The Expression And Methylation Of KISS-1 In The Malignant Pro-gression Of Esophageal Squamous Cell Carcinoma

Objectives:To detect the expression levels and promoter methylation status of KISS-1 in esophageal squamous cell carcinoma（ESCC）and adjacent normal tissues,ESCC and normal esophageal epithelial cells.To analyze the clinical significance of KISS-1 ex-pression and its promoter methylation in ESCC patients.To analyze the correlation between the promoter methylation of KISS-1 and its expression level in ESCC.To explore the effect of the promoter methylation status of KISS-1 on biological behavior of ESCC cells.Besides,to explore the potential mechanism of KISS-1 in the malig-nant progression of ESCC.Methods:1.Fresh ESCC and paratumor normal tissues were collected from the thoracic surgery room of Renmin Hospital of Wuhan University,and the clinicopathological information of patients with ESCC was also collected.q RT-PCR was used to investi-gate the m RNA expression levels of KISS-1 in ESCC and paratumor tissues.Correla-tion analysis was applied to reveal the association between KISS-1 m RNA expression and clinicopathological characteristics of ESCC patients.Immunocytochemistry（IHC）was utilized to detect the expression of KISS-1 protein in collected surgical specimens.Paraffin-embedded specimens of esophageal squamous epithelial dysplasia were col-lected from the pathology department of our hospital.After the paraffin specimens were cutting into sections,IHC was used to examine the protein expression of KISS-1in dysplastic tissues.The m RNA and protein expression levels of KISS-1 in five ESCC cell lines TE-1,Eca109,EC9706,KYSE30 and KYSE150 and one normal esophageal epithelial cell line HEEpi C were determined by q RT-PCR and western blotting,respectively.2.Bisulfite sequencing PCR（BSP）and methylation-specific PCR（MSP）were applied to investigated the methylation status of KISS-1 promoter region in five ESCC cell lines TE-1,Eca109,EC9706,KYSE30 and KYSE150,and normal esoph-ageal epithelial cell line HEEpi C.MSP was used to further examine the methylation pattern of KISS-1 promoter region in ESCC and paratumor tissues.Correlation analy-sis was employed to reveal the association between the methylation status of KISS-1promoter and clinicopathological characteristics of ESCC patients.q RT-PCR was used to detect the expression levels of KISS-1 m RNA in KISS-1-methylated and-unmethylated groups of ESCC tissues.Correlation analysis was further utilized to analyze the association between the promoter methylation of KISS-1 and its protein expression in ESCC tissues.KISS-1^low Eca109 and KYSE30 cells were selected to treated with hypomethylating agent 5-Aza-d C by different concentration groups（con-trol,1u M and 5u M）.After 5-Aza-d C treatment,BSP and MSP were used to examine the methylation status of KISS-1 promoter region in 2 ESCC cells,and immunoblot-ting was utilized to investigate the protein levels of KISS-1 in 2 ESCC cells.Follow-ing the treatment of 5-Aza-d C,CCK8 assay was used to investigate the proliferation levels of HEEpi C,Eca109 and KYSE30 cells,and Transwell assay was employed to determine the migration and invasion abilities of Eca109 and KYSE30 cells.3.Two ESCC cell lines Eca109 and KYSE30 were transfected with PIRES2-EGFP-KISS-1 plasmid to overexpress KISS-1.q RT-PCR was applied to determine the expression levels of KISS-1 m RNA in the two ESCC cells following transfection.CCK8,flow cytometry,wound healing and Transwell assays were utilized to examine the effects of KISS-1 overexpression on the biological behavior of Eca109 and KYSE30 cells.Western blotting was applied to investigate the effects of KISS-1overexpression on the protein expression levels of metal matrix proteases（MMPs）and epithelial-mesenchymal transformation（EMT）regulators in Eca109 and KYSE30cells.In addition,the two transfected ESCC cells were further treated with mito-gen-activated protein kinase（MAPK）/extracellular regulated protein kinase（ERK）agonist Honokiol and p38 MAPK agonist Hesperetin,respectively.Subsequently,the influences of KISS-1 overexpression alone or together with Honokiol/Hesperetin on the protein expression levels of ERK1/2,phosphorylated ERK1/2（p-ERK1/2）,p38MAPK and phosphorylated p38 MAPK（p-p38 MAPK）in Eca109 and KYSE30 cells were determined by immunoblotting.15 nude mice were randomly separated into three groups,including blank group,lentivirus