| Objective:With the constant discovery of microRNA, the function of microRNA is becoming thefocus of study.The correlation between microRNA-296and resistance to chemotherapy has beenconfirmed. This study will contribute a preliminary study to the correlation of P-gp, HER-2andmicroRNA-296expression with esophageal carcinoma radioresistant through the establishmentof radiation resistance esophageal carcinoma, and research the affection of X-Gay radiate the cellof esophageal carcinoma.Method:1. Divide human esophageal cancer cell lines Eca109into2groups: treatment group(induce to have radiation resistance) and control group (parent cells of esophageal carcinoma).Then give the former group repeated X-ray exposure, to the cumulative radiation dose60Gy, sothat it can have a certain degree of radiation resistance.2. Test the radiosensitivity of the two cells by MTT assay, the P-gp and HER-2expressiondifferences by immunocytochemical assay and the microRNA-296expression by Northern blotanalysis.3. Give8Gy X-ray irradiation to the esophageal carcinoma cell line Eca109, and thenincubate them for30min,2h,6h,12h,24h,48h,72h,7d,10d,14d,18d and then collect the cells.On the other hand, set up a control tube cell (ie, without any treatment of esophageal cancercells), test the expression of microRNA-296by Real Time-PCR;Result:1. Measurement of the proliferation inhibition rate of the two sets of cells: give the twogroups of cells0,2,4,6,8Gy X-ray irradiation at the same time, incubate for72hours, and thenmeasure the proliferation inhibition rate, of which the treatment group is significantly lower thanthat of the control group. The results suggest that, after a long ray induction, the treated cellshave begun to take the radiation resistance, and its sensitivity on ray is decreasing.2. Immunocytochemistry results: there is expression of P-gp and HER-2in both the twogroups of cells, with the P-gp’s staining intensity index0.718±0.123in the control group,1.524±0.423in the treatment group index, and HER-2’s staining intensity index0.402±0.089in thecontrol group,1.196±0.23in the treatment group. The results suggest that: the expressionof P-gp and HER-2has increased in the treatment group than the control group. There may be acorrelation between P-gp and HER-2and the esophageal carcinoma resistant. 3. Northern Blot results analysis: the density ratio analysis of the specific geneband microRNA-296and the gene band of U6in the control group and treatment group revealsthat the relative value of miR-296is0.0325±0.00159in control groupand0.034127±0.003151in treatment group. The results suggest that: there is no difference inthe expression of microRNA-296in two cell groups.4. Real Time-PCR results analysis: the detection employs8Gy X-ray to irradiate themiR-296expression levels of esophageal cancer cells at different time points, calculate the valueof Ct is1.17538±0.0482，1.4123±0.1409，0.9690±0.1063，1.0488±0.0999，0.9384±0.1669，0.8705±0.2138，0.6183±0.0951，1.6081±0.2230，3.0456±0.4146，1.0735±0.1102，0.9654±0.1294.The irradiation is completed after30min，2h，6h，12h，24h，48h，72h，7d，10d，14d，18d.The value of control is1.2902±0.1871.The miR-296expression level gradually reduced andreached the lowest expression level in3d. Then it gradually began to increase and in10d themiR-296expression was significantly higher than the control group and other groups. Then theexpression reduced to normal level. The results suggest that: X-rays affect the expression ofmiR-296in esophageal carcinoma cells.Conclusion:1. P-gp and HER-2may have a correlation with esophageal carcinoma resistance;2. It is unable to explain whether there is a correlation between miR-296and esophagealcarcinoma resistance;3. X-rays affect the expression of miR-296in esophageal carcinoma cells,and miR-296may be involved in esophageal cancer radiation damage repair mechanisms. |
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