The Mechanism Of HGF In The Development Of Esophagus Squamous Cell Carcinoma By Activating The PI3K/AKT Signaling Pathway

BackgroundEsophageal cancer is the eighth most common malignancy in the world,with the sixth highest mortality rate among all types of cancer,and the incidence varies by region.Histologically,esophageal cancer is mainly divided into squamous cell carcinoma(ESCC)and adenocarcinoma.In high-incidence areas,such as China,more than 90 percent of esophageal cancers are esophageal squamous cell carcinomas.Since the early symptoms of esophageal cancer are not obvious,most patients are already in the middle and late stage when they are diagnosed.Lymphatic metastasis and tissue infiltration have occurred in the tumor cells of esophageal cancer,which is also the main reason for the poor prognosis of patients with esophageal cancer.Despite some progress in treating esophageal cancer,overall survival remains low,at 15-34%over five years.Up to now,the factors that influence the development and prognosis of esophageal cancer remain unclear.Therefore,it is necessary to search for the key molecules of the pathogenesis and progression of esophageal cancer,improve the molecular mechanism of the occurrence and development of esophageal cancer,and provide a new target and direction for the diagnosis and treatment of esophageal cancer.Identification of biomarkers that can be used for diagnosis,treatment and prognosis of esophageal cancer is still urgently needed,and is of great significance for clinical diagnosis and treatment strategy.HGF/c-Met receptor tyrosine kinase(HGF/c-Met RTK)signaling pathway is inactivated in normal tissues but is abnormally activated in many types of cancer.Related studies have confirmed that the inhibition of HGF/c-Met signaling pathway can be an effective strategy for the treatment of various types of cancer,such as non-small cell lung cancer(NSCLC)liver cancer,gastric cancer,colorectal cancer,ovarian cancer,bladder cancer,head and neck cancer,endometrial cancer,etc.In clinical trials,c-Met inhibitors have been shown to inhibit the progression of NSCLC.Therefore,finding effective c-Met inhibitors and understanding the regulation of HGF/c-Met pathway are crucial to further inhibit HGF/c-Met pathway in human cancer.In our previous work,we found that hepatocyte growth factor(HGF),as a multifunctional cytokine,is highly expressed in esophageal cancer in collaboration with cyclin E,and closely related with ESCC degree of differentiation,TNM staging,affects the prognosis of patients.Therefore,this study mainly discusses the effect of HGF on the occurrence and development of esophageal cancer and the specific molecular mechanism involved,so as to provide a new target for clinical diagnosis and treatment of esophageal cancer.Part I:Expression and significance of HGF in esophageal cancerObjective:This part of the study mainly analyzed the gene expression map in esophageal cancer and the signal pathways that affect the occurrence and development of esophageal cancer.Methods:Firstly,RNA-seq was used to analyze the differences of gene expression in esophageal cancer tissues and adjacent tissues of clinically diagnosed esophageal cancer patients,and screen the genes related to esophageal cancer,and analyze the expression of HGF and cyclin E in esophageal cancer.KEGG signaling pathway analyzed and predicted the enrichment of screened genes in cancer-related signaling pathways,and Real-Time PCR was used to further verify the sequencing results.Furthermore,TCGA database was used to analyze whether the expression of HGF and cyclin E in esophageal cancer was synergistic and the relationship between HGF expression and tumor stage and prognosis.Finally,Real-Time PCR,Western blotting and immunohistochemistry were used to analyze the expression of HGF in esophageal and adjacent tissues.Results:We found that 29 genes were highly expressed in esophageal cancer tissues,and 4 genes were low-expressed in esophageal cancer tissues.Both HGF and cyclin E were highly expressed in esophageal cancer tissues.Analysis of the KEGG signaling pathway revealed that the PI3K/AKT signaling pathway was the most active signaling pathway in esophageal cancer tissues,including PPAR signaling pathway and NF-κB signaling pathway.Real-Time PCR further verified the differentially expressed genes analyzed by RNA-seq,HGF,cyclin E and other genes were up-regulated in esophageal cancer tissues.The expressions of HGF and cyclin E in esophageal cancer were analyzed by TCGA database.Compared with the corresponding paracancer tissues,HGF and cyclin E were highly expressed and synergistic in esophageal cancer tissues.The results of TCGA database analysis showed that the progression-free survival and overall survival time of esophageal cancer patients with low HGF expression were significantly better than that of the control group.The higher the HGF expression level in esophageal cancer tissues,the later the pathological stage of the tumor.We analyzed the clinical esophageal cancer tissues and the corresponding paracancer tissues by means of Real-Time PCR,Western blotting and immunohistochemistry.The results showed that compared with the corresponding