J Mol Hist (2015) 46:485–497 DOI 10.1007/s10735-015-9639-y
ORIGINAL PAPER
The RNA-binding protein Sam68 regulates tumor cell viability and hepatic carcinogenesis by inhibiting the transcriptional activity of FOXOs Tingting Zhang1 • Chunhua Wan2 • Weidong Shi3 • Jian Xu3 • Hui Fan3 Shusen Zhang1 • Zhipeng Lin1 • Runzhou Ni1 • Xiubing Zhang3
•
Received: 19 July 2015 / Accepted: 30 September 2015 / Published online: 5 October 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Src associated in mitosis (Sam68; 68 kDa) is a KH domain RNA-binding protein that belongs to the signal transduction and activation of RNA family, and has been implicated in the oncogenesis and progression of several human cancers. Our study aimed to investigated the clinicopathologic significance of Sam68 expression and its role in cell proliferation and the underlying molecular mechanism in hepatocellular carcinoma (HCC). We demonstrated that Sam68 expression was significantly increased in HCC and high expression of Sam68 was significantly associated with Edmondson grade, tumor size, tumor nodule number, HBsAg status and Ki-67 expression. The Kaplan–Meier survival curves showed that increased expression of Sam68 was correlated with poor prognosis in HCC patients and served as an independent prognostic marker of overall survival in a multivariable analysis. In addition, through serum starvation and refeeding assay, we demonstrated that Sam68 was lowly expressed in serum-starved HCC cells, and was progressively increased after serum-additioning. Furthermore, siRNA knockdown of endogenous Sam68 inhibited cell proliferation and tumourigenicity of HCC cells in vitro, through blocking the G1 to S phase transition. Moreover, we reported that the anti-proliferative effect of
& Runzhou Ni
[email protected] 1
Department of Digestion, Affiliated Hospital of Nantong University, Medical College of Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
2
Medical College, Nantong University, Nantong 226001, Jiangsu, People’s Republic of China
3
Department of Oncology, The Second Peoples Hospital of Nantong, Nantong 226001, Jiangsu, People’s Republic of China
silencing Sam68 was accompanied with up-regulated expression of cyclin-dependent kinase inhibitors, p21Cip1 and p27Kip1, enhanced transactivation of FOXO factors (FOXO4), and dysreuglation of Akt/GSK-3b signaling. Taken together, these findings provide a rational framework for the progression of HCC and thereby indicated that Sam68 might be a novel and useful prognostic marker and a potential target for human HCC treatment. Keywords Hepatocellular carcinoma Sam68 p27kip1 FOXOs RNA binding protein
Introduction Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide and the fourth leading cause of cancer-related death in many countries, especially in East Asia, with an increasing trend in incidence (Altekruse et al. 2014; Gramantieri et al. 2009; Zhao et al. 2011). Even with aggressive treatment, HCC usually has a poor prognosis, with a 5-year survival rate as low as 25–39 % after common treatments, such as surgery, radiotherapy and chemotherapy (Ding et al. 2009; Lu et al. 2015). Hepatocarcinogenesis is a multifactorial and multistep process that contains activating oncogenes and inactivating tumor suppressor genes in different stages of HCC progression (Hu et al. 2003). In the present study, the research on molecular mechanism of the occurrence and development of HCC is widely existed, although incentive induced hepatocarcinogenesis is different from one another, but the recent discovery considered that the biosynthesis and metabolism of RNA play important roles in regulating gene expression, it has been shown that RNA expression profiling is differentially distinct between normal and
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cancer cells, indicating the possibility that aberrant regulation of RNA metabolism might be related with the development and progression of cancer (Song et al. 2010). Clarifying and investigating the roles of dysregulated of the metabolism of RNA involved in HCC development would be valuable for further understanding the molecular mechanism underlying the hepatocarcinogenesis. Sam68 (Src associated in mitosis, 68 kDa) belongs to the signal transduction and activation of RNA (STAR) family of K homology (KH) domain-containing RNA binding proteins (Lukong and Richard 2003) and is originally identified as a substrate for tyrosine kinase c-Scr phosphorylation