miR-21在胃癌细胞化疗耐药中的作用及其机制研究
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摘要
胃癌是最常见的恶性肿瘤之一，其死亡率占全世界癌症死因的第二位。由于胃癌的筛查目前还没有得到普及和重视，同时早期胃癌无明显临床症状或症状不典型，大多数胃癌确诊时已到属中晚期，化疗是其主要的治疗方法。但是肿瘤耐药性的产生致使胃癌对化疗不敏感，最终导致胃癌治疗的失败。目前认为与肿瘤耐药相关的机制包括：ABC转运蛋白的跨膜转运、细胞解毒能力的增强、DNA损伤修复系统失调、凋亡相关通路受阻等。但是在实验和研究中，以这些分子靶点作为药物靶标进行耐药逆转，仍然存在着许多目前难以解决的问题。
近年来研究显示一类长度为22个核苷酸的单链非编码微小RNA(microRNA，miRNA)在肿瘤发生发展中发挥着重要的作用。生物信息学方法显示miRNA通过调节不同的靶基因而发挥重要的生物学效应。在肿瘤耐药方面，miRNA能够通过调控不同的靶点而影响肿瘤细胞对抗肿瘤药物的敏感性。
microRNA-21(miR-2l)是目前发现的唯一在多种恶性肿瘤中高表达的miRNA，miR-2l有癌基因性质，抑制miR-21的表达可抑制肿瘤细胞增殖、促进肿瘤细胞凋亡。目前已有研究发现miR-21与胆管细胞癌、乳腺癌、胰腺癌、恶性胶质瘤等恶性肿瘤的耐药相关，但尚无miR-21与胃癌化疗耐药相关的研究。本研究拟在前期研究和文献报道的基础上，进一步观察miR-21与胃癌化疗耐药之间的关系、并探讨其可能作用机制，从而为临床上胃癌化疗耐药的研究提供新的靶点。本研究分三部分。
第一部分miR-21与胃癌细胞顺铂化疗敏感性关系的研究
目的：研究miR-21与胃癌细胞顺铂敏感性的关系
方法：real-time PCR检测胃癌耐顺铂细胞SGC7901/DDP及耐长春新碱细胞SGC7901/VCR和亲本细胞SGC7901miR-21的表达水平；利用脂质体Lipofectamine2000将成熟miR-21的模拟物mimics、阻遏物inhibitors及阴性对照negative controlRNA（NC)转染至SGC7901和SGC7901/DDP细胞；real-time PCR技术验证转染结果；MTT法检测细胞增殖水平；Annexin Ⅴ/PI双染流式细胞术检测细胞凋亡率；PI流式细胞术检测细胞周期。
结果：1. miR-21在SGC7901/DDP中高表达，与SGC7901细胞相比，表达增高7.65±1.197倍，差异有统计学意义(P<0.01)；在SGC7901/VCR与SGC7901细胞之间未检测出miR-21表达的差异。2.转染miR-21mimics后SGC7901细胞对顺铂的敏感性下降， mimics组对顺铂的IC50为1.085±0.0639μg/mL，与单纯脂质体组（0.476±0.014μg/mL）相比有显著性差异(P<0.01)，NC组对顺铂的IC50为0.487±0.018μg/mL，与单纯脂质体组相比无差异；转染miR-21inhibitors后SGC7901/DDP细胞对顺铂的敏感性增加， inhibitors组对顺铂的IC50为3.116±0.0.165μg/mL，与单纯脂质体组（5.138±0.075μg/mL）相比有显著性差异(P<0.01)，NC组对顺铂的IC50为5.175±0.458μg/mL，与单纯脂质体组相比差异无统计学意义。3. SGC7901细胞转染miR-21mimics，NC及脂质体24h后加入终浓度为0.5μg/mL顺铂培养48h后，mimics组凋亡率明显减少，与NC及脂质体组相比有明显差异(P<0.05)；SGC7901/DDP细胞转染miR-21inhibitor，NC及脂质体24h后加入终浓度为5.0μg/mL顺铂培养48h后，inhibitors组凋亡率明显升高，与NC及脂质体组相比有显著差异(P<0.05)。4. SGC7901转染miR-21mimics，NC及脂质体后继续培养48h，mimics组S期细胞明显增多达（42.04%）与NC组相比有明显差异(P<0.05)。
结论：miR-21在胃癌耐顺铂细胞株中高表达，转染miR-21mimics后可以导致胃癌细胞SGC7901对顺铂的耐药；转染miR-21inhibitors后可以增加胃癌耐顺铂细胞SGC7901/DDP对顺铂的敏感性，部分逆转顺铂耐药。
第二部分miR-21与胃癌细胞阿霉素敏感性关系的研究
目的：研究miR-21与胃癌细胞阿霉素化疗敏感性的关系
方法：real-time PCR检测经IC50阿霉素、氟尿嘧啶作用72h胃癌细胞SGC7901、MGC803与未经处理相应胃癌细胞miR-21的表达水平；利用脂质体Lipofectamine2000将成熟miR-21的模拟物mimics及阴性对照negative controlRNA（NC)转染至SGC7901和MGC803细胞；real-time PCR技术验证转染结果；MTT检测细胞增殖；Annexin-V FITC/PI双染流式细胞术检测细胞凋亡率。
