DLC2表达调控结直肠癌发生、侵袭转移及预后的作用机制研究
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摘要
结直肠癌是世界上最常见的恶性肿瘤之一,由于其发病率、转移率、复发率以及死亡率均较高,使其成为一种严重威胁人类生命健康的恶性肿瘤。随着影像学、外科学的进步以及术后化疗的应用,结直肠癌的预后已经有所改善,但肿瘤的复发、转移仍是术后患者死亡的主要原因,且已成为提高患者生存期及改善预后的瓶颈。因此,深入研究结直肠癌发生、侵袭、转移的分子机制并努力探索有效的对抗复发、转移的治疗手段,对于进一步提高患者的生存期及改善预后有重要意义。
Rho GTP酶激活蛋白家族(GTPase-activating Proteins, GAPs)调控Rho GTP酶的活性。Rho GAPs的重要功能是作为信号传导的分子开关,将活化的Rho-GTP结合蛋白转变为失活的GDP结合状态,从而负性调节Rho GTP酶活性,由此参与粘附斑的形成和细胞骨架的重塑。
DLC蛋白亚家族属于Rho GAPs家族,由三个氨基酸序列高度同源的成员构成：DLCl、DLC2和DLC3。它们也都属于多结构域蛋白,含有SAM(sterile alpha motif)、RhoGAP(Rho GTPase-activating protein)和START(StAR-related lipid-transfer)三个结构域。DLC2(Deleted in liver cancer2)又被叫作STARD13,于2003年被成功克隆的一种抑癌基因,位于染色体13q12.3。目前对于DLC2的研究尚处起步阶段,至今还没有对DLC2在结直肠癌中的表达情况进行相关研究的报道；其次,作为一个与DLC1结构上高度同源的蛋白,DLC2是否在结直肠癌的发生、侵袭转移中也发挥重要作用,这仍是需要进一步研究的问题。
Rho家族蛋白是小G蛋白Ras超家族成员,它们是一组分子量为20KD～30KD的鸟苷酸结合蛋白,具有GTP酶活性。1985年,Rho作为Ras同源物首先被克隆出来,称Ras相关单体GTP酶,Rho具有RhoA、RhoB和RhoC三种异构体,Rho家族蛋白参与调节了细胞的多种生命过程,包括肌动蛋白的细胞骨架重组、细胞黏附、细胞运动、细胞周期进展、细胞分裂以及基因转录等。近年来研究发现,Rho家族蛋白与肿瘤发生、发展的多个方面均有联系,包括肿瘤的增殖、侵袭和转移、细胞凋亡、肿瘤新生血管的形成等。Rho家族,特别是RhoA在多种肿瘤组织中表达增加并和肿瘤恶性程度密切相关,提示其在肿瘤发生和发展中具有重要作用。目前,RhoA与恶性肿瘤侵袭与转移的关系已成为研究的热点之
由于DLC2蛋白的RhoGAP结构域能够激活Rho蛋白的GTP酶水解活性,使Rho蛋白由有活性的GTP结合态转化为无活性的GDP结合态,因此扮演了Rho蛋白家族负性调控因子的角色。同时近来研究已经证实DLC2可以通过抑制RhoA激酶的活性,调节基质金属蛋白酶(matrix metal protease, MMP)的产生,从而改变细胞形态,影响细胞迁移。这提示DLC2可能通过调控RhoA表达从而参与结直肠癌的发生、侵袭、转移过程,但其具体作用机制尚不清楚。
本课题首先检测结直肠癌组织及癌旁组织标本中DLC2mRNA及蛋白的表达情况,比较结直肠癌及癌旁组织之间表达差异,并且在临床病理变量中比较DLC2表达差异。然后,通过RNA干扰技术,抑制结直肠癌细胞系HT-29细胞中DLC2的表达水平,并综合利用一系列分子生物学和细胞生物学的方法研究DLC2调控结直肠癌发生、侵袭、转移的作用及分子机制。最后,我们通过检测RhoA mRNA及蛋白在结直肠癌组织、癌旁组织标本以及沉默DLC2表达的HT-29细胞系中的表达情况,初步阐述DLC2与RhoA对于结直肠癌的发生、侵袭、转移过程中可能发挥的作用。
DLC2在结直肠癌及癌旁组织中的表达情况及其与临床指标间的关系
目的了解DLC2在结直肠癌组织及其对应的癌旁组织中的表达情况并分析其与临床病理变量之间的关系。
方法(1)Real-time PCR方法检测DLC2mRNA在102例新鲜结直肠癌组织及其对应的癌旁组织中的表达水平。
(2) Western blot方法检测DLC2蛋白在102例新鲜结直肠癌及其对应的癌旁组织中的表达水平。
(3)免疫组化方法检测102例结直肠癌组织及其对应的癌旁组织中DLC2蛋白的分布情况和阳性表达率。
结果(1)Real-time PCR检测结果显示,在结直肠癌组织中DLC2mRNA表达水平低于相对应的癌旁组织,且具有统计学意义(p<0.05)。DLC2mRNA在临床病理变量中的表达情况：淋巴结转移阳性组低于淋巴结转移阴性组(p<0.05); TNM Ⅲ、Ⅳ期组低于TNMⅠ、Ⅱ期组(p<0.05)；低、未分化组明显低于中、高分化组(p<0.01)。
(2) Western blot印记检测结果显示,在结直肠癌组织中DLC2蛋白表达水平高于相对应的癌旁组织,但没有统计学差异(p>0.05)。DLC2蛋白在临床病理变量中的表达情况：淋巴结转移阳性组低于淋巴结转移阴性组(p<0.05); TNM Ⅲ、Ⅳ期组低于TNMⅠ、Ⅱ期组(p<0.05)。
(3)免疫组化法检测结果显示,DLC2阳性表达主要位于结直肠癌细胞浆内,结直肠癌石蜡组织中的DLC2蛋白表达阳性率及阳性评分均数高于癌旁组织,但无统计学差异(p>0.05)。
结论DLC2mRNA在结直肠癌组织中表达低于在癌旁组织；但是DLC2蛋白表达水平在结直肠癌和癌旁组织中无统计学差异；结直肠癌组织中DLC2mRNA和蛋白表达均与淋巴结转移和TNM分期相关；另外,DLC2mRNA表达与肿瘤组织学分级相关。
DLC2的过表达及低表达对结直肠癌细胞生物学行为的影响
目的利用RNA干扰技术抑制结直肠癌细胞中DLC2表达,研究DLC2对结直肠癌细胞生物学行为的影响。
方法(1)Western blot印记检测LOVO、SW-620、HT-29、CaCo-2、 HCT-8、HCT-116六株细胞中DLC2蛋白表达水平,筛选出DLC2表达最高的HT-29细胞株进行RNA干扰。
(2)脂质体LipofectamineTM2000转染HT-29细胞,获得HT-29、 HT-29DLC2RNAi-和HT-29DLC2RNAi+三组细胞用于后续试验。
(3) Real-time PCR检测HT-29、HT-29DLC2RNAi-和HT29DLC2RNAi+三组结直肠癌细胞株中DLC2mRNA表达情况。
(4) Western blot印记检测HT-29、HT-29DLC2RNAi-和DLC2RNAi+三组结直肠癌细胞株中DLC2蛋白表达量。
(5)细胞粘附实验、划痕愈合实验、Transwell侵袭实验分别比较HT-29、HT-29DLC2RNAi-和HT-29DLC2RNAi+三组结直肠癌细胞的粘附、迁移、侵袭能力。
(6)细胞免疫荧光实验比较HT-29、HT-29DLC2RNAi-和HT-29DLC2RNAi+三组结直肠癌细胞中DLC2蛋白表达情况并尝试比较三组细胞的形态差异。
(7)MTT、克隆形成实验和AnnexinV-FITC/PI双染色法、流式细胞技术比较HT-29、HT-29、DC2RNAi-和HT29DLC2RNAi+三组结直肠癌细胞增殖、凋亡能力。
结果(1)从LOVO、SW-620、HT-29、CaCo-2、HCT-8、HCT-116六株结直肠癌细胞中筛选出DLC2蛋白表达水平最高的HT-29细胞株,并获得满意的转染效率,成功获得DLC2表达沉默的HT-29DLC2RNAi+和无效对照的HT-29DLC2RNAi-以及空白对照HT-29细胞亚株。
(2)Real-time PCR检测结果显示,HT29DLC2RNAi+组细胞中的DLC2mRNA表达水平明显低于HT-29及HT-29DLC2RNAi-组细胞(p<0.01),证明HT-29DLC2RNAi+组抑制效率明显高于HT-29DLC2RNAi-组。
