| Part one Study on the expression of GRHL3 in esophageal squamouscancer tissuesObjective: To detect the expression of Grainyhead-like 3(GRHL3)and c-MYC in esophageal squamous cell cancer(ESCC)patients and the clinical significance.Methods:1 Tumor tissue,para-carcinoma tissue were collected from 64 cases of ESCC patients.Each specimen was divided to 2 pieces and treated as follows for different uses: put in liquid nitrogen for extracting RNA and fixed in 4% formaldehyde for making paraffin embedded blocks.2 Quantificational real-time polymerase chain reaction(q RT-PCR)was used to detect the mRNA expression of GRHL3 and c-MYC in tumor tissues and para-carcinoma tissues.The relationship between GRHL3 and c-MYC mRNA expression was analyzed.3 Immunohistochemistry was used to detect the protein expression of GRHL3 and c-MYC in tumor tissues and para-carcinoma tissues.In addition,the relationship between GRHL3 and c-MYC protein expression was analyzed.4 The associations of GRHL3 and c-MYC expression in ESCC tumor tissues with clinical parameters were analyzed.Results:1 The expression of GRHL3 mRNA in carcinoma tissue specimens was significantly higher than that in para-carcinoma tissues(2.85±2.83 vs.2.06±2.02,P<0.01).The expression of c-MYC mRNA in carcinoma tissue specimens was significantly higher than that in para-carcinoma tissues(5.13±5.11 vs.2.03±2.00,P<0.01).The expression of GRHL3 mRNA was positively correlated with c-MYC mRNA(P<0.001).2 The positive expression of GRHL3 protein in carcinoma tissue specimens is higher than that in para-carcinoma tissues(81.30% vs.25.00%,P<0.001).The positive expression of c-MYC protein in carcinoma tissue specimens is higher than that in para-carcinoma tissues(42.20% vs.20.30%,P<0.01).The expression of GRHL3 protein level was positively correlated with c-MYC protein level(P < 0.001).These results indicated that the expressions of GRHL3 and c-MYC were consistent at gene and protein level.3 The GRHL3 expression in tumor tissue was correlated with tumor differentiation,tumor invasion depth,lymph node metastasis and TNM stage(P<0.001,P=0.037,P=0.022,P=0.003,respectively)while not correlated with gender and age(P=0.604,P=0.904,respectively).Likewise,the c-MYC expression in tumor tissue was correlated with tumor differentiation,tumor invasion depth,lymph node metastasis and TNM stage(P=0.019,P=0.023,P=0.005,P=0.001,respectively)while not correlated with gender and age(P=0.403,P=0.747,respectively).Part two Study on the effects of GRHL3 on malignant biological behavior of ESCCObjective: To study the effect of GRHL3 on esophageal squamous cells malignant biological behavior by RNA interference(RNAi)method and to investigate the related mechanisms.Methods:1 GRHL3 expression level in 5 ESCC cell lines: TE13,TE1,KYSE30,Eca109 and KYSE170 were evaluated by qRT-PCR.KYSE30 cells which express highest level of GRHL3 gene were selected.RNA interference method was used to knock-down GRHL3 mRNA expression and set up interference group(si-GRHL3),negative control group(NC)and blank control group(Con).qRT-PCR and Western blot methods were used to identifiy the expression of GRHL3 mRNA and protein in three groups.2 MTS,colony formation,transwell migration and matrigel invasion chamber assays were used to detect the proliferation,migration and invasion abilities of KYSE30 cells.3 Flow cytometry assay was used to detect the cell cycle and cell apoptosis of KYSE30 cells.4 Western blot assay was used to detect expression levels of epithelial mesenchymal transition(EMT)related proteins of KYSE30 cells.Results:1 Among TE13,TE1,KYSE30,Eca109 and KYSE170 five esophageal squamous cancer cells,the highest expression level of GRHL3 mRNA were KYSE30 cells.RNAi method was used to knock down the expression of GRHL3 in KYSE30 cells.Compared with NC-GRHL3 group and Con group,the expression of GRHL3 mRNA and protein in si-GRHL3 group reduced significantly(P<0.05),which confirmed that the GRHL3 was knocked down in KYSE30 cells successfully.2 The ability of cell proliferation in si-GRHL3 group was decreased than NC-GRHL3 group and Con group detected by the colony formation assay [(59.28±3.64)vs.(87.33±1.90)vs.(88.87±0.36),P<0.05] and MTS assay [(0.87±0.01),(1.22±0.03),(1.37±0.25)vs.(1.10±0.01),(1.89±0.09),(2.10 ± 0.09)vs.(1.14 ± 0.04),(1.93 ± 0.01),(2.23 ± 0.05),P < 0.05,respectively].3 At 12 hours,the scratch width of si-GRHL3 group was significantly shorter than that in NC-GRHL3 group and Con group(24.5±3.2 vs.59.3±3.6 vs.64.2±3.2)μm,there was significant difference statistically(P<0.05).At 24 hours,the scratch width of si-GRHL3 group was significantly shorter than that in NC-GRHL3 group and Con group(55.4±4.8 vs.95.1±4.6 vs.92.3±3.5)μm,there was significant difference statistically(P < 0.05).Meanwhile,there was no significant difference between NC-GRHL3 group and Con group(P>0.05).The number of KYSE30 cells of si-GRHL3 group which pass through the matrigel membrane was significantly lower than that in NC-GRHL3 group and Con group(50.26±4.37 vs.120.00±4.01 vs.129.38±5.20),there was significant difference statistically(P<0.05).But there was no significant difference between NC-GRHL3 group and Con group(P>0.05).4 The percentage of G0/G1 phase of si-GRHL3 group was higher than that in NC-GRHL3 group and Con group detected by the FCM assay [(66.64± 1.31)% vs.(56.53 ± 2.15)% vs.(55.97 ± 1.36)%,P<0.05].There was no significant difference among three groups of apoptosis detected by the FCM assay [(5.36 ± 0.24)% vs.(6.32 ± 0.56)% vs.(6.53 ± 0.31)%,P>0.05].5 The expression levels of E-cadherin in si-GRHL3 group was higher than that in NC-GRHL3 group and Con group detected by the western blot [(0.81 ± 0.04)vs.(0.51 ± 0.05)vs.(0.53 ± 0.05),P<0.05].The expression of N-cadherin in si-GRHL3 group was decreased than that in NC-GRHL3 group and Con group detected by the western blot [(0.53 ± 0.06)vs.(1.12 ±0.10)vs.(0.90 ± 0.05),P<0.05].Conclusions:1 In ESCC tissue,the expressions of GRHL3 and c-MYC were higher than those in para-carcinoma tissues and were closely correlated with tumor differentiation,tumor invasion depth,lymph node metastasis and TNM stage,while not correlated with gender and age.2 GRHL3 could promote the development of ESCC by influencing cell proliferation,migration,invasion and promoting the epithelial mesenchymal transition. |
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