结果：1. miR-21在经IC50阿霉素作用72h SGC7901和MGC803中高表达，与未经处理SGC7901及MGC803相比，表达分别增高2.941±0.225倍(P<0.01)、2.164±0.167倍(P<0.01)，差异均有统计学意义；经IC50氟尿嘧啶作用72h后胃癌细胞SGC7901和MGC803与未经处理相应胃癌细胞之间miR-21表达无差异。2.SGC7901转染miR-21mimics后①细胞对阿霉素的敏感性下降，mimics组与NC组对阿霉素的IC50分别为0.942±0.027μg/mL、0.335±0.029μg/mL，前者是后者的2.8倍，差异有统计学意义(P<0.01)；②细胞凋亡率明显下降，与NC组相比差异有统计学意义(P<0.05)。3. MGC803转染miR-21mimics后对阿霉素的敏感性下降，mimics组与NC组IC50分别为1.430±0.124μg/mL、0.481±0.034μg/mL，前者是后者的3.0倍，差异有统计学意义(P<0.01)。
结论：经IC50阿霉素处理72h后胃癌细胞miR-21表达增加，转染miR-21mimics后，可以增加胃癌细胞对阿霉素的耐药性。
第三部分miR-21对胃癌细胞化疗敏感性影响的机制研究
目的：研究miR-21对胃癌细胞化疗敏感性影响的机制
方法：RT-PCR及real-time PCR检测SGC7901/DDP与SGC7901细胞间PTENmRNA表达差异，Western-blot法检测SGC7901/DDP与SGC7901细胞间PTEN蛋白表达差异；Western-blot法检测细胞转染miR-21mimics及inhibitors后PTEN、p-Akt、Akt蛋白的表达，MTT法检测细胞转染miR-21mimics联合PI3K通路阻滞剂LY294002对胃癌细胞增殖的影响。
结果：1. SGC7901/DDP PTEN mRNA及PTEN蛋白的表达较亲本细胞SGC7901明显减低。2. SGC7901细胞转染miR-21mimics后PTEN mRNA表达明显降低（P<0.01）；转染miR-21mimics后PTEN蛋白在miR-21mimics组表达明显降低，p-Akt/Akt蛋白比值明显增加（P<0.01）。3. SGC7901/DDP细胞转染miR-21inhibitor后PTEN mRNA表达明显增加（P<0.01）；PTEN蛋白在miR-21inhibitor组表达明显增加，p-Akt/Akt蛋白比值明显降低。4. SGC7901转染miR-21mimics后，对顺铂的IC50为1.097±0.031μg/mL，而miR-21mimics联合LY294002组对顺铂的IC50为0.428±0.015μg/mL，差异有统计学意义(P<0.01)，提示LY294002可抑制miR-21所导致的胃癌细胞耐药。
结论： SGC7901/DDP PTEN mRNA、PTEN蛋白表达均减少；miR-21诱导SGC7901/DDP细胞对顺铂的耐药与PTEN蛋白的表达下调、Akt通路的激活有关；LY294002可抑制miR-21所导致的胃癌细胞耐药。
Background Gastric cancer is one of the most commonly diagnosed cancer worldwideand ranks second in global cancer mortality. Surgery is the primary treatment for theearly stage of gastric cancer, but most patients are either diagnosed in an advanced stage,or develop a relapse after apparently curative operation. For these patients,chemotherapy is a main treatment option. Unfortunately, many gastric cancer patientsexperience a recurrence of cancer after initial therapy and become refractory tochemotherapy following treatment. Thus, the acquisition of chemo-resistance is a majorclinical obstacle to the successful treatment of gastric cancer. The process ofchemo-resistance appears to be multifactorial and includes changes in drug transportleading to decreasing drug accumulation and increasing drug detoxification, changes inDNA repair and damage and/or alterations in the apoptotic cell death pathways.However, the mechanisms underlying chemo-resistance have not been fullycharacterized.
