SIPA1通过下调Rap1-ERK通路抑制HCC的复发转移
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摘要
肝细胞癌(Hepatocellular carcinoma, HCC)是当前严重威胁我国人民生命健康的最主要恶性肿瘤之一。随着肝脏外科技术以及麻醉与围手术期处理水平的进步,肝癌的手术切除率显著提高,在一些大的肝脏外科中心肝癌手术死亡率已接近为零。尽管如此,以5年生存率为标志的肝癌整体治疗水平仍无显著改善,究其原因在于肝癌术后的复发转移率居高不下。据统计,肝癌切除术后5年的复发率高于60%,小肝癌也在40%以上。显然,肝癌的复发、转移已成为严重制约肝癌患者长期存活的关键因素。因此,深入研究肝癌复发转移机制,积极探索有效的抗复发治疗措施是当前肝癌诊治中的重点和难点,对于进一步改善肝癌患者长期存活率具有重要意义。
已有的研究证实,SIPA1 (Signal-induced Proliferation-associated Gene 1,信号诱导的增殖相关基因1)在血液系统肿瘤的发生发展过程中发挥了非常重要的作用。新近的研究表明小鼠sipa1是决定小鼠乳腺癌转移倾向的重要基因。众所周知,肝脏是胚胎时期的重要造血器官,血细胞(如血小板)与肿瘤的转移密切相关。根据以上的研究结果我们推测SIPA1基因很可能在HCC复发转移中发挥重要作用。因此,本课题在研究SIPA1在临床肝癌病例及肝癌细胞系中表达情况的基础上,进一步通过质粒构建转染技术上调SIPA1的表达,并综合利用一系列体外、体内方法研究SIPA1调控HCC侵袭转移的分子机制。我们获得了如下研究结果：
1.我们首先采用RT-PCR和Western blot等方法检测了SIPA1 mRNA和蛋白在30例新鲜HCC组织及相应邻近非癌肝组织(ANLT)中的表达水平,结果发现与ANLT比较,SIPA1 mRNA和蛋白水平在癌组织中表达均显著下调(P<0.05),且肝癌组织中SIPA1 mRNA和蛋白表达水平呈明显正相关(r=0.757,P<0.001)。进一步采用实时定量PCR检测显示ANLT中SIPA1 mRNA表达量为癌组织的4.17倍。进而,我们采用免疫组化技术检测了130例HCC组织中SIPA1的表达,结果显示SIPA1主要分布于细胞浆内,其在癌组织的阳性表达率为62.3%(81／130)。肝癌组织中SIPA1低表达与肿瘤多结节,分化差及静脉侵袭密切相关(P<0.05)。SIPA1阴性表达组HCC患者的总体生存时间显著短于SIPA1阳性表达组的患者(41.0±6.6个月vs.66.0±5.2个月,P=0.003),SIPA1阴性表达组的无瘤生存时间亦显著短于SIPA1阳性表达组(35.0±6.1个月vs.61.1±5.4个月,P=0.002)。多元Cox回归模型显示SIPA1阴性表达是HCC预后的独立危险因素。
2. RT-PCR及Western blot方法检测SIPA1 mRNA和蛋白在HepG2、MHCC97-L和HCCLM3这3种侵袭转移潜能依次升高的肝癌细胞系中的表达水平,并以正常肝细胞系L02、常氏肝细胞系CCL13作为对照。结果显示SIPA1 mRNA和蛋白在HepG2、HCCLM3和MHCC97-L肝癌细胞系中的表达水平显著低于CCL13和L02正常肝细胞系(P<0.05),且其表达水平在HepG2. MHCC97-L、HCCLM3细胞中依次降低(P<0.05),提示SIPA1表达水平与肝癌细胞的侵袭转移潜能呈负相关。同时确定以SIPA1表达水平最低的HCCLM3为过表达质粒的靶细胞。
3.我们构建了重组质粒pcDNA3.1-SIPA1,使用Lipofectamine 2000转染并行G418筛选,经RT-PCR和Western blot等方法验证SIPA1的表达,最终获得了稳定转染细胞系HCCLM3SIPA1+和空质粒对照组HCCLM3vector细胞。采用MTT法、粘附实验、划痕愈合实验和Transwell等实验方法分别检测两组细胞在细胞增殖与粘附能力、迁移运动能力和侵袭能力上的差别。MTT研究结果显示,HCCLM3SIPA1+的细胞增殖能力较HCCLM3vector显著下降(P<0.05)；粘附实验结果显示与HCCLM3vector细胞相比,HCCLM3SIPA1+细胞与FN的粘附能力明显减弱(P<0.05)；划痕愈合实验和Transwell侵袭实验显示HCCLM3SIPA1+与HCCLM3vector相比,细胞的迁移运动能力和侵袭能力均显著下降(P<0.05)。以上体外实验研究结果证实上调SIPA1的表达可显著抑制HCC细胞的增殖、粘附、迁移和侵袭能力。进一步采用免疫荧光技术检测HCCLM3SIPA1+细胞和HCCLM3vector细胞的骨架蛋白F-actin的形成情况,发现SIPA1能明显减少F-actin的形成。
