miR-138-5p在胃癌顺铂耐药中的作用及其机制研究

发布时间：2018-02-21 04:51

本文关键词： 胃癌 microRNA-138-5p 顺铂耐药核苷酸切除修复低钠血症　出处：《安徽医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:胃癌是世界范围内常见的恶性肿瘤之一,我国是胃癌高发国家。虽然近年来胃癌的诊疗技术已经取得了一些进步,但由于肿瘤早期症状表现无特异性,及高侵袭性和转移率,使得胃癌总体预后欠佳。晚期胃癌治疗以姑息性化疗为主,铂类作为胃癌化疗的基石类药物,大部分含铂方案的有效率均不超过50%,存在铂类原发或继发耐药现象限制了其在临床应用。近年来研究发现microRNAs(miRNAs)的表达异常与肿瘤化疗敏感性密切相关。miR-138-5p在肺癌化疗耐药中的作用已经得以证实,可通过负性调控ERCC1表达影响DNA损伤修复能力减少顺铂耐药。但迄今为止,关于miR-138-5p是否能调节胃癌细胞化疗耐药及其机制尚未明确,是否能真正用于指导胃癌患者临床治疗亦未明确。因此,本课题拟探讨miR-138-5p能否调控胃癌细胞株(SGC7901)对顺铂治疗敏感性,及其可能的作用机制。并进一步探讨晚期胃癌患者癌组织中及血浆中miR-138-5p的表达水平及其与一线含铂方案化疗疗效的相关性。以期更好的指导晚期胃癌患者临床个体化治疗。本研究在前期的临床病例资料收集过程中发现晚期胃癌伴有低钠血症患者预后明显变差,提示低钠血症为胃癌负性疗效预测因子。而目前关注晚期胃癌与低钠血症关系的研究较少,因此本研究探讨了晚期胃癌患者合并低钠血症发生情况对其一线治疗疗效、预后的影响,并进一步探讨低钠血症影响晚期胃癌患者化疗疗效及预后的原因,是否与miR-138-5p表达相关。以期将患者的实验室检验与miRNA检测结果相结合更好的指导临床个体化治疗。本研究共分三部分。第一部分:miR-138-5p在胃癌顺铂耐药细胞中的作用及机制研究目的:研究miR-138-5p在胃癌顺铂耐药细胞中表达情况及其介导顺铂耐药的可能机制。方法:采用基因芯片技术分析胃癌耐顺铂细胞SGC7901/DDP和亲本细胞SGC7901中表达差异miRNAs。生物信息学软件预测miR-138-5p作用靶点。采用qRT-PCR及Western blot分别检测胃癌耐顺铂细胞SGC7901/DDP和亲本细胞SGC7901中miR-138-5p及ERCC1、ERCC4的表达水平;利用包含干扰miR-138-5p表达的目的基因的慢病毒液及阴性对照negative control RNA(NC)转染至SGC7901细胞下调miR-138-5p表达,转染至SGC7901/DDP细胞上调miR-138-5p表达;荧光显微镜观察转染结果,qRT-PCR技术验证转染后miR-138-5p表达;Western blot验证转染后ERCC1,ERCC4的表达情况;MTT法检测细胞对顺铂治疗的敏感性;结果:1.基因芯片检测结果表明miR-138-5p在SGC7901/DDP细胞内的表达是SGC7901中的0.2686倍。进一步采用qRT-PCR方法验证miR-138-5p在SGC7901中的表达量是SGC7901/DDP的5.006倍,差异有统计学意义(P0.01)。两者结果一致,表明其在胃癌耐药细胞株(SGC7901/DDP)中表达明显下调;2.生物信息学软件预测miR-138-5p作用靶点为核苷酸切除修复通路中的ERCC1、ERCC4蛋白。Western blot结果表明ERCC1和ERCC4在SGC7901/DDP中表达较SGC7901细胞中明显升高,差异有统计学意义(P0.01);3.转染携带目的基因片段慢病毒液调节miR-138-5p的表达,在SGC7901细胞中下调miR-138-5p的表达,在SGC7901/DDP细胞中上调miR-138-5p的表达。荧光显微镜观察转染后SGC7901细胞呈现红色为表达下调,SGC7901/DDP细胞呈现绿色为表达上调。采用qRT-PCR方法检测慢病毒转染后miR-138-5p表达水平,结果证实其在SGC7901-LV-miR-138-5p-KD组中的表达下调,分别是SGC7901和NC-KD组的0.209倍和0.211倍,差异具有统计学意义(P0.05)。在SGC7901/DDP-LV-miR138-5p-OE中表达上调,分别是SGC-7901/DDP和SGC7901/DDP-LV-NC-OE组4.17和4.36倍,差异有统计学意义(P0.05);4.Western blot结果表明ERCC1、ERCC4在SGC7901-LV-miR138-5p-KD(miR-138-5p表达下调细胞)内的表达明显升高,在SGC7901/DDP-LV-miR138-5p-OE(miR-138-5p表达上调细胞)内的表达明显减少,差异有统计学意义(P0.01);5.MTT法结果表明SGC7901/DDP-LV-miR138-5p-OE组细胞,miR-138-5p表达上调后对顺铂的敏感性较SGC7901/DDP和SGC7901/DDP-LV-NC-OE增加,IC50分别是:3.14±0.32、6.81±0.12、6.89±0.14,差异有统计学意义(P0.01)。SGC7901-LV-miR138-5p-KD组细胞,miR-138-5p表达下调后对顺铂的耐药性增强,其IC50较SGC7901和SGC7901-LV-NC-KD细胞升高,IC50分别为:0.39±0.04、0.20±0.01、0.25±0.05差异有统计学意义(P0.01)。结论:miR-138-5p在胃癌耐药细胞株(SGC7901/DDP)中低表达,通过慢病毒转染使得miR-138-5p表达上调后,SGC7901/DDP细胞中ERCC1、ERCC4表达明显减少,核苷酸切除修复缺陷,增强胃癌耐药细胞株SGC7901/DDP对顺铂治疗的敏感性,起化疗增敏作用。下调miR-138-5p表达,SSGC7901细胞中ERCC1、ERCC4表达明显增加,核苷酸切除修复增加,导致胃癌细胞株SGC7901对顺铂耐药。推测其可能的作用机制为通过负性调控ERCC1、ERCC4表达干扰DNA损伤修复调节对顺铂治疗的敏感性。第二部分:miR-138-5p表达水平与晚期胃癌含铂方案化疗疗效的相关性研究目的:探讨晚期胃癌患者癌组织中及血浆中miR-138-5p的表达水平与一线含铂方案化疗疗效的相关性。方法:采用qRT-PCR方法检测51例晚期胃癌患者癌组织及血浆中miR-138-5p的表达水平,同时选取相匹配的癌旁组织标本20例及健康志愿者血浆20例作为实验对照。所有患者均接受含铂方案一线化疗,并评价其近期疗效及一线化疗后无进展生存时间、总生存。结果:胃癌组织中miR-138-5p的表达水平低于癌旁组织(t=4.30,P㩳0.01),胃癌患者血浆中miR-138-5p的表达水平低于健康对照组(t=3.04,P㩳0.01)。