MicroRNAs (miRNAs) are a class of22-nucleotide, non-coding RNA molecules thatnegatively regulate the expression of target genes post-transcriptionally by binding tothe3′untranslated region (3′UTR) of mRNA. It has been demonstrated that miRNAs mediate diverse physiological function, such as cell differentiation, proliferation,apoptosis, and metabolism. Deviations from normal pattern of expression may causehuman diseases. In particular, it has been shown that miRNAs are aberrantly expressedin a variety of human cancers compared with their normal counterparts, suggesting thatmiRNAs contribute to oncogenesis through modulation of key cellular processes byfunctioning as tumor suppressors or oncogenes. Furthermore, miRNAs also playimportant roles in the drug resistance of different tumors. Indeed, recent evidencesindicate that miRNAs can influence tumor cell response to chemotherapy in differentmalignancies.
MicroRNA-21(miR-21) has been shown to be implicated in multiple malignancyrelated processes including cell proliferation, apoptosis, invasion, and metastasis. ThismiRNA is frequently over-expressed in a wide variety of cancers. Previous studies havealso shown that miR-21was among the top miRNAs with increased expression ingastric cancer tissue. The oncogenic properties of miR-21are further supported byfunctional studies showing that inhibition of miR-21expression reduced proliferation orgenerated a proapoptotic response of several cancer cells. Recent studies also showed itsmultiple roles in chemoresistance in other cancers.
However, whether miR-21can lead to drug resistance in gastric cancer remainsunknown. Considering the critical role of miR-21in a variety of cancers includinggastric cancer and chemo-resistance in other cancers, we hypothesized that theacquisition of chemo-resistance by cancer cells may also be modulated via the changesin miR-21levels. This novel miR-21/PTEN/PI3K/Akt signaling pathway in gastriccancer cells may provide drug targets for the sensitivity of tumor cells and could beapplied to treat chemotherapy resistance in gastric cancer patients. This study containsthree parts.
Part l Expression of miR-21and relationship with cisplatin resistance in gastriccancer
Objective Investigate the expression of miR-21and relationship with cisplatinresistance in gastric cancer cells. Methods Real-time PCR was used to detectexpression of miR-21in gastric cancer cells. The SGC7901and SGC7901/DDP cellswere transfected with the mimics or inhibitors of miR-21or negative control RNA (NC)by lipofectamine2000. MTT was used to analyze drug sensitivity. Apoptosis analysisand cell cycle were measured by fluorescene activated cell sorter. Results Theexpression of miR-21was an average of (7.65±1.197)-fold higher in SGC7901/DDPcells than in SGC7901cells and there is a significant difference between the two groups(P<0.01). SGC7901cells transfection with mimics of miR-21showed that the IC50(1.085±0.0639μg/mL) was more than the NC (0.487±0.018μg/mL) and veicle group(0.476±0.014μg/mL), apoptosis rate was lower than the NC and veicle group, cell ratioin S phase was higer than the NC and veicle group, and there was a significantdifference between them (P<0.05). Conclusion The expression of miR-21wasupregulated in SGC7901/DDP cells. Transfection with miR-21inhibitors can reversecisplatin resistance in SGC7901/DDP cells. Transfection with miR-21mimics can leadto cisplatin resistance in SGC7901cells.
