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结直肠癌是常见的消化道恶性肿瘤之一,化疗仍是临床治疗不可缺少的方法。据报告白藜芦醇具有抑制肿瘤的发生、增殖和转移等抗肿瘤作用,但其抑制结直肠癌的相关分子机制尚不完全清楚。miR-34c作为抑癌基因,在结直肠癌组织表达较正常肠黏膜组织明显下调,故本研究主要关注白藜芦醇抑制结直肠细胞增殖与miR-34c相关机制。通过培养结直肠癌细胞系(HCT-116和HT-29)、建立裸鼠荷瘤模型,CCK8检测发现白藜芦醇明显抑制结直肠癌细胞的活性,具有时间和剂量依赖性。RTCA-DP实时无标记细胞功能分析结果亦可见白藜芦醇明显抑制结直肠癌细胞的迁移和侵袭能力。软琼脂集落形成实验显示白藜芦醇处理组形成克隆集落的数目明显少于对照组(HCT-116和HT-29细胞分别减少45.9%和57.4%,P<0.05)。流式细胞术发现白藜芦醇处理组细胞呈现明显的细胞周期阻滞,G0/G1期的HCT-116细胞百分比较对照组增加54.4%(P<0.05),G2/M期的HT-29细胞百分比较对照组增加37.1%(P<0.05),且凋亡细胞百分率也明显增高39.1%(HCT-116,P<0.05)和36.4%(HT-29,P<0.05)。另外,白藜芦醇明显提高结直肠癌细胞miR-34c的表达(HCT-116为3.5倍;HT-29为19.2倍),其靶基因KITLG(SCF)mRNA和蛋白水平均明显降低(P<0.05)。特异性敲降内源性miR-34c表达后,KITLG的表达明显增加,结直肠癌细胞的增殖、迁移和侵袭能力也得以恢复。将HCT-116细胞接种于裸鼠腋下皮下结缔组织成瘤1周后,静脉给予白藜芦醇(100mg/kg)或DMSO,可见白藜芦醇治疗组肿瘤体积明显小于DMSO组,且肿瘤组织内Ki67阳性细胞数明显少于DMSO组(P<0.05),TUNEL染色显示白藜芦醇组肿瘤组织凋亡细胞百分数高于DMSO组(P<0.05),Caspase-3表达增高。另外,我们发现白藜芦醇组肿瘤组织miR-34c表达较DMSO组增加1.8倍(P<0.05),而血浆中miR-34c的水平无明显差异。综上,本实验结果表明白藜芦醇具有促进结直肠癌细胞miR-34c的表达,发挥抑制其靶基因KITLG表达和结直肠癌细胞的增殖、迁移和侵袭的重要作用。
Colorectal cancer is one of the common gastrointestinal cancer, chemotherapy is still an indispensable method of clinical treatment. Resveratrol has been reported to inhibit the tumorigenesis, proliferation and metastasis and other anti-tumor effects, but its molecular mechanism of inhibiting colorectal cancer is not yet fully understood. As a tumor suppressor gene, miR-34c is significantly down-regulated in colorectal cancer tissues compared with normal intestinal mucosa tissues. Therefore, this study mainly focused on the mechanism of resveratrol in inhibiting colorectal cell proliferation and miR-34c. The nude mice tumor model was established by culturing colorectal cancer cell lines (HCT-116 and HT-29). CCK8 assay showed that resveratrol significantly inhibited the activity of colorectal cancer cells in a time-and dose-dependent manner. RTCA-DP real-time marker-free cell function analysis results also showed that resveratrol significantly inhibited colorectal cancer cell migration and invasion. Soft agar colony formation assay showed that the number of colonies formed by resveratrol treatment was significantly less than that of the control group (45.9% and 57.4%, respectively, HCT-116 and HT-29 cells, P <0.05). Flow cytometry showed that the cells treated with resveratrol exhibited obvious cell cycle arrest, the percentage of HCT-116 cells in G0 / G1 phase increased 54.4% (P <0.05) compared with the control group, while the G2 / M phase HT-29 The percentage of apoptotic cells increased by 37.1% (P <0.05), and the percentage of apoptotic cells increased by 39.1% (HCT-116, P <0.05) and 36.4% (HT-29, P <0.05), respectively. In addition, resveratrol significantly increased the expression of miR-34c in colorectal cancer cells (HCT-116 was 3.5-fold and HT-29 was 19.2-fold), and its target gene KITLG (SCF) mRNA and protein levels were significantly decreased (P < 0.05). Specific knockdown of endogenous miR-34c expression, KITLG expression was significantly increased, colorectal cancer cell proliferation, migration and invasion ability was also restored. After inoculation of resveratrol (100 mg / kg) or DMSO intravenously for 1 week, HCT-116 cells were inoculated into the subcutaneous connective tissue in nude mice and the tumor volume in the resveratrol group was significantly smaller than that in the DMSO group The number of Ki67 positive cells in the tissue was significantly lower than that in the DMSO group (P <0.05). The percentage of apoptotic cells in the resveratrol group was higher than that in the DMSO group (P <0.05), and the expression of Caspase-3 was increased by TUNEL staining. In addition, we found that the expression of miR-34c in resveratrol group increased 1.8-fold (P <0.05) compared with DMSO group, while the level of miR-34c in plasma did not change significantly. In summary, the results of this experiment show that resveratrol can promote the expression of miR-34c in colorectal cancer cells and play an important role in inhibiting the expression of its target gene KITLG and the proliferation, migration and invasion of colorectal cancer cells.