Objective:To clarify the function and molecular mechanisms of serpin family E member 2 (SERPINE2) in cellular migration and invasion of esophageal squamous cell carcinoma (ESCC).Methods:The expression of SERPINE2 in ESCC was analyzed by using online databases TCGA (http: //gepia.cancer-pku.cn/detail.php and http: //ualcan.path.uab. edu/index.html). The expressions of SERPINE2 mRNA in normal human esophageal epithelial cell line NE2, human ESCC cell lines KYSE30 and KYSE150 were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). SERPINE2-konckdown or SERPINE2-overexpressed plasmid was transfected into KYSE30 cells, and the efficiencies of the knockdown and overexpression system were tested by qRT-PCR. The relationships of SERPINE2 and ESCC migration and invasion were determined by migration and invasion assays in vitro. The associations between SERPINE2 expression and β-catenin as well as its target genes including c-Myc, cyclin D1 and CD44 were analyzed by immunofluorescence, qRT-PCR and western blot, respectively.Results:The expressions of SERPINE2 were significantly upregulated in both esophageal cancer (ESCA) and ESCC tissues compared to normal tissues by analyzing 182 and 95 cases, respectively ( P<0.01). SERPINE2 is highly expressed in both KYSE30 and KYSE150 cells ( P<0.05). The number of migrating and invading cells in control group were (212.66±24.11)/field and (136.00±14.42)/field, while were (88.33±9.71)/field and (77.00±9.53)/field in SERPINE2-knockdown 1 group, and (66.00±8.00)/field and (45.66±3.78)/field in SERPINE2-knockdown 2 group, respectively, and the differences were dramatically significant compared with the control group ( P<0.01). The number of migrating and invading cells in control group were (250.00±30.00)/field and (203.33±15.27)/field, while were (383.33±35.11)/field and (246.66±25.16)/field in SERPINE2-overpressed group, and the differences were strikingly significant compared with the control group ( P<0.01). The protein expression of β-catenin was upregulated while phosphorylated β-catenin protein expression was downregulated in SERPINE2-overexpressed KYSE30 cells when compared to control cells.The transcription activity of β-catenin was significantly upregulated and the mRNA expressions of its target genes including c-Myc, cyclin D1 and CD44 were all increased. After treated with 25 μM iCRT14, the number of migrated cells in the control and SERPINE2-overpressed groups were (200.00±36.05)/field and (258.33±22.54)/field, and the number of invaded cells were (160.00±17.32)/field and (188.33±25.65)/field, respectively, the differences were dramatically significant compared with the group without iCRT14 treatment ( P<0.01). Conclusion:SERPINE2 is significantly upregulated in ESCC cells and can promote cellular migration and invasion by activating β-catenin, which may provide a potential therapeutic target for patients with ESCC.

Objective:To clarify the function and molecular mechanisms of serpin family E member 2 (SERPINE2) in cellular migration and invasion of esophageal squamous cell carcinoma (ESCC).Methods:The expression of SERPINE2 in ESCC was analyzed by using online databases TCGA (http: //gepia.cancer-pku.cn/detail.php and http: //ualcan.path.uab. edu/index.html). The expressions of SERPINE2 mRNA in normal human esophageal epithelial cell line NE2, human ESCC cell lines KYSE30 and KYSE150 were detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). SERPINE2-konckdown or SERPINE2-overexpressed plasmid was transfected into KYSE30 cells, and the efficiencies of the knockdown and overexpression system were tested by qRT-PCR. The relationships of SERPINE2 and ESCC migration and invasion were determined by migration and invasion assays in vitro. The associations between SERPINE2 expression and β-catenin as well as its target genes including c-Myc, cyclin D1 and CD44 were analyzed by immunofluorescence, qRT-PCR and western blot, respectively.Results:The expressions of SERPINE2 were significantly upregulated in both esophageal cancer (ESCA) and ESCC tissues compared to normal tissues by analyzing 182 and 95 cases, respectively ( P<0.01). SERPINE2 is highly expressed in both KYSE30 and KYSE150 cells ( P<0.05). The number of migrating and invading cells in control group were (212.66±24.11)/field and (136.00±14.42)/field, while were (88.33±9.71)/field and (77.00±9.53)/field in SERPINE2-knockdown 1 group, and (66.00±8.00)/field and (45.66±3.78)/field in SERPINE2-knockdown 2 group, respectively, and the differences were dramatically significant compared with the control group ( P<0.01). The number of migrating and invading cells in control group were (250.00±30.00)/field and (203.33±15.27)/field, while were (383.33±35.11)/field