CARLo-5在子宫内膜癌中的作用及机制研究

发布时间：2018-05-30 12:19

本文选题：子宫内膜癌 + lncRNA　；参考：《河北医科大学》2017年博士论文

【摘要】：子宫内膜癌是最常见的妇科恶性肿瘤之一。2015年子宫内膜癌在中国的新发病例数约为63400人,死亡率约为21.8%,新发病例数逐年上升,且发病呈年轻化的趋势。2016年美国子宫内膜癌新发病例约为60050人,成为继乳腺癌之后的第二大妇科癌症,严重危害了女性的健康。尽管目前医学诊断和治疗水平已经非常发达,但子宫内膜癌的确切发病机制尚不明确,治疗手段也十分有限。最近,长链非编码RNA(long non-coding RNA,lncRNA)的发现为探索子宫内膜癌的发病机制提供了分子生物学基础。lncRNA是长度超过200nt不能编码蛋白质的转录本,大量的研究发现其在人类的生命活动及各种疾病的发生发展中发挥了重要的作用。CARLo-5是最近发现的长度为2628个核苷酸的lncRNA,位于原癌基因myc的增强子附近,据报道,CARLo-5在结肠癌、胃癌等多种癌组织中表达上调,在癌症的发生和进展中起着重要作用,对多种癌症的诊断、预后评估和治疗均具有潜在价值。然而,CARLo-5在子宫内膜癌中的表达情况及作用机制尚未见报道。本研究采用实时荧光定量PCR(Real-time PCR)的方法检测正常子宫内膜组织和子宫内膜癌组织中CARLo-5基因的表达水平,使用蛋白印迹法(Western-blot)检测细胞周期相关蛋白CDK2和CDKN1A的表达情况,深入分析了CARLo-5表达水平与子宫内膜癌患者临床病理特征和总生存期的相关性,并统计分析了CARLo-5基因表达水平与细胞周期相关蛋白CDK2和CDKN1A表达水平的相关性。体外敲低子宫内膜癌细胞中CARLo-5的表达后,通过MTS法、Transwell迁移、Matrigel侵袭实验、平板克隆形成实验及流式细胞术检测敲低CARLo-5对子宫内膜癌细胞生物学行为的影响。为了探究CARLo-5在子宫内膜癌中的作用机制,本研究通过Western-blot检测了敲低CARLo-5对子宫内膜癌细胞中CDK2、CDKN1A、基质金属蛋白酶MMP2和MMP9表达水平的影响。通过RNAi慢病毒技术构建了稳定敲低CARLo-5的子宫内膜癌细胞株,并进行裸鼠皮下荷子宫内膜癌细胞成瘤实验,检测敲低CARLo-5对子宫内膜癌细胞体内增殖能力的影响。本研究探索了CARLo-5在子宫内膜癌中的作用及机制,利于阐明子宫内膜癌发生发展的分子生物学机制,从而为子宫内膜癌的基因诊断和治疗提供前期实验基础。第一部分CARLo-5在子宫内膜癌组织中的表达及临床意义目的:研究lncRNA CARLo-5在子宫内膜癌组织中的表达情况,并探讨CARLo-5表达水平与子宫内膜癌患者临床病理特征及总生存期的关系。检测子宫内膜癌组织中细胞周期相关蛋白CDK2和CDKN1A的表达水平及其与CARLo-5表达水平的相关性。方法:1采用Real-time PCR法检测108例子宫内膜癌组织和66例正常子宫内膜组织中CARLo-5的表达水平。2采用Western-blot检测子宫内膜癌组织和正常子宫内膜组织中细胞周期相关蛋白CDK2和CDKN1A的表达情况。3分析CARLo-5的表达水平与子宫内膜癌患者临床病理特征和总生存期的相关性。4分析CARLo-5在子宫内膜癌组织中的表达与细胞周期相关蛋白CDK2和CDKN1A表达之间的相关性。结果:1 lncRNA CARLo-5在子宫内膜癌组织和正常子宫内膜组织中的表达水平Real-time PCR实验结果显示:CARLo-5在子宫内膜癌中的表达水平较正常子宫内膜组织明显升高(P0.001)。2 CARLo-5的表达水平与子宫内膜癌患者临床病理特征的相关性CARLo-5在子宫内膜癌组织中的表达水平与子宫内膜癌患者的临床分期和淋巴结转移密切相关(P0.05),与患者的年龄、病理类型、病理分级、肌层侵犯深度和脉管浸润无明显相关性(P0.05)。3 CARLo-5的表达水平与子宫内膜癌患者总生存期的相关性CARLo-5高表达组子宫内膜癌患者的平均生存时间(43.0±14.1月)较CARLo-5低表达组患者(52.5±9.1月)明显缩短(P0.05)。4子宫内膜癌组织中CDK2和CDKN1A蛋白的表达水平Western-blot结果显示:CDK2蛋白在子宫内膜癌组织中的表达水平(9.32±0.69)较正常子宫内膜组织(1.00±0.54)明显升高,而CDKN1A蛋白在子宫内膜癌组织中的表达水平(0.71±0.14)较正常子宫内膜组织(1.00±0.11)明显降低(P0.001)。5 CARLo-5的表达水平与CDK2和CDKN1A蛋白表达水平的相关性子宫内膜癌组织中CDK2蛋白的表达水平与CARLo-5的表达呈正相关关系(r=0.30,P=0.043)。CDKN1A蛋白的表达与CARLo-5的表达呈负相关关系(r=-0.28,P=0.046)。结论:1 CARLo-5在子宫内膜癌组织中的表达较正常子宫内膜明显升高,提示CARLo-5可能在子宫内膜癌发生发展中发挥了促进作用。2 CARLo-5高表达与子宫内膜癌患者的临床分期、淋巴结转移和不良预后相关,提示CARLo-5可能成为子宫内膜癌判断预后的分子标志物。3 CDK2蛋白在子宫内膜癌组织中的表达水平较正常子宫内膜明显升高,CDKN1A蛋白表达水平明显降低,且CDKN1A和CDK2蛋白的表达与CARLo-5的表达水平具有明显相关性。这些结果提示,CARLo-5可能部分通过调节CDK2和CDKN1A蛋白的表达而在子宫内膜癌中发挥促癌作用。第二部分CARLo-5对子宫内膜癌细胞生物学行为的影响及机制研究目的:通过调节CARLo-5在子宫内膜癌细胞中的表达情况来探讨其对子宫内膜癌细胞生物学行为的影响及其可能的机制。方法:1采用MTS法检测敲低CARLo-5对子宫内膜癌细胞体外增殖活性的影响。2采用Transwell迁移、划痕愈合实验和Matrigel侵袭实验检测敲低CARLo-5对子宫内膜癌细胞体外迁移和侵袭能力的影响。3采用平板克隆形成实验检测敲低CARLo-5对子宫内膜癌细胞克隆形成能力的影响。4采用流式细胞术检测敲低CARLo-5对子宫内膜癌细胞增殖周期的影响。5采用Real-time PCR和Western-blot检测敲低CARLo-5对子宫内膜癌细胞中CDK2和CDKN1A基因及蛋白表达水平的影响。6采用Western-blot检测敲低CARLo-5对子宫内膜癌细胞中MMP2和MMP9蛋白表达水平的影响。结果:1敲低CARLo-5对子宫内膜癌细胞体外增殖活性的影响MTS法检测结果显示:敲低CARLo-5后子宫内膜癌细胞HEC-1-B和KLE的体外增殖活性明显降低(P0.05)。