancer patients were divided into high expression groups 37 cases,36 cases in low expression group.Kaplan-Meier was used to analyze the 5-year survival of patients with esophageal cancer,and the relationship between HOTTIP expression and clinicopathological factors in patients with esophageal cancer was analyzed by Spearman.HOTTIP and si-HOTTIP vectors were transfected into KYSE410 and ECA109 cells,respectively;qRT-PCR method was used to detect transfection efficiency;MMT method was used to detect cell proliferation activity;cell cloning experiment was used to detect cell clone formation ability;flow cytometry was used to detect apoptotic rate;Transwell chamber experiment was used to detect cell migration and invasion ability;Western blot was used to detect E-cadherin,N-cadherin,Vimentin,PD-L1 protein expression.The esophageal cancer cells transfected with HOTTIP and si-HOTTIP vectors were co-cultured with T lymphocytes,and the levels of IFNy,TNFα,and IL-2 were detected by enzyme-linked immunosorbent assay(ELISA).Results:(1)The expression of HOTTIP in esophageal cancer tissues and esophageal cancer cells was significantly higher than that in normal tissues and normal esophageal epithelial cells(P<0.01).(2)High expression of HOTTIP was associated with tumor size,lymph node metastasis,and TNM stages in esophageal cancer patients was significantly related(P<0.01);the overall survival rate of esophageal cancer patients in the HOTTIP high expression group was significantly lower than that in the low expression group(P<0.01).(3)After over-expression of HOTTIP,the proliferation activity of KYSE410 cells was significantly increased,the number of cell clones was significantly increased,the number of cell migration and invasion was significantly increased,the expression of E-cadherin protein was significantly reduced,and the expressions of N-cadherin and Vimentin proteins were significantly increased.PD-L1 mRNA and protein expressions were significantly increased;the levels of IFNγ,TNFα,and IL-2 released by T lymphocytes co-cultured with esophageal cancer cells were significantly reduced(P<0.01).(4)After HOTTIP was silenced,the proliferation activity of ECA109 cells was significantly reduced,the number of cell clones was significantly reduced,the rate of apoptosis was significantly increased,the number of cell migration and invasion was significantly reduced,the expression of E-cadherin protein was significantly increased,and the expressions of N-cadherin and Vimentin proteins were significantly reduced,the levels of PD-L1 mRNA and protein were significantly reduced,and the levels of IFNy,TNFα,and IL-2 released by T lymphocytes co-cultured with esophageal cancer cells were significantly increased(P<0.01).Conclusion:The expression of HOTTIP in esophageal cancer tissues and cells was increased,which is related to the survival of patients.It can be used as an important molecular marker for judging the prognosis of patients.Silencing HOTTIP can inhibit the proliferation,colony formation,migration,invasion and EMT of esophageal cancer cells,and induce apoptosis and immune escape of tumor cells.Chapter 2:Effect of lncRNA HOTTIP on the process of esophageal cancer by regulating miR-516b-5pBackground:MicroRNA(microRNA,miRNA)is a type of small molecule RNA of about 22 nucleotides in length,which is highly conservative.Studies have shown that miRNA not only regulates various life activities of the body,but also participates in the occurrence and development of tumors.Abnormal miRNA expression in cells can provide new ideas for the diagnosis and treatment of tumors.The biological information website predicted that lncRNA HOTTIP may adsorb miRNA,and found that HOTTIP may adsorb miR-516b-5p.miR-516b can inhibit the proliferation,migration and invasion of non-small cell lung cancer cells.However,the role of miR-516b-5p in esophageal cancer and whether miR-516b-5p is regulated by HOTTIP to affect the occurrence and development of esophageal squamous cell carcinoma have not yet Knowable.Aim:To investigate whether miR-516b-5p is involved in the process of esophageal squamous cell carcinoma,and whether miR-516b-5p is regulated by HOTTIP to affect the occurrence and development of esophageal squamous cell carcinomaMethods:Double-luciferase reporter gene experiments and RIP experiments were used to verify the targeted regulatory relationship between HOTTIP and miR-516b-5p.qRT-PCR was used to detect the expression of miR-516b-5p in esophageal cancer tissues and esophageal cancer cell lines.Co-transfect HOTTIP and miR-516b-5p overexpression vectors into KYSE410 cells,co-transfect si-HOTTIP and anti-miR-516b-5p into ECA109 cells;use the experimental methods in Chapter 1 to measure cell proliferation activity,the ability to form clones,the rate of apoptosis,the number of cells migrating and invading,and the expression of E-cadherin,N-cadherin,Vimentin,and PD-L1 proteins.Esophageal cancer transfected with HOTTIP and miR-516b-5p,si-HOTTIP and anti-miR-516b-5p were co-cultured with T lymphocytes,and IFNy,TNFα and IL-2 levels were detected by ELISA.Results:(1)HOTTIP can target and bind to miR-516b-5p.The expression of miR-516b-5p in esophageal cancer tissues and esophageal cancer cells was significantly reduced(P<0.01).