stathmin表达及其磷酸化状态在肝癌发生发展中的作用
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摘要
Stathmin表达及其磷酸化状态在肝癌发生发展中的作用中文摘要
肝细胞肝癌(hepatocellular carcinoma, HCC)是一种常见的恶性肿瘤,死亡率在我国恶性肿瘤中居第二位。在我国,流行病学研究发现HCC的发展基本上遵循着这样一个发展途径：正常→乙型肝炎(HBV)→肝硬化→肝癌。肝癌的发生发展是一个多基因、多步骤参与的复杂过程,其发病机制仍不太清楚。故而阐明肝癌的发生发展的分子机制,对寻找有效的肝癌治疗方法,降低肝癌患者的发病率和复发率,提高患者的生存率和预后具有重要意义。
人stathmin位于染色体lp36.11,分子量为19KD,是一种广泛分布的胞浆磷蛋白。在其N末端包括了四个丝氨酸磷酸化位点,分别为Ser16、Ser25、Ser38和Ser63,由不同的蛋白激酶进行调控。作为一种重要的微管解离蛋白,stathmin还参与纺锤体的形成；调节细胞形态、细胞分裂、分化等过程；在调节细胞增殖、细胞周期和细胞运动中发挥着重要作用。研究还发现stathmin在白血病、乳腺癌、卵巢癌、前列腺癌、肉瘤等多种肿瘤中高表达,并与肿瘤的进程相关,这提示stathmin可能参与了肿瘤的发生发展,可能成为一个肿瘤潜在的诊断标志物和治疗靶点。
本文研究了肝癌相关不同肝病组织中stathmin的表达及其不同位点磷酸化状态；首次探讨了stathmin及其磷酸化状态对肝癌细胞增殖、凋亡及侵袭转移能力的影响,初步揭示stathmin参与HCC病变过程的分子机制。
第一部分肝癌相关不同肝病组织及不同转移潜能肝癌细胞系中Stathmin表达及其位点磷酸化状态的研究
本部分研究通过半定量RT-PCR、Western Blot和IHC的方法结合应用stathmin及其相关的位点磷酸化抗体检测stahmin在肝癌相关肝病组织和不同转移潜能肝癌细胞株中的表达及其不同位点的磷酸化水平,初步探讨stathmin的表达及其不同位点磷酸化状态与肝癌发生发展的关系。
应用半定量RT-PCR和Western Blot的检测16对肝癌及其配对癌旁组织中stathmin的表达,结果显示肝癌组织中,11例可检出stathmin mRNA表达,阳性率为68.75%,9例呈现蛋白质表达,阳性率56.25%,二者表达一致率达到81.81%；而在配对癌旁组织里面未见stathmin的表达。在包含48例肝癌及其癌旁组织的组织芯片上,IHC检测结果显示stathmin在肝癌组织中的阳性率64.58%(31/48),在配对的癌旁组织为6.25%(3/48)。通过RT-PCR技术检测正常肝、肝硬化、不伴转移和伴转移肝癌组织中stathmin的表达,结果显示：stathmin mRNA在正常肝组织不表达,在肝硬化组织中表达低(stathmin相对表达量0.13±0.03),伴转移的肝癌组织较不伴转移的肝癌组织中表达高(0.56±0.13 vs 0.41±0.09)。正常肝、肝炎、肝硬化、不伴转移和伴转移肝癌组织的组织芯片上,用免疫组化检测stathmin的表达,IPP软件分析结果显示：stathmin在正常肝组织、肝炎、肝硬化组织中表达低(其积分光密度分别为38.04±6.46、73.69±15.22、84.35±21.95)；在肝癌组织尤其是伴转移的肝癌组织中表达高(387.66±77.41 vs605.23±99.03)。在不同转移潜能人肝癌细胞系Hep3B、SMMC-7721、MHCC97L、MHCC97H、HCCLM3和HCCLM6中采用RT-PCR和Western Blot方法检测stathmin的表达,发现stahtmin的表达与肝癌细胞转移潜能正相关。其中stathmin在高转移潜能的HCCLM3和HCCLM6中表达最高；在不转移的Hep3B和SMMC-7721中表达最低。
应用免疫组化结合stathmin位点特异性磷酸化抗体的方法在组织芯片(肝癌与癌旁组织、肝癌相关不同肝病组织、伴转移与不伴转移肝癌组织)上的检测stathmin的不同磷酸化状态。在肝癌和癌旁组织芯片检测发现肝癌组织中stathmin pS25的阳性率为31.25%(15/48),配对的癌旁组织中未见stathmin pS25的表达；肝癌组织中stathmin pS38的阳性率为37.50%(18/48),配对的癌旁组织中也未见stathmin pS38的表达。肝癌相关不同肝病组织芯片检测结果发现stathmin pS25在正常肝组织、肝炎和肝硬化组织中表达低(73.52±13.75、84.39±18.12、89.96±18.83),在肝癌组织尤其伴转移的肝癌组织中表达高(263.77±51.38 vs 399.64±89.59); stathmin pS38的结果和pS25的结果相似,在正常肝组织、肝炎和肝硬化组织中表达低(68.23±18.01、88.62±17.99、90.05±21.52),在肝癌组织尤其伴转移的肝癌组织中表达高(305.48±58.33 vs455.89±82.74)。同时stathmin pS25和pS38位的磷酸化可随着肝癌细胞转移潜能的增加而增强。而stathmin pS 16和pS63磷酸化状态在肝癌相关不同肝病组织和肝癌细胞系中未见以上趋势。
第二部分Stathmin基因表达缺失对肝癌细胞生物行为的影响
本部分研究是在前面研究的基础上,应用RNA干扰技术,观察stathmin基因表达沉默对肝癌细胞增殖、凋亡及侵袭运动的改变,了解stathmin与肝癌发生发展的关系。
应用RNA干扰技术,设置三个平行实验组：control组(HCCLM3细胞)、Mock组(转染非特异性stathmin siRNA,不针对任何基因,经验证转染后不抑制基因表达)和RNA干扰组(转染stathmin特异性siRNA)。采用LipotamineTM2000瞬时转染stathmin siRNA,分别筛选三对stathmin siRNA的作用浓度和作用时间,优化stathmin RNAi的体系。Stathmin RNAi之后,半定量RT-PCR和WB检测结果显示,在stathmin siRNA-1,80nmol/L,转染48 hr的条件下,HCCLM3细胞中stathmin的表达被有效抑制,抑制率达90%。免疫荧光实验显示stathmin在HCCLM3肝癌细胞中表达,主要定位于胞浆,RNAi后细胞中stathmin的绿色荧光几不可见,证实siRNA的转染效果效率良好。
CCK8实验发现转染stathmin siRNA的HCCLM3细胞的增殖受到明显抑制,在作用时间为24h,48h和72h时,细胞增殖抑制率分别为13.04±0.10%,28.10±0.41%和37.36±2.15%(P<0.05)。联用Annexin和PI检测细胞凋亡,和Mock组比较,HCCLM3 RNAi组的凋亡明显增加,从9.20±0.64%明显增加至25.11±1.62%(P<0.05)。细胞粘附实验发现,在20min、40min和60min不同作用时间点,与Mock组细胞粘附能力(0.94±0.17、1.18±0.09和1.46±0.27)相比较,RNAi组细胞粘附能力(0.67±0.14、0.75±0.13和0.96±0.09)明显抑制(P<0.05)。采用体外Transwell实验检测stathmin RAN干扰后HCCLM3细胞运动侵袭能力的变化,实验发现与Mock组细胞(运动实验细胞穿膜数：48.44±5.31；侵袭实验细胞穿膜数：28.00±4.41)相比较,RNAi组细胞(运动实验细胞穿膜数：29.4±4.62；侵袭实验细胞穿膜数：10.36±2.97)都显著减少(P<0.05)。这些结果提示stathmin的表达下调可使细胞增殖抑制,诱导凋亡；细胞黏附、运动和侵袭受到抑制,表明stathmin可能参与了肝癌的发生发展过程。