Part2Expression of miR-21and relationship with Adriamycin resistance in gastriccancer
Objective Investigate the expression of miR-21and relationship with Adriamycin(ADM) resistance in gastric cancer cells. Methods Real-time PCR was used to detectexpression of miR-21in gastric cancer cells treated with ADM at IC50. The SGC7901and MGC803cells were transfected with the mimics of miR-21or negative controlRNA (NC) by lipofectamine2000. MTT was used to analyze drug sensitivity. Apoptosis analysis measured by fluorescene activated cell sorter. Results The expression ofmiR-21was an average of (2.941±0.225)-fold and (2.164±0.167)-fold higher inSGC7901and MGC803cells treated with ADM at IC50than untreated cells and there isa significant difference between the two groups (P<0.01). SGC7901cells transfectionwith miR-21mimics showed that the IC50(0.942±0.027μg/mL) was more than the NCgroup (0.335±0.029μg/mL), apoptosis rate was lower than the NC group (P<0.05).MGC803cells transfection with miR-21mimics showed that the IC50(1.430±0.124μg/mL) was more than the NC group (0.481±0.034μg/mL)(P<0.05).Conclusion The expression of miR-21was upregulated in SGC7901and MGC803cellstreated with IC50ADM. Transfection with miR-21mimics can lead to ADM resistancein SGC7901and MGC803.
Part3the mechanism of miR-21in chemoresistance of gastric cancer cell
Objective Investigate the mechanism of miR-21in chemoresistance of gastric cancercell. Methods The expresstion of PTEN mRNA was measured by real-time PCR. Theexpresstion of PTEN, p-Akt, Akt protein was measured by western-blot. The SGC7901and SGC7901/DDP cells were transfected with the mimics or inhibitors of miR-21ornegative control RNA (NC) by lipofectamine2000. MTT was used to analyze drugsensitivity. Results The expression of PTEN mRNA and protein was downreagulated inSGC7901/DDP cells. Overexpression of miR-21decreased the PTEN mRNAexpression and PTEN protein level in SGC7901cells, whereas knockdown of miR-21increased the PTEN mRNA expression and PTEN protein level (P<0.01) inSGC7901/DDP cells. SGC7901cells that were transfected with miR-21increased Aktphosphorylation (P<0.001). Knockdown of miR-21in the SGC7901/DDP cellsdecreased Akt phosphorylation (P<0.01). miR-21transfected SGC7901cells weretreated with serial dilutions of cisplatin and/or PI3K inhibitor LY294002. LY294002abrogated miR-21–activated Akt and significantly inhibited miR-21–induced cell survival and cisplatin resistance. Conclusion The expression of PTEN wasdownregulated in SGC7901/DDP cells. Overexpression of miR-21can activate thePI3K/Akt pathway by decreasing the PTEN protein level.
近年来研究显示一类长度为22个核苷酸的单链非编码微小RNA(microRNA，miRNA)在肿瘤发生发展中发挥着重要的作用。生物信息学方法显示miRNA通过调节不同的靶基因而发挥重要的生物学效应。在肿瘤耐药方面，miRNA能够通过调控不同的靶点而影响肿瘤细胞对抗肿瘤药物的敏感性。
microRNA-21(miR-2l)是目前发现的唯一在多种恶性肿瘤中高表达的miRNA，miR-2l有癌基因性质，抑制miR-21的表达可抑制肿瘤细胞增殖、促进肿瘤细胞凋亡。目前已有研究发现miR-21与胆管细胞癌、乳腺癌、胰腺癌、恶性胶质瘤等恶性肿瘤的耐药相关，但尚无miR-21与胃癌化疗耐药相关的研究。本研究拟在前期研究和文献报道的基础上，进一步观察miR-21与胃癌化疗耐药之间的关系、并探讨其可能作用机制，从而为临床上胃癌化疗耐药的研究提供新的靶点。本研究分三部分。