4.为进一步体内观察上调SIPA1对肝癌侵袭转移的影响,我们建立了裸鼠原位肝癌肺转移模型。结果显示,HCCLM3SIPA1+细胞在肝脏中成瘤体积明显较HCCLM3vector细胞小,分别为1.57±0.08cm3和6.28±0.13cm3,(P<0.05)。通过裸鼠肺组织连续切片观察肺转移灶的发生率,发现与HCCLM3vector组相比,HCCLM3SIPA1+组的肺转移率显著下降(16.7%vs.83.3%,P=0.021)。由此证明,上调SIPA1可显著抑制肝癌细胞的体内成瘤能力和侵袭转移能力。
5.综合已有的研究,我们推测SIPA1可能通过Rapl-ERK信号传导通路调控肝癌细胞的侵袭转移。为证实上述假说,我们首先检测了HCCLM3SIPA1+细胞和HCCLM3Vector细胞中Rap1的活性,研究发现与后者相比,过表达SIPA1的HCCLM3SIPA1+细胞中,Rap1的活性显著降低(P<0.05)；免疫共沉淀技术亦证实HCCLM3SIPA1+细胞中SIPA1与Rap1存在直接的相互作用。进而,我们采用Rap1激活因子8CPT-2Me-cAMP(终浓度100μM),恢复HCCLM3SIPA1+细胞中Rap1的活性,反向验证Rap1在SIPA1调控肝癌侵袭转移中的作用。结果发现,恢复Rap1的活性后,细胞的迁移运动能力和和侵袭能力得到显著恢复(P<0.05)且细胞骨架蛋白F-actin表达亦得到明显恢复。Western blot证实HCCLM3SIPA1+细胞中Rap1下游基因ERK的磷酸化水平显著下降(P<0.05),恢复Rap1活性后p-ERK的表达得到恢复。上述研究结果证实,SIPA1可通过下调Rap1-ERK信号传导通路抑制肝癌细胞的侵袭转移。
综上所述,我们首次发现SIPA1在肝细胞癌组织中的表达显著下调,其表达水平与肝癌分化差、多结节、静脉侵犯等临床病理特征密切相关,同时与肝细胞癌预后差和术后早期发生复发转移等密切相关。SIPA1是肝细胞癌预后的独立危险因素,可作为预测肝细胞癌术后生存和复发转移的分子标志物。体内、体外研究证明,SIPA1通过下调Rap1-ERK信号通路抑制肝细胞癌的增殖和侵袭转移能力。
Hepatocellular carcinoma (HCC) is a main malignancy that threaten our people now. With the improvement of liver surgical technique and management level of perioperation, the resection rate of HCC is increasing and the operation mortality rate is approaching zero at large liver surgery centers. But the overall mortality of HCC patients remains high and the 5-year survial rate of patients is still low. The postoperation recurrence and metastasis of HCC is the main cause of high overall mortality. The 5-year recurrence rate of HCC after operation is more than 60%, even the small HCC the 5-year recurrence rate is above 40%. Obviously the recurrence and metastasis after HCC resection is the bottleneck problem that severely restricts the improvement of long survival rate of HCC patients. So deep study of the mechanism of recurrence and metastasis of HCC and explore the methods of anti-metastasis are focus of HCC research, which are very meaningful for improvement of long survival rate of HCC patients.