of negative control（Lv-NC）group and lentivirus of KISS-1 overexpression（Lv-KISS-1）group,and each group contained 5mice.ESCC cell line Eca109 was transfected with KISS-1 overexpression lentiviral plasmid,and puromycin was utilized to screen stable transfected cell lines.The Eca109 cell suspension,empty loading stable cell lines and KISS-1 overexpression stable cell lines were injected into in the left anterior axillary of mice in relevant group respectively to establish the subcutaneous xenograft tumor model of Eca109cells.The volume of xenograft tumor was measured regularly following subcutaneous injection,5 days per time.After the collection of xenograft tumors from nude mice,their weights in each group were calculated.IHC was used to examine the protein ex-pression of KISS-1 and Ki67 in xenograft tumor tissues from relevant group.Another15 nude mice were randomly divided into three groups,including blank group,Lv-NC group and Lv-KISS-1 group,and each group contained 5 mice.The Eca109 cell sus-pension,empty loading stable cell lines and KISS-1 overexpression stable cell lines were injected into the tail vein of nude mice in relevant group respectively to establish the lung metastasis model of Eca109 cells.After the collection of lungs from nude mice,their weights in each group were calculated.Then,all the collected lungs were subjected to hematoxylin-eosin（H&E）staining.Subsequently,the number of lung metastases in each group was counted under microscope.Results:1.64 paired ESCC and paratumor tissues were collected from the thoracic sur-gery department of our hospital.q RT-PCR results found that the expression of KISS-1m RNA level in ESCC tissues was conspicuously lower than paratumor tissues（P<0.05）.Correlation analysis uncovered that KISS-1 expression was correlated with lymph node metastasis（P=0.005）,distant metastasis（P=0.015）and TNM stage（P=0.025）of ESCC,but not correlated with the other clinicopathological characteristics such as gender,age,tumor size and differentiation（P>0.05）.IHC results indicated that the positive expression rate of KISS-1 protein in ESCC tissues（28.13%,18/64）was markedly lower than paratumor tissues（82.81%,53/64）（P<0.01）.20 paraffin-embedded specimens of esophageal squamous epithelial dysplasia were collected from the pathology department of our hospital,including 5 low grade,5 middle grade and 10 high grade dysplastic tissues.Results of IHC score showed that the expression of KISS-1 protein in high grade dysplastic tissues（1.70±1.10）was remarkably lower than middle grade dysplastic tissues（5.00±1.79）（P<0.01）.Furthermore,its expres-sion in middle grade dysplastic tissues（5.00±1.79）was also obviously lower than low grade dysplastic tissues（9.40±1.36）（P<0.01）.q RT-PCR and western blotting results unveiled that the m RNA and protein expression levels of KISS-1 in five ESCC cell lines TE-1,Eca109,EC9706,KYSE30 and KYSE150 were evidently lower than normal esophageal epithelial cell line HEEpi C（P<0.01）.2.BSP assay showed that the Cp G sites in Cp G island of KISS-1 promoter re-gion were hypermethylated in five ESCC cell lines TE-1,Eca109,EC9706,KYSE30and KYSE150,while they were hypomethylated or unmethylated in normal esopha-geal epithelial cell line HEEpi C.Moreover,compared with HEEpi C cells,the methyl-ation rate of Cp G island in KISS-1 promoter region of ESCC cells was dramatically increased（P<0.01）.MSP assay uncovered different degrees of methylation of KISS-1promoter region in 5 ESCC cell lines,while it was unmethylated in normal cell line HEEpi C.In addition,MSP results of clinical samples suggested that the methylation rate of KISS-1 promoter in ESCC tissues（70.31%,45/64）was remarkably increased in comparison with paratumor tissues（17.19%,11/64）（P<0.01）.What’s more,the methylation of KISS-1 promoter was correlated with lymph node metastasis（P=0.029）and distant metastasis（P=0.042）of ESCC,but not correlated with the other clinico-pathological characteristics such as age,sex,tumor size,tumor differentiation and TNM stage（P>0.05）.q RT-PCR results found that the m RNA expression level of KISS-1 in methylated ESCC tissues was markedly lower than unmethylated ESCC tissues（P<0.01）.In addition,the positive expression rate of KISS-1 protein in un-methylated ESCC tissues（57.89%,11/19）was notably higher than methylated ESCC tissues（15.56%,7/45）（P=0.001）.BSP assay confirmed that the hypomethylating agent 5-Aza-d C