2敲低CARLo-5对子宫内膜癌细胞体外迁移和侵袭能力的影响Transwell迁移和划痕愈合实验结果显示:敲低CARLo-5后子宫内膜癌细胞HEC-1-B和KLE的体外迁移能力明显降低(P0.05);Matrigel侵袭实验结果显示:敲低CARLo-5后子宫内膜癌细胞HEC-1-B和KLE的体外侵袭能力明显降低(P0.05)。3敲低CARLo-5对子宫内膜癌细胞克隆形成能力的影响平板克隆形成实验结果显示:敲低CARLo-5后子宫内膜癌细胞HEC-1-B和KLE的克隆形成能力明显下降(P0.05)。4敲低CARLo-5对子宫内膜癌细胞增殖周期的影响流式细胞术结果显示:敲低CARLo-5后子宫内膜癌HEC-1-B和KLE细胞G0/G1期细胞数明显增多,S期细胞数明显减少(P0.05)。5敲低CARLo-5对子宫内膜癌细胞CDK2和CDKN1A的基因及蛋白表达水平的影响Real-time PCR和Western-blot结果显示:敲低CARLo-5后子宫内膜癌细胞中CDK2 m RNA和蛋白的表达水平明显降低(P0.05),而CDKN1A m RNA和蛋白的表达水平明显增高(P0.05)。6敲低CARLo-5对子宫内膜癌细胞MMP2和MMP9蛋白表达水平的影响Western-blot结果显示:敲低CARLo-5后子宫内膜癌细胞中MMP2和MMP9蛋白表达水平明显降低(P0.05)。结论:1敲低CARLo-5能抑制子宫内膜癌细胞的体外增殖、迁移和侵袭能力,提示CARLo-5的表达能够促进子宫内膜癌细胞的增殖、侵袭和转移,CARLo-5有望成为子宫内膜癌治疗的新的生物学靶标。2敲低CARLo-5将子宫内膜癌细胞阻滞在G0/G1期,并抑制CDK2 m RNA和蛋白水平的表达,促进CDKN1A m RNA和蛋白水平的表达,提示CARLo-5可能通过调控CDK2和CDKN1A的表达,抑制细胞G0/G1期阻滞,进而促进子宫内膜癌细胞的增殖。3敲低CARLo-5能够抑制子宫内膜癌细胞中MMP2和MMP9蛋白的表达,提示CARLo-5可能通过上调MMP2和MMP9蛋白的表达来促进子宫内膜癌的转移。第三部分敲低CARLo-5对子宫内膜癌细胞裸鼠移植瘤生长的影响及机制研究目的:检测稳定敲低CARLo-5对子宫内膜癌细胞在裸鼠体内的成瘤能力的影响,通过裸鼠体内实验来探讨其在子宫内膜癌发生发展中的作用机制。方法:1通过RNAi慢病毒技术构建稳定敲低CARLo-5的子宫内膜癌细胞株。2裸鼠皮下成瘤实验检测敲低CARLo-5对子宫内膜癌细胞体内成瘤能力的影响。3免疫组织化学法实验检测裸鼠移植瘤组织中CDK2和CDKN1A蛋白的表达水平。结果:1敲低CARLo-5在体内对子宫内膜癌细胞的影响sh RNA-1组、sh RNA-2组和对照组裸鼠移植瘤的平均体积分别为(157.66±37.93 mm3)、(102.81±58.33mm3)、(499.74±98.61 mm3),敲低CARLo-5组裸鼠移植瘤的生长速度和重量较未敲低组均明显降低(P0.05)。2敲低CARLo-5对裸鼠移植瘤组织中CDK2和CDKN1A蛋白的表达水平的影响免疫组织化学结果显示:敲低CARLo-5组裸鼠移植瘤组织中CDK2的蛋白表达水平较未敲低组明显降低(P0.05),而CDKN1A的蛋白表达水平较未敲低组明显增高(P0.05)。结论:敲低CARLo-5明显抑制子宫内膜癌细胞的体内增殖活性,其作用机制可能为CARLo-5抑制CDKN1A并促进CDK2蛋白表达,进而促进子宫内膜癌细胞的体内增殖,CARLo-5有望成为子宫内膜癌治疗的新的靶点。
[Abstract]:Endometrial carcinoma is one of the most common gynecologic malignancies in.2015 years. The number of new cases of endometrial cancer in China is about 63400, the mortality rate is about 21.8%. The number of new cases is rising year by year, and the incidence is younger in.2016. The new incidence of endometrial cancer in the United States is about 60050, and the second major gynecologic cancer after breast cancer. In spite of the serious harm to the health of women, although the level of medical diagnosis and treatment is very advanced, the exact pathogenesis of endometrial cancer is not clear and the treatment is very limited. Recently, the discovery of long chain non coded RNA (long non-coding RNA, lncRNA) provides molecular biology for the pathogenesis of endometrial carcinoma. Basic.LncRNA is a transcriptional transcript with a length of more than 200nt for proteins that can not be encoded. A large number of studies have found that it plays an important role in human life activities and the development of various diseases..CARLo-5 is a recently discovered lncRNA of 2628 nucleotides, located near the enhancer of the proto oncogene myc. It is reported that CARLo-5 is in the colon. Cancer, gastric cancer and other cancer tissues are up-regulated and play an