第一部分miR-21与胃癌细胞顺铂化疗敏感性关系的研究
目的：研究miR-21与胃癌细胞顺铂敏感性的关系
方法：real-time PCR检测胃癌耐顺铂细胞SGC7901/DDP及耐长春新碱细胞SGC7901/VCR和亲本细胞SGC7901miR-21的表达水平；利用脂质体Lipofectamine2000将成熟miR-21的模拟物mimics、阻遏物inhibitors及阴性对照negative controlRNA（NC)转染至SGC7901和SGC7901/DDP细胞；real-time PCR技术验证转染结果；MTT法检测细胞增殖水平；Annexin Ⅴ/PI双染流式细胞术检测细胞凋亡率；PI流式细胞术检测细胞周期。
结果：1. miR-21在SGC7901/DDP中高表达，与SGC7901细胞相比，表达增高7.65±1.197倍，差异有统计学意义(P<0.01)；在SGC7901/VCR与SGC7901细胞之间未检测出miR-21表达的差异。2.转染miR-21mimics后SGC7901细胞对顺铂的敏感性下降， mimics组对顺铂的IC50为1.085±0.0639μg/mL，与单纯脂质体组（0.476±0.014μg/mL）相比有显著性差异(P<0.01)，NC组对顺铂的IC50为0.487±0.018μg/mL，与单纯脂质体组相比无差异；转染miR-21inhibitors后SGC7901/DDP细胞对顺铂的敏感性增加， inhibitors组对顺铂的IC50为3.116±0.0.165μg/mL，与单纯脂质体组（5.138±0.075μg/mL）相比有显著性差异(P<0.01)，NC组对顺铂的IC50为5.175±0.458μg/mL，与单纯脂质体组相比差异无统计学意义。3. SGC7901细胞转染miR-21mimics，NC及脂质体24h后加入终浓度为0.5μg/mL顺铂培养48h后，mimics组凋亡率明显减少，与NC及脂质体组相比有明显差异(P<0.05)；SGC7901/DDP细胞转染miR-21inhibitor，NC及脂质体24h后加入终浓度为5.0μg/mL顺铂培养48h后，inhibitors组凋亡率明显升高，与NC及脂质体组相比有显著差异(P<0.05)。4. SGC7901转染miR-21mimics，NC及脂质体后继续培养48h，mimics组S期细胞明显增多达（42.04%）与NC组相比有明显差异(P<0.05)。
结论：miR-21在胃癌耐顺铂细胞株中高表达，转染miR-21mimics后可以导致胃癌细胞SGC7901对顺铂的耐药；转染miR-21inhibitors后可以增加胃癌耐顺铂细胞SGC7901/DDP对顺铂的敏感性，部分逆转顺铂耐药。
第二部分miR-21与胃癌细胞阿霉素敏感性关系的研究
目的：研究miR-21与胃癌细胞阿霉素化疗敏感性的关系
方法：real-time PCR检测经IC50阿霉素、氟尿嘧啶作用72h胃癌细胞SGC7901、MGC803与未经处理相应胃癌细胞miR-21的表达水平；利用脂质体Lipofectamine2000将成熟miR-21的模拟物mimics及阴性对照negative controlRNA（NC)转染至SGC7901和MGC803细胞；real-time PCR技术验证转染结果；MTT检测细胞增殖；Annexin-V FITC/PI双染流式细胞术检测细胞凋亡率。
结果：1. miR-21在经IC50阿霉素作用72h SGC7901和MGC803中高表达，与未经处理SGC7901及MGC803相比，表达分别增高2.941±0.225倍(P<0.01)、2.164±0.167倍(P<0.01)，差异均有统计学意义；经IC50氟尿嘧啶作用72h后胃癌细胞SGC7901和MGC803与未经处理相应胃癌细胞之间miR-21表达无差异。2.SGC7901转染miR-21mimics后①细胞对阿霉素的敏感性下降，mimics组与NC组对阿霉素的IC50分别为0.942±0.027μg/mL、0.335±0.029μg/mL，前者是后者的2.8倍，差异有统计学意义(P<0.01)；②细胞凋亡率明显下降，与NC组相比差异有统计学意义(P<0.05)。3. MGC803转染miR-21mimics后对阿霉素的敏感性下降，mimics组与NC组IC50分别为1.430±0.124μg/mL、0.481±0.034μg/mL，前者是后者的3.0倍，差异有统计学意义(P<0.01)。
结论：经IC50阿霉素处理72h后胃癌细胞miR-21表达增加，转染miR-21mimics后，可以增加胃癌细胞对阿霉素的耐药性。
第三部分miR-21对胃癌细胞化疗敏感性影响的机制研究
目的：研究miR-21对胃癌细胞化疗敏感性影响的机制
方法：RT-PCR及real-time PCR检测SGC7901/DDP与SGC7901细胞间PTENmRNA表达差异，Western-blot法检测SGC7901/DDP与SGC7901细胞间PTEN蛋白表达差异；Western-blot法检测细胞转染miR-21mimics及inhibitors后PTEN、p-Akt、Akt蛋白的表达，MTT法检测细胞转染miR-21mimics联合PI3K通路阻滞剂LY294002对胃癌细胞增殖的影响。
结果：1. SGC7901/DDP PTEN mRNA及PTEN蛋白的表达较亲本细胞SGC7901明显减低。2. SGC7901细胞转染miR-21mimics后PTEN mRNA表达明显降低（P<0.01）；转染miR-21mimics后PTEN蛋白在miR-21mimics组表达明显降低，p-Akt/Akt蛋白比值明显增加（P<0.01）。3. SGC7901/DDP细胞转染miR-21inhibitor后PTEN mRNA表达明显增加（P<0.01）；PTEN蛋白在miR-21inhibitor组表达明显增加，p-Akt/Akt蛋白比值明显降低。4. SGC7901转染miR-21mimics后，对顺铂的IC50为1.097±0.031μg/mL，而miR-21mimics联合LY294002组对顺铂的IC50为0.428±0.015μg/mL，差异有统计学意义(P<0.01)，提示LY294002可抑制miR-21所导致的胃癌细胞耐药。
结论： SGC7901/DDP PTEN mRNA、PTEN蛋白表达均减少；miR-21诱导SGC7901/DDP细胞对顺铂的耐药与PTEN蛋白的表达下调、Akt通路的激活有关；LY294002可抑制miR-21所导致的胃癌细胞耐药。