(3) Western blot印记检测结果显示,HT-29DLC2RNAi+组细胞中的DLC2蛋白表达水平明显低于HT-29及HT-29DLC2RNAi-组细胞(p<0.01),再次证实HT-29DLC2RNAi+组抑制效率明显高于HT29DLC2KNAi-组。
(4)细胞粘附实验、划痕愈合实验、Transwell侵袭实验结果显示,HT-29DLC2RNAi+组细胞较HT-29和HT-29DLC2RNAi-组细胞的粘附能力增强(p<0.05)；细胞迁移能力增强(p<0.05)；侵袭能力显著增强(p<0.01)。
(5)细胞免疫荧光检测结果显示,DLC2主要表达于细胞浆,且HT-29DLC2RNAi+组细胞与HT-29和HT-29DLC2RNAi-两组细胞比较,细胞膜表面有类似片状伪足样突起。
(6)MTT、克隆形成实验和Annexin V-FITC/PI双染色法、流式细胞技术检测结果显示,HT-29、HT-29DLC2RNAi-和HT-29DLC2RNAi+三组细胞增殖能力没有统计学差异(p>0.05)；而HT-29DLC2RNAi+组细胞的凋亡率以及凋亡细胞所占各周期细胞比例均明显低于HT-29、HT-29DLC2RNAi-两组细胞(p<0.01)
结论体外抑制DLC2的表达水平可显著增强结直肠癌细胞HT-29的侵袭、转移能力和凋亡能力,但并不影响结直肠癌细胞的增殖能力。
DLC2与RhoA在结直肠癌发生、侵袭、转移过程中的相互关系
目的了解RhoA在结直肠癌及癌旁组织中的表达情况以及其与临床病理变量之间的关系；通过RNA干扰技术,检测RhoA在DLC2表达抑制的HT-29细胞株中的表达情况,以初步研究DLC2与RhoA两者在结直肠癌发生、侵袭、转移过程中的相互关系。
方法
(1) Real-time PCR方法检测RhoA mRNA在102例结直肠癌组织及其对应的癌旁组织中的表达水平；同时检测RhoA mRNA在结直肠癌细胞株HT-29、HT-29DLC2RNAi-及HT-29DLC2RNAi+三组细胞中的表达水平。
(2) Western blot印记方法检测RhoA蛋白在102例结直肠癌组织及其对应的癌旁组织中的表达水平；同时检测RhoA蛋白在结直肠癌细胞株HT-29、HT-29DLC2RNAi-及HT-29DLC2RNAi+三组细胞中的表达水平。
(3)免疫组化方法检测102例结直肠癌组织及其对应的癌旁组织中RhoA蛋白的分布情况及阳性表达率。
结果
(1) Real-time PCR检测结果显示,在结直肠癌组织中RhoA mRNA表达水平明显高于相对应的癌旁组织(p<0.01)。RhoA mRNA在临床病理变量中的表达情况：淋巴结转移阳性组明显高于淋巴结转移阴性组(p<0.01); TNMⅢ、Ⅳ期组高于TNMⅠ、Ⅱ期组(p<0.05)；低、未分化组明显高于中、高分化组(p<0.01)。RhoA mRNA在HT-29DLC2RNAi+组细胞中的表达水平明显高于HT-29及HT-29DLC2RNAi-两组细胞(p<0.01)。
(2) Western blot印记检测结果显示,在结直肠癌组织中RhoA蛋白表达水平高于相对应的癌旁组织(p<0.01)。RhoA mRNA在临床病理变量中的表达情况：淋巴结转移阳性组高于淋巴结转移阴性组(p<0.05); TNMⅢ、Ⅳ期组明显高于TNMⅠ、Ⅱ期组(p<0.01)；低、未分化组明显高于中、高分化组(p<0.01)。RhoA蛋白在HT-29DLC2RNAi+组细胞中的表达水平高于HT-29及HT-29DLC2RNAi-两组细胞(p<0.05)。
(3)免疫组化检测结果显示,RhoA阳性表达主要位于结直肠癌细胞浆内,偶见细胞膜染色；结直肠癌组织与癌旁组织相比较,RhoA阳性表达率及阳性评分均数高于对应的癌旁组织,其中阳性表达率有统计学差异(p<0.05)；阳性评分均数有显著统计学差异(p<0.01)。
结论
RhoA mRNA及蛋白在结直肠癌组织中表达明显高于癌旁组织；RhoA mRNA和蛋白表达与淋巴结转移、TNM分期以及肿瘤分级相关；在结直肠癌细胞HT-29中,沉默DLC2表达能增加RhoA mRNA及蛋白的表达。
Colorectal cancer (CRC) is one of the most common malignant tumors in the world, because of its higher recurrence metastasis incidence and mortality rate, CRC becomes a serious threat to human life and health. With the progress of imageology, surgery and adjuvant chemotherapy, the prognosis of colorectal cancer has improved. However, the postoperative recurrence and metastasis are still the leading causes of death in patients with CRC, and have become the bottleneck of improving survival and prognosis of the patients. Therefore, further study in tumorigenesis, invasion and metastasis molecular mechanism of CRC and trying to explore the effective treatment against its recurrence and metastasis, are important to improve the long-term survival rate and prognosis of CRC patients.
Rho GTPase activating protein family (GTPase activating Proteins, GAPs) regulated of Rho GTPase, which act by switching between an inactive GDP-bound and an active GTP-bound conformation, with the latter form could negatively regulate Rho GTPase activity, which involved in the formation of the focal adhesion and the cytoskeletal reorganization.
DLC (Deleted in liver cancer) protein family, as a member of Rho GAPs family, has three members DLC1DLC2and DLC3, which have the highly relevant amino acid sequence. They all contain SAM (sterile alpha motif), RhoGAP (Rho GTPase activating protein) and START (StAR related lipid transfer) domains. DLC2(Deleted in liver cancer2), also called STARD13, was cloned in2003as a tumor suppressor gene, located in chromosome13q12.3. First, researches about DLC2were still in infancy, and there were no researches about DLC2expression in colorectal cancer; Second, as a protein closely related to DLC1, whether DLC2plays an important role in CRC invasion and metastasis is still need further research.