during mitosis (Fumagalli et al. 1994; Taylor and Shalloway 1994). Sam68 is ubiquitously expressed and involved in a wide range of cellular processes including signal transduction, gene transcription, cell cycle progression, proliferation, apoptosis and alternative splicing (Bielli et al. 2011; Lukong and Richard 2003). Sam68 has been suggested to act as an adaptor in signal transduction by binding to SH2- and SH3-containing proteins, through its proline-rich regions (Richard et al. 2005). Moreover, Sam68 has been found to interact with a number of signalling proteins, such as Src, BRK, P59fyn, PI3K, PRMT, FBP21, and FBP309, through the SH2, SH3 and WW domains, further suggesting that Sam68 might act as an adaptor protein in signal transduction involved in various biological processes (Bedford et al. 2000; Derry et al. 2000; Espejo et al. 2002; Fumagalli et al. 1994; Richard et al. 1995; Taylor and Shalloway 1994). Additionally, alternative splicing of multiple genes, such as Bclxl, CD44, Sgce, Centrophilin, and Cyclin D1, has been certified to be regulated by Sam68 (Chawla et al. 2009; Paronetto et al. 2010). Meanwhile, Sam68 play a major effect in the regulation of RNA metabolism through RNA processing and export (Chawla et al. 2009; Chen et al. 1999). Furthermore, alternative splicing of multiple genes, including CD44, Bcl-xl, Sgce and Cyclin D1, has been reported to be regulated by Sam68 (Chawla et al. 2009; Matter et al. 2002; Paronetto et al. 2010). A series of published reports stated that Sam68 expression was upregulated in a variety of human cancers, including prostate cancer (Busa et al. 2007), renal cell carcinoma (Zhang et al. 2009), non-small cell lung cancer (NSCLC) (Zhang et al. 2014), colorectal cancer (Liao et al. 2013) and breast cancer (Busa et al. 2007; Song et al. 2010) indicating that Sam68 might act as a oncogene that promotes tumor progression (Li et al. 2012). All of these published studies suggest that the Sam68 might be mechanistically involved in cancer development or progression. However, no evidence is available on the possible role of Sam68 in HCC. Our observations point to a conserved and critical role for Sam68 in the regulation of the progress in HCC, the findings indicated that Sam68 was elevated during HCC
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progression using western blotting and immunohistochemistry, then testified the relationship between the expression of Sam68 and several clinicopathological features of HCC and evaluated the prognostic value of Sam68 expression for the survival of HCC patients. We also demonstrated that Sam68 had the ability of enhancing HCC cells proliferation in vitro. Finally, siRNA silencing of Sam68 suppressed cell proliferation and induced apoptosis of Huh7 and HepG2 cell lines, in accompaniment with the increased p21Cip1 and p27Kip1, as well as up-regulation of FOXO transcriptional activity and deactivation of the PI3K/Akt pathway. These studies suggest that Sam68, in addition to serving as a potential prognostic marker, may also serve as a novel therapeutic target and/or a target for inhibiting malignant tumor progression.
Materials and methods Cell lines and culture conditions Normal hepatocyte cell line (LO2) and HCC cell lines (Huh7, HepG2, 97L, 97H and 7404) were obtained from the Shanghai Institute of Cell Biology, Academic Sinica and cultured in DMEM supplemented with 10 % fetal bovine serum (Hyclone, Logan, UT, USA) (Wang et al. 2013; Wu et al. 2013). All the cells were cultured in 37 °C in a humidified chamber containing 5 % CO2. Patients and tissue samples 105 samples of HCC patients with their corresponding adjacent nontumor liver tissues were obtained from patients who had undergone curative partial hepatic resection at the Affiliated Hospital of Nantong University between 2005 and 2007. The study group consisted of 73 male and 32 female patients. Histological grades were classified to well (grades I and II; n = 44) and poorly (grades III and IV; n = 61) differentiated. The follow-up time was ranging from 1 to 86 months. The main clinicopathologic characteristics of the patients are summarized in Table 1. Eight paired HCC and adjacent normal fresh samples were frozen in liquid nitrogen immediately after surgical removal and maintained at -80 °C until use for Western blotting. Both patient tissues collected for research purposes and correlated methods obtained patient’s consent and approval from the Ethics Committee of Affiliated Hospital of Nantong University (Chang et al. 2015; Lan et al. 2014). Antibodies Antibodies for Western blotting included: rabbit antiSam68 (Santa Cruz Biotechnology, USA, 1:800), mouse