晚期胃癌患者癌组织中及血浆中miR-138-5p的表达水平与患者性别、年龄、转移部位、转移器官数目、ECOG评分、癌组织分化程度等均无相关性(P0.05)。51例患者均可评价近期疗效,其中11例PR、23例SD、17例PD,ORR率为21.6%,DCR率为66.7%。癌组织及血浆中miR-138-5p的相对表达量均与化疗近期疗效呈正相关(P㩳0.05)。采用ROC曲线判断miR-138-5p表达水平预测化疗有效率的最佳截断值,取癌组织中miR-138-5p表达量0.104为截点,AUC值为0.907,预测含铂方案化疗后疾病控制的灵敏度为90.91%,特异度为75%。超过0.104为高表达组,低于0.104为低表达组。取血浆中miR-138-5p表达量0.091为截点,AUC值为0.973,预测含铂方案化疗后疾病控制的灵敏度为100%,特异度为85%。超过0.091为高表达组,低于0.091为低表达组。51例患者中位PFS时间为4.0月(95%CI:3.7~4.3月),中位OS为9.9月(95%CI:9.2~10.6月)。胃癌组织中miR-138-5p高表达与低表达组的中位PFS分别为5.5月(95%CI:3.5~7.5月)及3.2月(95%CI:1.7~4.7月)(P0.05);中位OS分别为11.2月(95%CI:10.9~11.5月)及9.2月(95%CI:8.7~9.7月),采用Log-rank检验,差异均有统计学意义(P0.01)。血浆中miR-138-5p高表达与低表达组的中位PFS分别为5.5月(95%CI:3.0~8.1月)及3.5月(95%CI:1.5~5.5月)(P0.05)。中位OS分别为11.4月(95%CI:11.0~11.8月)及9.5月(95%CI:8.6~10.4月),采用Log-rank检验,差异均有统计学意义(P0.01)。线性相关分析表明,胃癌肿瘤组织与血浆中miR-138-5p的表达具有正相关性(r=0.899,P0.05)。结论:胃癌组织及血浆中miR-138-5p的表达水平具有正相关性,且癌组织及血浆中miR-138-5p表达水平高低与晚期胃癌患者一线含铂化疗方案的近期疗效、一线PFS、OS具有一定的正相关性。第三部分:低钠血症与晚期胃癌化疗疗效及miR-138-5p表达水平的相关性研究目的:探讨晚期胃癌合并低钠血症患者的临床特点及预后分析,及低钠血症发生与miR-138-5p表达水平的相关性;方法:收集2009/06/01-2015/12/31期间在安徽医科大学第一附属医院住院的胃癌合并低钠血症患者信息,从中筛选出44例晚期胃癌伴有低钠血症并接受一线化疗患者,并将45例晚期胃癌血钠正常患者作为对照组,进行回顾性对照分析。收集这些患者一线化疗的近期疗效,一线PFS及毒副反应信息。采用qRT-PCR方法检测了低钠组共5例、对照组共10例患者组织中miR-138-5p表达水平。结果:晚期、女性、肝脏及腹膜转移、ECOG评分低的胃癌患者更易合并低钠血症。低钠组及对照组一线化疗的ORR率分别为:25%(11/44)和40.0%(18/45)(P=0.131);DCR率分别为40.9%(18/44)和73.3%(33/45)(P=0.002),疾病控制率的差异具有统计学意义。低钠组一线化疗中位PFS为3.0个月(95%CI:2.8~3.7月),对照组一线化疗中位PFS为4.5个月(95%CI:4.3~6.2月),(χ2=14.618,P0.001),两组差异具有统计学意义。低钠血症纠正者一线化疗PFS优于低钠血症持续存在患者,差异具有统计学意义。低钠组一线化疗后贫血、恶心、呕吐、乏力、纳差的发生率更高,差异具有统计学意义。低钠组胃癌组织中miR-138-5p表达水平低于对照组,但差异并无统计学意义。结论:低钠血症可作为晚期胃癌患者不良的化疗疗效预测因素之一,可结合miR-138-5p表达水平共同预测晚期胃癌铂类化疗疗效及判断预后。
[Abstract]:Background: gastric cancer is one of the most common malignant tumors in the world, China is a country with high incidence of gastric cancer. Although in recent years, the technology of diagnosis and treatment of gastric cancer has made some progress, but because the early symptoms of cancer are nonspecific, and high invasion and metastasis of gastric cancer, the prognosis is poor overall in palliative treatment of advanced gastric cancer. Chemotherapy, platinum drugs as a cornerstone of gastric cancer chemotherapy, the effective rate of most platinum containing regimens was less than 50%, there are platinum primary or secondary drug resistance limit its clinical application. Recent studies have found that microRNAs (miRNAs) effect are closely related to the abnormal expression and chemosensitivity of.MiR-138-5p in lung cancer chemotherapy in has been confirmed through the negative regulation of ERCC1 ability to repair DNA damage effects of decreased expression of cisplatin resistance. But so far, about whether miR-138-5p can regulate the gastric cancer Cell resistance to chemotherapy and its mechanism is not yet clear whether can really be used to guide the clinical treatment of patients with gastric cancer is not clear. Therefore, this thesis intends