Background Gastric cancer is one of the most commonly diagnosed cancer worldwideand ranks second in global cancer mortality. Surgery is the primary treatment for theearly stage of gastric cancer, but most patients are either diagnosed in an advanced stage,or develop a relapse after apparently curative operation. For these patients,chemotherapy is a main treatment option. Unfortunately, many gastric cancer patientsexperience a recurrence of cancer after initial therapy and become refractory tochemotherapy following treatment. Thus, the acquisition of chemo-resistance is a majorclinical obstacle to the successful treatment of gastric cancer. The process ofchemo-resistance appears to be multifactorial and includes changes in drug transportleading to decreasing drug accumulation and increasing drug detoxification, changes inDNA repair and damage and/or alterations in the apoptotic cell death pathways.However, the mechanisms underlying chemo-resistance have not been fullycharacterized.
MicroRNAs (miRNAs) are a class of22-nucleotide, non-coding RNA molecules thatnegatively regulate the expression of target genes post-transcriptionally by binding tothe3′untranslated region (3′UTR) of mRNA. It has been demonstrated that miRNAs mediate diverse physiological function, such as cell differentiation, proliferation,apoptosis, and metabolism. Deviations from normal pattern of expression may causehuman diseases. In particular, it has been shown that miRNAs are aberrantly expressedin a variety of human cancers compared with their normal counterparts, suggesting thatmiRNAs contribute to oncogenesis through modulation of key cellular processes byfunctioning as tumor suppressors or oncogenes. Furthermore, miRNAs also playimportant roles in the drug resistance of different tumors. Indeed, recent evidencesindicate that miRNAs can influence tumor cell response to chemotherapy in differentmalignancies.
MicroRNA-21(miR-21) has been shown to be implicated in multiple malignancyrelated processes including cell proliferation, apoptosis, invasion, and metastasis. ThismiRNA is frequently over-expressed in a wide variety of cancers. Previous studies havealso shown that miR-21was among the top miRNAs with increased expression ingastric cancer tissue. The oncogenic properties of miR-21are further supported byfunctional studies showing that inhibition of miR-21expression reduced proliferation orgenerated a proapoptotic response of several cancer cells. Recent studies also showed itsmultiple roles in chemoresistance in other cancers.
However, whether miR-21can lead to drug resistance in gastric cancer remainsunknown. Considering the critical role of miR-21in a variety of cancers includinggastric cancer and chemo-resistance in other cancers, we hypothesized that theacquisition of chemo-resistance by cancer cells may also be modulated via the changesin miR-21levels. This novel miR-21/PTEN/PI3K/Akt signaling pathway in gastriccancer cells may provide drug targets for the sensitivity of tumor cells and could beapplied to treat chemotherapy resistance in gastric cancer patients. This study containsthree parts.