and (246.66±25.16)/field in SERPINE2-overpressed group, and the differences were strikingly significant compared with the control group ( P<0.01). The protein expression of β-catenin was upregulated while phosphorylated β-catenin protein expression was downregulated in SERPINE2-overexpressed KYSE30 cells when compared to control cells.The transcription activity of β-catenin was significantly upregulated and the mRNA expressions of its target genes including c-Myc, cyclin D1 and CD44 were all increased. After treated with 25 μM iCRT14, the number of migrated cells in the control and SERPINE2-overpressed groups were (200.00±36.05)/field and (258.33±22.54)/field, and the number of invaded cells were (160.00±17.32)/field and (188.33±25.65)/field, respectively, the differences were dramatically significant compared with the group without iCRT14 treatment ( P<0.01). Conclusion:SERPINE2 is significantly upregulated in ESCC cells and can promote cellular migration and invasion by activating β-catenin, which may provide a potential therapeutic target for patients with ESCC.

Objective:To explore the function and mechanism of transcription factor En1 in esophageal squamous cell carcinoma (ESCC).Methods:The correlations of En1 with prognosis were analyzed using the overall survival data of 9 397 pan-cancer patients and progression-free survival data of 4 349 pan-cancer patients from The Cancer Genome Atlas (TCGA) database. The En1 expression data in 53 and 155 cases of ESCC and their paired adjacent tissues were from Gene Expression Omnibus (GEO) database and National Genomics Data Center-Genome Sequence Archive(NGDC-GSA)database. Lentivirus was used to generate En1 stable knockout cell lines KYSE180 and KYSE450. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The migration ability of the cells was detected by Transwell assay. The effect of En1 on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of En1, glioma-associated oncogene family zinc finger 1 (GLI1), glioma-associated oncogene family zinc finger 2 (GLI2) and smoothened (SMO).Results:Pan-cancer data from TCGA showed that patients with low En1 expression had longer overall survival and progression-free survival than patients with high En1 expression ( P< 0.001). Data from GEO and GSA databases also showed a high expression level of En1 in ESCC tissues compared with paired tissues ( P＜0.001). Proliferation was inhibited after knockout of En1 in KYSE180 and KYSE450 cells ( P＜0.001). The colony formation numbers decreased. The colony formation numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 138.33±23.07 and 127.00±19.70, respectively, significantly lower than that of the shNC group 340.67±12.06 ( P<0.001). The colony formation numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 65.33±2.52 and 9.00±3.00, respectively, significantly lower than that of the shNC group 139.00±13.00 ( P<0.001). The migration numbers was inhibited after knockout of En1 [the Transwell numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 66.67±12.66 and 71.33±11.02, respectively, significantly lower than that of the shNC group 334.67±16.56 ( P<0.001). The Transwell numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 112.33±14.57 and 54.33±5.51, respectively, significantly lower than that of the shNC group 253.33±21.03 ( P<0.001)]. Xenograft model showed a slower growth rate of shEn1#1 and shEn1#2 cell lines ( P＜0.001). The tumor weights of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were (0.046±0.026)g and (0.047±0.025)g, respectively, significantly lower than that of the shNC group (0.130±0.038)g ( P＜0.001). After knockdown of En1, the relative expression levels of GLI1 in KYSE180 cells of the shEn1#1 group and the shEn1#2 group were 0.326±0.162 and 0.322±0.133, and the relative expression levels of GLI1 in KYSE450 cells of the shEn1#1 and shEn1#2 groups were 0.131±0.006 and 0.352±0.050, respectively, which were all lower than that in the shNC group ( P＜0.01). After knockdown of En1, overexpression of GLI1 attenuated the inhibitory effect of knockdown of En1 on cell proliferation ( P＜0.001), colony formation[the colony formation numbers of the shEn1#1-GLI1 group were 151.00±9.54, higher than 102.33±10.02 ( P=0.004) of the shEn1#1-vector group] and migration [the migration numbers of the shEn1#1-GLI1 group were 193.67±10.07, higher than 109.33±11.50 ( P<0.001) in the shEn1#1-vector group]. In clinical samples of ESCC, major regulatory factors of the Hedgehog pathway were up-regulated and the pathway was activated. Conclusion:En1 promotes the proliferation and migration of ESCC cells by regulating the Hedgehog pathway and can be used as a new potential target for targeted therapy of ESCC.