Rho family proteins are small G proteins of the Ras superfamily members. Rho proteins are guanylic acid binding protein. Their molecular weights are about20kd-30kd and have GTPase activity. In1985, Rho, as a Ras homologue, was first cloned and called Ras related monomer GTPase. Rho has RhoA, RhoB and RhoC three isomers, Rho family proteins were involved in regulating a variety of cell life processes, including actin cytoskeleton reorganization, cell adhesion, cell movement, cell cycle progression, cell division, and gene transcription. In recent years, studies had found that Rho family proteins were associated with many aspects of both tumorigenesis and tumor development, including tumor growth, proliferation, invasion, metastasis, cell apoptosis and tumor new blood vessels formation, etc. Rho family, especially RhoA, highly expressed in a wide variety of tumor tissues and associated with tumor malignant degree, this implied that Rho plays an important role in the tumorigenesis and metastasis. At present, RhoA and its relationship with tumor invasion and metastasis has become one of the hot spot.
Because of the RhoGAP domain of DLC2can activate the GTPase hydrolysis activity of Rho protein, which transform the Rho protein by GTP combinated active state into GDP combinated with inactive state. Thus, DLC2plays a negative regulatory factor of the Rho protein family. Recent studies have confirmed that the role of DLC2can inhibit the activity of RhoA kinase and adjust the generation of MMP (matrix metal proteases), this may interfere the cell shape changes and migration. It also implied that DLC2might through the regulation of RhoA expression level to participate the tumorigenesis, invasion and metastasis process of CRC, but the specific mechanism is still unclear.
In this study, we first detected DLC2mRNA and protein expression in CRC and PCIT (pericarcinomatous intestine tissues) specimens and investigated the relationships between DLC2expressions and clinicopathological parameters. Second, we investigated the role and molecular mechanism of DLC2in regulating of tumorigenesis, invasion and metastasis of CRC, comprehensive utilization of a series of molecular biology and cell biology methods after inhibiting the expression level of DLC2in HT-29by RNA interference technology. Last, we detected RhoA expression level in CRC and PCIT tissue specimens and investigated the relationships between RhoA expressions and clinicopathological parameters. Even more, we also detected the RhoA expression levels in HT-29cell lines, which DLC2expression were inhibit by RNA interference technology. Classification R656.9
The expression of DLC2in human colorectal cancer and the relationship between DLC2expression and clinic pathological parameters
Objective To investigate the expressions of DLC2in CRC tissues and PCIT, even more, to evaluated the expression levels of DLC2in subgroups by clinic pathological parameters.
Methods
(1) Real-time PCR was employed to detect DLC2mRNA expression in102CRC and PCIT specimens.
(2) Western blot was employed to detect DLC2protein expression in102CRC and PCIT specimens.
(3) Immunohistochemistry was employed to detect DLC2expressions in CRC and PCIT specimens.