Signal-induced Proliferation-associated Gene 1 (SIPA1) was found play an important role in the blood system tumors in the past decade. Recent research found that mouse sipal gene was an important gene that control the metastasis of mouse breast cancer. It is well-known that the liver is a hematogenic organ in embryonic period and blood cells (for example platelet) had close relationship with tumor metastasis. These facts urge us to explore the role of SIPA1 gene in the HCC which had high potential of metastasis. Up to now there is no report about this both at home and abroad. So in this study we first examine the expression of SIPA1 gene in HCC specimens and HCC cell lines, then overexpress the gene by cotransfection the recombinant plasmid (pcDNA3.1-SIPA1) with HCC cell line. Molecullar mechenism of the regulation of SIPA1 to HCC metastasis was studied by in vitro and in vivo. The results as follows was observed:
1. The SIPA1 mRNA and protein in 30 fresh HCC specimens, which consisted of cancer tissue and corresponding ANLT(adjacent non-tumor liver tissue), was detected by Reverse Transcription PCR(Polymerase chain reaction) and Western blot.The expression of mRNA and protein of SIPA1 in fresh HCC tissues was lower than in ANLTs significantly(P<0.05). The expression of SIPA1 mRNA was positively correlated with SIPA1 protein(r=0.757, P<0.001). The real time PCR was used to detect the relative accurate exprssion of SIPA1 mRNA, and the expression of SIPA1 mRNA in ANLT was found to be 4.17 times of that in cancer tissue. The expression of SIPA1 in 130 paraffin HCC specimens was dectected by immunohistochemistry, the positive expression rate of SIPA1 protein in cancer tissue was 62.3% (81/130). Low expression of SIPA1 in cancer tissue of HCC had close relationship with multiple tumor nodules, poor differentiation and vein invasion (P<0.05). Patients with negative expression of SIPA1 in cancer tissue had a shorter overall survial (OS) than the positive group (41.0±6.6 months vs. 66.0±5.2 months, P=0.003), the disease-free survial (DFS) of negative group of SIPA1 was shorter than the positive group(35.0±6.1 months vs.61.1±5.4 months, P=0.002). Multiple cox regression showed negative expression of SIPA1 in cancer tissue was a independent risk factor for HCC patients. Thus results suggested that negative expression of SIPA1 gene had relationship with tumor invasion and metastasis.
2. Through reverse transcriptase-PCR and Western blot the expression of SIPA1 mRNA and protein was detected in L02 cell line, CCL13 cell line and three HCC cell lines (HepG2, MHCC97-L, HCCLM3), their invasion ability increases in order. The expression of SIPA1 mRNA and protein in HCCLM3 was found to be lowest among the five cell lines (P<0.05).Thus results indicated that low expression of SIPA1 gene had relationship with invasion of HCC cells. In addition, HCCLM3 cell line was chosen as the target cell for overexpression of SIPA1.
3. To analyze the clinical findings, overexpression SIPA1 plasmid was constructed with gene cloning and recombinant DNA technique. The HCCLM3SIPA1+cell line overexpressing SIPA1 stably was established successfully through Lipofectamine 2000 transfection and G418 screening. Through reverse transcriptase PCR and Western blot, the SIPA1 mRNA and protein was proved expressing higher in HCCLM3SIPA1+cell line than in HCCLM3vector(P< 0.05). MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to detect the proliferation and adhesion ability of HCCLM3SIPA1+cells, and overexpression of SIPA1 gene was found to inhibit the proliferation ability and adhesion ability to fibronectin of HCC cells (P<0.05). The abilities of Wound-healing, Transwell invasion, formation of cytoskeleton of HCCLM3SIPA1+cell line were all weaker than that of HCCLM3vector cell line (P<0.05). Thus results all showed that overexpression of SIPA1 gene could inhibit the proliferaton, adhesion, motility and invasion ability of HCC cells in vitro.
