| Objectives:This study is to explore the role and mechanism of P4HA1 in esophageal squamous cell carcinoma,and to explore whether STAT1 can transcript and activate P4HA1 to promote the progression of esophageal carcinoma.Search for new biomarkers and new therapeutic targets for esophageal cancer.Method:1)The complete clinical and pathological data of patients by surgical resection of esophageal squamous cell carcinoma and pathologically confirmed in the First Affiliated Hospital of Xinjiang Medical University from March 2008 to December 2017 were collected.A total of 120 specimens of tumor and adjacent normal esophageal tissues from ESCC patients were collected and made into tissue microarray.Histological specimens were used for HE section and immunohistochemical staining,and clinical and pathological data were used for correlation analysis between P4HA1 and clinicopathological indexes.Follow-up was conducted by telephone or review of medical records for 24 to 85 months.2)The expression of P4HA1 in each cell line was detected by qRT-PCR and Western Blot.P4HA1 stable low expression cell line was constructed and screened.Cell proliferation was detected by CCK8 assay,colony formation assay and EdU assay.Detection of apoptosis by flow cytometry.The expressions of collagens(COL1A1 and COL4A1)were detected by Western Blot.Tumorigenesis assay in nude mice was used to detect the effect of P4HA1 knockdown on tumorigenesis ability of different groups of cells.The expression of P4HA1 and COL4A1and Ki67 proliferation index were detected by immunohistochemistry.Masson trichome staining was used to detect the level of collagen fibers in tumorigenic tissues,and TUNEL staining was used to detect the apoptosis of tumorigenic tissues.3)The interaction between STAT1 and P4HA4 was analyzed using ChIP,dual luciferase reporter gene and Y1H assays.Construction of STAT1 knockdown and P4HA1 overexpression cell lines,and the expressions of STAT1 and P4HA1 after STAT1 knockdown and P4HA1 overexpression cell lines were detected by qRT-PCR and Western Blot.CCK8 assay,EdU assay and plate cloning assay were used to detect the proliferation ability of STAT1 knockdown and P4HA1 overexpression.Flow cytometry was used to detect cell apoptosis after STAT1 knockdown and P4HA1 overexpression.Western Blot assay was used to detect the expression of CoL1A1 and CoL4A1 proteins after STAT1 knockdown and P4HA1 overexpression.Result:1)The positive rate of P4HA1 in esophageal cancer tissues was 68.7%(163/237),and the expression of P4HA1 was negative in all adjacent tissues,and its expression was significantly increased in patients with esophageal cancer.In the comparison of the expression level of P4HA1 in esophageal cancer and its relationship with clinical pathological parameters,it was found that the expression of P4HA1 is related to the degree of tumor differentiation(χ2=14.973,P=0.01),the site of tumor occurrence(χ2=6.328,P=0.042),lymph node metastasis(χ2=31.625,P<0.01),and TNM staging(χ2=7.892,P=0.048).Univariate survival analysis showed that prognostic factors affecting OS in esophageal cancer patients include tumor differentiation(χ2=7.117,P=0.028),lymph node metastasis(χ2=16.328,P=0.01),and high expression of P4HA1(χ2=16.529,P=0.01).Multivariate COX regression survival analysis showed that independent prognostic factors affecting OS in esophageal cancer patients included high expression of P4HA1(HR=2.234,P=0.001,95.0%CI:1.310-3.810)and lymph node metastasis(HR=1.677,P=0.001,95.0%CI:1.146-2.454).The Kaplan Meier method was used for univariate survival analysis.Prognostic factors affecting PFS in esophageal cancer patients include tumor differentiation(χ2=7.293,P=0.026),lymph node metastasis(χ2=18.869,P=0.001),and high expression of P4HA1(χ2=19.278,P=0.001).Multivariate COX regression analysis suggests that independent prognostic factors for PFS include lymph node metastasis(HR=1.753,P=0.004,95.0%CI:1.1498-2.564)and high expression of P4HA1(HR=2.342,P=0.002,95.0%CI:1.378-3.980).2)P4HA1 expression was highly in esophageal cancer cell lines.After knockout of P4HA1,the proliferation level of EC cells decreased significantly and the apoptosis rate increased.The levels of collagen COL1A1 and COL4A1 were significantly reduced.The growth of subcutaneous tumor tissue in mice was significantly inhibited.The expressions of Ki67,P4HA1 and COL4A1 in tumorigenic tissue were significantly decreased.The collagen fibers decreased tremendously andThe apoptosis rate was significantly increased.3)STAT1 can bind to the P4HA1 promoter to activate and promote the expression of P4HA1.STAT3 can be directly bound to the P4HA1 promoter.The protein expression levels of STAT1 and P4HA1 were significantly decreased after knockout of STAT1 in cells.After knockout of ST AT1 and high expression of P4HA1,the down-regulation of P4HA1 expression caused by si-STAT1 can be reversed.The down-regulation of cell proliferation induced by si-STAT1 can be reversed.The down-regulation of CoL1A1 and CoL4A1 protein expression caused by si-STAT1 can be reversed.It can reverse the up-regulation of apoptosis rate caused by si-STAT1.Conclusion:P4HA1 is highly expressed in esophageal squamous cell carcinoma,and the expression of P4HA1 is relevanted to tumor differentiation,tumor location,lymph node metastasis,and TNM stage.P4HA1 is an independent prognostic factor affecting overall survival and progression-free survival in patients with esophageal squamous cell carcinoma.P4HA1 was significantly up-regulated in EC cells,and P4HA1 silencing significantly inhibited EC cell proliferation and collagen synthesis,while increasing apoptosis.Subcutaneous injection of sh-P4HA1 cells significantly inhibited the growth of subcutaneous tumor tissue in mice.The collagen fibers in tumorigenic tissues decreased significantly and the apoptosis of tumor cells increased.STAT1 can transcriptionally activate P4HA1,promote EC cell proliferation and collagen generation,inhibit cell apoptosis,and promote the progression of ESCC.STAT1 may positively regulate the expression of P4HA1. |
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