important role in the development and progression of cancer. The diagnosis, evaluation and treatment of various cancers are of potential value. However, the expression and mechanism of CARLo-5 in endometrial carcinoma have not yet been reported. This study uses real-time fluorescent quantitative PCR (Real-time PCR). The expression of CARLo-5 gene in normal endometrium and endometrial carcinoma tissues was detected. The expression of cell cycle related proteins CDK2 and CDKN1A were detected by Western blot (Western-blot). The correlation between the expression level of CARLo-5 and the clinicopathological features and the total survival time of endometrial cancer patients was analyzed. The correlation between the expression level of CARLo-5 gene and the expression level of cell cycle related protein CDK2 and CDKN1A was analyzed. After the expression of CARLo-5 in low endometrial carcinoma cells in vitro, MTS, Transwell migration, Matrigel invasion test, flat clone formation experiment and flow cytometry were used to detect the biological line of endometrial cancer by knocking low CARLo-5 to endometrial carcinoma. In order to explore the mechanism of CARLo-5 in endometrial carcinoma, this study examined the effect of Western-blot on the expression of CDK2, CDKN1A, matrix metalloproteinase and MMP9 in endometrial carcinoma cells by knocking low CARLo-5. Through RNAi lentivirus technology, we constructed a stable knockout low CARLo-5 endometrial carcinoma cell line. The effect of knockout CARLo-5 on the proliferation of endometrial carcinoma cells in nude mice was tested. This study explored the role and mechanism of CARLo-5 in endometrial carcinoma in order to elucidate the molecular mechanism of endometrial cancer and the genetic diagnosis and treatment of endometrial carcinoma. The expression of CARLo-5 in endometrial carcinoma and its clinical significance: To investigate the expression of lncRNA CARLo-5 in endometrial carcinoma and to explore the relationship between the expression of CARLo-5 and the clinicopathological features and the total survival period of endometrial carcinoma. Expression level of cyclin related protein CDK2 and CDKN1A and their correlation with CARLo-5 expression. Methods: 1 the expression level of CARLo-5 in 108 endometrial carcinoma tissues and 66 normal endometrium tissues was detected by Real-time PCR, and Western-blot was used to detect the cell cycle phase in endometrial carcinoma and normal endometrium by Western-blot Expression of protein CDK2 and CDKN1A.3 analysis of the correlation between the expression level of CARLo-5 and the clinicopathological features and the total survival period of endometrial carcinoma.4 analysis of the correlation between the expression of CARLo-5 in endometrial carcinoma and the expression of cell cycle related protein CDK2 and