Part l Expression of miR-21and relationship with cisplatin resistance in gastriccancer
Objective Investigate the expression of miR-21and relationship with cisplatinresistance in gastric cancer cells. Methods Real-time PCR was used to detectexpression of miR-21in gastric cancer cells. The SGC7901and SGC7901/DDP cellswere transfected with the mimics or inhibitors of miR-21or negative control RNA (NC)by lipofectamine2000. MTT was used to analyze drug sensitivity. Apoptosis analysisand cell cycle were measured by fluorescene activated cell sorter. Results Theexpression of miR-21was an average of (7.65±1.197)-fold higher in SGC7901/DDPcells than in SGC7901cells and there is a significant difference between the two groups(P<0.01). SGC7901cells transfection with mimics of miR-21showed that the IC50(1.085±0.0639μg/mL) was more than the NC (0.487±0.018μg/mL) and veicle group(0.476±0.014μg/mL), apoptosis rate was lower than the NC and veicle group, cell ratioin S phase was higer than the NC and veicle group, and there was a significantdifference between them (P<0.05). Conclusion The expression of miR-21wasupregulated in SGC7901/DDP cells. Transfection with miR-21inhibitors can reversecisplatin resistance in SGC7901/DDP cells. Transfection with miR-21mimics can leadto cisplatin resistance in SGC7901cells.
Part2Expression of miR-21and relationship with Adriamycin resistance in gastriccancer
Objective Investigate the expression of miR-21and relationship with Adriamycin(ADM) resistance in gastric cancer cells. Methods Real-time PCR was used to detectexpression of miR-21in gastric cancer cells treated with ADM at IC50. The SGC7901and MGC803cells were transfected with the mimics of miR-21or negative controlRNA (NC) by lipofectamine2000. MTT was used to analyze drug sensitivity. Apoptosis analysis measured by fluorescene activated cell sorter. Results The expression ofmiR-21was an average of (2.941±0.225)-fold and (2.164±0.167)-fold higher inSGC7901and MGC803cells treated with ADM at IC50than untreated cells and there isa significant difference between the two groups (P<0.01). SGC7901cells transfectionwith miR-21mimics showed that the IC50(0.942±0.027μg/mL) was more than the NCgroup (0.335±0.029μg/mL), apoptosis rate was lower than the NC group (P<0.05).MGC803cells transfection with miR-21mimics showed that the IC50(1.430±0.124μg/mL) was more than the NC group (0.481±0.034μg/mL)(P<0.05).Conclusion The expression of miR-21was upregulated in SGC7901and MGC803cellstreated with IC50ADM. Transfection with miR-21mimics can lead to ADM resistancein SGC7901and MGC803.
Part3the mechanism of miR-21in chemoresistance of gastric cancer cell
Objective Investigate the mechanism of miR-21in chemoresistance of gastric cancercell. Methods The expresstion of PTEN mRNA was measured by real-time PCR. Theexpresstion of PTEN, p-Akt, Akt protein was measured by western-blot. The SGC7901and SGC7901/DDP cells were transfected with the mimics or inhibitors of miR-21ornegative control RNA (NC) by lipofectamine2000. MTT was used to analyze drugsensitivity. Results The expression of PTEN mRNA and protein was downreagulated inSGC7901/DDP cells. Overexpression of miR-21decreased the PTEN mRNAexpression and PTEN protein level in SGC7901cells, whereas knockdown of miR-21increased the PTEN mRNA expression and PTEN protein level (P<0.01) inSGC7901/DDP cells. SGC7901cells that were transfected with miR-21increased Aktphosphorylation (P<0.001). Knockdown of miR-21in the SGC7901/DDP cellsdecreased Akt phosphorylation (P<0.01). miR-21transfected SGC7901cells weretreated with serial dilutions of cisplatin and/or PI3K inhibitor LY294002. LY294002abrogated miR-21–activated Akt and significantly inhibited miR-21–induced cell survival and cisplatin resistance. Conclusion The expression of PTEN wasdownregulated in SGC7901/DDP cells. Overexpression of miR-21can activate thePI3K/Akt pathway by decreasing the PTEN protein level.
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