第三部分Stathmin磷酸化状态对肝癌细胞生物学性能的影响
本部分研究的目的是应用定点突变技术,构建stathmin S25A和stathminS38A重组质粒,并进一步从体内外检测stathmin位点磷酸化水平和状态的改变对肝癌细胞增殖、凋亡、侵袭运动和体内外成瘤能力的影响,探讨stathmin磷酸化状态和肝癌的关系。
采用基因定点突变的技术,在Flag-pcDNA3.1-stathmin wt重组质粒上点突变stathmin M25 S→A和stathmin M38 S→A,经测序鉴定突变正确。单克隆筛选方法建立稳定表达Flag-pcDNA3.1, Flag-pcDNA3.1-stathmin wt、Flag-pcDNA3.1-stathmin S25A和Flag-pcDNA3.1-stathmin S38A的SMMC-7721细胞株,分别记为SMMC-7721 control、stathmin wt、stathminS25A和stathmin S38A细胞。采用Western Blot的方法检测四个细胞株中Flag、stathmin的表达及其pS25和pS38磷酸化水平。结果证明四个细胞株中Flag蛋白相对表达量分别为0.48±0.09、0.52±0.09、0.51±0.10和0.53±0.10,没有统计学差异(P>0.05),提示转染效率一致。检测stathmin磷酸化状态结果发现,与stathminwt细胞(pS25:0.41±0.04; pS38:0.78±0.02)比较,stathmin S25A细胞中的pS25磷酸化水平显著降低(0.18±0.03)；stathmin S38A细胞中的pS38磷酸化状态明显降低(0.36±0.04)。而与SMMC-7721 control细胞(0.16±0.05)比较,stathmin wt细胞中stathmin的表达明显增加(0.76±0.12)(P<0.05)。
用细胞计数试剂盒(CCK8)检测stathmin的细胞增殖,发现和stathmin wt细胞比较,stathmin S25A和stathmin S38A细胞增殖分别被抑制到38.02±1.45%和25.10±0.79%(P<0.05)。联用Annexin V和PI,流式细胞术测定细胞凋亡,发现和stathmin wt细胞凋亡率(5.80±0.33%)相比,stathmin S25A和stathminS38A细胞凋亡率分别为8.82±0.30%和7.35±0.38%,均增高(P<0.05)。运用粘附实验检测细胞粘附能力,发现stathmin wt(1.01±0.08)较stathmin S25A (0.71±0.06)、stathmin S38A (0.83±0.05)细胞粘附能力显著增加(P<0.05)。用体外Transwell实验检测细胞的运动侵袭能力的变化,发现较stathmin wt组(细胞运动实验细胞穿膜数130.45±14.13；侵袭实验透膜细胞数57.76±8.50),stathmin S25A、S38A突变株细胞侵袭运动能力显著降低(细胞运动实验细胞穿膜数分别是96.00±11.80和111.00±9.54；侵袭实验透膜细胞数分别是35.74±5.51和42.00±7.21),P均<0.05。软琼脂克隆形成实验发现较stathmin wt细胞克隆形成数,stathmin S25A被抑制42.82±1.72%(P<0.01),而stathmin S38A抑制为14.58±1.30%,没有显著变化。裸鼠体内实验发现皮下成瘤stathmin wt (2.94±0.81 g)瘤重明显重于stathmin S25A (1.82±0.26 g)和stathmin S38A(2.05±1.03g),和stathmin wt细胞成瘤相比较,stathmin S25A和S38A肿瘤明显抑制51.61%和19.26%。肝膜下原位种植瘤stathmin wt(2.35±0.42g)肿瘤显著重于stathmin S25A (1.62±0.69g)和S38A(2.12±0.39g)(P<0.05)。每组6只裸鼠中stathmin wt组有4只裸鼠发生肝转移,4只裸鼠发生肺转移；而stathmin S25A成瘤组有2只裸鼠发生肝转移,3只裸鼠发生肺转移；stathmin S38A成瘤组有2只裸鼠发生肝转移,4只裸鼠发生肺转移。综合以上实验结果,提示stathmin S25和S38磷酸化水平和状态可能与肝癌的发展和转移过程有关联。
1.与正常肝、肝炎和肝硬化组织相比,stathmin在肝癌中高表达,且stathmin的表达与肝癌细胞的转移潜能呈正相关。
2.在肝癌中stathmin pS25和stathmin pS38呈高磷酸化状态,肝癌细胞中它们的高磷酸化状态与转移潜能呈正相关。
3.肝癌细胞HCCLM3中stathmin的表达下调,明显促进细胞凋亡,抑制细胞增殖、粘附和运动侵袭,提示stathmin可能参与了肝癌的发生发展过程。
4.与stathmin wt细胞比较,stathmin S25A和stathmin S38A突变株中stathminpS25和stathmin pS38分别呈低磷酸化状态,但能明显诱导肝癌细胞细胞凋亡,抑制体内外细胞增殖、粘附、运动侵袭,并致成瘤能力下降。提示stathmin S25和S38磷酸化水平和状态可能与肝癌的发展和转移过程有关联。
创新点
1.首次检测肝癌相关疾病肝组织中stathmin及不同位点磷酸化水平的差异,探讨其临床病理意义,评价其作为肝癌分子标记物的可能性。
2.首次研究发现stathmin S25A和S38A位点突变导致stathmin位点的磷酸化水平下降,引发肝癌细胞生物学功能的明显改变,提示这两个位点在肝癌的发生发展中的重要意义。
潜在应用价值
1. stathmin是一个潜在的肝癌诊断分子和治疗靶点。
2.为深入研究肝癌发生和转移过程中stathmin的表达及位点磷酸化的分子调控规律、途径及其与肝癌的发生发展分子机制中的地位和作用提供实验基础。
Hepatocellular carcinoma (HCC) is the most common malignant cancers in the world and the second leading cause of cancer mortality in China. As we can see from the epidemiology, hepatocarcinogenesis is linked tightly to the development of chronic HBV infection firstly, and associated with the evolution of fibrosis and cirrhosis in China. However, hepatocarcinogenesis is believed a complex multisteps process and multi-gene participation. Hence, the mechanism of HCC is still unclear. It is important to elucidate the molecular mechanism of HCC for searching effective therapy, eliminating patient morbidity and recurrence, and improving survival rate and prognosis.