conspicuously reduced the methylation level of Cp G island in KISS-1promoter region in Eca109 and KYSE30 cells compared with controls in a dose-pendent manner（P<0.05）.MSP results uncovered that the extent of decreased methylation induced by 5-Aza-d C differed considerably in the two cell lines.Western blotting results revealed that 5-Aza-d C significantly increased the expression levels of KISS-1 protein in Eca109 and KYSE30 cells in a dose-dependent manner（P<0.01）.CCK8 and Transwell assays unveiled that 5-Aza-d C had no effect on the viability of HEEpi C cells,but could obviously inhibit the proliferation,migration and invasion abilities of Eca109 and KYSE30 cells in a dose-dependent manner（P<0.01）.3.After transfection,q RT-PCR results showed that in comparison with empty vector group and blank group,the expression levels of KISS-1 m RNA in Eca109 and KYSE30 cells were observably elevated in KISS-1 overexpression group（P<0.01）.CCK8 assay revealed that the proliferation levels of the two ESCC cells in KISS-1overexpression group was conspicuously lower than empty vector group and blank group（P<0.01）.Flow cytometry data unveiled that in comparison with empty vector group and blank group,KISS-1 overexpression notably increased the G1 phase and decreased G2 phase of the two ESCC cells（P<0.05）,but no significant change was observed in S phase.Furthermore,the apoptosis levels of Eca109 and KYSE30 cells in KISS-1 overexpression group were considerably increased compared with control groups（P<0.01）.Wound healing and Transwell assays uncovered that the migration and invasion abilities of Eca109 and KYSE30 cells in KISS-1 overexpression group were evidently reduced compared with empty vector group and blank group（P<0.01）.Immunoblotting results showed that in comparison with empty vector group and blank group,KISS-1 overexpression markedly decreased the protein expression levels of MMP2 and MMP9 in Eca109 and KYSE30 cells（P<0.05）.In addition,the expres-sion level of E-cadherin protein in KISS-1 overexpression group was notably elevated（P<0.05）,while the protein levels of Vimentin and N-cadherin were remarkably de-creased（P<0.05）.Moreover,KISS-1 overexpression remarkably decreased the ex-pression levels of p-ERK1/2 and p-P38 MAPK in Eca109 and KYSE30 cells,without affecting the total ERK1/2 and P38 MAPK protein levels.Furthermore,the suppres-sive effects of KISS-1 overexpression on p-ERK1/2 and p-P38 MAPK in 2 ESCC cells were reversed by Honokiol and Hesperetin agonists respectively（P<0.05）.Re-sults of xenograft tumor model showed that all 15 nude mice formed xenograft tumor,and the tumor formation rate was 100%,indicating the successful establishment of subcutaneous xenograft tumor model of Eca109 cell in nude mice.Statistical results revealed that,compared with Lv-NC group and blank group,the volume and weight of xenograft tumors in Lv-KISS-1 group were conspicuously decreased（P<0.01）.IHC score unveiled that the expression of KISS-1 protein in Lv-KISS-1 group（8.8±1.94）was markedly higher than Lv-NC group（1.8±1.60）and blank group（2.2±1.47）（P<0.01）.However,the expression of Ki67 protein in Lv-KISS-1 group（2.80±0.98）was notably decreased compared with Lv-NC group（9.80±1.83）and blank group（8.60±1.96）（P<0.01）.Results of lung metastasis model showed that the lung weight in Lv-KISS-1 group was observably lower than Lv-NC group and blank group（P<0.01）.H&E staining results confirmed that in comparison with control groups,the number of lung metastases in Lv-KISS-1 group was conspicuously de-creased（P<0.01）.Conclusions:1.KISS-1 was down-regulated in ESCC and correlated with tumor progression,which suggest that KISS-1 is a tumor suppressor gene in ESCC.2.KISS-1 promoter region was hypermethylated in ESCC,and correlated with tumor metastasis.3.The methylation status of KISS-1 promoter is correlated with its expression level,and the hypermethylation of KISS-1 promoter may be one of the reasons for its downregula-tion in ESCC.4.The decreasing of KISS-1 promoter methylation level induced by5-Aza-d C inhibited the proliferation,migration and invasion phenotypes of ESCC cells.5.KISS-1 inhibits the proliferation,and induces cell cycle arrest and apoptosis of ESCC cells.6.KISS-1 inhibits the metastasis of ESCC cells through suppressing MMP2 and MMP9 via the inactivation of MAPK/ERK and P38 MAPK pathways.7.KISS-1 suppresses the growth and metastasis of ESCC cells in vivo.