| Objective:Through cellular experiment in vitro, we explored the roles and mechanism of miR-21and ERK1/2in esophageal squamous carcinoma (ESCC). Via histological experiment in vivo, we detected miR-21and ERK1/2expression in tumor tissues and corresponding adjacent normal tissues of80cases of ESCC patients and analyzed the association of miR-21and ERK1/2, and then examed the relationship between miR-21, ERK1/2and clinic pathological parameters of80cases of ESCC patients and discussed the clinical significance. Methods:MiR-21expression was detected by qRT-PCR in ESCC cell lines:EC9706.Eca109and KYSE510. We transfected miR-21mimics, inhibitor, scramble sequences, pCMV-miR-21plasmid to the Eca109cell by Lipofectamine2000. U0126treated Eca109cells and inhibited ERK1/2MAPK signal pathway activation. Cell proliferation were determined by Cell counting and MTT tests. Cell migration was detected by wound-healing assay. Cell cycle and apoptosis were determined by flow cytometry. MiR-21and ERK1/2mRNA were detected by qRT-PCR. Western-blot detected total ERK1/2and phosphorylated ERK1/2. MiR-21and ERK1/2expression in80cases of ESCC tumor tissues and adjacent normal tissues detected by in situ hybridization and immunohistochemical methods respectively. relationship between miR-21, ERK1/2and clinical pathological parameters of80cases of ESCC, then discussed the clinical significance. Results:1) miR-21expression was highest in EC9706cell line, lowest in KYSE510cell line, moderate expression in Eca109cell line, Eca109cell line was choosed as research cell for subsequent experiments. Eca109cell was transfected with miR-21mimics (miR-21-M), miR-21inhibitor (miR-21-I) and scramble oligonucleotide sequence which carrying green fluorescence, the efficiency was up to90%, miR-21expression decreased to0.15-fold in miR-21-I group, increased to8.85-fold in miR-21-M group (P<0.05). Cell counting results showed, cell proliferation promoted6.15%(24h, P>0.05),19.69%(48h, P<0.05),11.69%(72h, P<0.05),17.97%(96h, P<0.05) in miR-21-M group, inhibited10.35%(24h, P<0.05).9.23%(48h, P<0.05),16.03%(72h, P<0.05),15.23%(96h, P<0.05) in miR-21-I group. MTT comfirmed the results, cell proliferation increased1.89%(24h, P>0.05)、9.6%(48h, P<0.05),14.55%(72h, P<0.05)、9.62%(96h,P<0.05) in miR-21-M group, decreased2.50%(24h, P>0.05)、43.32%(48h, P<0.05)、34.52%(72h, P<0.05).38.57%(96h,<0.05) in miR-21-I group. Colony formation results showed, cell colony formation ability increased11.03%(P<0.05) in miR-21-M group, decreased17.23%(P <0.05) in miR-21-I group. In wound-healing assays, wound-healing ability increased14.50%(24h, P<0.05),15.77%(48h, P<0.05),24.35%(72h, P<0.05) in miR-21-M group, decreased9.16%(24h, P>0.05).14.93%(48h, P<0.05).18.13%(72h, P<0.05) in miR-21-I group. Apoptosis assays showed, cell apoptosis decreased82.61%(P<0.05) in miR-21-M group, increased107.38%(P<0.05) in miR-21-I group. In cell cycle assays, Gl-phase increased6.98%(P<0.05), S-phase decreased33.33%(P<0.05), G2-phase decreased16.04%(P<0.05) in miR-21-I group, G1-phase decreased30.26%(P<0.05), S-phase increased136.60%(P0.05), G2-phase increased75.93%(P<0.05) in miR-21-M group.(8) Western-blot results indicated, p-ERK1/2protein expression increased in miR-21-M group, but decreased in miR-21-I group.2) We pharmacologically treated Eca109cells with ERK1/2inhibitor (U0126), cell counting showed U0126can inhibit cell proliferation in concentration-dependent manner,20μmol/L U0126can obviously and moderately depress cell proliferation and meet the need of our research, so20μmol/L U0126was choosed for subsequent experiment. MTT decided the cell proliferation, inhibited18.93%(24h, P>0.05)、23.17%(48h, P>0.05)、43.08%(72h, P<0.05).56.05%(96h, P<0.05) in U0126group; compared with U0126group, U0126+miR-21group cell proliferation enhanced19.81%(24h, P>0.05),22.76%(48h, P<0.05、44.48%(72h, P<0.05)、58.40%(96h, P<0.05); compared with miR-21group, U0126+miR-21group cell proliferation