Stathmin, a ubiquitous 19kDa cytosolic phosphoprotein, located in chromosome 1p36.11. There are four serine phosphorylation sites in N-terminal of stathmin, including Ser16, Ser25, Ser38 and Ser63, which are regulated by different protein kinase. Stathmin, which is an important microtubule destabilizing protein, can regulate formation of the mitotic spindle, control cell shaping, cell division and cell differentiation, and play a key role in cell proliferation, cell cycle, cell motility. Studies conducts that stathmin is overexpressed in many tumor types (e.g., leukemia, breast cancer, ovarian cancer, prostate cancer and sarcoma) and is related closely with tumor progression, which implied that stathmin may contribute to hepatocarcinogenesis and might be a potential diagnostic and therapeutic target.
In the current study, we detected stathmin expression and its site phosphorylation statuses in liver tissues represented the different HCC related liver diseases, and in a series HCC cell lines with different metastatic potential; we firstly investigated how stathmin and its phosphorylation statuses affected on HCC cell functions such as proliferation, apoptosis, motility and invasion both in vitro and in vivo, and elucidated inner molecular mechanism that stathmin and its phosphorylation statuses contribute to hepatocarcinogenesis.
Stathmin expression and its phosphorylation statuses in liver tissues represented HCC related liver diseases and in human HCC cell lines with different metastatic potential
To research initially the relationship of stathmin and its phosphorylation statuses with hepacarcinogenesis, stathmin and its phosphorylation statuses were detected in tissues represented HCC related liver diseases and in human hepatocellular carcinoma cell lines with different metastatic potential, using semi-quantitive RT-PCR, Western Blot and immunohistochemistry (IHC) by anti-stathmin antibody and antibodies against related site phosphrylation.
In 16 pairs of HCC and the adjacent liver tissues, stathmin mRNA expression (11/16, 68.75%) and protein expression (9/16,56.25%) was induced in HCC tissues, which the mRNA and protein expression coincidence up to 81.81%, while stathmin was not detective in the adjacent liver tissues. IHC results showed that more than 64.58% cases displayed overexpression of stathmin in tumor specimens, compared with 6.25 % in the adjacent liver tissues using tissue microarray (48 pairs of HCC tissues and adjacent tissues). Using RT-PCR, stathmin mRNA expression was evaluated and the result showed that stathmin was overexpressed in HCC tissue(stathmin/GAPDH relative ratio 0.41±0.09), especially in HCC tissues with metastatic (0.56±0.13), with comparison to the low expression level in liver cirrhosis specimens(0.13±0.03) and couldn't be detected in normal liver tissues. Using IHC and Image-Pro Plus software, stathmin expression was detected further in TMA(containing the liver tissue from healthy, hepatitis, cirrhosis, HCC and HCC with metastasis specimens).The data showed that stathmin expression was highly up-regulated in HCC (387.66±77.41) especially in HCC tissues with metastasis (605.23±99.03), compared with that in normal liver, hepatitis and cirrhosis samples(38.04±6.46、73.69±15.22、84.35±21.95). In hepatocellur carcinoma cell lines with different metastatic potential (Hep3B、SMMC-7721、MHCC97L、MHCC97H、HCCLM3 and HCCLM6), The results from RT-PCR and Western Blot indicated that stathmin expression was consistently up regulated with increased HCC metastatic potential. And the expression of stathmin was the highest in HCCLM3 and HCCLM6 cells with highest metastatic potential, but the lowest in Hep3B and SMMC-7721 without metastatic potential.
Stathmin different phosphorylation statuses were detected by tissue microarray (TMA) technique (pairs HCC and adjacent normal tissues/healthy, hepatitis, cirrhosis, HCC and metastatic HCC tissues) and IHC. In TMA, stathmin pS25 (phosphorylation status) was positive of 31.25%(15/48) in HCC cases, while couldn't detect in the adjacent tissue. And stathmin pS38 (phosphorylation status) was positive of 37.50% (18/48) in HCC cases, while didn't detect in the adjacent tissue. TMA of healthy, hepatitis B, cirrhosis and HCC specimens showed that stathmin pS25 phosphorylation status was increased in HCC (263.77±51.38), especially in metastatic HCC tissues(399.64±89.59), compared with normal liver, hepatitis and cirrhosis samples (73.52±13.75、84.3±8.12、89.96±18.83, respectively).And the data showed stathmin pS38 was the similar as stathmin pS25, was increased in HCC (305.48±58.33), especially in metastatic HCC tissues(455.89±82.74), compared with normal liver, hepatitis and cirrhosis samples (68.23±18.01、88.62±17.99、90.05±21.52). In hepatocellur carcinoma cell lines with different metastatic potential (Hep3B、SMMC-7721、MHCC97L. MHCC97H、HCCLM3和HCCLM6), Western Blot results indicated that stathmin pS25 and pS38 was consistently increased with HCC metastatic potential. From other point of view, there was no statistic significance between tissues represented HCC related liver diseases or various human hepatocellular carcinoma cell lines with different metastatic potential in the phosphorylation statuses of stathmin pS16 and pS63,
Part two
Stathmin RNA interference influences the biological characteristic in the hepatocellular carcinoma cell
Based on the mentioned above, the alterations of cell proliferation, apoptosis, motility and cell invasion of stathmin RNA interferenced HCCLM3 cells were detected, and investigated further the role of stathmin RNA silencing to hepatocarcigenesis.
In the present study, stathmin siRNA mediated transient transfection by LipotamineTM2000 liposome was used to screen three pairs of siRNA, different si RNA concentrations and different time points for optimizing stathmin RNA interference system. Stathmin expression was effectively inhibited up to 90% under the optimized condition (transfected 80nmol/L stathmin siRNA-1 for 48 hr) in HCCLM3 cells by semi-quantitive RT-PCR, Western Blot and fluorescence aided cell immunochemistry.
