| OBJECTIVE:To observe the effect of paclitaxel combined with miR-200a mimics on proliferation and invasion ability of gastric adenocarcinoma cell line SGC-7901in vitro and in vivo and explore the possible mechanism as well.METHODS:1.Experiments in vitroThere were five groups of cells in the study:the200a group, the paclitaxel group, the combined group, the scramble group, the control group. The200a group was transfected with miR-200a mimics for48h. The paclitaxel group was treated with50nmol/L paclitaxel for48h. The combined group was given the two treatments above. The scramble group was transfected with negative control RNA. No treatment was done to the control group. The level of miR-200a was detected by RT-PCR. The proliferation capacity was detected by the cell cycle, the percentage of apoptotic cells measured by flow cytometry and the cell cloning. The invasive ability of SGC-7901cells was determined by Transwell assay and wound healing test. The different effect between using paclitaxel alone or combined with miR-200a on proliferation and invasion of SGC-7901cells was contrasted through all the experiments above. Western-blot was carried out to find the variation of β-catenin.2.Experiments in vivoSubcutaneous tumor xenograft in nude mice was adopted to evaluate the influence of paclitaxel in combination with miRNA-200a on SGC-7901cell in vivo.0.1mL of suspension contained1×107SGC-7901cells was injected hypodermically. Tumors of first generation were cut into small pieces and then were transplanted to the new nude mouses. When tumor volume reached to about130mm3, mouse were grouped at random(totally5groups, n=6) and treated with miR-200a and paclitaxel alone or in combination.20mg/kg paclitaxel was delivered to each mice via intraperitoneal injection in the paclitaxel group every7days(totally three times). MiR-200a mimics(or negative control RNA, scramble group) and Lipofectamine2000mixture(20μL per time) was given via multipoint in situ injection in the miR-200a group(or the scramble group) every two days. No treatment was given to the control group. The length and width of tumor were measured regularly(volume of tumor=lengthxwidth2/2).21days after the treatment, all the mouse were sacrificed. Curve of tumor growth was plotted to describe the proliferation of SGC-7901cell in vivo. Immunohistochemistry analysis and TUNEL staining were carried out to evaluate the apoptosis and proliferation state of SGC-7901cells in vivo.RESULTS:The experiments showed that the group treated both with paclitaxel and miR-200a mimics had significantly higher cell apoptotic rate[(22.82±1.43)%vs.(18.74±1.27)%, P<0.05] and higher percentage of cells blocked at G2/M phase [(63.74±1.76)%vs.(51.75±2.01)%, P<0.01], lower percentage of cell cloning [(4.03±0.25)%vs.(7.80±0.30)%, P<0.01], less transmembrane cell number[(22.00±2.00) vs.(56.33±5.13), P<0.01], lower migrating rate after48h [(21.43±2.34)%vs.(54.26±2.49)%, P<0.01] than the group treated with paclitaxel alone. Protein level of β-catenin decreased sharply in the miR-200a group and the combined group. Level of mir-200a in the200a and combined group increased obviously than the other groups(P<0.01) which indicated that transfection of miR-200a mimic was successful.The study also revealed a significantly slower growth of tumor in the combined group than the other groups since the15th day after treatment. Immunohistochemistry analysis showed the highest protein level of Caspase-3and lowest level of PCNA in the combined group. A decline of β-catenin was found among the tissue from miR-200a group and combined group. TUNEL staining indicated higher apoptic index[(26.55±6.09)%] in the combined group than the others(P<0.01).CONCLUTION:Paclitaxel combined with miR-200a has a better effect on inhibiting proliferation and invasion of human gastric adenocarcinoma cell line SGC-7901than the paclitaxel alone in vitro and in vivo, which may provide a new way of gastric cancer chemotherapy and a potential gene therapeutic target. |
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