Results
(1) CRC tissues revealed significantly lower level of DLC2mRNA than PCIT (p<0.05). The expression of DLC2mRNA was correlated with lymph node metastasis, tumor TNM stage and tumor histopathological degree significantly (p<0.05).
(2) The DLC2protein expression level had no significant difference between CRC and PCIT. The expression of DLC2protein was correlated with lymph node metastasis and tumor TNM stage significantly (p<0.05).
(3) According to the results of immunohistochemistry, the distribution of DLC2protein was mainly in the cytoplasm, and the positive expression rate and scores mean of DLC2in CRC were both higher than PCIT, however, there were no significant difference between them (p>0.05).
Conclusion
Our data strongly suggested that decreased DLC2expression in CRC correlates with lymph node metastasis and clinicopathologic stage of CRC, underexpression of DLC2is probably associated with poor prognosis in CRC patients.
Target to DLC2expression effect on the biological behaviour of colorectal cancer cells by small interfering RNAi
Objective To investigate the DLC2expression effect on the biological behaviours of the CRC cells.
Methods
(1) Western blot was used to detect the expression of DLC2in CRC cells:LOVO, SW-620, HT-29, CaCo-2, HCT-8and HCT-116, chosing the highest one HT-29cell in further RNAi.
(2) Using LipofectamineTM2000transfected the small interference RNA into HT-29cell, and acquired three groups of cells:HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
(3) Real-time PCR was employed to detect the expression of DLC2mRNA in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
(4) Western blot was employed to detect the expression of DLC2protein in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
(5) Wound-healing assay, transwell invasion assay, adhesion test were employed to detect migration, invasion and adhesion in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines
(6) Immunofluorescence assay was employed to detect the expression of DLC2and cell morphology in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
(7) MTT, colony formation and flow ctyometry assay were employed to detect proliferation and apoptosis in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
Results
(1) We chosed the HT-29cell line because its expression level of DLC2was the highest in LOVO, SW-620, HT-29, CaCo-2, HCT-8and HCT-116. In addition, we got satisfactory transfection efficiency and successfully acquired HT-29DLC2RNAi+with downregulated expression of DLC2and HT-29DLC2RNAi-as invalid control, HT-29as blank control.
(2) Real-time PCR results showed that the expression of DLC2mRNA in HT-29DLC2RNAi+cell line was much lower than HT-29and HT-29DLC2RNAi- cell lines (p<0.01), and suggested inhibition efficiency of DLC2in HT-29DLC2RNAi+
(3) Western blot results indicated that the expression of DLC2protein in HT-29DLC2RNAi+cell line was much lower than HT-29and HT-29DLC2RNAi-cell lines (p<0.01), also confirmed that the inhibition efficiency of DLC2in HT-29DLC2RNAi+was apparently higher than HT-29DLC2RNAi-cell line.
(4) The adhesion test demonstrated HT-29DLC2RNAi+cells had significantly higher adhesion ability than HT-29and HT-29DLC2RNAi-cells (p<0.05). Wound-healing assay showed that the closure of HT-29DLC2RNAi+was significantly more quickly than that of HT-29and HT-29DLC2RNAi-(p<0.05). Transwell invasion assay showed that the number of HT-29DLC2RNAi+cells passed through matrigel was much more as compared with HT-29and HT-29DLC2RNAi-cells (p<0.01).
(5) Cell immunofluorescence assay showed that DLC2positive expression was in cytoplasma and there was some protuberance likes pseudopodium in HT-29DLC2RNAi+cell membrane.
(6) There was no significant difference in cell proliferation between HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cells through MTT and colony formation experiments. However, the result of flow cytometry assay showed that the HT-29DLC2RNAi+cells apoptosis ratio and the ratio of the apoptosis cells in cell cycle was much lower than HT-29and HT-29DLC2RNAi-cells (p<0.01).
Conclusion
The down-regulate expression of DLC2may obviously stimulate invasion, metastasis and apoptosis ability of colorectal cancer cell lines in vitro, but may not affect the proliferation of colorectal cancer cell lines.
Association between DLC2and RhoA in tumorigenesis, invasion and metastasis of CRC
Objective To investigate the expression of RhoA in CRC tissues and PCIT and to evaluate the expression levels of DLC2in subgroups by clinic pathological parameters.Meanwhile, investigated the expression levels of RhoA in DLC2down-regulated HT-29cells by using RNAi.
Methods
(1) Real-time PCR was employed to detect RhoA mRNA expression in102CRC and PCIT specimens, as well as HT-29, HT-29DLC2RNAi" and HT-29DLC2RNAi+cell lines.
(2) Western blot was employed to detect DLC2protein expression in CRC and PCIT specimens, as well as HT-29, HT-29DLC2RNAi" and HT-29DLC2RNAi+cell lines.
(3) Immunohistochemistry was employed to detect DLC2expressions in CRC and PCIT specimens.
Results
(1) CRC tissues revealed significantly higher level of RhoA mRNA than PCIT (p<0.01). The expression of RhoA mRNA was correlated with lymph node metastasis (p<0.01), tumor TNM stage (p<0.05) and tumor histopathological degree significantly (p<0.01).Even more, RhoA mRNA expression level in HT-29DLC2RNAi+was significantly higher than HT-29and HT-29DLC2RNAi-cell lines (p<0.01).
(2) RhoA protein expression in CRC tissues also revealed significantly higher level than PCIT (p<0.01). The expression of RhoA protein was correlated with lymph node metastasis (p<0.05), tumor TNM stage (p<0.01) and tumor histopathological degree significantly (p<0.01). Even more, RhoA protein expression level in HT-29DLC2RNAi+was significantly higher than HT-29and HT-29DLC2RNAi-cell lines (p<0.05).
(3) According to the results of immunohistochemistry, the distribution of RhoA protein was mainly in cytoplasm, cytomembrane was not common. The positive expression rate and scores mean of RhoA protein in CRC were both higher than PCIT, and there are significant differences between them (p<0.05,p<0.01).