Using cell count kit (CCK8), it was shown that the HCCLM3 cell proliferation was obviously depressed by 13.04±0.10%,28.10±0.41% and 37.36±2.15% at the time point of 24 hr,48hr and 72hr with the comparison to Mock group. Flow cytometry was used to analyze cells apoptosis by FITC-AnnexinV+PI apoptosis assay kit, the results demonstrated that the percentage of apoptotic cells was increased to 25.11±1.62% in RNAi group, compared with 9.20±0.64% in Mock group. Using cell adhesion assay, the results showed that the ratio of the cell adhered coated FN (i.e.0.67±0.14、0.75±0.13 and 0.96±0.09 at the time point of 20 min,40 min and 60 min) in RNAi treated group was obviously less than that in Mock group (0.94±0.17、1.18±0.09 and 1.46±0.27) at the time points mentioned above. Cell migration and invasion assay in vitro revealed that the average number of invaded and migrated HCCLM3 RNAi cells (cells of outer surface in migrant assay: 29.40±4.62; cells in invasive assay:10.36±2.97) significantly decreased in comparison with Mock group cells (cells of outer surface in migrant assay: 48.44±5.31; cells in invasive assay:28.00±4.41). All the results implied that stathmin may play a role in HCC progression and metastasis.
Part three
Alteration of stathmin phosphorylation statuses has effect on hepatocellular carcinoma cells
The object of the part is to construct the recombination plasmid containing stathmin S25A or stathmin S38A by site-directed mutation techniques, to study the alteration of HCC cell proliferation, apoptosis, motility, invasion and tumor forming ability in soft agar effected by the changes of stathmin phosphorylation statuses in vivo and in vitro, and to deep insight in the relationship between stathmin phosphorylation statuses and hepatocarconogenesis.
Sequencing reports showed that the point mutation in recombinant plasmids of Flag-pcDNA3.1-stathmin M25 S→A and Flag-pcDNA3.1-stathmin M38 S→A were correct. Using monoclone screening method, established stable SMMC-7721 cell lines were transfected with Flag-pcDNA3.1, Flag-pcDNA3.1-stathmin wt, Flag-pcDNA3.1-stathmin S25A and Flag-pcDNA3.1-stathmin S38A plasmid, which named as SMMC-7721 control、stathmin wt、stathmin S25A and stathmin S38A cell line respectively. Flag/GAPDH relative ratio in above four cell lines were 0.48±0.09、0.52±0.09、0.51±0.10 and 0.53±0.10, determined by Western Blot analysis respectively (P>0.05).This implied that the transfection efficacy of transfected cells was similar. Site phosphorylation statuses of stathmin were detected, and the data showed stathmin S25 phosphorylation status in stathmin S25A cells (0.18±0.03) was decrease compared with stathmin wt cells (0.41±0.04), meanwhile stathmin S38 phosphorylation status in stathmin S38A cells (0.36±0.04) was decrease compared with stathmin wt cells (0.78±0.02) also. And compared with SMMC-7721 control cells (0.16±0.05), stathmin expression was induced obviously in stathmin wt cells (0.76±0.12)
Using cell count kit (CCK8), the results indicated that cell proliferation in stathmin S25A and stathmin S38A cell lines were obviously depressed by 38.02±1.45% and 25.10±0.79% respectively as compared with stathmin wt cells(P<0.05). FITC-AnnexinV and PI labeling followed by Flow cytometry determination was used to analyze cells apoptosis, it was found that the cell apoptotic rate was increased to 8.82±0.30% and 7.35±0.38% in stathmin S25A and stathmin S38A cell lines, compared with that in stathmin wt cells(5.80±0.33%) (P<0.05). The results showed that ratio of stathmin S25A and stathmin S38A cells adhesion to coated FN (0.71±0.06,0.83±0.05) was markedly less than that of stathmin wt cells (1.01±0.08) (P<0.05) using cell adhesion assay. Migration and invasion assay in vitro revealed that the average number of invaded and migrated stathmin S25A and S38A cells (migrant assay:96.00±11.80,111.00±9.54; invasive assay:35.74±5.51 and 42.00±7.21 respectively) significantly decreased in comparison with stathmin wt cells (migrant assay:130.45±14.13; invasive assay:57.76±8.50) (P<0.05). Using soft agar clone forming assay, the results displayed that clone forming ability was inhibited by 42.82±1.72%in stathmin S25A cells(P< 0.01), while by 14.58±1.30 % in stathmin S38 A cells(P>0.05) in comparison with the count of cell clones in stathmin wt cells. Nude mice experiments displayed that transplanted tumor weight of statmin wt group (2.35±0.42g) was obviously increased than stathmin S25A group (1.62±0.69g)and stathmin S38A group(2.12±0.39g) (P<0.05). The incidence of lung metastasis of nude mice in stathmin S25A group(2/6) and stathmin S38A group(3/6) was significant decreased compared with stathmin wt group(4/6). And the incidence of liver metastasis of nude mice in stathmin S25A group (2/6) and stathmin S38A group (2/6) was significant decreased in compared with stathmin wt group(4/6).
Conclusion
1. Stathmin expression was highly up-regulated in HCC tissues, compared with normal liver, hepatitis and cirrhosis tissue. And stathmin expression was increased in hepatocellular carcinoma cells with increasing metastastic potential.
2. Stathmin phosphorylation statuses of S25 and S38 were regulated in HCC tissues, compared with normal liver, hepatitis and cirrhosis tissue. And stathmin S25 and S38 phosphorylation statuses were increased in hepatocellular carcinoma cells with increasing metastastic potential.
3. Stathmin RNA silencing in HCCLM3 promoted obviously apoptosis, and inhibited cell proliferation, adhesion and motility significantly, it was implying stathmin may be as a crucial factor during hepatocarcinogenesis.
4. Suppressed cell proliferation, adhesion, motility and tumor forming ability and induced cell apoptosis were observed in site mutational stathmin S25A and S38A cell lines in vitro and in vivo, compared with stathmin wt cells. It implied that stathmin S25 and S38 phosphorylation statuses may be contribute hepatocarcinogenesis.
Novelty
1. Stathmin expression and its phosphorylation statuses in tissues represented HCC related liver diseases were dynamic studied firstly, which revealed the highly expression level and the alteration of site phosporylation of stathmin may link to HCC development and metastasis.
2. Stathmin S25 and S38 phosphorylation statuses was related closely to the biological characteristics of hepatocellular carcinoma cells, it implied that stathmin phosphorylation statuses may play an important contribution to hepatocarcinogenesis.
The potential application of this work
1. Stathmin may be a potential and valuable biomarker and therapeutic target in HCC.
2. Our study provides experimental data to research further for molecular mechanism of stathmin expression and its phosphorylation statuses linked to HCC development and metastasis.