to explore whether miR-138-5p can regulate the gastric cancer cell line (SGC7901) on cisplatin sensitivity and its possible mechanism. And to further explore the correlation between the expression of miR-138-5p in tumor tissues of patients with advanced gastric cancer and in plasma and the efficacy of first-line platinum based chemotherapy. In order to better guide clinical individualized treatment for patients with advanced gastric cancer. The prognosis of patients with advanced gastric cancer found hyponatremia was significantly worse on the pre clinical data collection process, suggesting that hyponatremia is the negative effect of gastric cancer predictors. At present, paying attention to relationship advanced gastric cancer with hyponatremia research, this study of the advanced gastric cancer patients with hyponatremia in the first-line treatment The curative effect and prognosis, and to further explore the causes of hyponatremia in patients with gastric cancer undergoing chemotherapy efficacy and prognosis of advanced, is associated with expression of miR-138-5p. In order to make the laboratory test and miRNA test results were combined with the clinical individual treatment better. This research is divided into three parts. The first part: To study in the cisplatin resistant cell in gastric cancer: effect and mechanism of miR-138-5p expression and its mechanism may be mediated by cisplatin resistance of miR-138-5p in gastric cancer cells resistant to cisplatin resistant human gastric cancer. Methods: analysis of differentially expressed miRNAs. were predicted by bioinformatics software miR-138-5p target cells SGC7901/DDP and cisplatin SGC7901 parental cells by gene chip technology. Using qRT-PCR and Western blot was used to detect the miR-138-5p and ERCC1 of cisplatin resistant human gastric cancer cell SGC7901/DDP and its parental cell line SGC7901, the expression level of ERCC4; Using lentiviral solution and negative expression of miR-138-5p gene contains interference control of negative control RNA (NC) was transfected into SGC7901 cells by down regulating the expression of miR-138-5p was transfected into SGC7901/DDP cells by upregulation of miR-138-5p expression; transfection results by fluorescence microscopy, the expression of miR-138-5p qRT-PCR Western blot to verify the verification after transfection; ERCC1 after transfection, the expression of ERCC4 MTT; method to detect the sensitivity of cells to cisplatin therapy; results: the testing results showed that the expression of miR-138-5p 1. gene chip in SGC7901/DDP cells is 0.2686 times of SGC7901. A qRT-PCR method was used to verify the expression of miR-138-5p in SGC7901 was 5.006 times of SGC7901/DDP, the difference was statistically significant (P0.01). The two results are consistent, that the resistant gastric cancer cell lines (SGC7901/DDP) in gene expression was significantly decreased; 2. were predicted by bioinformatics