MicroRNA-430靶向调控CXCR7对膀胱癌细胞生物学行为的影响及其机制的研究
摘要
目的：
膀胱癌是人类常见的恶性肿瘤,然而其增殖和侵袭的分子机制依旧不明。
MicroRNAs (miRNAs)是一类微小的内源性非编码的RNA,通过与靶位点mRNA的3’端非翻译区UTR相互作用来抑制基因的表达。新近的数据表明,miRNAs在各种生物过程,包括人类癌症的发生发展侵袭转移的调控中发挥着重要的作用。mirna-430的研究主要是在斑马鱼上进行研究,miR-430已被证明与斑马鱼的早期胚胎发育有重要关系。然而,mir-430在涉及人类恶性肿瘤的调控上暂时没有报道。CXCR7是趋化因子SDF-1α即CXCL12的受体,其是一种具有7次跨膜的基质蛋白。CXCR7的高表达被很多研究报道在肿瘤的发生发展侵袭转移中有增强作用,建议CXCR7作为癌症治疗中一个有吸引力的目标。Yates等发现CXCR7的表达在膀胱癌组织中增高,并且其增高的水平伴随着高级别的肿瘤及肿瘤转移。此外,CXCR7已被证实通过多种途径与膀胱癌的增殖、迁移、侵袭有关,其中包括ERK,Stat3与AKT等信号通路.。本研究目的为验证miR-430靶向作用于CXCR7,并进一步阐述这种靶向作用对膀胱癌细胞的影响及对其作用机制进行初步的研究。
方法：
1、采用Real-time PCR检测miR-430在正常膀胱组织、癌旁组织及不同分期的膀胱癌组织中的表达情况。采用双荧光素酶报告系统验证miR-430与CXCR7之间的靶向调控关系。
2、构建miR-430过表达慢病毒载体和CXCR7过表达真核质粒载体。
3、采用MTT法、流式细胞检测法、TransWell法等常用方法对miR-430过表达慢病毒感染和CXCR7过表达质粒转染的人膀胱癌5637细胞进行实验,从细胞生长、细胞周期、侵袭能力、克隆形成的方面来检测miR-430靶向调控CXCR7对膀胱癌生物学行为的影响。
4、用Western-Blot方法来检测转染了miR-430过表达慢病毒和CXCR7过表达质粒的膀胱癌5637细胞中ERK、p-ERK、MMP-2、 MMP-9的表达情况,来初步探寻其对膀胱癌细胞生物学影响的机制。
结果：
1、miR-430在膀胱癌组织中表达较正常组织组和癌旁组织组降低,且在膀胱癌组织中随病理分期的增加而降低明显。通过双荧光素酶报告基因系统验证了miR-430与CXCR7之间存在靶向调控关系,miR-430能够降低CXCR7在膀胱癌细胞中的表达。
2.成功建立了miR-430过表达慢病毒载体及CXCR7过表达真核质粒载体。
3、在人膀胱癌5637细胞株中使mir-430的强制过表达明显抑制细胞增殖,迁移侵袭和克隆形成的能力,与CXCR7强制过表达的结果截然相反。
4.在人膀胱癌5637细胞株中miR-430的过表达能抑制ERK、 p-ERK、MMP-2、MMP-9的表达,而上调TFAM表达却促进ERK、 p-ERK、MMP-2、MMP-9的表达。
结论：
本研究首先发现在正常膀胱、癌旁组织和膀胱癌组织中的mir-430的异常表达。此外,在人膀胱癌5637细胞株中使mi-430的强制过表达明显抑制细胞增殖,迁移侵袭和克隆形成的效率,与CXCR7强制过表达的结果截然相反,而CXCR7在本研究中已经被验证是miR-430的直接靶向目标。我们进一步的研究表明,在膀胱癌5637细胞株中过表达miR-430后与细胞增殖和迁移相关的基因包括ERK, p-ERK, MMP2和MMP9均明显下调,而在过表达CXCR7的膀胱癌5637细胞株中这些基因的表达显著上调。我们的研究首次揭示了mi-430在膀胱癌细胞中靶向调控CXCR7从而对膀胱癌细胞的生物学行为进行调控的潜在机制。
Objective:
Bladder cancer is a common malignant tumor in human; however, the molecular mechanism underlying its growth and invasion remains unclear. MicroRNAs (miRNAs) are small, endogenous and non-coding RNAs that inhibit gene expression via interaction with target sites in the3'-untranslational region (UTRs) of mRNA.. Emerging data has shown that miRNAs play crucial roles in the regulation of various biological processes including the development of human cancers. MiRNA-430(miR-430) has mainly been researched in zebrafish, and has been shown to be associated with early embryo development. However, whether miR-430involves in the development of malignant tumors has not been reported.