肝细胞肝癌(hepatocellular carcinoma, HCC)是一种常见的恶性肿瘤,死亡率在我国恶性肿瘤中居第二位。在我国,流行病学研究发现HCC的发展基本上遵循着这样一个发展途径：正常→乙型肝炎(HBV)→肝硬化→肝癌。肝癌的发生发展是一个多基因、多步骤参与的复杂过程,其发病机制仍不太清楚。故而阐明肝癌的发生发展的分子机制,对寻找有效的肝癌治疗方法,降低肝癌患者的发病率和复发率,提高患者的生存率和预后具有重要意义。
人stathmin位于染色体lp36.11,分子量为19KD,是一种广泛分布的胞浆磷蛋白。在其N末端包括了四个丝氨酸磷酸化位点,分别为Ser16、Ser25、Ser38和Ser63,由不同的蛋白激酶进行调控。作为一种重要的微管解离蛋白,stathmin还参与纺锤体的形成；调节细胞形态、细胞分裂、分化等过程；在调节细胞增殖、细胞周期和细胞运动中发挥着重要作用。研究还发现stathmin在白血病、乳腺癌、卵巢癌、前列腺癌、肉瘤等多种肿瘤中高表达,并与肿瘤的进程相关,这提示stathmin可能参与了肿瘤的发生发展,可能成为一个肿瘤潜在的诊断标志物和治疗靶点。
本文研究了肝癌相关不同肝病组织中stathmin的表达及其不同位点磷酸化状态；首次探讨了stathmin及其磷酸化状态对肝癌细胞增殖、凋亡及侵袭转移能力的影响,初步揭示stathmin参与HCC病变过程的分子机制。
第一部分肝癌相关不同肝病组织及不同转移潜能肝癌细胞系中Stathmin表达及其位点磷酸化状态的研究
本部分研究通过半定量RT-PCR、Western Blot和IHC的方法结合应用stathmin及其相关的位点磷酸化抗体检测stahmin在肝癌相关肝病组织和不同转移潜能肝癌细胞株中的表达及其不同位点的磷酸化水平,初步探讨stathmin的表达及其不同位点磷酸化状态与肝癌发生发展的关系。
应用半定量RT-PCR和Western Blot的检测16对肝癌及其配对癌旁组织中stathmin的表达,结果显示肝癌组织中,11例可检出stathmin mRNA表达,阳性率为68.75%,9例呈现蛋白质表达,阳性率56.25%,二者表达一致率达到81.81%；而在配对癌旁组织里面未见stathmin的表达。在包含48例肝癌及其癌旁组织的组织芯片上,IHC检测结果显示stathmin在肝癌组织中的阳性率64.58%(31/48),在配对的癌旁组织为6.25%(3/48)。通过RT-PCR技术检测正常肝、肝硬化、不伴转移和伴转移肝癌组织中stathmin的表达,结果显示：stathmin mRNA在正常肝组织不表达,在肝硬化组织中表达低(stathmin相对表达量0.13±0.03),伴转移的肝癌组织较不伴转移的肝癌组织中表达高(0.56±0.13 vs 0.41±0.09)。正常肝、肝炎、肝硬化、不伴转移和伴转移肝癌组织的组织芯片上,用免疫组化检测stathmin的表达,IPP软件分析结果显示：stathmin在正常肝组织、肝炎、肝硬化组织中表达低(其积分光密度分别为38.04±6.46、73.69±15.22、84.35±21.95)；在肝癌组织尤其是伴转移的肝癌组织中表达高(387.66±77.41 vs605.23±99.03)。在不同转移潜能人肝癌细胞系Hep3B、SMMC-7721、MHCC97L、MHCC97H、HCCLM3和HCCLM6中采用RT-PCR和Western Blot方法检测stathmin的表达,发现stahtmin的表达与肝癌细胞转移潜能正相关。其中stathmin在高转移潜能的HCCLM3和HCCLM6中表达最高；在不转移的Hep3B和SMMC-7721中表达最低。
应用免疫组化结合stathmin位点特异性磷酸化抗体的方法在组织芯片(肝癌与癌旁组织、肝癌相关不同肝病组织、伴转移与不伴转移肝癌组织)上的检测stathmin的不同磷酸化状态。在肝癌和癌旁组织芯片检测发现肝癌组织中stathmin pS25的阳性率为31.25%(15/48),配对的癌旁组织中未见stathmin pS25的表达；肝癌组织中stathmin pS38的阳性率为37.50%(18/48),配对的癌旁组织中也未见stathmin pS38的表达。肝癌相关不同肝病组织芯片检测结果发现stathmin pS25在正常肝组织、肝炎和肝硬化组织中表达低(73.52±13.75、84.39±18.12、89.96±18.83),在肝癌组织尤其伴转移的肝癌组织中表达高(263.77±51.38 vs 399.64±89.59); stathmin pS38的结果和pS25的结果相似,在正常肝组织、肝炎和肝硬化组织中表达低(68.23±18.01、88.62±17.99、90.05±21.52),在肝癌组织尤其伴转移的肝癌组织中表达高(305.48±58.33 vs455.89±82.74)。同时stathmin pS25和pS38位的磷酸化可随着肝癌细胞转移潜能的增加而增强。而stathmin pS 16和pS63磷酸化状态在肝癌相关不同肝病组织和肝癌细胞系中未见以上趋势。
第二部分Stathmin基因表达缺失对肝癌细胞生物行为的影响
本部分研究是在前面研究的基础上,应用RNA干扰技术,观察stathmin基因表达沉默对肝癌细胞增殖、凋亡及侵袭运动的改变,了解stathmin与肝癌发生发展的关系。
应用RNA干扰技术,设置三个平行实验组：control组(HCCLM3细胞)、Mock组(转染非特异性stathmin siRNA,不针对任何基因,经验证转染后不抑制基因表达)和RNA干扰组(转染stathmin特异性siRNA)。采用LipotamineTM2000瞬时转染stathmin siRNA,分别筛选三对stathmin siRNA的作用浓度和作用时间,优化stathmin RNAi的体系。Stathmin RNAi之后,半定量RT-PCR和WB检测结果显示,在stathmin siRNA-1,80nmol/L,转染48 hr的条件下,HCCLM3细胞中stathmin的表达被有效抑制,抑制率达90%。免疫荧光实验显示stathmin在HCCLM3肝癌细胞中表达,主要定位于胞浆,RNAi后细胞中stathmin的绿色荧光几不可见,证实siRNA的转染效果效率良好。
CCK8实验发现转染stathmin siRNA的HCCLM3细胞的增殖受到明显抑制,在作用时间为24h,48h和72h时,细胞增殖抑制率分别为13.04±0.10%,28.10±0.41%和37.36±2.15%(P<0.05)。联用Annexin和PI检测细胞凋亡,和Mock组比较,HCCLM3 RNAi组的凋亡明显增加,从9.20±0.64%明显增加至25.11±1.62%(P<0.05)。细胞粘附实验发现,在20min、40min和60min不同作用时间点,与Mock组细胞粘附能力(0.94±0.17、1.18±0.09和1.46±0.27)相比较,RNAi组细胞粘附能力(0.67±0.14、0.75±0.13和0.96±0.09)明显抑制(P<0.05)。采用体外Transwell实验检测stathmin RAN干扰后HCCLM3细胞运动侵袭能力的变化,实验发现与Mock组细胞(运动实验细胞穿膜数：48.44±5.31；侵袭实验细胞穿膜数：28.00±4.41)相比较,RNAi组细胞(运动实验细胞穿膜数：29.4±4.62；侵袭实验细胞穿膜数：10.36±2.97)都显著减少(P<0.05)。这些结果提示stathmin的表达下调可使细胞增殖抑制,诱导凋亡；细胞黏附、运动和侵袭受到抑制,表明stathmin可能参与了肝癌的发生发展过程。