decreased2.95%(24h, P>0.05)、10.20%(48h, P>0.05).11.73%(72h,.P<0.05)、13.74%(96h, P<0.05).20μmol/L U0126significantly destroyed normal morphology of Eca109cells. Colony-formation in plate test found, U0126and DDP group cells formed the colonies lately, less in number and diameter. The number of colonies decreased15.783%in U0126group (P<0.05), increased11.03%(P<0.05) in miR-21-M group. Compared with U0126group, the number of colonies in U0126+miR-21group increased14.96%(P<0.05); compared with miR-21group, the number of colonies in U0126+miR-21group decreased12.11%(P< 0.05). In wound-healing test, the wound-healing ability in U0126group inhibited23.83%(24h, P<0.05),59.50%(48h, P<0.05)>180.52%(72h, P<0.05). Compared with U0126group, the wound-healing ability in U0126+miR-21group increased24.83%(24h, P<0.05)、40.07%(48h, P<0.05)、67.17%(72h, P<0.05). Compared with miR-21group, the wound-healing ability in U0126+miR-21group decreased7.52%(24h, P>0.05)、15.72%(48h, P<0.05),93.54%(72h, P<0.05). Apoptosis assay indicated, cell apoptosis of U0126group increased357.24%(P<0.05).Compared with U0126, cell apoptosis of U0126+miR-21group decreased76.14%(P<0.05); Compared with miR-21group, cell apoptosis of U0126+miR-21group increased410.13%(P<0.05). The results of cell cycle test showed, G1-phase increased6.42%(P<0.05), G2-phase decreased18.45%(P<0.05), S-phase reduced26.15%(P<0.05) in U0126group; Compared with U0126group, G1-phase decreased6.36%(P<0.05), G2-phase increased26.88%(P<0.05), S-phase increased33.19%(P<0.05) in U0126+miR-21group; Compared with miR-21group, G1-phase promoted42.89%(P<0.05), G2-phase reduced41.18%(P<0.05), S-phase decreased58.43%(P<0.05) in U0126+miR-21group. Western-blot showed p-ERK1/2expression was significantly inhibited (P<0.05) in U0126group; compared with U0126group, p-ERK1/2expression was significantly enhanced (P<0.05) in U0126+miR-21group; compared with miR-21group, p-ERK1/2expression was significantly inhibited (P<0.05) in U0126+miR-21group. qRT-PCR test found miR-21expression decreased93.77%(P<0.05) in U0126group; compared with U0126group, miR-21expression increased14.48-fold (P<0.05) in U0126+miR-21group; compared with miR-21group, miR-21expression decreased93.26%(P<0.05) in U0126+miR-21group.3) positive miR-21expression rate was96.25%in80ESCC tumor tissues,71.25%in adjacent normal tusses (P<0.05); miR-21expression in ESCC tumor tissues (131.25±83.20) was more higer (174.50%) than in adjacent normal tusses (47.81±44.51, P<0.05). Positive p-ERK1/2expression rate was91.25%in80ESCC tumor tissues,77.50%in adjacent normal tusses (P<0.05); p-ERK1/2expression in ESCC tumor tissues (135.00±94.94) was more higer (163.41%) than in adjacent normal tusses (51.25±44.63, P<0.05). Pearson correlation analysis indicated that miR-21and p-ERK1/2expression correlated positively in80ESCC patients (r=0.57, P<0.05). Both miR-21and p-ERK1/2expression increased in ESCC patients with lymph nodes metastasis (P<0.05), but have no obvious association with gender, age, nationality, pathologic type, invasion and tumor volume (P>0.05). Conclusion:1) miR-21promoted Eca109cell proliferation, migration, colony formation, induced G1-phase cells enter into S-phase and G2-phase, speed up the cell cycle and inhibited cell apoptosis by activation of ERK1/2MAPK signal pathway.(2) Inhibition of ERK1/2MAPK pathway depressed cell proliferation, migration, colony formation, induced G1-phase arrest extented the cell cycle and increased cell apoptosis by inhibition of miR-21expression.3) miR-21and p-ERK1/2expression correlated positively in80ESCC patients. Both miR-21and p-ERKl/2expression increased in ESCC patients with lymph nodes metastasis, but have no obvious association with gender, age, nationality, pathologic type, invasion and tumor volume.4) In conclusion, miR-21and p-ERKl/2MAPK have synergistic oncogenic roles and regulate each other in ESCC development. |
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