CXCR7is7-transmembrane chemokine receptors of the stroma-derived factor (SDF-1α). Its expression has been reported to be enhanced during tumor development, suggesting that CXCR7is an attractive therapeutic target for cancer. Yates. et al. found that CXCR7expression was elevated in bladder cancer tissues and was associated with high-grade and metastasis. Moreover, CXCR7has been demonstrated to be associated with proliferation, migration and invasion of bladder cancer through several signaling pathways including ERK, Stat3and AKT signaling.
The present study firstly revealed a miR-430expression pattern in normal bladder, adjacent tissue and bladder cancer tissue. Moreover, forced overexpression of miR-430in human bladder cancer5637cells significantly inhibited cell proliferation, migration and colony-formation efficiency, entirely contrary to the results of forced overexpression of CXCR7, which was validated to be a direct target of miR-430in this study. Further analysis showed that cell proliferation-and migration-related genes including ERK, p-ERK, MMP2and MMP9were significantly downregulated in miR-430overexpressed5637cells, while markedly upregulated in CXCR7overexpressed5637cells. Our study reveals a novel role as well as a potential regulation mechanism of miR-430in bladder cancer cells. Methods:
1.The expression of miR-430was detected by Real-time PCR in the
bladder cancer, adjacent tissues and normal tissues.The regulatory relation between miR-430and CXCR7was tested and verified by the Dual-Luciferase reporter assays.2. Construct the vectors of miR-430and CXCR7overexpression
3. Effects of miR-430and CXCR7overexpression on proliferation,
cell cycle, colony-formation efficiency and migration of5637cells were detected by MTT assays, flow cytometry assays and Trans-Well.4. The expressions of ERK、p-ERK、MMP-2and MMP-9in cells
which were transfected with miR-430lentiviral vectors and CXCR7over-expression vectors were detected, to seek the signal control paths of their regulatory relation.
Results:
1.The bladder cancer tissues showed lower expression of miR-430th an normal tissue and adjacent tissues. Moreover, the higher stage of blad der cancer, the lower expression of miR-430. Dual Luciferase reporter assays Show that CXCR7was the direct target of miR-430.and miR-430can induce the expression of CXCR7.
2. The vectors of miR-430and CXCR7overexpression were constructed.
3. forced overexpression of miR-430in human bladder cancer5637cells significantly inhibited cell proliferation, migration and colony-formation efficiency, entirely contrary to the results of forced overexpression of CXCR7, which was validated to be a direct target of miR-430in this study.
4.The results indicated that the expressions of ERK,p-ERK, MMP-2and MMP-9was decreased in cells transfected with miR-430lentiviral v ectors, while increased in the cells transfected with CXCR7overexpression vectors when compared with controls.
膀胱癌是人类常见的恶性肿瘤,然而其增殖和侵袭的分子机制依旧不明。
MicroRNAs (miRNAs)是一类微小的内源性非编码的RNA,通过与靶位点mRNA的3’端非翻译区UTR相互作用来抑制基因的表达。新近的数据表明,miRNAs在各种生物过程,包括人类癌症的发生发展侵袭转移的调控中发挥着重要的作用。mirna-430的研究主要是在斑马鱼上进行研究,miR-430已被证明与斑马鱼的早期胚胎发育有重要关系。然而,mir-430在涉及人类恶性肿瘤的调控上暂时没有报道。CXCR7是趋化因子SDF-1α即CXCL12的受体,其是一种具有7次跨膜的基质蛋白。CXCR7的高表达被很多研究报道在肿瘤的发生发展侵袭转移中有增强作用,建议CXCR7作为癌症治疗中一个有吸引力的目标。Yates等发现CXCR7的表达在膀胱癌组织中增高,并且其增高的水平伴随着高级别的肿瘤及肿瘤转移。此外,CXCR7已被证实通过多种途径与膀胱癌的增殖、迁移、侵袭有关,其中包括ERK,Stat3与AKT等信号通路.。本研究目的为验证miR-430靶向作用于CXCR7,并进一步阐述这种靶向作用对膀胱癌细胞的影响及对其作用机制进行初步的研究。
方法：
1、采用Real-time PCR检测miR-430在正常膀胱组织、癌旁组织及不同分期的膀胱癌组织中的表达情况。采用双荧光素酶报告系统验证miR-430与CXCR7之间的靶向调控关系。
2、构建miR-430过表达慢病毒载体和CXCR7过表达真核质粒载体。
3、采用MTT法、流式细胞检测法、TransWell法等常用方法对miR-430过表达慢病毒感染和CXCR7过表达质粒转染的人膀胱癌5637细胞进行实验,从细胞生长、细胞周期、侵袭能力、克隆形成的方面来检测miR-430靶向调控CXCR7对膀胱癌生物学行为的影响。
4、用Western-Blot方法来检测转染了miR-430过表达慢病毒和CXCR7过表达质粒的膀胱癌5637细胞中ERK、p-ERK、MMP-2、 MMP-9的表达情况,来初步探寻其对膀胱癌细胞生物学影响的机制。
结果：
1、miR-430在膀胱癌组织中表达较正常组织组和癌旁组织组降低,且在膀胱癌组织中随病理分期的增加而降低明显。通过双荧光素酶报告基因系统验证了miR-430与CXCR7之间存在靶向调控关系,miR-430能够降低CXCR7在膀胱癌细胞中的表达。
2.成功建立了miR-430过表达慢病毒载体及CXCR7过表达真核质粒载体。
3、在人膀胱癌5637细胞株中使mir-430的强制过表达明显抑制细胞增殖,迁移侵袭和克隆形成的能力,与CXCR7强制过表达的结果截然相反。