J Mol Hist (2015) 46:485–497 Table 1 Sam68 or Ki-67 expression and clinicopathologic characteristics in 105 HCC specmens
487
Characteristics
Total
Sam68 expression Low
High
Gender
p value
Ki-67 expression Low
High
0.541
Female
32
10
22
Male
73
22
51
Age (years)
0.023* 3
29
21
52
0.420
0.200
B45
36
10
26
6
30
[45
69
22
47
18
51
\50
34
14
20
11
23
C50
71
18
53
13
58
Serum AFP (ng/ml)
0.079
Edmondson grade I–II III–IV
22 10
22 51
No
22
7
Yes
83
\4.5 C4.5
0.053 14 10
30 51
15
8
14
25
58
16
67
29
21
8
15
14
76
11
65
9
67
9
26
15
55
11
44
13
37
15
21
9
60
6
13
18
68
Liver cirrhosis
0.534
Maximal tumor size (cm)
0.082
0.000*
Tumor capsula
0.000*
0.351
Absent
35
12
23
Present
70
20
50
Distant metastasis
0.397
0.231
No
55
19
36
Yes
50
13
37
Tumor nodule number Multiple HBsAg status
0.089
0.000* 44 61
Single
p value
0.309
0.000* 36
20
16
69
12
57
0.001*
0.000*
Negative
19
13
6
Positive
86
19
67
Microvascular invasion
0.237
0.570
0.441
Absent
43
13
30
9
34
Present
62
19
43
15
47
Low
24
16
8
High
81
16
65
Ki-67 expression
0.000*
Statistical analyses were performed by the Pearson v2 test * p \ 0.05 was considered significant
anti-PCNA (Santa Cruz Biotechnology, USA, 1:1000), rabbit anti-cyclin A (Santa Cruz Biotechnology, USA, 1:1000), rabbit anti-p21Cip1 (Santa Cruz Biotechnology; 1:500), mouse anti-p27kip1 (Santa Cruz Biotechnology; 1:500), rabbit anti-GAPDH (Santa Cruz Biotechnology, USA, 1:1000). The antibodies used for IHC in this study included: rabbit anti-Sam68 (Santa Cruz Biotechnology, USA,
1:100) and mouse anti-Ki-67 (Santa Cruz Biotechnology, USA, 1:100). Protein extraction and western blotting analysis Frozen liver tissues and harvested cells were carried out for western blot analysis. The tissues and cell proteins were promptly homogenized in a homogenization buffer
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containing 1 M Tris–HCl pH 7.5, 1 % TritonX-100, 1 % nonidet p-40 (NP-40), 10 % sodium dodecyl sulfate (SDS), 0.5 % sodium deoxycholate, 0.5 M EDTA, 10 lg/ml leupeptin, 10 lg/ml aprotinin, and 1 mM PMSF, then centrifuged at 12,000 g for 30 min to collect the supernatant. Before gel electrophoresis, total protein concentration determined with a Bio-Rad protein assay (BioRad, Hercules, CA, USA). The proteins were resolved by 10 % SDS—polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidine diflouride filter (PVDF) membranes (Millipore, Bedford, MA). After blocked the non-specific binding sites for 60 min with 5 % dried skim milk in TBST (20 mM Tris, 150 mM NaCl, 0.05 % Tween-20), the membranes were incubated overnight at 4 °C with proper antibody against using the primary antibodies. Then the membranes were washed with TBST for three 5 min. Subsequently, the membranes were incubated with second antibody goat-anti-mouse or goat-anti-rabbit conjugated horseradish peroxidase (1:2000; Southern-Biotech) for 1.5 h and visualized using an enhanced chemiluminescence system (ECL; PierceCompany, USA). At last, the membranes were subjected to three 8 min washes with TBST, signals were detected using enhanced chemiluminescence system (ECL, Cell Signaling Technologies) (Liu et al. 2013). Immunohistochemical evaluation Two pathologists independently scored the results of the staining and similar results were obtained in these samples. For assessment of Sam68 and Ki-67, at least five highpower fields were randomly chosen and cytoplasma (nuclear) staining was also examined under high power magnification. More than 600 cells were counted to determine the mean percent, which represented the percentage of immunostained cells relative to the total number of cells. We defined Sam68 expression levels according to the final score of each sample (low or high) by adding the scores for the intensity and extent of staining. The intensity of staining was scored as 0 (negative), 1 (weak), 2 (moderate) or 3 (strong). The extent of staining was scored based on the percentage of positive tumor cells: 0 (B10 %), 1 (10–30 %), 2 (30–50 %), 3 (50–75 %), and 4 (75–100 %). Each case was finally considered low if the final score was 0 to 3 and high if the final score was 4–7. As for assessing the expression of Ki-67, the specific experimental methods can be found in the study by Ke et al. (Wan et al. 2015).