Conclusion
Our data strongly suggested that RhoA expression was increased in CRC. Its expressions correlated with lymph node metastasis, TNM stage and clinicopathologic stage of CRC. Down-regulate expression of DLC2probably stimulate the up-regulate expression of RhoA in HT-29cell line.
Rho GTP酶激活蛋白家族(GTPase-activating Proteins, GAPs)调控Rho GTP酶的活性。Rho GAPs的重要功能是作为信号传导的分子开关,将活化的Rho-GTP结合蛋白转变为失活的GDP结合状态,从而负性调节Rho GTP酶活性,由此参与粘附斑的形成和细胞骨架的重塑。
DLC蛋白亚家族属于Rho GAPs家族,由三个氨基酸序列高度同源的成员构成：DLCl、DLC2和DLC3。它们也都属于多结构域蛋白,含有SAM(sterile alpha motif)、RhoGAP(Rho GTPase-activating protein)和START(StAR-related lipid-transfer)三个结构域。DLC2(Deleted in liver cancer2)又被叫作STARD13,于2003年被成功克隆的一种抑癌基因,位于染色体13q12.3。目前对于DLC2的研究尚处起步阶段,至今还没有对DLC2在结直肠癌中的表达情况进行相关研究的报道；其次,作为一个与DLC1结构上高度同源的蛋白,DLC2是否在结直肠癌的发生、侵袭转移中也发挥重要作用,这仍是需要进一步研究的问题。
Rho家族蛋白是小G蛋白Ras超家族成员,它们是一组分子量为20KD～30KD的鸟苷酸结合蛋白,具有GTP酶活性。1985年,Rho作为Ras同源物首先被克隆出来,称Ras相关单体GTP酶,Rho具有RhoA、RhoB和RhoC三种异构体,Rho家族蛋白参与调节了细胞的多种生命过程,包括肌动蛋白的细胞骨架重组、细胞黏附、细胞运动、细胞周期进展、细胞分裂以及基因转录等。近年来研究发现,Rho家族蛋白与肿瘤发生、发展的多个方面均有联系,包括肿瘤的增殖、侵袭和转移、细胞凋亡、肿瘤新生血管的形成等。Rho家族,特别是RhoA在多种肿瘤组织中表达增加并和肿瘤恶性程度密切相关,提示其在肿瘤发生和发展中具有重要作用。目前,RhoA与恶性肿瘤侵袭与转移的关系已成为研究的热点之
由于DLC2蛋白的RhoGAP结构域能够激活Rho蛋白的GTP酶水解活性,使Rho蛋白由有活性的GTP结合态转化为无活性的GDP结合态,因此扮演了Rho蛋白家族负性调控因子的角色。同时近来研究已经证实DLC2可以通过抑制RhoA激酶的活性,调节基质金属蛋白酶(matrix metal protease, MMP)的产生,从而改变细胞形态,影响细胞迁移。这提示DLC2可能通过调控RhoA表达从而参与结直肠癌的发生、侵袭、转移过程,但其具体作用机制尚不清楚。
本课题首先检测结直肠癌组织及癌旁组织标本中DLC2mRNA及蛋白的表达情况,比较结直肠癌及癌旁组织之间表达差异,并且在临床病理变量中比较DLC2表达差异。然后,通过RNA干扰技术,抑制结直肠癌细胞系HT-29细胞中DLC2的表达水平,并综合利用一系列分子生物学和细胞生物学的方法研究DLC2调控结直肠癌发生、侵袭、转移的作用及分子机制。最后,我们通过检测RhoA mRNA及蛋白在结直肠癌组织、癌旁组织标本以及沉默DLC2表达的HT-29细胞系中的表达情况,初步阐述DLC2与RhoA对于结直肠癌的发生、侵袭、转移过程中可能发挥的作用。
DLC2在结直肠癌及癌旁组织中的表达情况及其与临床指标间的关系
目的了解DLC2在结直肠癌组织及其对应的癌旁组织中的表达情况并分析其与临床病理变量之间的关系。
方法(1)Real-time PCR方法检测DLC2mRNA在102例新鲜结直肠癌组织及其对应的癌旁组织中的表达水平。
(2) Western blot方法检测DLC2蛋白在102例新鲜结直肠癌及其对应的癌旁组织中的表达水平。
(3)免疫组化方法检测102例结直肠癌组织及其对应的癌旁组织中DLC2蛋白的分布情况和阳性表达率。
结果(1)Real-time PCR检测结果显示,在结直肠癌组织中DLC2mRNA表达水平低于相对应的癌旁组织,且具有统计学意义(p<0.05)。DLC2mRNA在临床病理变量中的表达情况：淋巴结转移阳性组低于淋巴结转移阴性组(p<0.05); TNM Ⅲ、Ⅳ期组低于TNMⅠ、Ⅱ期组(p<0.05)；低、未分化组明显低于中、高分化组(p<0.01)。
(2) Western blot印记检测结果显示,在结直肠癌组织中DLC2蛋白表达水平高于相对应的癌旁组织,但没有统计学差异(p>0.05)。DLC2蛋白在临床病理变量中的表达情况：淋巴结转移阳性组低于淋巴结转移阴性组(p<0.05); TNM Ⅲ、Ⅳ期组低于TNMⅠ、Ⅱ期组(p<0.05)。
(3)免疫组化法检测结果显示,DLC2阳性表达主要位于结直肠癌细胞浆内,结直肠癌石蜡组织中的DLC2蛋白表达阳性率及阳性评分均数高于癌旁组织,但无统计学差异(p>0.05)。
结论DLC2mRNA在结直肠癌组织中表达低于在癌旁组织；但是DLC2蛋白表达水平在结直肠癌和癌旁组织中无统计学差异；结直肠癌组织中DLC2mRNA和蛋白表达均与淋巴结转移和TNM分期相关；另外,DLC2mRNA表达与肿瘤组织学分级相关。
DLC2的过表达及低表达对结直肠癌细胞生物学行为的影响
目的利用RNA干扰技术抑制结直肠癌细胞中DLC2表达,研究DLC2对结直肠癌细胞生物学行为的影响。
方法(1)Western blot印记检测LOVO、SW-620、HT-29、CaCo-2、 HCT-8、HCT-116六株细胞中DLC2蛋白表达水平,筛选出DLC2表达最高的HT-29细胞株进行RNA干扰。
(2)脂质体LipofectamineTM2000转染HT-29细胞,获得HT-29、 HT-29DLC2RNAi-和HT-29DLC2RNAi+三组细胞用于后续试验。
(3) Real-time PCR检测HT-29、HT-29DLC2RNAi-和HT29DLC2RNAi+三组结直肠癌细胞株中DLC2mRNA表达情况。
(4) Western blot印记检测HT-29、HT-29DLC2RNAi-和DLC2RNAi+三组结直肠癌细胞株中DLC2蛋白表达量。
(5)细胞粘附实验、划痕愈合实验、Transwell侵袭实验分别比较HT-29、HT-29DLC2RNAi-和HT-29DLC2RNAi+三组结直肠癌细胞的粘附、迁移、侵袭能力。
(6)细胞免疫荧光实验比较HT-29、HT-29DLC2RNAi-和HT-29DLC2RNAi+三组结直肠癌细胞中DLC2蛋白表达情况并尝试比较三组细胞的形态差异。