CDKN1A. Fruit: 1 lncRNA CARLo-5 in endometrial carcinoma tissue The expression level of Real-time PCR in endometrium and normal endometrium showed that the expression level of CARLo-5 in endometrial carcinoma was significantly higher than that of normal endometrium (P0.001), the expression level of.2 CARLo-5 and the clinicopathological features of endometrial carcinoma patients, the expression level of CARLo-5 in endometrial carcinoma tissue The clinical stage of endometrial carcinoma is closely related to lymph node metastasis (P0.05). There is no significant correlation with age, pathological type, pathological grade, muscular layer invasion depth and vascular infiltration (P0.05), the correlation between the expression level of.3 CARLo-5 and the total survival period of endometrial cancer patients with endometrial carcinoma, the average of CARLo-5 high expression group of endometrium cancer patients The survival time (43 + 14.1 months) was significantly shorter than that of the CARLo-5 low expression group (52.5 + 9.1 months). The expression level of CDK2 and CDKN1A protein in endometrial carcinoma tissue of (P0.05).4 showed that the expression level of CDK2 protein in endometrial carcinoma (9.32 + 0.69) was significantly higher than that of normal endometrium (1 + 0.54), and CDKN1A The expression level of protein in endometrial carcinoma (0.71 + 0.14) was significantly lower than that of normal endometrium (1 + 0.11). The expression level of.5 CARLo-5 and the expression level of CDK2 and CDKN1A protein were correlated with the expression level of CDK2 protein in endometrial carcinoma and the expression of CARLo-5 was positively correlated (r=0.30, P=0.043).CDKN1A. The expression of protein was negatively correlated with the expression of CARLo-5 (r=-0.28, P=0.046). Conclusion: the expression of 1 CARLo-5 in endometrial carcinoma is significantly higher than that of normal endometrium, suggesting that CARLo-5 may play a role in the development of endometrial carcinoma, which may promote the clinical stage of.2 CARLo-5 high and endometrial cancer patients. Metastasis is associated with poor prognosis, suggesting that CARLo-5 may be a molecular marker for predicting the prognosis of endometrial carcinoma. The expression level of.3 CDK2 protein in endometrial carcinoma is significantly higher than that of normal endometrium, and the expression of CDKN1A protein is significantly reduced, and the expression of CDKN1A and CDK2 protein is significantly related to the expression level of CARLo-5. These results suggest that CARLo-5 may play a role in promoting cancer by regulating the expression of CDK2 and CDKN1A protein in endometrial carcinoma. Second the effect of part CARLo-5 on the biological behavior of endometrial carcinoma cells and its mechanism: To investigate the expression of