4.在人膀胱癌5637细胞株中miR-430的过表达能抑制ERK、 p-ERK、MMP-2、MMP-9的表达,而上调TFAM表达却促进ERK、 p-ERK、MMP-2、MMP-9的表达。
结论：
本研究首先发现在正常膀胱、癌旁组织和膀胱癌组织中的mir-430的异常表达。此外,在人膀胱癌5637细胞株中使mi-430的强制过表达明显抑制细胞增殖,迁移侵袭和克隆形成的效率,与CXCR7强制过表达的结果截然相反,而CXCR7在本研究中已经被验证是miR-430的直接靶向目标。我们进一步的研究表明,在膀胱癌5637细胞株中过表达miR-430后与细胞增殖和迁移相关的基因包括ERK, p-ERK, MMP2和MMP9均明显下调,而在过表达CXCR7的膀胱癌5637细胞株中这些基因的表达显著上调。我们的研究首次揭示了mi-430在膀胱癌细胞中靶向调控CXCR7从而对膀胱癌细胞的生物学行为进行调控的潜在机制。
Objective:
Bladder cancer is a common malignant tumor in human; however, the molecular mechanism underlying its growth and invasion remains unclear. MicroRNAs (miRNAs) are small, endogenous and non-coding RNAs that inhibit gene expression via interaction with target sites in the3'-untranslational region (UTRs) of mRNA.. Emerging data has shown that miRNAs play crucial roles in the regulation of various biological processes including the development of human cancers. MiRNA-430(miR-430) has mainly been researched in zebrafish, and has been shown to be associated with early embryo development. However, whether miR-430involves in the development of malignant tumors has not been reported.
CXCR7is7-transmembrane chemokine receptors of the stroma-derived factor (SDF-1α). Its expression has been reported to be enhanced during tumor development, suggesting that CXCR7is an attractive therapeutic target for cancer. Yates. et al. found that CXCR7expression was elevated in bladder cancer tissues and was associated with high-grade and metastasis. Moreover, CXCR7has been demonstrated to be associated with proliferation, migration and invasion of bladder cancer through several signaling pathways including ERK, Stat3and AKT signaling.
The present study firstly revealed a miR-430expression pattern in normal bladder, adjacent tissue and bladder cancer tissue. Moreover, forced overexpression of miR-430in human bladder cancer5637cells significantly inhibited cell proliferation, migration and colony-formation efficiency, entirely contrary to the results of forced overexpression of CXCR7, which was validated to be a direct target of miR-430in this study. Further analysis showed that cell proliferation-and migration-related genes including ERK, p-ERK, MMP2and MMP9were significantly downregulated in miR-430overexpressed5637cells, while markedly upregulated in CXCR7overexpressed5637cells. Our study reveals a novel role as well as a potential regulation mechanism of miR-430in bladder cancer cells. Methods:
1.The expression of miR-430was detected by Real-time PCR in the
bladder cancer, adjacent tissues and normal tissues.The regulatory relation between miR-430and CXCR7was tested and verified by the Dual-Luciferase reporter assays.2. Construct the vectors of miR-430and CXCR7overexpression
3. Effects of miR-430and CXCR7overexpression on proliferation,
cell cycle, colony-formation efficiency and migration of5637cells were detected by MTT assays, flow cytometry assays and Trans-Well.4. The expressions of ERK、p-ERK、MMP-2and MMP-9in cells
which were transfected with miR-430lentiviral vectors and CXCR7over-expression vectors were detected, to seek the signal control paths of their regulatory relation.
Results:
1.The bladder cancer tissues showed lower expression of miR-430th an normal tissue and adjacent tissues. Moreover, the higher stage of blad der cancer, the lower expression of miR-430. Dual Luciferase reporter assays Show that CXCR7was the direct target of miR-430.and miR-430can induce the expression of CXCR7.
2. The vectors of miR-430and CXCR7overexpression were constructed.
3. forced overexpression of miR-430in human bladder cancer5637cells significantly inhibited cell proliferation, migration and colony-formation efficiency, entirely contrary to the results of forced overexpression of CXCR7, which was validated to be a direct target of miR-430in this study.
4.The results indicated that the expressions of ERK,p-ERK, MMP-2and MMP-9was decreased in cells transfected with miR-430lentiviral v ectors, while increased in the cells transfected with CXCR7overexpression vectors when compared with controls.
引文
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