[摘 要] 目的：探究西贝素通过调控Cyclin D1介导的CDK4/6-Rb-E2F1信号轴对人胃腺癌细胞（AGS）的增殖抑制效果及其分子机制。方法：利用不同浓度的西贝素处理AGS和人正常胃黏膜上皮细胞（GES-1），通过CCK-8检测和结晶紫染色实验确定西贝素的最适抑制剂量和作用时间；采用划痕实验和Transwell迁移实验与侵袭实验检测西贝素对AGS细胞迁移与侵袭能力的影响；利用流式细胞术和WB检测西贝素对AGS细胞周期及细胞周期相关蛋白表达的影响；利用质粒过表达Cyclin D1，并通过WB检测、结晶紫染色实验、划痕实验和Transwell迁移实验与侵袭实验进一步明确西贝素的抑制机制；构建AGS移植瘤裸鼠模型，采用免疫荧光染色法和免疫组化检测Cyclin D1、CDK4、CDK6、Rb、p-Rb、E2F1的表达水平。结果：与对照组相比，西贝素药物浓度为100 μg/mL时，对GES-1细胞的增殖无影响（P > 0.05），但能够显著性抑制AGS细胞增殖、迁移和侵袭能力（P < 0.01），并诱导AGS细胞周期G0/G1期阻滞，显著抑制周期相关蛋白Cyclin D1、CDK4、CDK6、p-Rb、E2F1的表达水平（P < 0.05或P < 0.01）；Cyclin D1过表达能够显著降低西贝素对AGS细胞的抑制效果（P < 0.05或P < 0.01）；荷瘤裸鼠实验结果显示，西贝素能够抑制AGS移植瘤的增殖，影响肿瘤组织中Cyclin D1、CDK4、CDK6、p-Rb、E2F1的表达（P < 0.01）。结论：西贝素可能通过下调Cyclin D1介导的CDK4/6-Rb-E2F1信号轴活化抑制AGS增殖。

[Abstract] Objective: To analyze the expression level of miR-224 in cancer tissues and plasma of hepatocellular carcinoma (HCC) patients, and its correlation with clinicopathological characteristics, diagnosis and prognosis of HCC patients, and to further analyze its mechanism of action in the occurrence and development of liver cancer through bioinformatics analysis and in vitro experiments. Methods: The expression level of miR-224 in HCC tissues and normal tissues was analyzed using large sample data from Gene Expression Omnibus (GEO). qPCR method was used to verify the expression level of miR-224 in the tumor tissues and corresponding adjacent tissues that surgically resected from 80 HCC patients in Hebei Provincial People ’s Hospital from January 2017 to January 2020; in addition, the miR-224 level was also examined in plasma samples from 30 HCC patients. The Kaplan-Meier plotter database was used to analyze the correlation between the miR-224 expression and the overall survival time of HCC patients. The biological processes and signal pathways involving miR-224 were analyzed using bioinformatics tools. Hepatocellular carcinoma HepG2 cells were transfected with miR-224 inhibitor, and then Clone formation experiment, Transwell chamber experiment, qPCR and WB methods were used to detect the effect of miR-224 knockdown on the proliferation and invasion of HepG2 cells and the expression level of EMT-related molecules. Results: The results of GEO database analysis showed that the expression level of miR-224 in HCC tissues was significantly higher than that in normal tissues. The results of clinical specimen verification showed that the expression level of miR-224 in the tumor tissues and plasma of HCC patients was significantly higher than that in the corresponding adjacent tissues and plasma from healthy controls (all P<0.01). The expression level of miR-224 was significantly correlated with the TNM stage, lymph node metastasis status and tumor size of HCC patients (P<0.05 or P<0.01). ROC analysis indicated that miR-224 showed a prominent diagnostic value in liver cancer, and the increased expression level of miR-224 was significantly related to the poor prognosis of HCC patients (P<0.05). Functional enrichment analysis revealed that miR-224 was mainly involved in the mTOR signaling pathway, AGE-RAGE signaling pathway, Rap1 signaling pathway, Ras signaling pathway, ErbB signaling pathway, HIF-1 signaling pathway and p53 signaling pathway and other signaling pathways related to tumor occurrence and development. Knockdown of miR-224 could significantly inhibit the colony formation and invasion of HepG2 cells and affect the expression of EMT-related markers (P<0.05 or P<0.01). Conclusion: miR-224 is highly expressed in HCC tissues and plasma and is significantly related to the poor prognosis of HCC patients. Knockdown of miR-224 expression can inhibit the colony formation, invasion and EMT process of liver cancer HepG2 cells.

Objective: To explore the difference in the proliferation inhibition of doxorubicin and dual specific oncolytic adenoviruses (Ad-VT, Ad-T, Ad-VP3 and d-Mock) on breast cancer cells and normal mammary cells. Methods: The proliferation inhibition rates of doxorubicin and recombinant adenovirus(Ad-VT, Ad-T, Ad-VP3and Mock) on breast cancer cells were detected through WST-1 experiment, and the effects of two drugs on the inhibitory rates of normal mammary epithelial cells were also detected. Moreover, the apoptosis rates of doxorubicin and oncolytic adenoviruses on breast cancer cells and normal mammary epithelial cells were evaluated by Annexin V flow cytometry, Hoechst and JC-1 staining, and the difference in the apoptosis rates were also compared. Results: All the recombinant adenovirus could effectively suppress the proliferation of breast cancer cells (P<0.05 or P<0.01), the inhibition effects followed the order ofAd-VT>Ad-T>Ad-VP3>Ad-MOCK, and the inhibition effect was positively correlated with time. Doxorubicin could also effectively suppress the proliferation of breast cancer cells (P<0.05 or P<0.01), and the inhibition effect was markedly enhanced with the increases in does and time. However, doxorubicin also showed strong inhibition effect on the normal mammary epithelial cells, and the inhibition rate achieved 80% under 72 h and 5 ug/ml doxorubicin, while that of oncolytic adenovirus Ad-VT on MCF-10A was 20% at 72 h. The apoptosis effects of oncolytic adenoviruses-induced breast cancer cellwere increased with time, and the apoptosis rate efficiency followed the order of Ad-VT>Ad-T>Ad-VP3>Ad-MOCK, but they displayed low ability to induce normal mammary cell apoptosis. The apoptosis effects of doxorubicin-induced breast cancer cell were similar to that of the normal mammary epithelial cell (P <0.05 or P<0.01), which followed the dose of 0.05<0.5<5 μg/ml. Conclusion: Dual specific oncolytic adenoviruses can effectively suppress the proliferation of breast cancer cells, but they have low inhibition on normal mammary cells, which have displayed superior safety and provide a new method for the biotherapy of tumor.