第三部分Stathmin磷酸化状态对肝癌细胞生物学性能的影响
本部分研究的目的是应用定点突变技术,构建stathmin S25A和stathminS38A重组质粒,并进一步从体内外检测stathmin位点磷酸化水平和状态的改变对肝癌细胞增殖、凋亡、侵袭运动和体内外成瘤能力的影响,探讨stathmin磷酸化状态和肝癌的关系。
采用基因定点突变的技术,在Flag-pcDNA3.1-stathmin wt重组质粒上点突变stathmin M25 S→A和stathmin M38 S→A,经测序鉴定突变正确。单克隆筛选方法建立稳定表达Flag-pcDNA3.1, Flag-pcDNA3.1-stathmin wt、Flag-pcDNA3.1-stathmin S25A和Flag-pcDNA3.1-stathmin S38A的SMMC-7721细胞株,分别记为SMMC-7721 control、stathmin wt、stathminS25A和stathmin S38A细胞。采用Western Blot的方法检测四个细胞株中Flag、stathmin的表达及其pS25和pS38磷酸化水平。结果证明四个细胞株中Flag蛋白相对表达量分别为0.48±0.09、0.52±0.09、0.51±0.10和0.53±0.10,没有统计学差异(P>0.05),提示转染效率一致。检测stathmin磷酸化状态结果发现,与stathminwt细胞(pS25:0.41±0.04; pS38:0.78±0.02)比较,stathmin S25A细胞中的pS25磷酸化水平显著降低(0.18±0.03)；stathmin S38A细胞中的pS38磷酸化状态明显降低(0.36±0.04)。而与SMMC-7721 control细胞(0.16±0.05)比较,stathmin wt细胞中stathmin的表达明显增加(0.76±0.12)(P<0.05)。
用细胞计数试剂盒(CCK8)检测stathmin的细胞增殖,发现和stathmin wt细胞比较,stathmin S25A和stathmin S38A细胞增殖分别被抑制到38.02±1.45%和25.10±0.79%(P<0.05)。联用Annexin V和PI,流式细胞术测定细胞凋亡,发现和stathmin wt细胞凋亡率(5.80±0.33%)相比,stathmin S25A和stathminS38A细胞凋亡率分别为8.82±0.30%和7.35±0.38%,均增高(P<0.05)。运用粘附实验检测细胞粘附能力,发现stathmin wt(1.01±0.08)较stathmin S25A (0.71±0.06)、stathmin S38A (0.83±0.05)细胞粘附能力显著增加(P<0.05)。用体外Transwell实验检测细胞的运动侵袭能力的变化,发现较stathmin wt组(细胞运动实验细胞穿膜数130.45±14.13；侵袭实验透膜细胞数57.76±8.50),stathmin S25A、S38A突变株细胞侵袭运动能力显著降低(细胞运动实验细胞穿膜数分别是96.00±11.80和111.00±9.54；侵袭实验透膜细胞数分别是35.74±5.51和42.00±7.21),P均<0.05。软琼脂克隆形成实验发现较stathmin wt细胞克隆形成数,stathmin S25A被抑制42.82±1.72%(P<0.01),而stathmin S38A抑制为14.58±1.30%,没有显著变化。裸鼠体内实验发现皮下成瘤stathmin wt (2.94±0.81 g)瘤重明显重于stathmin S25A (1.82±0.26 g)和stathmin S38A(2.05±1.03g),和stathmin wt细胞成瘤相比较,stathmin S25A和S38A肿瘤明显抑制51.61%和19.26%。肝膜下原位种植瘤stathmin wt(2.35±0.42g)肿瘤显著重于stathmin S25A (1.62±0.69g)和S38A(2.12±0.39g)(P<0.05)。每组6只裸鼠中stathmin wt组有4只裸鼠发生肝转移,4只裸鼠发生肺转移；而stathmin S25A成瘤组有2只裸鼠发生肝转移,3只裸鼠发生肺转移；stathmin S38A成瘤组有2只裸鼠发生肝转移,4只裸鼠发生肺转移。综合以上实验结果,提示stathmin S25和S38磷酸化水平和状态可能与肝癌的发展和转移过程有关联。
1.与正常肝、肝炎和肝硬化组织相比,stathmin在肝癌中高表达,且stathmin的表达与肝癌细胞的转移潜能呈正相关。
2.在肝癌中stathmin pS25和stathmin pS38呈高磷酸化状态,肝癌细胞中它们的高磷酸化状态与转移潜能呈正相关。
3.肝癌细胞HCCLM3中stathmin的表达下调,明显促进细胞凋亡,抑制细胞增殖、粘附和运动侵袭,提示stathmin可能参与了肝癌的发生发展过程。
4.与stathmin wt细胞比较,stathmin S25A和stathmin S38A突变株中stathminpS25和stathmin pS38分别呈低磷酸化状态,但能明显诱导肝癌细胞细胞凋亡,抑制体内外细胞增殖、粘附、运动侵袭,并致成瘤能力下降。提示stathmin S25和S38磷酸化水平和状态可能与肝癌的发展和转移过程有关联。
创新点
1.首次检测肝癌相关疾病肝组织中stathmin及不同位点磷酸化水平的差异,探讨其临床病理意义,评价其作为肝癌分子标记物的可能性。
2.首次研究发现stathmin S25A和S38A位点突变导致stathmin位点的磷酸化水平下降,引发肝癌细胞生物学功能的明显改变,提示这两个位点在肝癌的发生发展中的重要意义。
潜在应用价值
1. stathmin是一个潜在的肝癌诊断分子和治疗靶点。
2.为深入研究肝癌发生和转移过程中stathmin的表达及位点磷酸化的分子调控规律、途径及其与肝癌的发生发展分子机制中的地位和作用提供实验基础。
Hepatocellular carcinoma (HCC) is the most common malignant cancers in the world and the second leading cause of cancer mortality in China. As we can see from the epidemiology, hepatocarcinogenesis is linked tightly to the development of chronic HBV infection firstly, and associated with the evolution of fibrosis and cirrhosis in China. However, hepatocarcinogenesis is believed a complex multisteps process and multi-gene participation. Hence, the mechanism of HCC is still unclear. It is important to elucidate the molecular mechanism of HCC for searching effective therapy, eliminating patient morbidity and recurrence, and improving survival rate and prognosis.