4. We further examined the in vivo relevance of the potential role for SIPA1 in HCC tumorigenesis and metastasis by using a orthotopic nude mice implantation liver cancer model. The tumors of mouse livers were measured, we found that the average volume of primary tumors in HCCLM3SIPA1+group was dramatically smaller than that of HCCLM3vector group (1.57±0.08cm3 vs.6.28±0.13cm3), which suggested that up-regulation of SIPA1 in vivo could inhibit growth of HCC. The expression of SIPA1 in liver tumors was determined by immunohistochemistry to ensure the difference of SIPA1 expression in vivo. The pulmonary metastasis was observed in the lung tissue sections, and the ratio of pulmonary metastasis in HCCLM3SIPA1+cell group was found significantly less than in HCCLM3vector cell group (16.7% vs.83.3%, P=0.021). Together, these data supported that SIPA1 played an important role in inhibiting HCC tumorigenesis and metastasis in vivo.
5. Many researches have proved that SIPA1 gene play an import role in bood system tumors through regulating the Rap1 small G protein in hematopoietic progenitor cells. So we speculate that SIPA1 may regulate the Rap1 protein in HCC cells. Pull-down assay showed the level of Rap1-GTP was lower in HCCLM3SIPA1+cells than in HCCLM3vector cells (P< 0.05). Next, co-immunoprecipitations confirmed the direct interaction of SIPA1 and Rap1 proteins in HCC cells. When HCCLM3SIPA1+cells were exposed in chemical factor 8CPT-2Me-cAMP (final concentration 100μM), the Rap1 activity recovered, and the motility, invasion ability and the formation of F-actin cytoskeletal protein of HCCLM3SIPA1+cells also restored. Western blot showed the expression of p-ERK decreased when the SIPA1 was up-regulated (P<0.05), and the expression of p-ERK restored when Rap1 activity was recoverd by adding chemical factor 8CPT-2Me-cAMP(final concentration 100μM) to the HCCLM3SIPA1+ cells.Thus results indicated that SIPA1 inhibited the invasion and metastasis of HCC cells probably through down-regulating the activity of Rap 1-ERK pathway.
Conclusions:SIPA1 is firstly found significantly down-regulated in HCC tissue. Low expression of SIPA1 in HCC is related with multiple tumor nodules, poor differentiation, vein invasion and early recurrence and metastasis. SIPA1 is an independent risk factor for the prognosis of HCC and may became a molecular marker of survival and recurrence after operaton. In vitro and in vivo exprements prove that SIPA1 plays an important role in inhibiting the proliferation ability, invasion and metastasis of HCC cells through down-regulating Rap1-ERK pathway.
已有的研究证实,SIPA1 (Signal-induced Proliferation-associated Gene 1,信号诱导的增殖相关基因1)在血液系统肿瘤的发生发展过程中发挥了非常重要的作用。新近的研究表明小鼠sipa1是决定小鼠乳腺癌转移倾向的重要基因。众所周知,肝脏是胚胎时期的重要造血器官,血细胞(如血小板)与肿瘤的转移密切相关。根据以上的研究结果我们推测SIPA1基因很可能在HCC复发转移中发挥重要作用。因此,本课题在研究SIPA1在临床肝癌病例及肝癌细胞系中表达情况的基础上,进一步通过质粒构建转染技术上调SIPA1的表达,并综合利用一系列体外、体内方法研究SIPA1调控HCC侵袭转移的分子机制。我们获得了如下研究结果：