(7)MTT、克隆形成实验和AnnexinV-FITC/PI双染色法、流式细胞技术比较HT-29、HT-29、DC2RNAi-和HT29DLC2RNAi+三组结直肠癌细胞增殖、凋亡能力。
结果(1)从LOVO、SW-620、HT-29、CaCo-2、HCT-8、HCT-116六株结直肠癌细胞中筛选出DLC2蛋白表达水平最高的HT-29细胞株,并获得满意的转染效率,成功获得DLC2表达沉默的HT-29DLC2RNAi+和无效对照的HT-29DLC2RNAi-以及空白对照HT-29细胞亚株。
(2)Real-time PCR检测结果显示,HT29DLC2RNAi+组细胞中的DLC2mRNA表达水平明显低于HT-29及HT-29DLC2RNAi-组细胞(p<0.01),证明HT-29DLC2RNAi+组抑制效率明显高于HT-29DLC2RNAi-组。
(3) Western blot印记检测结果显示,HT-29DLC2RNAi+组细胞中的DLC2蛋白表达水平明显低于HT-29及HT-29DLC2RNAi-组细胞(p<0.01),再次证实HT-29DLC2RNAi+组抑制效率明显高于HT29DLC2KNAi-组。
(4)细胞粘附实验、划痕愈合实验、Transwell侵袭实验结果显示,HT-29DLC2RNAi+组细胞较HT-29和HT-29DLC2RNAi-组细胞的粘附能力增强(p<0.05)；细胞迁移能力增强(p<0.05)；侵袭能力显著增强(p<0.01)。
(5)细胞免疫荧光检测结果显示,DLC2主要表达于细胞浆,且HT-29DLC2RNAi+组细胞与HT-29和HT-29DLC2RNAi-两组细胞比较,细胞膜表面有类似片状伪足样突起。
(6)MTT、克隆形成实验和Annexin V-FITC/PI双染色法、流式细胞技术检测结果显示,HT-29、HT-29DLC2RNAi-和HT-29DLC2RNAi+三组细胞增殖能力没有统计学差异(p>0.05)；而HT-29DLC2RNAi+组细胞的凋亡率以及凋亡细胞所占各周期细胞比例均明显低于HT-29、HT-29DLC2RNAi-两组细胞(p<0.01)
结论体外抑制DLC2的表达水平可显著增强结直肠癌细胞HT-29的侵袭、转移能力和凋亡能力,但并不影响结直肠癌细胞的增殖能力。
DLC2与RhoA在结直肠癌发生、侵袭、转移过程中的相互关系
目的了解RhoA在结直肠癌及癌旁组织中的表达情况以及其与临床病理变量之间的关系；通过RNA干扰技术,检测RhoA在DLC2表达抑制的HT-29细胞株中的表达情况,以初步研究DLC2与RhoA两者在结直肠癌发生、侵袭、转移过程中的相互关系。
方法
(1) Real-time PCR方法检测RhoA mRNA在102例结直肠癌组织及其对应的癌旁组织中的表达水平；同时检测RhoA mRNA在结直肠癌细胞株HT-29、HT-29DLC2RNAi-及HT-29DLC2RNAi+三组细胞中的表达水平。
(2) Western blot印记方法检测RhoA蛋白在102例结直肠癌组织及其对应的癌旁组织中的表达水平；同时检测RhoA蛋白在结直肠癌细胞株HT-29、HT-29DLC2RNAi-及HT-29DLC2RNAi+三组细胞中的表达水平。
(3)免疫组化方法检测102例结直肠癌组织及其对应的癌旁组织中RhoA蛋白的分布情况及阳性表达率。
结果
(1) Real-time PCR检测结果显示,在结直肠癌组织中RhoA mRNA表达水平明显高于相对应的癌旁组织(p<0.01)。RhoA mRNA在临床病理变量中的表达情况：淋巴结转移阳性组明显高于淋巴结转移阴性组(p<0.01); TNMⅢ、Ⅳ期组高于TNMⅠ、Ⅱ期组(p<0.05)；低、未分化组明显高于中、高分化组(p<0.01)。RhoA mRNA在HT-29DLC2RNAi+组细胞中的表达水平明显高于HT-29及HT-29DLC2RNAi-两组细胞(p<0.01)。
(2) Western blot印记检测结果显示,在结直肠癌组织中RhoA蛋白表达水平高于相对应的癌旁组织(p<0.01)。RhoA mRNA在临床病理变量中的表达情况：淋巴结转移阳性组高于淋巴结转移阴性组(p<0.05); TNMⅢ、Ⅳ期组明显高于TNMⅠ、Ⅱ期组(p<0.01)；低、未分化组明显高于中、高分化组(p<0.01)。RhoA蛋白在HT-29DLC2RNAi+组细胞中的表达水平高于HT-29及HT-29DLC2RNAi-两组细胞(p<0.05)。
(3)免疫组化检测结果显示,RhoA阳性表达主要位于结直肠癌细胞浆内,偶见细胞膜染色；结直肠癌组织与癌旁组织相比较,RhoA阳性表达率及阳性评分均数高于对应的癌旁组织,其中阳性表达率有统计学差异(p<0.05)；阳性评分均数有显著统计学差异(p<0.01)。
结论
RhoA mRNA及蛋白在结直肠癌组织中表达明显高于癌旁组织；RhoA mRNA和蛋白表达与淋巴结转移、TNM分期以及肿瘤分级相关；在结直肠癌细胞HT-29中,沉默DLC2表达能增加RhoA mRNA及蛋白的表达。
Colorectal cancer (CRC) is one of the most common malignant tumors in the world, because of its higher recurrence metastasis incidence and mortality rate, CRC becomes a serious threat to human life and health. With the progress of imageology, surgery and adjuvant chemotherapy, the prognosis of colorectal cancer has improved. However, the postoperative recurrence and metastasis are still the leading causes of death in patients with CRC, and have become the bottleneck of improving survival and prognosis of the patients. Therefore, further study in tumorigenesis, invasion and metastasis molecular mechanism of CRC and trying to explore the effective treatment against its recurrence and metastasis, are important to improve the long-term survival rate and prognosis of CRC patients.