Stathmin, a ubiquitous 19kDa cytosolic phosphoprotein, located in chromosome 1p36.11. There are four serine phosphorylation sites in N-terminal of stathmin, including Ser16, Ser25, Ser38 and Ser63, which are regulated by different protein kinase. Stathmin, which is an important microtubule destabilizing protein, can regulate formation of the mitotic spindle, control cell shaping, cell division and cell differentiation, and play a key role in cell proliferation, cell cycle, cell motility. Studies conducts that stathmin is overexpressed in many tumor types (e.g., leukemia, breast cancer, ovarian cancer, prostate cancer and sarcoma) and is related closely with tumor progression, which implied that stathmin may contribute to hepatocarcinogenesis and might be a potential diagnostic and therapeutic target.
In the current study, we detected stathmin expression and its site phosphorylation statuses in liver tissues represented the different HCC related liver diseases, and in a series HCC cell lines with different metastatic potential; we firstly investigated how stathmin and its phosphorylation statuses affected on HCC cell functions such as proliferation, apoptosis, motility and invasion both in vitro and in vivo, and elucidated inner molecular mechanism that stathmin and its phosphorylation statuses contribute to hepatocarcinogenesis.
Stathmin expression and its phosphorylation statuses in liver tissues represented HCC related liver diseases and in human HCC cell lines with different metastatic potential
To research initially the relationship of stathmin and its phosphorylation statuses with hepacarcinogenesis, stathmin and its phosphorylation statuses were detected in tissues represented HCC related liver diseases and in human hepatocellular carcinoma cell lines with different metastatic potential, using semi-quantitive RT-PCR, Western Blot and immunohistochemistry (IHC) by anti-stathmin antibody and antibodies against related site phosphrylation.
In 16 pairs of HCC and the adjacent liver tissues, stathmin mRNA expression (11/16, 68.75%) and protein expression (9/16,56.25%) was induced in HCC tissues, which the mRNA and protein expression coincidence up to 81.81%, while stathmin was not detective in the adjacent liver tissues. IHC results showed that more than 64.58% cases displayed overexpression of stathmin in tumor specimens, compared with 6.25 % in the adjacent liver tissues using tissue microarray (48 pairs of HCC tissues and adjacent tissues). Using RT-PCR, stathmin mRNA expression was evaluated and the result showed that stathmin was overexpressed in HCC tissue(stathmin/GAPDH relative ratio 0.41±0.09), especially in HCC tissues with metastatic (0.56±0.13), with comparison to the low expression level in liver cirrhosis specimens(0.13±0.03) and couldn't be detected in normal liver tissues. Using IHC and Image-Pro Plus software, stathmin expression was detected further in TMA(containing the liver tissue from healthy, hepatitis, cirrhosis, HCC and HCC with metastasis specimens).The data showed that stathmin expression was highly up-regulated in HCC (387.66±77.41) especially in HCC tissues with metastasis (605.23±99.03), compared with that in normal liver, hepatitis and cirrhosis samples(38.04±6.46、73.69±15.22、84.35±21.95). In hepatocellur carcinoma cell lines with different metastatic potential (Hep3B、SMMC-7721、MHCC97L、MHCC97H、HCCLM3 and HCCLM6), The results from RT-PCR and Western Blot indicated that stathmin expression was consistently up regulated with increased HCC metastatic potential. And the expression of stathmin was the highest in HCCLM3 and HCCLM6 cells with highest metastatic potential, but the lowest in Hep3B and SMMC-7721 without metastatic potential.
Stathmin different phosphorylation statuses were detected by tissue microarray (TMA) technique (pairs HCC and adjacent normal tissues/healthy, hepatitis, cirrhosis, HCC and metastatic HCC tissues) and IHC. In TMA, stathmin pS25 (phosphorylation status) was positive of 31.25%(15/48) in HCC cases, while couldn't detect in the adjacent tissue. And stathmin pS38 (phosphorylation status) was positive of 37.50% (18/48) in HCC cases, while didn't detect in the adjacent tissue. TMA of healthy, hepatitis B, cirrhosis and HCC specimens showed that stathmin pS25 phosphorylation status was increased in HCC (263.77±51.38), especially in metastatic HCC tissues(399.64±89.59), compared with normal liver, hepatitis and cirrhosis samples (73.52±13.75、84.3±8.12、89.96±18.83, respectively).And the data showed stathmin pS38 was the similar as stathmin pS25, was increased in HCC (305.48±58.33), especially in metastatic HCC tissues(455.89±82.74), compared with normal liver, hepatitis and cirrhosis samples (68.23±18.01、88.62±17.99、90.05±21.52). In hepatocellur carcinoma cell lines with different metastatic potential (Hep3B、SMMC-7721、MHCC97L. MHCC97H、HCCLM3和HCCLM6), Western Blot results indicated that stathmin pS25 and pS38 was consistently increased with HCC metastatic potential. From other point of view, there was no statistic significance between tissues represented HCC related liver diseases or various human hepatocellular carcinoma cell lines with different metastatic potential in the phosphorylation statuses of stathmin pS16 and pS63,
Part two
Stathmin RNA interference influences the biological characteristic in the hepatocellular carcinoma cell
Based on the mentioned above, the alterations of cell proliferation, apoptosis, motility and cell invasion of stathmin RNA interferenced HCCLM3 cells were detected, and investigated further the role of stathmin RNA silencing to hepatocarcigenesis.
In the present study, stathmin siRNA mediated transient transfection by LipotamineTM2000 liposome was used to screen three pairs of siRNA, different si RNA concentrations and different time points for optimizing stathmin RNA interference system. Stathmin expression was effectively inhibited up to 90% under the optimized condition (transfected 80nmol/L stathmin siRNA-1 for 48 hr) in HCCLM3 cells by semi-quantitive RT-PCR, Western Blot and fluorescence aided cell immunochemistry.
