| 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 potentialTo 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 twoStathmin RNA interference influences the biological characteristic in the hepatocellular carcinoma cellBased 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 threeAlteration of stathmin phosphorylation statuses has effect on hepatocellular carcinoma cellsThe 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).Conclusion1. 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.Novelty1. 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 work1. 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. |
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