paracancer tissues,HGF was indeed highly expressed in esophageal cancer tissues at both mRNA and protein levels(p<0.05).Conclusion:HGF is widely found in esophageal cancer tumor cells and may affect the development of esophageal cancer by participating in the PI3K/AKT signaling pathway,and the expression of HGF gradually increases with the progress of tumor,which is negatively correlated with disease-free survival and overall survival time of patients.Part Ⅱ:The effect of HGF on the biological function of esophageal carcinoma cellsObjective:This part of the study mainly explores the effect of HGF on the biological functions of esophageal cancer cells,thus providing a new theoretical basis for clinical use of HGF as a therapeutic target for esophageal cancer.Methods:This section explores the effects of HGF on cell survival,proliferation,invasion and metastasis,apoptosis and tumor formation at the level of esophageal cancer cells.First,two ESCC cell lines were selected and the expression of HGF gene was analyzed by Real-Time PCR.The cell lines were divided into low expression and high expression of HGF,and the expression and localization of HGF in the two cell lines were analyzed by Real-Time PCR,Western blotting and immunofluorescence from the mRNA and protein levels respectively.The expression of E-cadherin and N-cadherin and vimentin in EC9706 and KYSE-2 were detected by Western blotting.Then shRNA was used to knock down the expression of HGF in HGF highly expressed cell lines,and cDNA was used to express HGF highly in HGF low expressed cell lines.CCK-8 test was used to detect the effect of HGF expression changes on the activity of esophageal cancer cells.The effect of HGF expression changes on the proliferation of esophageal cancer cells was detected by cloning formation test.The cell cycle of two groups of cell lines was detected by flow cytometry.Western blotting was used to detect the effect of HGF expression on the expression of cell cycle-related proteins in esophageal carcinoma.Annexin V assay was performed to detect apoptosis in two cell lines.Western blotting was used to detect the effect of HGF expression on the expression of apoptosis-related proteins in esophageal cancer cells.The expression of E-cadherin,N-cadherin and vimentin in the two groups were detected by Real-Time PCR and Western blotting.Transwell test was used to detect the invasion and metastasis of esophageal cancer cells in two groups of cell lines.The tumor formation of esophageal carcinoma cells in two groups of cell lines was investigated in subcutaneous xenograft model of nude mice.HE staining and Tunel assay were used to observe the proliferation and inhibition of cell apoptosis in tumor tissues of mice.The effects of HGF expression changes on cell survival,proliferation,invasion and metastasis,apoptosis and tumor formation were analyzed.Results:Real-Time PCR results showed that HGF was poorly expressed in KYSE-2 cell line and highly expressed in EC9706 cell line(p<0.01).Western blotting showed that the expression level of HGF in EC9706 was significantly higher than that of KYSE-2(p<0.05).Subsequently,we used immunofluorescence to analyze the expression and localization of HGF in two cell lines,and found that in EC9706 with high expression of HGF,HGF was mainly located in the nucleus.In KYSE-2 with low expression of HGF,HGF is mainly located in the cytoplasm,suggesting that HGF may be activated in EC9706 and affect cell proliferation by regulating the expression of downstream genes.Western blotting test results show that compared with the low HGF expression KYSE-2,HGF high expression of EC9706 epithelioid cells of molecular markers in E-cadherin expression is low,ectomesenchymal cells and the molecular markers of N-cadherin and vimentin expression is higher(p<0.05),suggesting high HGF expression of esophageal cancer cell lines are more likely to happen EMT,this process could be due to the HGF regulation of cancer related signaling pathway.We used HGF cDNA and shRNA to detect the influence of HGF expression changes on the activity of esophageal cancer cells by overexpression and knockdown of HGF expression in KYSE-2 and EC9706 cell lines with high HGF expression,respectively.Compared with the control group,the high expression of HGF can significantly improve the cell activity(p<0.05).In contrast,the knockdown of HGF significantly inhibited the cell activity(p<0.05).The results of clone formation test showed that,compared with the control group,the high expression of HGF could significantly promote cell proliferation,and the difference was statistically significant(p<0.01).Compared with the control group,HGF knockdown can significantly inhibit cell proliferation,and the difference is statistically significant(p<0.01).Through the above studies,we proved that HGF can promote the proliferation and clone formation of esophageal cancer cells.The results of flow cytometry showed that,compared with the control group,the proportion of HGF cells in the S phase was significantly increased and the proportion in the G1 phase was significantly reduced after the high expression of HGF,and the difference was statistically significant(p<0.01).On the contrary,lentivirus