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with 50 mg/ml of propidium iodide. The DNA contents were analyzed by a FACScan flow cytometer (Becton– Dickinson, Lincoln Park, NJ, USA). Cell proliferation assay Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) assay in accordance with the manufacturer’s instructions. Briefly, cells were seeded in a 96-well plate at a density of 2 9 103 cells/well in a volume of 50 ll, and grown overnight. CCK-8 reagents were added to each well at the indicated time after the treatments, and incubated for another 2 h at 37 °C. The absorbancy was measured at a test wavelength of 490 nm and a reference wavelength of 650 nm using a microplate reader (Bio-Rad). Each experiment was repeated at least three times. Apoptosis assay HepG2 and Huh7 cells were cultured and harvested at 48, 72 and 96 h after the cells infected with Sam68-siRNA or the control siRNA. Then add 60 ll of MuseTM Annexin V & Dead Cell Reagent (Part No. 4700-1485, 100 tests/bottle) to each tube and add 60 ll of cells in suspension to each tube. After incubated for 20 min at room temperature in the dark, then the apoptosis assay was performed by MuseTM Cell Analyser (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. siRNA and transfection Small interference RNAs (siRNA) of Sam68 and negative control were purchased from Gene Pharma (Shanghai). The Sam68-targeting sequences of siRNA#1 oligos were: 5-ACCGGATATGATGGATGAT-3, sequences of siRNA #2 oligos were: 5-ACAAGGGAATACAATCAAA-3. The control-siRNA sense sequence were: 5-UUC UCC GAA CGU GUC ACG UTT-3. Huh7 and HepG2 cells were seeded the day before transfection using DMEM with 10 % FBS without antibiotics. The HCC cells were transfected with the Sam68-siRNA oligos or the control siRNA oligos using lipofectamine 2000 in accordance with the manufacturer’s protocol. 8 h after transfection, the media were replaced with DMEM supplemented with 10 % FBS. Transfected cells were subjected to succedent experiments at 48 h after transfection.
Flow cytometric analysis
Statistical analysis
For flow cytometric analysis, cells were fixed with 70 % methanol in PBS at -20 °C overnight, then incubated with 1 mg/ml RNase A for 30 min at 37 °C in PBS and stained
The results were expressed as mean ± SE. All statistical analysis was performed using the SPSS 17.0 software package. The association between Sam68 and Ki-67
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489
Results
significant correlation between patient gender, age, serum AFP, liver cirrhosis, distant metastasis, tumor capsula or microvascular invasion. Meanwhile, we also found that the expression of Ki-67 was significantly related to gender (p = 0.023), tumor nodule number (p \ 0.001) and tumor size (p \ 0.001), but not related to other clinicopathologic parameters. Furthermore, linear regression analysis confirmed that expression of Sam68 in HCC tissues was positively correlated with that of Ki-67 (Fig. 2, p = 0.001).
Protein expression of Sam68 in primary HCC tissue samples and HCC cells
High Sam68 expression correlates with patient poor survival
To explore whether Sam68 was aberrantly expressed and played a role in the formation of hepatocelluar cancer, Western blotting analysis was performed on 8 paired clinical samples from HCC cancerous tissues and matched adjacent non-tumor liver tissues and HCC cell lines to determine their Sam68 protein levels. In the clinical samples, the Sam68 expression was markedly elevated in the tumor tissues than in comparison with the corresponding adjacent non-tumor liver tissues (Fig. 1a). Furthermore, the 7404, Huh7, 97L, HepG2 and 97H HCC cell lines showed up-regulated Sam68 transcript levels relative to the LO2 normal liver cell line. Additionally, Sam68 expression was significantly higher in Huh7 and HepG2 HCC cells (Fig. 1b).