Rho GTPase activating protein family (GTPase activating Proteins, GAPs) regulated of Rho GTPase, which act by switching between an inactive GDP-bound and an active GTP-bound conformation, with the latter form could negatively regulate Rho GTPase activity, which involved in the formation of the focal adhesion and the cytoskeletal reorganization.
DLC (Deleted in liver cancer) protein family, as a member of Rho GAPs family, has three members DLC1DLC2and DLC3, which have the highly relevant amino acid sequence. They all contain SAM (sterile alpha motif), RhoGAP (Rho GTPase activating protein) and START (StAR related lipid transfer) domains. DLC2(Deleted in liver cancer2), also called STARD13, was cloned in2003as a tumor suppressor gene, located in chromosome13q12.3. First, researches about DLC2were still in infancy, and there were no researches about DLC2expression in colorectal cancer; Second, as a protein closely related to DLC1, whether DLC2plays an important role in CRC invasion and metastasis is still need further research.
Rho family proteins are small G proteins of the Ras superfamily members. Rho proteins are guanylic acid binding protein. Their molecular weights are about20kd-30kd and have GTPase activity. In1985, Rho, as a Ras homologue, was first cloned and called Ras related monomer GTPase. Rho has RhoA, RhoB and RhoC three isomers, Rho family proteins were involved in regulating a variety of cell life processes, including actin cytoskeleton reorganization, cell adhesion, cell movement, cell cycle progression, cell division, and gene transcription. In recent years, studies had found that Rho family proteins were associated with many aspects of both tumorigenesis and tumor development, including tumor growth, proliferation, invasion, metastasis, cell apoptosis and tumor new blood vessels formation, etc. Rho family, especially RhoA, highly expressed in a wide variety of tumor tissues and associated with tumor malignant degree, this implied that Rho plays an important role in the tumorigenesis and metastasis. At present, RhoA and its relationship with tumor invasion and metastasis has become one of the hot spot.
Because of the RhoGAP domain of DLC2can activate the GTPase hydrolysis activity of Rho protein, which transform the Rho protein by GTP combinated active state into GDP combinated with inactive state. Thus, DLC2plays a negative regulatory factor of the Rho protein family. Recent studies have confirmed that the role of DLC2can inhibit the activity of RhoA kinase and adjust the generation of MMP (matrix metal proteases), this may interfere the cell shape changes and migration. It also implied that DLC2might through the regulation of RhoA expression level to participate the tumorigenesis, invasion and metastasis process of CRC, but the specific mechanism is still unclear.
In this study, we first detected DLC2mRNA and protein expression in CRC and PCIT (pericarcinomatous intestine tissues) specimens and investigated the relationships between DLC2expressions and clinicopathological parameters. Second, we investigated the role and molecular mechanism of DLC2in regulating of tumorigenesis, invasion and metastasis of CRC, comprehensive utilization of a series of molecular biology and cell biology methods after inhibiting the expression level of DLC2in HT-29by RNA interference technology. Last, we detected RhoA expression level in CRC and PCIT tissue specimens and investigated the relationships between RhoA expressions and clinicopathological parameters. Even more, we also detected the RhoA expression levels in HT-29cell lines, which DLC2expression were inhibit by RNA interference technology. Classification R656.9
The expression of DLC2in human colorectal cancer and the relationship between DLC2expression and clinic pathological parameters
Objective To investigate the expressions of DLC2in CRC tissues and PCIT, even more, to evaluated the expression levels of DLC2in subgroups by clinic pathological parameters.
Methods
(1) Real-time PCR was employed to detect DLC2mRNA expression in102CRC and PCIT specimens.
(2) Western blot was employed to detect DLC2protein expression in102CRC and PCIT specimens.
(3) Immunohistochemistry was employed to detect DLC2expressions in CRC and PCIT specimens.
Results
(1) CRC tissues revealed significantly lower level of DLC2mRNA than PCIT (p<0.05). The expression of DLC2mRNA was correlated with lymph node metastasis, tumor TNM stage and tumor histopathological degree significantly (p<0.05).
(2) The DLC2protein expression level had no significant difference between CRC and PCIT. The expression of DLC2protein was correlated with lymph node metastasis and tumor TNM stage significantly (p<0.05).