1.我们首先采用RT-PCR和Western blot等方法检测了SIPA1 mRNA和蛋白在30例新鲜HCC组织及相应邻近非癌肝组织(ANLT)中的表达水平,结果发现与ANLT比较,SIPA1 mRNA和蛋白水平在癌组织中表达均显著下调(P<0.05),且肝癌组织中SIPA1 mRNA和蛋白表达水平呈明显正相关(r=0.757,P<0.001)。进一步采用实时定量PCR检测显示ANLT中SIPA1 mRNA表达量为癌组织的4.17倍。进而,我们采用免疫组化技术检测了130例HCC组织中SIPA1的表达,结果显示SIPA1主要分布于细胞浆内,其在癌组织的阳性表达率为62.3%(81／130)。肝癌组织中SIPA1低表达与肿瘤多结节,分化差及静脉侵袭密切相关(P<0.05)。SIPA1阴性表达组HCC患者的总体生存时间显著短于SIPA1阳性表达组的患者(41.0±6.6个月vs.66.0±5.2个月,P=0.003),SIPA1阴性表达组的无瘤生存时间亦显著短于SIPA1阳性表达组(35.0±6.1个月vs.61.1±5.4个月,P=0.002)。多元Cox回归模型显示SIPA1阴性表达是HCC预后的独立危险因素。
2. RT-PCR及Western blot方法检测SIPA1 mRNA和蛋白在HepG2、MHCC97-L和HCCLM3这3种侵袭转移潜能依次升高的肝癌细胞系中的表达水平,并以正常肝细胞系L02、常氏肝细胞系CCL13作为对照。结果显示SIPA1 mRNA和蛋白在HepG2、HCCLM3和MHCC97-L肝癌细胞系中的表达水平显著低于CCL13和L02正常肝细胞系(P<0.05),且其表达水平在HepG2. MHCC97-L、HCCLM3细胞中依次降低(P<0.05),提示SIPA1表达水平与肝癌细胞的侵袭转移潜能呈负相关。同时确定以SIPA1表达水平最低的HCCLM3为过表达质粒的靶细胞。
3.我们构建了重组质粒pcDNA3.1-SIPA1,使用Lipofectamine 2000转染并行G418筛选,经RT-PCR和Western blot等方法验证SIPA1的表达,最终获得了稳定转染细胞系HCCLM3SIPA1+和空质粒对照组HCCLM3vector细胞。采用MTT法、粘附实验、划痕愈合实验和Transwell等实验方法分别检测两组细胞在细胞增殖与粘附能力、迁移运动能力和侵袭能力上的差别。MTT研究结果显示,HCCLM3SIPA1+的细胞增殖能力较HCCLM3vector显著下降(P<0.05)；粘附实验结果显示与HCCLM3vector细胞相比,HCCLM3SIPA1+细胞与FN的粘附能力明显减弱(P<0.05)；划痕愈合实验和Transwell侵袭实验显示HCCLM3SIPA1+与HCCLM3vector相比,细胞的迁移运动能力和侵袭能力均显著下降(P<0.05)。以上体外实验研究结果证实上调SIPA1的表达可显著抑制HCC细胞的增殖、粘附、迁移和侵袭能力。进一步采用免疫荧光技术检测HCCLM3SIPA1+细胞和HCCLM3vector细胞的骨架蛋白F-actin的形成情况,发现SIPA1能明显减少F-actin的形成。
4.为进一步体内观察上调SIPA1对肝癌侵袭转移的影响,我们建立了裸鼠原位肝癌肺转移模型。结果显示,HCCLM3SIPA1+细胞在肝脏中成瘤体积明显较HCCLM3vector细胞小,分别为1.57±0.08cm3和6.28±0.13cm3,(P<0.05)。通过裸鼠肺组织连续切片观察肺转移灶的发生率,发现与HCCLM3vector组相比,HCCLM3SIPA1+组的肺转移率显著下降(16.7%vs.83.3%,P=0.021)。由此证明,上调SIPA1可显著抑制肝癌细胞的体内成瘤能力和侵袭转移能力。
5.综合已有的研究,我们推测SIPA1可能通过Rapl-ERK信号传导通路调控肝癌细胞的侵袭转移。为证实上述假说,我们首先检测了HCCLM3SIPA1+细胞和HCCLM3Vector细胞中Rap1的活性,研究发现与后者相比,过表达SIPA1的HCCLM3SIPA1+细胞中,Rap1的活性显著降低(P<0.05)；免疫共沉淀技术亦证实HCCLM3SIPA1+细胞中SIPA1与Rap1存在直接的相互作用。进而,我们采用Rap1激活因子8CPT-2Me-cAMP(终浓度100μM),恢复HCCLM3SIPA1+细胞中Rap1的活性,反向验证Rap1在SIPA1调控肝癌侵袭转移中的作用。结果发现,恢复Rap1的活性后,细胞的迁移运动能力和和侵袭能力得到显著恢复(P<0.05)且细胞骨架蛋白F-actin表达亦得到明显恢复。Western blot证实HCCLM3SIPA1+细胞中Rap1下游基因ERK的磷酸化水平显著下降(P<0.05),恢复Rap1活性后p-ERK的表达得到恢复。上述研究结果证实,SIPA1可通过下调Rap1-ERK信号传导通路抑制肝癌细胞的侵袭转移。
综上所述,我们首次发现SIPA1在肝细胞癌组织中的表达显著下调,其表达水平与肝癌分化差、多结节、静脉侵犯等临床病理特征密切相关,同时与肝细胞癌预后差和术后早期发生复发转移等密切相关。SIPA1是肝细胞癌预后的独立危险因素,可作为预测肝细胞癌术后生存和复发转移的分子标志物。体内、体外研究证明,SIPA1通过下调Rap1-ERK信号通路抑制肝细胞癌的增殖和侵袭转移能力。
Hepatocellular carcinoma (HCC) is a main malignancy that threaten our people now. With the improvement of liver surgical technique and management level of perioperation, the resection rate of HCC is increasing and the operation mortality rate is approaching zero at large liver surgery centers. But the overall mortality of HCC patients remains high and the 5-year survial rate of patients is still low. The postoperation recurrence and metastasis of HCC is the main cause of high overall mortality. The 5-year recurrence rate of HCC after operation