The prognostic value of Sam68 for overall survival in HCC patients was evaluated by comparing the patients with high and low Sam68 expression. According to a Kaplan–Meier survival analysis, high Sam68 expression was significantly associated with poor prognosis compared with those with low Sam68 expression (Fig. 3, p \ 0.001). Further univariate and multivariate analyses were conducted using a Cox proportional-hazards model to examine the impact of Sam68 expression and other clinical pathological parameters in HCC patients. Edmondson grade, serum AFP, maximal tumor size, tumor nodule number, Sam68 and Ki67 expression were significant prognostic factors in the univariate analysis (Table 2). Multivariate Cox regression analyses showed that Sam68 was an independent predictor along with Edmondson grade (p = 0.025), tumor nodule number (p = 0.030), maximal tumor size (p = 0.034) and Ki-67 expression (p = 0.012). Thus, the expression of Sam68 may be useful for predicting the overall survival of HCC patients (p = 0.044, Table 3).
expression and clinicopathological features were analyzed using the v2 test. The Kaplan–Meier curves and the logrank test were performed to analyze the survival data. Multivariate analysis was constructed using Cox’s proportional hazards model. p \ 0.05 was considered statistically significant (Cui et al. 2014).
Immunohistochemical analysis of Sam68 and Ki-67 expression and its association with clinicopathological variables in HCC clinical samples Ki-67 protein is a marker of cell proliferation, since this protein is expressed in all phases of the cell cycle except the resting phase (G0), making it a marker for cycling cells (Gerdes et al. 1984). To investigate the relationship to clinicopathological variables of HCC, Sam68 and Ki-67 expression were deginited in 105 HCC surgical specimens using IHC staining, in the positive cases, Sam68 was majority located in the nucleus of the HCC cells. Likewise, Ki-67 was mainly detected in nucleus (Fig. 1c). In cases with differented differentiated HCC tissue, we also observed the stain of Sam68 and Ki-67 showed a sharp contrast between the infiltrative areas of positively staining representing the tumor tissue, negative stained the nontumor and the adjacent (Fig. 1c). The relationship between Sam68 expression and various clinicopathological parameters is described in Table 1. Sam68 expression was significantly correlated with Edmondson grade (p \ 0.001), maximal tumor size (p \ 0.001), HBsAg status (p \ 0.001), tumor nodule number (p \ 0.001) and the Ki67 expression (p \ 0.001). There was no statistically
Sam68 increase hepatocellular carcinoma cell proliferation Given the fact that the above data have demonstrated the critical involvement of Sam68 in the regulation of the prognosis and survival of HCC patients, we employed cultured HCC cells to evaluate the regulatory role of Sam68 in HCC development. To analyze the role of Sam68 on cell viability, the Huh7 and HepG2 HCC cell lines with higher expression were choosed (Fig. 1b). First, we detected the expression of Sam68 during cell cycle progression in Huh7 and HepG2 cells. The Huh7 cell line was arrested in G1 phase by serum deprivation for 72 h and the G1 phase increased from 49.95 to 69.95 %. Similarly, the G1 phase of HepG2 cell increased from 50.45 to 70.02 %. Upon serum addition, the cells were released from G1 phase and reentered S phase (Fig. 4a). In parallel experiments, Western blotting analysis showed the expression of Sam68 in Huh7 and HepG2 cells were relatively low after serum starvation for 72 h, and were progressively
123
490
Fig. 1 Sam68 protein expression in primary HCC tissue samples and HCC cells. a Expression of Sam68 in eight representative matched samples of HCC tissue (T) and adjacent nontumor liver tissues (N). The bar chart demonstrates the ratio of Sam68 protein to GAPDH for the left by densitometry. The same Experiment was repeated at least three times. The data are mean ± SEM (#p \ 0.05, compared with adjacent normal tissues). b Western blotting analysis shows that Sam68 protein expression is increased in HCC cells compared with