CARLo-5 in endometrial carcinoma cells to explore the intrauterine The effects of membrane cancer cell biological behavior and its possible mechanisms. Methods: 1 the effects of MTS on the proliferation of endometrial carcinoma cells in vitro were detected by MTS method. Transwell migration, scratch healing experiment and Matrigel invasion test were used to detect the effect of CARLo-5 on the migration and invasion ability of endometrial carcinoma cells in vitro and.3 mining. The effect of knockout low CARLo-5 on the clone formation ability of endometrial cancer cells using a flat cloned clone.4 the effect of.4 on the proliferation cycle of endometrial carcinoma cells by flow cytometry;.5 and Real-time PCR and Western-blot were used to detect the CDK2 and CDKN1A genes and protein tables in endometrial cancer cells with Real-time PCR and Western-blot Effect of.6 on the expression of MMP2 and MMP9 protein in endometrial carcinoma cells by Western-blot. Results: 1 the effect of knockout low CARLo-5 on the proliferation activity of endometrial carcinoma cells in vitro MTS assay results showed that the proliferation activity of HEC-1-B and KLE in endometrial carcinoma cells after knocking low CARLo-5 was obvious. The effect of reduced (P0.05).2 knockout CARLo-5 on the migration and invasion ability of endometrial carcinoma cells in vitro, Transwell migration and scratch healing experiment showed that the migration ability of HEC-1-B and KLE in endometrial carcinoma cells decreased significantly after the knock at low CARLo-5 (P0.05); Matrigel invasion experiment results showed that endometrial cancer cells were H after low CARLo-5 The invasiveness of EC-1-B and KLE in vitro significantly decreased (P0.05).3 knockdown CARLo-5 effect on the clone formation ability of endometrial carcinoma cells. Experimental results showed that the clone formation ability of HEC-1-B and KLE in endometrial carcinoma cells decreased significantly after low CARLo-5 (P0.05).4 knockdown CARLo-5 on the proliferation cycle of endometrial carcinoma cells The results of flow cytometry showed that the number of G0/G1 cells in the endometrial carcinoma HEC-1-B and KLE cells increased significantly after the knockout of CARLo-5, and the number of cells in the S phase decreased significantly (P0.05) the effect of.5 knocking low CARLo-5 on the gene and protein expression level of CDK2 and CDKN1A in endometrial carcinoma cells The expression level of CDK2 m RNA and protein in endometrium cancer cells decreased significantly (P0.05), while the expression level of CDKN1A m RNA and protein increased significantly (P0.05).6 knock low CARLo-5 on endometrial carcinoma cells MMP2 and MMP9 protein expression level P0.05. Conclusion: 1 knockout low CARLo-5 can inhibit the proliferation, migration and invasion ability of endometrial