is more than 60%, even the small HCC the 5-year recurrence rate is above 40%. Obviously the recurrence and metastasis after HCC resection is the bottleneck problem that severely restricts the improvement of long survival rate of HCC patients. So deep study of the mechanism of recurrence and metastasis of HCC and explore the methods of anti-metastasis are focus of HCC research, which are very meaningful for improvement of long survival rate of HCC patients.
Signal-induced Proliferation-associated Gene 1 (SIPA1) was found play an important role in the blood system tumors in the past decade. Recent research found that mouse sipal gene was an important gene that control the metastasis of mouse breast cancer. It is well-known that the liver is a hematogenic organ in embryonic period and blood cells (for example platelet) had close relationship with tumor metastasis. These facts urge us to explore the role of SIPA1 gene in the HCC which had high potential of metastasis. Up to now there is no report about this both at home and abroad. So in this study we first examine the expression of SIPA1 gene in HCC specimens and HCC cell lines, then overexpress the gene by cotransfection the recombinant plasmid (pcDNA3.1-SIPA1) with HCC cell line. Molecullar mechenism of the regulation of SIPA1 to HCC metastasis was studied by in vitro and in vivo. The results as follows was observed:
1. The SIPA1 mRNA and protein in 30 fresh HCC specimens, which consisted of cancer tissue and corresponding ANLT(adjacent non-tumor liver tissue), was detected by Reverse Transcription PCR(Polymerase chain reaction) and Western blot.The expression of mRNA and protein of SIPA1 in fresh HCC tissues was lower than in ANLTs significantly(P<0.05). The expression of SIPA1 mRNA was positively correlated with SIPA1 protein(r=0.757, P<0.001). The real time PCR was used to detect the relative accurate exprssion of SIPA1 mRNA, and the expression of SIPA1 mRNA in ANLT was found to be 4.17 times of that in cancer tissue. The expression of SIPA1 in 130 paraffin HCC specimens was dectected by immunohistochemistry, the positive expression rate of SIPA1 protein in cancer tissue was 62.3% (81/130). Low expression of SIPA1 in cancer tissue of HCC had close relationship with multiple tumor nodules, poor differentiation and vein invasion (P<0.05). Patients with negative expression of SIPA1 in cancer tissue had a shorter overall survial (OS) than the positive group (41.0±6.6 months vs. 66.0±5.2 months, P=0.003), the disease-free survial (DFS) of negative group of SIPA1 was shorter than the positive group(35.0±6.1 months vs.61.1±5.4 months, P=0.002). Multiple cox regression showed negative expression of SIPA1 in cancer tissue was a independent risk factor for HCC patients. Thus results suggested that negative expression of SIPA1 gene had relationship with tumor invasion and metastasis.