was used to knock down the expression of HGF in EC9706,and the results showed that compared with the control group,the proportion of HGF knockdown cells in the S phase was significantly reduced,and the proportion in the G1 phase was significantly increased,and the difference was statistically significant(p<0.05).The effect of HGF expression on the expression of cell cyclin E-related proteins in esophageal carcinoma was detected by Western blotting.The results showed that,compared with the control group,the high expression of HGF significantly promoted the expression of PCNA and CyclinD1 in KYSE-2(p<0.05).Compared with the control group,HGF knockdown significantly inhibited the expression of PCNA and CyclinD1 in EC9706(p<0.05).Through the above studies,we proved that HGF can promote the division of esophageal cancer cells and promote the expression of cell cycle-related proteins.To demonstrate the effect of HGF expression on apoptosis of esophageal cancer cells,AnnexinV assay was used to detect the effect of HGF expression changes on apoptosis of esophageal cancer cells.The results showed that compared with the control group,the proportion of apoptosis after HGF high expression was significantly reduced,and the difference was statistically significant(p<0.05).The apoptosis rate of HGF knockdown cells was significantly increased,and the difference was statistically significant(p<0.05).Subsequently,Western blotting was used to detect the effect of HGF expression on the expression of apoptosis-related proteins in esophageal cancer cells.The results showed that,compared with the control group,the high expression of HGF could significantly inhibit the expression of Caspase-7 and Caspase-9 in KYSE-2(p<0.05).The knockdown of HGF significantly promoted the expression of Caspase-7 and Caspase-9 in EC9706(p<0.05).Through the above studies,we proved that HGF can inhibit the apoptosis of esophageal cancer cells and promote the expression of apoptosis-related proteins.In order to demonstrate the effect of HGF expression on EMT in esophageal cancer cells,molecular biological methods such as Real-Time PCR,Western blotting and immunofluorescence were used to detect the effect of HGF expression changes on emt-related protein expression in esophageal cancer cells.Compared with the control group,HGF mainly concentrated in the KYSE-2 nucleus after high expression of HGF,and inhibited the expression of E-cadherin,promoting the expression of N-cadherin and vimentin(p<0.05).After the knockdown of HGF,HGF was mainly distributed in the cytoplasm of EC9706 cells and could promote the expression of E-cadherin and inhibit the expression of N-cadherin and vimentin(p<0.05).Transwell test detected the invasion and metastasis of esophageal cancer cells in the two groups,and found that the high expression of HGF could significantly promote the invasion and metastasis of cells,and the difference was statistically significant(p<0.05).Knockdown of HGF can significantly inhibit cell invasion and metastasis,and the difference is statistically significant(p<0.05).In vitro cell experiments proved that HGF can promote the survival,proliferation,division,invasion and metastasis of esophageal cancer cells and inhibit cell apoptosis.We investigated the effect of HGF expression changes on esophageal cancer cell tumor formation in nude mouse subcutaneous tumor transplantation model.The results showed that,compared with the control group,the high expression of HGF could significantly promote the formation and growth of tumors,and the difference was statistically significant(p<0.01).HE staining and Tunel test showed that overexpression of HGF could significantly promote cell proliferation and inhibit cell apoptosis in tumor tissues,and the difference was statistically significant(p<0.01).Knockdown of HGF can significantly inhibit tumor formation and growth,and the difference is statistically significant(p<0.01).HE staining and Tunel test showed that the knockdown of HGF could significantly inhibit the proliferation of cells in tumor tissues and promote the apoptosis of cells,and the difference was statistically significant(p<0.01).Through the above studies,we proved that HGF can promote the formation of esophageal cancer cell tumors and promote the proliferation of tumor cells.Conclusion:In this part of the study,we proved that HGF can promote the proliferation,survival,invasion and metastasis of esophageal cancer cells,inhibit cell apoptosis and promote the formation of esophageal cancer tumors through in vitro cell experiments and in vivo tumor transplantation model.Part Ⅲ:The molecular mechanism by which HGF affects the biology of esophageal carcinoma cellsObjective:This part of the study mainly explores the molecular mechanism of HGF affecting the biological characteristics of esophageal cancer cells.Methods:Western blotting and immunohistochemistry were used to detect the expression of HGF and PI3K/AKT proteins in esophageal carcinoma and adjacent carcinoma tissues.Western blotting was used to detect the expression of HGF and PI3K/AKT signaling pathway in esophageal carcinoma cell lines.Subsequently,we altered the expression of HGF in two cell lines and detected