Using cell count kit (CCK8), it was shown that the HCCLM3 cell proliferation was obviously depressed by 13.04±0.10%,28.10±0.41% and 37.36±2.15% at the time point of 24 hr,48hr and 72hr with the comparison to Mock group. Flow cytometry was used to analyze cells apoptosis by FITC-AnnexinV+PI apoptosis assay kit, the results demonstrated that the percentage of apoptotic cells was increased to 25.11±1.62% in RNAi group, compared with 9.20±0.64% in Mock group. Using cell adhesion assay, the results showed that the ratio of the cell adhered coated FN (i.e.0.67±0.14、0.75±0.13 and 0.96±0.09 at the time point of 20 min,40 min and 60 min) in RNAi treated group was obviously less than that in Mock group (0.94±0.17、1.18±0.09 and 1.46±0.27) at the time points mentioned above. Cell migration and invasion assay in vitro revealed that the average number of invaded and migrated HCCLM3 RNAi cells (cells of outer surface in migrant assay: 29.40±4.62; cells in invasive assay:10.36±2.97) significantly decreased in comparison with Mock group cells (cells of outer surface in migrant assay: 48.44±5.31; cells in invasive assay:28.00±4.41). All the results implied that stathmin may play a role in HCC progression and metastasis.
Part three
Alteration of stathmin phosphorylation statuses has effect on hepatocellular carcinoma cells
The object of the part is to construct the recombination plasmid containing stathmin S25A or stathmin S38A by site-directed mutation techniques, to study the alteration of HCC cell proliferation, apoptosis, motility, invasion and tumor forming ability in soft agar effected by the changes of stathmin phosphorylation statuses in vivo and in vitro, and to deep insight in the relationship between stathmin phosphorylation statuses and hepatocarconogenesis.
Sequencing reports showed that the point mutation in recombinant plasmids of Flag-pcDNA3.1-stathmin M25 S→A and Flag-pcDNA3.1-stathmin M38 S→A were correct. Using monoclone screening method, established stable SMMC-7721 cell lines were transfected with Flag-pcDNA3.1, Flag-pcDNA3.1-stathmin wt, Flag-pcDNA3.1-stathmin S25A and Flag-pcDNA3.1-stathmin S38A plasmid, which named as SMMC-7721 control、stathmin wt、stathmin S25A and stathmin S38A cell line respectively. Flag/GAPDH relative ratio in above four cell lines were 0.48±0.09、0.52±0.09、0.51±0.10 and 0.53±0.10, determined by Western Blot analysis respectively (P>0.05).This implied that the transfection efficacy of transfected cells was similar. Site phosphorylation statuses of stathmin were detected, and the data showed stathmin S25 phosphorylation status in stathmin S25A cells (0.18±0.03) was decrease compared with stathmin wt cells (0.41±0.04), meanwhile stathmin S38 phosphorylation status in stathmin S38A cells (0.36±0.04) was decrease compared with stathmin wt cells (0.78±0.02) also. And compared with SMMC-7721 control cells (0.16±0.05), stathmin expression was induced obviously in stathmin wt cells (0.76±0.12)
Using cell count kit (CCK8), the results indicated that cell proliferation in stathmin S25A and stathmin S38A cell lines were obviously depressed by 38.02±1.45% and 25.10±0.79% respectively as compared with stathmin wt cells(P<0.05). FITC-AnnexinV and PI labeling followed by Flow cytometry determination was used to analyze cells apoptosis, it was found that the cell apoptotic rate was increased to 8.82±0.30% and 7.35±0.38% in stathmin S25A and stathmin S38A cell lines, compared with that in stathmin wt cells(5.80±0.33%) (P<0.05). The results showed that ratio of stathmin S25A and stathmin S38A cells adhesion to coated FN (0.71±0.06,0.83±0.05) was markedly less than that of stathmin wt cells (1.01±0.08) (P<0.05) using cell adhesion assay. Migration and invasion assay in vitro revealed that the average number of invaded and migrated stathmin S25A and S38A cells (migrant assay:96.00±11.80,111.00±9.54; invasive assay:35.74±5.51 and 42.00±7.21 respectively) significantly decreased in comparison with stathmin wt cells (migrant assay:130.45±14.13; invasive assay:57.76±8.50) (P<0.05). Using soft agar clone forming assay, the results displayed that clone forming ability was inhibited by 42.82±1.72%in stathmin S25A cells(P< 0.01), while by 14.58±1.30 % in stathmin S38 A cells(P>0.05) in comparison with the count of cell clones in stathmin wt cells. Nude mice experiments displayed that transplanted tumor weight of statmin wt group (2.35±0.42g) was obviously increased than stathmin S25A group (1.62±0.69g)and stathmin S38A group(2.12±0.39g) (P<0.05). The incidence of lung metastasis of nude mice in stathmin S25A group(2/6) and stathmin S38A group(3/6) was significant decreased compared with stathmin wt group(4/6). And the incidence of liver metastasis of nude mice in stathmin S25A group (2/6) and stathmin S38A group (2/6) was significant decreased in compared with stathmin wt group(4/6).
Conclusion
1. Stathmin expression was highly up-regulated in HCC tissues, compared with normal liver, hepatitis and cirrhosis tissue. And stathmin expression was increased in hepatocellular carcinoma cells with increasing metastastic potential.
2. Stathmin phosphorylation statuses of S25 and S38 were regulated in HCC tissues, compared with normal liver, hepatitis and cirrhosis tissue. And stathmin S25 and S38 phosphorylation statuses were increased in hepatocellular carcinoma cells with increasing metastastic potential.
3. Stathmin RNA silencing in HCCLM3 promoted obviously apoptosis, and inhibited cell proliferation, adhesion and motility significantly, it was implying stathmin may be as a crucial factor during hepatocarcinogenesis.
4. Suppressed cell proliferation, adhesion, motility and tumor forming ability and induced cell apoptosis were observed in site mutational stathmin S25A and S38A cell lines in vitro and in vivo, compared with stathmin wt cells. It implied that stathmin S25 and S38 phosphorylation statuses may be contribute hepatocarcinogenesis.
Novelty
1. Stathmin expression and its phosphorylation statuses in tissues represented HCC related liver diseases were dynamic studied firstly, which revealed the highly expression level and the alteration of site phosporylation of stathmin may link to HCC development and metastasis.
2. Stathmin S25 and S38 phosphorylation statuses was related closely to the biological characteristics of hepatocellular carcinoma cells, it implied that stathmin phosphorylation statuses may play an important contribution to hepatocarcinogenesis.
The potential application of this work
1. Stathmin may be a potential and valuable biomarker and therapeutic target in HCC.
2. Our study provides experimental data to research further for molecular mechanism of stathmin expression and its phosphorylation statuses linked to HCC development and metastasis.
引文
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