software miR-138-5p targets As the nucleotide excision repair pathway of ERCC1, ERCC4 protein.Western blot results showed that the expression of ERCC1 and ERCC4 in SGC7901/DDP was significantly higher than that in SGC7901 cells, the difference was statistically significant (P0.01); 3. GFP gene fragment lentiviral solution regulating miR-138-5p expression, down-regulation of miR-138-5p expression in SGC7901 cells, upregulation of miR-138-5p the expression in SGC7901/DDP cells. After transfection, SGC7901 cells showed expression of red fluorescence microscopy, SGC7901/DDP cells showed green expression. The expression of miR-138-5p was detected by qRT-PCR lentiviral transfection results confirmed the down-regulation of its expression in the SGC7901-LV-miR-138-5p-KD group, SGC7901 and NC-KD were 0.209 times and 0.211 times group, with statistically significant difference (P0.05). The expression of SGC7901/DDP-LV-miR138-5p-OE, SGC-7901/DDP and SGC790 respectively. Group 1/DDP-LV-NC-OE was 4.17 and 4.36 times, the difference was statistically significant (P0.05); 4.Western blot ERCC1 ERCC4 in SGC7901-LV-miR138-5p-KD showed that, (the down-regulation of miR-138-5p expression in cells) was significantly increased in SGC7901/DDP-LV-miR138-5p-OE (upregulation of miR-138-5p expression in the cells) decreased significantly, the difference was statistically significant (P0.01); 5.MTT assay results showed that SGC7901/DDP-LV-miR138-5p-OE group cells, miR-138-5p expression after sensitivity to cisplatin than SGC7901/DDP and SGC7901/DDP-LV-NC-OE, IC50 respectively is: 3.14 + 0.32,6.81 + 0.12,6.89 + 0.14, the difference was statistically significant (P0.01) cells in.SGC7901-LV-miR138-5p-KD group, miR-138-5p expression was down regulated after cisplatin resistance increased, the IC50 compared with SGC7901 and SGC7901-LV-NC-KD cells increased, IC50 were statistically 0.39 + 0.04,0.20 + 0.01,0.25 + 0.05 difference (P0.01). Conclusion: miR- 138-5p in gastric cancer multidrug resistance cell line (SGC7901/DDP) low expression, makes the expression of miR-138-5p by lentivirus transfected ERCC1 SGC7901/DDP cells, the expression of ERCC4 was significantly reduced, nucleotide excision repair defects, enhance the sensitivity of gastric cancer SGC7901/DDP cells to cisplatin treatment, chemotherapy sensitization. Expression of miR-138-5p ERCC1 in SSGC7901 cells. The expression of ERCC4 increased obviously, nucleotide excision repair, leading to gastric cancer cell line SGC7901 to cisplatin resistance. Its possible mechanism was speculated through negative regulation of ERCC1 expression, ERCC4 interference DNA damage repair regulation on sensitivity of cisplatin treatment. The second part: the expression level of miR-138-5p and the efficacy of chemotherapy for advanced gastric cancer platinum correlation study the expression level of miR-138-5p and the first: To investigate the effect of advanced gastric cancer tissues