Objective:To explore the function and mechanism of transcription factor En1 in esophageal squamous cell carcinoma (ESCC).Methods:The correlations of En1 with prognosis were analyzed using the overall survival data of 9 397 pan-cancer patients and progression-free survival data of 4 349 pan-cancer patients from The Cancer Genome Atlas (TCGA) database. The En1 expression data in 53 and 155 cases of ESCC and their paired adjacent tissues were from Gene Expression Omnibus (GEO) database and National Genomics Data Center-Genome Sequence Archive(NGDC-GSA)database. Lentivirus was used to generate En1 stable knockout cell lines KYSE180 and KYSE450. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The migration ability of the cells was detected by Transwell assay. The effect of En1 on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the expressions of En1, glioma-associated oncogene family zinc finger 1 (GLI1), glioma-associated oncogene family zinc finger 2 (GLI2) and smoothened (SMO).Results:Pan-cancer data from TCGA showed that patients with low En1 expression had longer overall survival and progression-free survival than patients with high En1 expression ( P< 0.001). Data from GEO and GSA databases also showed a high expression level of En1 in ESCC tissues compared with paired tissues ( P＜0.001). Proliferation was inhibited after knockout of En1 in KYSE180 and KYSE450 cells ( P＜0.001). The colony formation numbers decreased. The colony formation numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 138.33±23.07 and 127.00±19.70, respectively, significantly lower than that of the shNC group 340.67±12.06 ( P<0.001). The colony formation numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 65.33±2.52 and 9.00±3.00, respectively, significantly lower than that of the shNC group 139.00±13.00 ( P<0.001). The migration numbers was inhibited after knockout of En1 [the Transwell numbers of KYSE180 cells in the shEn1#1 group and the shEn1#2 group were 66.67±12.66 and 71.33±11.02, respectively, significantly lower than that of the shNC group 334.67±16.56 ( P<0.001). The Transwell numbers of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were 112.33±14.57 and 54.33±5.51, respectively, significantly lower than that of the shNC group 253.33±21.03 ( P<0.001)]. Xenograft model showed a slower growth rate of shEn1#1 and shEn1#2 cell lines ( P＜0.001). The tumor weights of KYSE450 cells in the shEn1#1 group and the shEn1#2 group were (0.046±0.026)g and (0.047±0.025)g, respectively, significantly lower than that of the shNC group (0.130±0.038)g ( P＜0.001). After knockdown of En1, the relative expression levels of GLI1 in KYSE180 cells of the shEn1#1 group and the shEn1#2 group were 0.326±0.162 and 0.322±0.133, and the relative expression levels of GLI1 in KYSE450 cells of the shEn1#1 and shEn1#2 groups were 0.131±0.006 and 0.352±0.050, respectively, which were all lower than that in the shNC group ( P＜0.01). After knockdown of En1, overexpression of GLI1 attenuated the inhibitory effect of knockdown of En1 on cell proliferation ( P＜0.001), colony formation[the colony formation numbers of the shEn1#1-GLI1 group were 151.00±9.54, higher than 102.33±10.02 ( P=0.004) of the shEn1#1-vector group] and migration [the migration numbers of the shEn1#1-GLI1 group were 193.67±10.07, higher than 109.33±11.50 ( P<0.001) in the shEn1#1-vector group]. In clinical samples of ESCC, major regulatory factors of the Hedgehog pathway were up-regulated and the pathway was activated. Conclusion:En1 promotes the proliferation and migration of ESCC cells by regulating the Hedgehog pathway and can be used as a new potential target for targeted therapy of ESCC.