the activation of the PI3K/AKT signaling pathway.Western blotting was used to detect the expression of HGF and PI3K/AKT signaling pathway related proteins in oesophageal carcinoma cell lines that were overexpressed or underexpressed with HGF.Immunohistochemistry was used to detect the expression of HGF and PI3K/AKT signaling pathway related proteins in subcutaneous xenograft tumors of nude mice in the second part.We overexpressed HGF in cell lines with low HGF expression,treated cells with PI3K inhibitor,or treated cells with low HGF expression in cell lines with high HGF expression,treated cells with PI3K agonist,and detected the effect of HGF expression changes on the proliferation of esophageal cancer cells by cloning formation test.Transwell test was used to detect the invasion and metastasis of esophageal cancer cells in two groups of cell lines.The tumor formation of the above-mentioned cell lines of esophageal carcinoma cells was investigated in the subcutaneous transplanted tumor model of nude mice.To observe the ability of cells to proliferate,invade,and form tumorsResults:Compared with the corresponding paracancer tissues,Western blotting and immunohistochemical experiments showed that when HGF expression in esophageal cancer tissues was abnormally high,the phosphorylation levels of AKT and mTOR in the PI3K/AKT signaling pathway significantly increased(p<0.05),and the expression of the upstream negative regulatory protein PTEN in the PI3K/AKT signaling pathway significantly decreased(p<0.05).Western blotting results showed that compared with KYSE-2,the phosphorylation levels of AKT and mTOR were significantly increased and the expression of PTEN was significantly decreased in the esophageal cancer cell line EC9706 with high HGF expression(p<0.05).In addition,we also found that the expression of cycinE in HGF highly expressed cell lines was also abnormally increased(p<0.05).Western blotting results showed that HGF overexpression significantly promoted the phosphorylation levels of AKT and mTOR,significantly inhibited the expression of PTEN,and promoted the expression of cyclin E(p<0.05).When the expression of HGF was lowered,we found that the phosphorylation levels of AKT and mTOR were significantly reduced,the expression levels of PTEN were significantly increased(p<0.05),and the expression of cyclin E was inhibited(p<0.05).Subcutaneous tumor-forming experiments in nude mice demonstrated that HGF overexpression can promote the phosphorylation of AKT in tumors and inhibit the expression of PTEN(p<0.05).Knockdown of HGF can inhibit the phosphorylation level of AKT in tumor and promote the expression of PTEN(p<0.05).We overexpressed HGF in the low expression of HGF in esophageal cancer cell line KYSE-2,and treated cells with appropriate concentration of PI3K inhibitor to inhibit the activity of PI3K/AKT signaling pathway.In the high expression of HGF in esophageal cancer cell line EC9706,the expression of HGF was decreased,and the activity of PI3K/AKT signaling pathway was activated by the treatment of cells with appropriate concentration of PI3K agonist.The results showed that compared with the control group,HGF overexpression significantly promoted the formation of cell cloning,while HGF overexpression+PI3K inhibitor significantly inhibited the formation of cell cloning,and the difference was statistically significant(p<0.01).HGF knockdown significantly inhibited the formation of cell cloning,while HGF knockdown+PI3K agonist significantly promoted the formation of cell cloning,and the difference was statistically significant(p<0.01).Transwell test results showed that HGF overexpression could significantly promote cell invasion,while HGF overexpression+PI3K inhibitor could significantly inhibit cell invasion,and the difference was statistically significant(p<0.05).HGF knockdown significantly inhibited cell invasion,while HGF knockdown+PI3K agonist significantly promoted cell invasion,and the difference was statistically significant(p<0.05).Experimental results of subcutaneous tumor transplantation in nude mice showed that HGF overexpression can significantly promote the formation of cell tumors,while HGF overexpression+PI3K inhibitor can significantly inhibit the formation of cell tumors,and the difference was statistically significant(p<0.01).HGF knockdown significantly inhibited the formation of cell tumors,while HGF knockdown+PI3K agonist significantly promoted the formation of cell tumors,and the difference was statistically significant(p<0.01).The above results indicate that HGF ACTS on the upstream of the PI3K/AKT signaling pathway and promotes the formation of esophageal cancer cell tumors by activating the PI3K/AKT signaling pathway.Conclusion:In this part of the study,we proved that HGF mainly ACTS on the upstream of the PI3K/AKT signaling pathway,activates the PI3K/AKT signaling pathway by inhibiting the expression of PTEN and promoting the phosphorylation of AKT and mTOR,thus promoting the proliferation,invasion and tumor formation of esophageal cancer cells.Therefore,HGF and its regulation of the PI3K/AKT signaling pathway can be potential targets for the treatment of esophageal cancer in clinical practice.