(3) According to the results of immunohistochemistry, the distribution of DLC2protein was mainly in the cytoplasm, and the positive expression rate and scores mean of DLC2in CRC were both higher than PCIT, however, there were no significant difference between them (p>0.05).
Conclusion
Our data strongly suggested that decreased DLC2expression in CRC correlates with lymph node metastasis and clinicopathologic stage of CRC, underexpression of DLC2is probably associated with poor prognosis in CRC patients.
Target to DLC2expression effect on the biological behaviour of colorectal cancer cells by small interfering RNAi
Objective To investigate the DLC2expression effect on the biological behaviours of the CRC cells.
Methods
(1) Western blot was used to detect the expression of DLC2in CRC cells:LOVO, SW-620, HT-29, CaCo-2, HCT-8and HCT-116, chosing the highest one HT-29cell in further RNAi.
(2) Using LipofectamineTM2000transfected the small interference RNA into HT-29cell, and acquired three groups of cells:HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
(3) Real-time PCR was employed to detect the expression of DLC2mRNA in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
(4) Western blot was employed to detect the expression of DLC2protein in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
(5) Wound-healing assay, transwell invasion assay, adhesion test were employed to detect migration, invasion and adhesion in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines
(6) Immunofluorescence assay was employed to detect the expression of DLC2and cell morphology in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
(7) MTT, colony formation and flow ctyometry assay were employed to detect proliferation and apoptosis in HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cell lines.
Results
(1) We chosed the HT-29cell line because its expression level of DLC2was the highest in LOVO, SW-620, HT-29, CaCo-2, HCT-8and HCT-116. In addition, we got satisfactory transfection efficiency and successfully acquired HT-29DLC2RNAi+with downregulated expression of DLC2and HT-29DLC2RNAi-as invalid control, HT-29as blank control.
(2) Real-time PCR results showed that the expression of DLC2mRNA in HT-29DLC2RNAi+cell line was much lower than HT-29and HT-29DLC2RNAi- cell lines (p<0.01), and suggested inhibition efficiency of DLC2in HT-29DLC2RNAi+
(3) Western blot results indicated that the expression of DLC2protein in HT-29DLC2RNAi+cell line was much lower than HT-29and HT-29DLC2RNAi-cell lines (p<0.01), also confirmed that the inhibition efficiency of DLC2in HT-29DLC2RNAi+was apparently higher than HT-29DLC2RNAi-cell line.
(4) The adhesion test demonstrated HT-29DLC2RNAi+cells had significantly higher adhesion ability than HT-29and HT-29DLC2RNAi-cells (p<0.05). Wound-healing assay showed that the closure of HT-29DLC2RNAi+was significantly more quickly than that of HT-29and HT-29DLC2RNAi-(p<0.05). Transwell invasion assay showed that the number of HT-29DLC2RNAi+cells passed through matrigel was much more as compared with HT-29and HT-29DLC2RNAi-cells (p<0.01).
(5) Cell immunofluorescence assay showed that DLC2positive expression was in cytoplasma and there was some protuberance likes pseudopodium in HT-29DLC2RNAi+cell membrane.
(6) There was no significant difference in cell proliferation between HT-29, HT-29DLC2RNAi-and HT-29DLC2RNAi+cells through MTT and colony formation experiments. However, the result of flow cytometry assay showed that the HT-29DLC2RNAi+cells apoptosis ratio and the ratio of the apoptosis cells in cell cycle was much lower than HT-29and HT-29DLC2RNAi-cells (p<0.01).
Conclusion
The down-regulate expression of DLC2may obviously stimulate invasion, metastasis and apoptosis ability of colorectal cancer cell lines in vitro, but may not affect the proliferation of colorectal cancer cell lines.
Association between DLC2and RhoA in tumorigenesis, invasion and metastasis of CRC
Objective To investigate the expression of RhoA in CRC tissues and PCIT and to evaluate the expression levels of DLC2in subgroups by clinic pathological parameters.Meanwhile, investigated the expression levels of RhoA in DLC2down-regulated HT-29cells by using RNAi.
Methods
(1) Real-time PCR was employed to detect RhoA mRNA expression in102CRC and PCIT specimens, as well as HT-29, HT-29DLC2RNAi" and HT-29DLC2RNAi+cell lines.
(2) Western blot was employed to detect DLC2protein expression in CRC and PCIT specimens, as well as HT-29, HT-29DLC2RNAi" and HT-29DLC2RNAi+cell lines.
(3) Immunohistochemistry was employed to detect DLC2expressions in CRC and PCIT specimens.
Results
(1) CRC tissues revealed significantly higher level of RhoA mRNA than PCIT (p<0.01). The expression of RhoA mRNA was correlated with lymph node metastasis (p<0.01), tumor TNM stage (p<0.05) and tumor histopathological degree significantly (p<0.01).Even more, RhoA mRNA expression level in HT-29DLC2RNAi+was significantly higher than HT-29and HT-29DLC2RNAi-cell lines (p<0.01).
(2) RhoA protein expression in CRC tissues also revealed significantly higher level than PCIT (p<0.01). The expression of RhoA protein was correlated with lymph node metastasis (p<0.05), tumor TNM stage (p<0.01) and tumor histopathological degree significantly (p<0.01). Even more, RhoA protein expression level in HT-29DLC2RNAi+was significantly higher than HT-29and HT-29DLC2RNAi-cell lines (p<0.05).
(3) According to the results of immunohistochemistry, the distribution of RhoA protein was mainly in cytoplasm, cytomembrane was not common. The positive expression rate and scores mean of RhoA protein in CRC were both higher than PCIT, and there are significant differences between them (p<0.05,p<0.01).
Conclusion
Our data strongly suggested that RhoA expression was increased in CRC. Its expressions correlated with lymph node metastasis, TNM stage and clinicopathologic stage of CRC. Down-regulate expression of DLC2probably stimulate the up-regulate expression of RhoA in HT-29cell line.
引文
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