carcinoma cells in vitro, suggesting that CARLo-5 expression can promote the proliferation, invasion and metastasis of endometrial cancer cells. CARLo-5 is expected to be a new biological target for the treatment of endometrial cancer,.2 knockdown CARLo-5 to make endometrial cancer cells Blocking the expression of CDK2 m RNA and protein levels and promoting the expression of CDKN1A m RNA and protein levels, suggesting that CARLo-5 may inhibit the G0/G1 phase block by regulating the expression of CDK2 and CDKN1A, and then promote the proliferation of endometrial cancer cells to inhibit the proliferation of endometrial carcinoma cells and inhibit the G0/G1 and the protein of endometrial cancer cells. Expression, suggesting that CARLo-5 may promote the metastasis of endometrial carcinoma by up regulation of the expression of MMP2 and MMP9 protein. Third the effect of low CARLo-5 on the growth of nude mice with endometrial cancer cells and its mechanism: to detect the effect of low CARLo-5 on the tumorigenicity of endometrial cancer cells in nude mice The mechanism of action in the development of endometrial carcinoma in mice in vivo. Methods: 1 the effect of RNAi Lentivirus on endometrial cancer cell line that stably knockdown CARLo-5 was constructed in.2 nude mice, the effects of low CARLo-5 on the tumorigenicity of endometrial carcinoma cells were detected by.3 immuno histochemical test in nude mice The expression level of CDK2 and CDKN1A protein in the transplanted tumor tissue. Results: 1 the effect of low CARLo-5 on the endometrial cancer cells in the body sh RNA-1 group, the average volume of the transplanted tumor in the SH RNA-2 group and the control group was (157.66 + 37.93 mm3), (102.81 + 58.33mm3), (499.74 + 98.61 mm3), and the growth rate of nude mice in the low CARLo-5 group was the growth rate and the growth rate of the nude mice. The effect of.2 knock low CARLo-5 on the expression level of CDK2 and CDKN1A protein in transplanted tumor tissues of nude mice was significantly lower than that in the lower group (P0.05). The immunohistochemical results showed that the protein expression level of CDK2 in the transplanted tumor tissues of the low CARLo-5 group was significantly lower than that in the lower group (P0.05), while the level of the CDKN1A protein expression was not knockout. The lower group was significantly increased (P0.05). Conclusion: the knock down CARLo-5 obviously inhibited the proliferation of endometrial carcinoma cells in vivo. The mechanism may be that CARLo-5 inhibits CDKN1A and promotes the expression of CDK2 protein, thus promoting the proliferation of endometrial cancer cells in vivo. CARLo-5 may be a new target for the treatment of endometrium carcinoma.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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