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the normal hepatocyte cell lines (LO2). The bar chart demonstrates the ratio of Sam68 protein to GAPDH by densitometry. c Immunohistochemical analysis of Sam68 and Ki-67 expression in HCC tissues. Paraffin-embedded tissue sections were stained with antibodies for Sam68 and Ki-67 and counterstained with hematoxylin. High expression of Sam68 and Ki-67 is showed in HCC tissue samples. Low concentrations of Sam68 and Ki-67 in adjacent non-tumourous tissue
increased upon serum refeeding. Further more, we found that the expression of Sam68 had a accordance trend with that of cyclin A and PCNA both in Huh7 and HepG2 cell lines (Fig. 4b). Down-regulation of Sam68 inhibits cellular viability and promotes cell cycle arrest
Fig. 2 Correlation between Sam68 expression and Ki-67 index in HCC. Scatterplot of Sam68 versus Ki-67 with regression line showing a positive correlation using the Spearman’s correlation coefficient
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To further elucidate the biological functions of Sam68 in HCC, we used siRNA to knockdown Sam68 expression in the Huh7 and HepG2 cells. The efficiency of two Sam68specific siRNA-mediated down-regulation were assessed by Western blotting analysis. As predicted, siRNA knocked down the protein expression of Sam68 as compared with control treatment and negative control siRNA (Fig. 5a). Withwise, it showed that the PCNA protein correlated positively with Sam68 expression (Fig. 5a). To determine the effect of Sam68 knock-down on cell cycle, we then examined the possible effects of Sam68 expression on the
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491
The previous finding that the expression of p27Kip1 could be transcriptionally regulated by FOXO family transcriptional factors prompted us to test whether Sam68 could modulate the transactivation activities of FOXO factors (Brunet et al. 1999; Cardozo and Pagano 2004; Seoane et al. 2004) and the transcriptional activities of FOXO factors have previously been found to be modulated by Akt activation (Brunet et al. 1999), we assessed FOXO4 phosphorylation and found that the phosphorylation levels of FOXO4 were down-regulated in Sam68 siRNA-infected cells, compared with those in the control-siRNA cells. It is also of worth note that the level of phosphorylated Akt in silencing Sam68 cells were dramatically lower than that in control-siRNA cells (Fig. 6b). Taken together, these data demonstrated that the observed up-regulation of cell cycle inhibitors p21Cip1 and p27Kip1 caused by lower expressed Sam68 was correlated with inhibition of the Akt kinase activity and subsequently increased the transactivation activities of FOXO factors. Fig. 3 Kaplan–Meier survival curves for low versus high Sam68 expression in 105 patients with HCC show a highly significant separation between curves (p \ 0.001, log-rank test). On the basis of final score of each sample, the patients were divided into two groups: low (scores 0–3) and high (scores 4–7)
cell cycle by flow cytometric analysis. We found that silencing Sam68 in the Huh7 and HepG2 cell lines inhibited the G1/S transition (Fig. 5b). Furthermore, we investigated the role of Sam68 in the proliferation of HCC cells. Using CCK-8 assay, we found that cell proliferation rate of Huh7 and HepG2 cells treated with siRNA exhibited a significant decrease compared with the negative control siRNA (Fig. 5c). In parallel experiments, we investigated the role of Sam68 in the apoptosis of HCC cells. More apoptotic cells were found in the Sam68-low expressing HCC lines than in control lines (Fig. 5d). Collectively, our data indicated that the Sam68 might be involved in a specific advancing effect on cell proliferation and tumourigenicity associated with cell cycle progress in HCC cells. Silencing Sam68 promoted the transcriptional activity of FOXOs via inhibiting Akt activation To investigate the mechanism that mediates Sam68 silenced is related to cell cycle arrest, we examined the expression of mitotic cyclins (cyclin A), p21Cip1 and p27Kip1 by Western blotting. Knocking down of Sam68 by siRNA lead failure to accumulate cyclin A, while the levels of p21Cip1 and p27Kip1 has a slightly increasing (Fig. 6a). We next examined the mechanisms of that mediates the level of p21Cip1 and p27Kip1 function in HCC cell lines.
