| ObjectiveTo explore the expression of HCCR, pERK, pJNK, pP38, pELK1 in gastric tumor and its relationship. To study the molecular mechanism of HCCR expression upregulation in SGC-7901 cell line via a ERK/ELK1 pathway treated induced with EGF.Methods1. HCCR, pERK, pJNK, pP38 and pELK1 expression was detected by immunohistochemistry in 50 human gastric cancers and their corresponding paraneoplastic tissuses.2. Cells were pretreated with the specific inhibitor of MAPK then cultured with EGF (100ng/ml) for 24h, the expression of HCCR protein in these cells were measured by Western blot and RT-PCR. Reporter plasmids containing HCCR promoter region and ELK1 were transfected into SGC-7901 by lipofectamine 2000, HCCR promoter activity were detected.3. pGL3-210~+30 report gene plasmid for pGL3-ELK1 and pGL3-mELK1 were constructed,and transfected into gastric cancer cell line. Then cells were treated with concentration 100ng/ml of EGF, luciferase activity of pGL3-ELK1 and pGL3-mELK1 report gene were detected.4. Four siRNA ELK1 plasmid and NE plasmid were constructed. HEK/293 cells were transfected with pcDNA?6.2-GW/EmGFP-NE and Four pcDNA?6.2-GW/EmGFP-siELK1 by lipofectmine 2000, respectively. The valid siRNA pcDNA3.1-siRNA were selected and confirmed. Then SGC-7901 cells were transfected ELK1-pcDNA3.1-siRNA, NE-pcDNA3.1 by lipofectmine 2000, stable transfectants were selected and confirmed using RT-PCR technique. The constructs of pGL3-ELK1 and pGL3-mELK1 were co-transfected to the ELK1-deficient SGC-7901 cells, then cells were treated with concentration 100ng/ml of EGF, report gene activity of pGL3-ELK1 and pGL3-mELK1 were detected.Results1. The expression of HCCR, MAPKs and pELK in gastric cancer and its relationship. The expression of HCCR, pERK, pJNK, pP38 and pELK1 were 75%(45/60), 83.3% (50/60), 12%(5/60), 1.2%(19/60) and 78.3%(47/60), respectively, and the expression of HCCR, pERK and pELK1 were higher in gastric tumor than that in paraneoplastic tissues (P<0.05). The expression of HCCR had no relationship with both lymphnode metastasis and nerve infiltration. The HCCR, pERK and pELK1 were co-expressed in gastric cancer (P<0.05).2. EGF upregulated HCCR via a MAPK/ERK signal transduction pathway in SGC-7901 cells. Treatment of the cells with MAPKs inhibitor, only ERK inhibitor can significantly suppress EGF induced HCCR expression. And the reporter assay got the same result. These results showed that the MAPK/ERK pathway is involved in the stimulation of HCCR production by EGF.3. The HCCR promoter activity were reduced by transfected with pGL3-mELK1 The HCCR promoter activity were reduced by transfected with pGL3-mELK1 in NIH3T3 and SGC-7901(p<0.05).4. The report gene assay of pGL3-mELK1 were higher than that of pGL3 ELK in stable transfected pcDNA?6.2-GW/EmGFP-siELK1 SGC-7901 cellsThe stable transfected SGC-7901 cells were successful confirmed; Treated with EGF(100ng/ml) can upregulated the reporter gene activity both in pGL3-ELK1 and pGL3-mELK1, however, the reporter gene activity of pGL3-ELK1 were higher than that of pGL3-mELK1.Conclusion1. The expression HCCR, pERK and pELK1 were higher in gastric tumor than that in paraneoplastic tissues; The expression of HCCR pERK and pELK1 coordinated in gastric cancer. It showed that the ERK/Elk1 pathway may be involved in the regulation of HCCR expression.2. EGF induces HCCR expression in SGC-7901 cells, but the ERK inhibitor can reduce the effect.3. HCCR promoter and ELK1 reporter gene can be upregulated by EGF, however, the effect of EGF also can be suppressed by ERK inhibitor.4. The ELK1 binding in the HCCR promoter region plays an important role in transcription of HCCR; The stbale transfectants with pcDNA?6.2-GW/EmGFP-siELK1 can reduce the reporter activityof pGL3-ELK1. |
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