(2)Compared with the HOTTIP+miR-NC group,the cell proliferation activity of HOTTIP+miR-516b-5p group was significantly reduced,the number of cell clones was significantly reduced,the number of cell migration and invasion was significantly reduced,the expression of E-cadherin protein was significantly increased,the expressions of N-cadherin and Vimentin proteins were significantly reduced,and PD-L1 mRNA and protein expressions were significantly reduced,and the levels of IFNy,TNFα,and IL-2 released by T lymphocytes co-cultured with esophageal cancer cells were significantly increased(P<0.01).(3)Compared with the si-HOTTIP+anti-miR-NC group,the cell proliferation activity of the si-HOTTIP+anti-miR-516b-5p group was significantly increased,the number of cell clones was significantly increased,the apoptosis rate was significantly reduced,and the number of cell migration and invasion was significantly increased,the expression of E-cadherin protein was significantly reduced,the expressions of N-cadherin and Vimentin protein were significantly increased,and the expressions of PD-L1 mRNA and protein were significantly increased;the levels of IFNy,TNFa and IL-2 released by T lymphocytes co-cultured with esophageal cancer cells were significantly reduced(P<0.01).Conclusion:HOTTIP may participate in biological processes such as proliferation,clone formation,apoptosis,migration,invasion,EMT and tumor cell immune response through targeted regulation of miR-516b-5p expression.Chapter 3:Effect of miR-516b-5p on the process of esophageal cancer by regulating SPIN1Background:miRNA can be completely or incompletely paired with messenger RNA(mRNA)to regulate the translation or degradation of target mRNA,thereby regulating the expression of genes at the post-transcriptional level,thereby playing a role in biological activities.Spindlin1(SPIN1)is an important member of the SPIN/SSTY gene family and is expressed in a variety of cancers,and its abnormal activation is closely related to the occurrence and development of tumors.However,the role of SPIN1 in the proliferation,apoptosis,migration,invasion and EMT of esophageal cancer,and whether SPIN1 is regulated by miR-516b-5p and thus affects the occurrence and development of esophageal cancer are still unknown.Aim:To explore the role of SPIN1 in the proliferation,apoptosis,migration,invasion and EMT of esophageal cancer and whether SPIN1 is regulated by miR-516b-5p to affect the occurrence and development of esophageal cancer.Methods:Double-luciferase reporter gene experiments were used to verify the targeted regulatory relationship between miR-516b-5p and SPIN1.qRT-PCR and Western blot were used to detect the expressions of SPIN1 mRNA and protein in esophageal cancer tissue and esophageal cancer cell line,respectively.Co-transfect miR-516b-5p with SPIN1 overexpression vector into KYSE410 cells,co-transfect anti-miR-516b-5p and si-SPIN1 into ECA109 cells;use the experimental method in Chapter 1 to detect cell proliferation activity,colony forming ability,apoptotic rate,number of migrating cells and invading cells,and expression of E-cadherin,N-cadherin,Vimentin,PD-L1 protein,respectively.Esophageal cancer transfected with miR-516b-5p and SPIN1,anti-miR-516b-5p and si-SPIN1 were co-cultured with T lymphocytes,and IFNy,TNFa and IL-2 levels were detected by ELISA.Results:(1)miR-516b-5p could target and regulate SPIN1.The expression of SPIN1 in esophageal cancer tissues and esophageal cancer cells was significantly increased(P<0.01).(2)Compared with the miR-516b-5p+vector group,the cell proliferation activity of the miR-516b-5p+SPIN1 group was significantly increased,the number of cell clones was significantly increased,the apoptosis rate was significantly reduced,and the number of cell migration and invasion was significantly increased;E-cadherin protein expression was significantly reduced,N-cadherin and Vimentin protein expressions were significantly increased,PD-L1 mRNA and protein expressions were significantly increased,and the levels of IFNy,TNFα,and IL-2 released by T lymphocytes co-cultured with esophageal cancer cells were decreased significantly(P<0.01).