作者：刘雪娇1，张雪云1，张勇2

单位：

1.110000辽宁省沈阳市，辽宁省人民医院呼吸与危重症内一科　2.110801辽宁省沈阳市，辽宁省肿瘤医院病理科

Units：

1.Department of Respiratory and Critical Care, Liaoning Provincial People's Hospital, Shenyang 110000, China2.Department of Pathology, Liaoning Cancer Hospital, Shenyang 110801, China

关键词：

肺癌；顺铂；天南星提取物；微小RNA-320；E2F转录因子1；药理作用分子作用机制（中药）

Keywords：

Lung cancer; Cisplatin; Araceae extract; microRNA-320; E2F transcription factor 1; Molecular mechanisms of pharmacological action（TCD）

CLC：

DOI：

10.12114/j.issn.1008-5971.2022.00.172

Funds：

辽宁省自然科学基金资助项目（20170540559）

摘要：

目的　基于miR-320/E2F转录因子1（E2F1）轴探讨天南星提取物对肺癌细胞顺铂耐药的影响及相关机制。方法　本实验时间为2020年9月至2021年6月。取对数期A549/DDP细胞分为空白组和天南星组，空白组置于RPMI 1640培养基中常规培养，天南星组置于加入天南星提取物的RPMI 1640培养基中常规培养。另取对数期A549/DDP细胞，转染miR-320抑制质粒后随机分为转染组、转染联合天南星组，转染组置于RPMI 1640培养基中常规培养，转染联合天南星组置于加入天南星提取物的RPMI 1640培养基中常规培养。培养48 h后进行后续实验。采用qRT-PCR检测不同细胞系及四组miR-320相对表达量，采用MTT法检测四组不同浓度顺铂处理后细胞增殖抑制率，并计算半数抑制浓度（IC50）；采用流式细胞术检测细胞凋亡率，采用双荧光素酶实验验证miR-320与E2F1的靶向关系，采用Western blotting法检测E2F1蛋白相对表达量。结果　16HBE细胞系miR-320相对表达量高于A549/DDP细胞系（P ＜0.001）。与空白组比较，天南星组miR-320相对表达量、不同浓度顺铂处理后细胞增殖抑制率、细胞凋亡率升高，IC50、E2F1蛋白相对表达量降低，转染组miR-320相对表达量、不同浓度顺铂处理后细胞增殖抑制率、细胞凋亡率降低，IC50、E2F1蛋白相对表达量升高（P ＜0.05）；与天南星组比较，转染联合天南星组miR-320相对表达量、不同浓度顺铂处理后细胞增殖抑制率、细胞凋亡率降低，IC50、E2F1蛋白相对表达量升高（P ＜0.05）；与转染组比较，转染联合天南星组miR-320相对表达量、不同浓度顺铂处理后细胞增殖抑制率、细胞凋亡率升高，IC50、E2F1蛋白相对表达量降低（P ＜0.05）。双荧光素酶实验结果显示，E2F1是miR-320的一个靶基因，两者结合位点是E2F1的3'-UTR片段。结论　天南星提取物可逆转肺癌细胞顺铂耐药，促进肺癌细胞凋亡，其机制可能与调控miR-320，进而靶向下调E2F1表达有关。

Abstract：

Objective　To investigation the impact and mechanism of araceae extract against cisplatin resistance onlung cancer cells based on miR-320/E2F transcription factor 1 axis. Methods　The experiment time was from September 2020 toJune 2021. The logarithmic A549/DDP cells were selected and divided into blank group (group A) and araceae extract group (groupB) . The group A was routinely cultured in RPMI 1640 medium, and the group B was routinely cultured in RPMI 1640 medium addedwith araceae extract. In addition, the logarithmic A549/DDP cells were transfected with miR-320 inhibitor plasmid and thenrandomly divided into transfection group (group C) and transfection combined with araceae extract group (group D) . The group Cwas routinely cultured in RPMI 1640 medium, and the group D was routinely cultured in RPMI 1640 medium added with araceaeextract extract. The follow-up experiment was carried out after 48 hours of culture. qRT-PCR was used to detect the relativeexpression of miR-320 in different cell series and four groups, respectively. MTT method was used to detect the inhibition rate ofcell proliferation after different concentrations of cisplatin treatment in four groups, and the half inhibition concentration (IC50) wascalculated; the apoptosis rate was detected by flow cytometry, the targeting relationship between miR-320 and E2F1 was verifiedby double luciferase assay, and the relative expression of E2F1 protein was detected by Western blotting. Results　The relativeexpression of miR-320 in 16HBE cell series was higher than that in A549/DDP cell series (P < 0.001) . Compared with the groupA, the relative expression of miR-320, cell proliferation after different concentrations of cisplatin treatment and apoptosis rate inthe group B were higher, and the IC50 and relative expression of E2F1 protein in the group B were lower, the relative expression ofmiR-320, cell proliferation after different concentrations of cisplatin treatment and apoptosis rate in the group C were lower, andthe IC50 and relative expression of E2F1 protein in the group C were higher (P < 0.05) . Compared with the group B, the relativeexpression of miR-320, cell proliferation after different concentrations of cisplatin treatment and apoptosis rate in the group Dwere lower, and the IC50 and relative expression of E2F1 protein in the group D were higher (P < 0.05) . Compared with the groupC, the relative expression of miR-320, cell proliferation after different concentrations of cisplatin treatment and apoptosis rate inthe group D were higher, and the IC50 and relative expression of E2F1 protein in the group D were lower (P < 0.05) . The results ofdual luciferase show that E2F1 was a target gene of miR-320, and the binding site of the two was the 3'-UTR fragment of E2F1.Conclusion　Araceae extract can reverse the cisplatin resistance of lung cancer cells and promote lung cancer cell apoptosis. Itsmechanism may be related to the regulation of miR-320 to further target down-regulation of E2F1 expression.

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