2. Through reverse transcriptase-PCR and Western blot the expression of SIPA1 mRNA and protein was detected in L02 cell line, CCL13 cell line and three HCC cell lines (HepG2, MHCC97-L, HCCLM3), their invasion ability increases in order. The expression of SIPA1 mRNA and protein in HCCLM3 was found to be lowest among the five cell lines (P<0.05).Thus results indicated that low expression of SIPA1 gene had relationship with invasion of HCC cells. In addition, HCCLM3 cell line was chosen as the target cell for overexpression of SIPA1.
3. To analyze the clinical findings, overexpression SIPA1 plasmid was constructed with gene cloning and recombinant DNA technique. The HCCLM3SIPA1+cell line overexpressing SIPA1 stably was established successfully through Lipofectamine 2000 transfection and G418 screening. Through reverse transcriptase PCR and Western blot, the SIPA1 mRNA and protein was proved expressing higher in HCCLM3SIPA1+cell line than in HCCLM3vector(P< 0.05). MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to detect the proliferation and adhesion ability of HCCLM3SIPA1+cells, and overexpression of SIPA1 gene was found to inhibit the proliferation ability and adhesion ability to fibronectin of HCC cells (P<0.05). The abilities of Wound-healing, Transwell invasion, formation of cytoskeleton of HCCLM3SIPA1+cell line were all weaker than that of HCCLM3vector cell line (P<0.05). Thus results all showed that overexpression of SIPA1 gene could inhibit the proliferaton, adhesion, motility and invasion ability of HCC cells in vitro.
4. We further examined the in vivo relevance of the potential role for SIPA1 in HCC tumorigenesis and metastasis by using a orthotopic nude mice implantation liver cancer model. The tumors of mouse livers were measured, we found that the average volume of primary tumors in HCCLM3SIPA1+group was dramatically smaller than that of HCCLM3vector group (1.57±0.08cm3 vs.6.28±0.13cm3), which suggested that up-regulation of SIPA1 in vivo could inhibit growth of HCC. The expression of SIPA1 in liver tumors was determined by immunohistochemistry to ensure the difference of SIPA1 expression in vivo. The pulmonary metastasis was observed in the lung tissue sections, and the ratio of pulmonary metastasis in HCCLM3SIPA1+cell group was found significantly less than in HCCLM3vector cell group (16.7% vs.83.3%, P=0.021). Together, these data supported that SIPA1 played an important role in inhibiting HCC tumorigenesis and metastasis in vivo.
5. Many researches have proved that SIPA1 gene play an import role in bood system tumors through regulating the Rap1 small G protein in hematopoietic progenitor cells. So we speculate that SIPA1 may regulate the Rap1 protein in HCC cells. Pull-down assay showed the level of Rap1-GTP was lower in HCCLM3SIPA1+cells than in HCCLM3vector cells (P< 0.05). Next, co-immunoprecipitations confirmed the direct interaction of SIPA1 and Rap1 proteins in HCC cells. When HCCLM3SIPA1+cells were exposed in chemical factor 8CPT-2Me-cAMP (final concentration 100μM), the Rap1 activity recovered, and the motility, invasion ability and the formation of F-actin cytoskeletal protein of HCCLM3SIPA1+cells also restored. Western blot showed the expression of p-ERK decreased when the SIPA1 was up-regulated (P<0.05), and the expression of p-ERK restored when Rap1 activity was recoverd by adding chemical factor 8CPT-2Me-cAMP(final concentration 100μM) to the HCCLM3SIPA1+ cells.Thus results indicated that SIPA1 inhibited the invasion and metastasis of HCC cells probably through down-regulating the activity of Rap 1-ERK pathway.
Conclusions:SIPA1 is firstly found significantly down-regulated in HCC tissue. Low expression of SIPA1 in HCC is related with multiple tumor nodules, poor differentiation, vein invasion and early recurrence and metastasis. SIPA1 is an independent risk factor for the prognosis of HCC and may became a molecular marker of survival and recurrence after operaton. In vitro and in vivo exprements prove that SIPA1 plays an important role in inhibiting the proliferation ability, invasion and metastasis of HCC cells through down-regulating Rap1-ERK pathway.
引文
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