Discussion The biosynthesis and metabolism of RNA represent one of the capital steps in gene regulation. Mounting evidence has suggested that RNA expression profiles in cancer cells are significantly different from those in benign cells, suggesting the possibility that an aberrant regulation of RNA metabolism might be associated with the initiation and progression of tumour (Cooper et al. 2009; Johnson and Johnson 2008; Perrotti and Neviani 2007). In consistence with this notion, multiple proteins related to the metabolism of RNA have been found to be dysregulated in primary tumours (Lee et al. 2007; Michlewski et al. 2008; Ross et al. 2001). Sam68 is a multifunctional protein, known to govern RNA metabolism, cellular signal transduction, transcription, proliferation, apoptosis and HIV-1 replication (Modem et al. 2011). Previous researches suggested that the functions of Sam68 were mixed in different cellular contexts. A few studies indicated that Sam68 acted as a tumor suppressor. For example, Sam68 deficiency resulted in neoplastic transformation of murine NIH3T3 fibroblasts. Reduction of Sam68 was associated with anchorage—independent growth, defective contact inhibition, and the ability to form metastatic tumors in nude mice (Liu et al. 2000), while overexpression of Sam68 in NIH-3T3 fibroblasts led to both cell cycle arrest and apoptosis (Taylor et al. 2004). On the contrary, a large proportion of recent reports demonstrated that Sam68 played an oncogenic role. Sam68 knockdown in polyoma middle T-antigen (PyMT) oncogene transformed cell lines delayed tumorigenesis and metastasis formation in nude mice
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492 Table 2 Contribution of various potential prognostic factors to survival by univariate analysis in 105 HCC specimens
J Mol Hist (2015) 46:485–497
Characteristics
Total
Survival status Died n = 71
p value Alive n = 34
Gender
0.096
Female
32
25
7
Male
73
46
27
Age (years)
0.308
B45
36
26
10
[45
69
45
24
\50
34
16
18
C50
71
55
16
Serum AFP (ng/ml)
0.002*
Edmondson grade I–II III–IV
0.000* 44 61
20 51
24 10
No
22
15
7
Yes
83
56
27
\4.5
29
2
27
C4.5
76
69
7
No
55
37
18
Yes
50
34
16
Single
36
9
27
Multiple
69
62
7
35
22
13
70
49
21
Negative
19
10
9
Positive
86
61
25
Liver cirrhosis
0.583
Maximal tumor size (cm)
0.000*
Distant metastasis
0.552
Tumor nodule number
0.000*
Tumor capsula Absent Present HBsAg status
0.301
0.103
Microvascular invasion
0.274
Absent
43
31
12
Present
62
40
22
Low
24
6
18
High
81
65
16
Low
32
8
24
High
73
63
10
Ki-67 expression
0.000*
Sam68 expression
0.000*
2
Statistical analyses were performed by the Pearson v test * p \ 0.05 was considered significant
(Richard et al. 2008). Additionally, downregulation of Sam68 in prostate cancer cells delayed cell cycle progression and reduced the proliferation rate (Busa et al. 2007). But the expression that promote translocations in hepatocellular cancer has not been fully elucidated.
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In the present study, we examined Sam68 protein expression in paired primary HCC samples. We found that Sam68 expression was elevated at translational levels in most primary HCC tumor tissues and HCC cell lines. Consistent with these observations, IHC analyses also
J Mol Hist (2015) 46:485–497 Table 3 Contribution of various potential prognostic factors to survival by Cox regression analysis in 105 HCC specimens
493
Hazard ratio
95 % confidence interval
p value
Gender
0.133
0.876–2.732
1.547
Age (years)
0.897
0.536–1.501
0.680
Serum AFP (ng/ml)
1.647
0.849–3.194
0.140
Edmondson grade
2.039
1.091–3.809
0.025*
Liver cirrhosis
1.340
0.705–2.545
0.372
Maximal tumor size (cm)
6.005
1.144–31.528
0.034*
Tumor capsula
0.879
0.508–1.520
0.644
Distant metastasis
0.772
0.443–1.343
0.359
Tumor nodule number
2.578
1.096–6.064
0.030*
HBsAg status
0.501
0.205–1.221
0.128
Microvascular invasion
1.297
0.736–2.285
0.369
Ki-67 expression
3.362
1.307–8.646
0.012*
Sam68 expression
2.929
1.307–8.328
0.044*
Statistical analyses were performed using log-rank test * p \ 0.05 was considered significant
Fig. 4 Expression of Sam68 and cell cycle related molecules in proliferating HCC cells. a Flow cytometry quantitation of cell cycle progress in Huh7 and HepG2 cells. Cells were synchronized at G1 after serum starvation for 72 h, then progressed into cell cycle by adding medium containing 10 % FBS for the indicated times. b Huh7 and HepG2 cells were synchronized by serum starvation for 72 h and upon serum releasing, cell lysates were prepared and analyzed by Western blotting using antibodies directed against Sam68, PCNA and cyclin A. GAPDH was used as a control for protein load and integrity. The bar chart below demonstrates the ratio of Sam68, cyclin A and PCNA protein to GAPDH for each time point by densitometry. The data are mean ± SEM (n = 3, *,#,^p \ 0.05, compared with control: S72h). S serum starvation, R serum release