and plasma in the treatment with platinum based chemotherapy Methods: using the qRT-PCR method. Correlation between the expression level of miR-138-5p in tumor tissues of patients with advanced gastric cancer and 51 cases of plasma detection, and selected matched noncancerous tissues and 20 cases of healthy volunteers plasma in 20 cases as the control. All the patients were treated with platinum based first-line chemotherapy, and to evaluate its efficacy and after first-line chemotherapy progression free survival and overall survival. Results: the expression of miR-138-5p in gastric cancer tissue than that of adjacent tissues (t=4.30, P? 0.01), the expression level of miR-138-5p in plasma of gastric cancer patients were lower than the healthy control group (t=3.04, P? 0.01). The expression level of miR-138-5p in tumor tissues of patients with advanced gastric cancer and plasma in patients with gender, age, metastasis, number of metastatic organs, ECOG scores, there was no correlation between cancer tissue differentiation degree (P0.05) of.51 patients were evaluable for efficacy, including 11 cases of PR, 23 cases SD, 17 cases PD, ORR rate was 21. 6%, the DCR rate of miR-138-5p 66.7%. in cancer tissue and plasma relative expression were positively associated with the recent curative effect of chemotherapy (P? 0.05). Using ROC curve to determine the expression of miR-138-5p optimal truncation level prediction of chemotherapy efficiency value, miR-138-5p expression of the 0.104 cutoff point in cancer tissue, AUC value was 0.907, the sensitivity of the forecasts the disease control of platinum based chemotherapy was 90.91%, the specificity was 75%. more than 0.104 high expression group and low expression group were less than 0.104. The expression of miR-138-5p in 0.091 as the cut-off point in plasma, the AUC value is 0.973, the prediction of disease control sensitivity of platinum based chemotherapy was 100%, the specificity was more than 0.091 85%. for high expression group and low expression group was less than 0.091.51 PFS patients the median time was 4 months (95%CI:3.7~4.3 months), the median OS was 9.9 months (95%CI:9.2~10.6 months). The high expression of miR-138-5p in gastric cancer tissues respectively with a median of PFS low expression group was 5.5 months (9 5%CI:3.5~7.5 months) and 3.2 months (95%CI:1.7~4.7 months) (P0.05); the median OS was 11.2 months (95%CI:10.9~11.5 months) and 9.2 months (95%CI:8.7~9.7 months), the Log-rank test, the differences were statistically significant (P0.01). Plasma miR-138-5p high expression and low PFS expression respectively in the group was 5.5 months (95%CI:3.0~8.1 months) and 3.5 months (95%CI:1.5~5.5 months) (P0.05). The median OS was 11.4 months (95%CI:11.0~11.8 months) and 9.5 months (95%CI:8.6~10.4 months), the Log-rank test, the differences were statistically significant (P0.01). The correlation analysis of linear table Ming, the expression of