(3)Compared with the anti-miR-516b-5p+si-NC group,the cell proliferation activity of the anti-miR-516b-5p+si-SPIN1 group was significantly reduced,the number of cell clones was significantly reduced,the number of cell migration and invasion was significantly reduced,and the expression of E-cadherin protein was significantly increased,the expressions of N-cadherin and Vimentin proteins were significantly decreased,and the expressions of PD-L1 mRNA and protein were significantly reduced;the levels of IFNγ,TNFα and IL-2 released by T lymphocytes co-cultured with esophageal cancer cells were significantly increased(P<0.01).Conclusion:miR-516b-5p may participate in the biological processes of esophageal cancer cell proliferation,colony formation,apoptosis,migration,invasion,EMT and tumor cell immune response by regulating the expression of SPIN1.Chapter 4:lncRNA HOTTIP regulates the expression of SPIN1 through sponge miR-516b-5p and influences the PI3K/AKT/mTOR signaling pathwayBackground:Studies have shown that non-coding RNA molecules can regulate gene expression at various levels through multiple pathways,thereby participating in the life activities of cells,tissues and even the entire body.LncRNA and miRNA,miRNA and mRNA,and lncRNA and mRNA all have a mutually regulated relationship.Formed a mutual regulation network of lncRNA-miRNA-mRNA.The PI3K/AKT/mTOR signaling pathway is closely related to the occurrence and progression of various cancers.The relationship between HOTTIP and SPIN1 and whether HOTTIP regulates the expression of SPIN1 through miR-516b-5p are still unclear,and whether HOTTIP affects the PI3K/AKT/mTOR signaling pathway and the development and progression of esophageal cancer by regulating miR-516b-5p/SPIN1 not yet known.Aim:Whether HOTTIP affects the PI3K/AKT/mTOR signaling pathway through the regulation of miR-516b-5p/SPIN1 and then the occurrence and development of esophageal cancer.Methods:Spearman was used to analyze the correlation between HOTTIP,miR-516b-5p,and SPIN1;HOTTIP and miR-516b-5p overexpression vectors were co-transfected into KYSE410 cells,and si-HOTTIP and anti-miR-516b-5p were co-transfected into ECA109 cells,and detected the expression of SPIN1 protein by Western blot;HOTTIP,HOTTIP and miR-516b-5p,HOTTIP and si-SPIN1 were co-transfected into esophageal cancer cell KYSE410,respectively;si-HOTTIP,si-HOTTIP and anti-miR-516b-5p,si-HOTTIP and SPIN1 were co-transfected into esophageal cancer cell ECA109,respectively.Western blot was used to detect the expression of PI3K/AKT/mTOR signaling pathway related proteins.Results:(1)LncRNA HOTTIP was negatively correlated with miR-516b-5p,SPIN1 was negatively correlated with miR-516b-5p,and lncRNA HOTTIP was positively correlated with SPIN 1(P<0.01).(2)Compared with the HOTTIP+miR-NC group,the expression of SPIN1 protein in the HOTTIP+miR-516b-5p group was significantly reduced,and compared with the si-HOTTIP+anti-miR-NC group,the expression of SPIN1 protein was significantly increased in the si-HOTTIP+anti-miR-516b-5p group(P<0.01).(3)Compared with the HOTTIP+miR-NC group,the expressions of p-PI3K,p-AKT,and p-mTOR proteins in the HOTTIP+miR-516b-5p group were significantly reduced;compared with the HOTTIP+si-NC group,the expressions of p-PI3K,p-AKT,and p-mTOR proteins in the HOTTIP+si-SPIN1 group were significantly reduced(P<0.01);compared with the si-HOTTIP+anti-miR-NC group,the expressions of p-PI3K,p-AKT,and p-mTOR proteins in the si-HOTTIP+anti-miR-516b-5p group were significantly increased;compared with the si-HOTTIP+vector group,p-PI3K,p-AKT,p-mTOR protein expressions were significantly increased in the si-HOTTIP+SPIN1 group(P<0.01).Conclusion:HOTTIP may regulate the PI3K/AKT/mTOR signaling pathway by regulating the expression of SPIN1 through sponge miR-516b-5p.General conclusion:(1)HOTTIP is highly expressed in tissue and cell lines of esophageal cancer;(2)The prognosis of patients with high expression of HOTTIP is poorer;(3)HOTTIP may promote esophageal cancer cell proliferation,clone formation,migration,invasion,EMT,inhibit apoptosis and assist tumor immune escape by down-regulating miR-516b-5p and then up-regulating the expression of SPIN1.(4)HOTTIP may regulate the PI3K/AKT/mTOR signaling pathway through the miR-516b-5p/SPIN1 axis. |
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