miR-138-5p in tumor tissue and plasma in gastric cancer with a positive correlation (r=0.899, P0.05). Conclusion: the expression of miR-138-5p in gastric cancer tissue and plasma in a positive correlation, and miR-138-5p expression in cancer tissue and plasma level of efficacy, and late first-line platinum based chemotherapy in patients with gastric cancer line PFS, OS has a The positive correlation between the objective. The third part: the relationship between hyponatremia and advanced gastric cancer chemotherapy and the expression level of miR-138-5p: To investigate the clinical characteristics and prognostic analysis of hyponatremia in patients with advanced gastric cancer complicated with hyponatremia and the level of correlation with the expression of miR-138-5p; methods: in the First Affiliated Hospital of Medical University Of Anhui were collected during 2009/06/01-2015/12/31 gastric cancer complicated with hyponatremia, screened from 44 patients with advanced gastric cancer patients with hyponatremia and received first-line chemotherapy, and 45 cases of advanced gastric cancer patients with normal serum sodium as control group, were analyzed retrospectively. These patients collected short-term efficacy of first-line chemotherapy, and toxicity of first-line PFS information detection of the low sodium group. A total of 5 cases with qRT-PCR method, the expression level of miR-138-5p in control group of 10 patients in tissues. Results: the late female, liver and Peritoneal metastasis, low ECOG score of patients with gastric cancer more easily hyponatremia. Low sodium group and control group in the first-line chemotherapy ORR rate was 25% (11/44) and 40% (18/45) (P=0.131); the DCR rate was 40.9% (18/44) and 73.3% (33/45) (P=0.002), the difference was statistically significant the disease control rate. Low sodium group first-line chemotherapy, the median PFS was 3 months (95%CI:2.8~3.7 months), the control group first-line chemotherapy. The median PFS was 4.5 months (95%CI:4.3~6.2 months), (2=14.618, P0.001), statistically significant differences between the two groups. Hyponatremia corrected first-line chemotherapy is better than low sodium PFS persist in patients, the difference was statistically significant. Anemia, low sodium group after first-line chemotherapy nausea, vomiting, fatigue, anorexia, the incidence is higher, the difference was statistically significant. The level of miR-138-5p expression in gastric cancer tissues than in the control group low sodium group, but the difference was not statistically significant. Conclusion: Hyponatremia can As one of the predictors of poor chemotherapy efficacy in advanced gastric cancer, it can be combined with miR-138-5p expression level to predict prognosis and prognosis of platinum based chemotherapy for advanced gastric cancer.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2

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