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Fig. 5 Sam68 knockdown inhibited cell proliferation and effects of altered Sam68 expression on cell cycle in HCC cells. a Western blotting analysis showed that siRNA treatment of Sam68 markedly decreased Sam68 and PCNA levels 48 h after siRNA transfection in Huh7 and HepG2 cells. The bar chart below demonstrates the ratio of Sam68 and PCNA protein to GAPDH by densitometry. The data are mean ± SEM (*,#p \ 0.05, compared with the negative control). b Sam68 knockdown resulted in the delay of G1-S transition and significant arrest in G2 phase of Huh7 and HepG2 cells, after these
HCC cells were released from the synchronous status. The data are showed as mean ± SE for three experiments. c CCK-8 assay showed that Sam68 knockdown inhibited cell proliferation. Cell Counting Kit-8 reagents were added to the medium and incubated for additional 2 h. Absorbance was measured at each indicated time (0, 1, 2 and 3 days). Each time point was derived from three independent experiments. d The effect of Sam68 expression on apoptosis in the Huh7 and HepG2 cells at 48, 72, and 96 h after the infection (*,#p \ 0.05, compared with the negative control)
Fig. 6 Depletion of Sam68 enhances the transcriptional activity of FOXO factors and inhibits Akt signalling. a Western blotting analysis of p21Cip1, p27Kip1 and cell cycle related protein cyclin A in Sam68 depletion Huh7 and HepG2 cells. b Western blotting analysis of
phosphorylated Akt (p-Akt), total Akt, phosphorylated GSK-3b (pGSK-3b), total GSK-3b, phosphorylated FOXO4 (p-FOXO4-Ser193), and total FOXO4 protein in the indicated HCC cell lines
showed that Sam68 expression was increased in most HCC tumor tissues compared with the adjacent non-tumorous liver tissues. These results indicated that high-regulated Sam68 expression may play an important role in HCC
development. Furthermore, Sam68 was found to localize to both the cytoplasm and nuclei of cancer cells. It is particularly noteworthy that the subgroup of patients with well differentiated or early stage tumors often displayed
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cytoplasmic Sam68 staining, while HCC patients with advanced clinical stage HCC often exhibited nuclear localization of Sam68. In addition, patients with cytoplasmic Sam68 localization had a better clinical outcome than patients with Sam68 nuclear localization. These researches suggested that nuclear Sam68 might play a dominant role in oncogenesis of HCC. However, distinguished from our results, cytoplasmic localization of Sam68 was significantly correlated with cancer progression and poor prognosis in human renal cell carcinoma and breast cancer (Li et al. 2012; Song et al. 2010). In the IHC analysis, upregulated Sam68 was significantly associated with Edmondson grade, maximal tumor size, HBsAg status, tumor nodule number and Ki-67 expression. The relationship between high Sam68 expression, larger tumor size, tumor nodule number and Edmondson grade suggested that the increase in Sam68 expression may help facilitate the rapid expansion of HCC. Additionally, most of the welldifferentiated HCC samples were weaker for Sam68 and Ki-67 expression, but Sam68 and Ki-67 expression was profoundly positive in the moderately and poorly differentiated tumor samples. Furthermore, Sam68 expression was an independent prognostic factor for overall survival in the multivariate analysis. Kaplan–Meier survival analysis revealed that high Sam68 expression was significantly linked to poor prognosis in the HCC patients. These results suggest that Sam68 can serve as a new predictor of prognosis in HCC patients after surgical resection. This finding strengthens the hypothesis that Sam68 acts as a novel HCC promoter. Although intrinsic mechanisms that modulate Sam68 function are beginning to emerge, the regulatory events contributing to its expression remain elusive. Then we detected the expression of Sam68 during cell cycle progression in HCC cells and found that the expression of Sam68 was promoted the HCC cells proliferation. This heightened proliferation was associated with a sharp decrease in G0/G1 and a corresponding increase in S and G2/M phases in exponentially growing cultures. The increased S phase population in turn correlated with enhanced expression of proliferating cell nuclear antigen (PCNA) and cell cycle regulatory protein such as cyclin A, which are required for the transition of cells from G1 to S phase. Therefore, to further determine whether Sam68 plays an effect on the proliferation of HCC cell lines, we knocked down the expression of Sam68 in Huh7 and HepG2 cells using specific siRNA. Western blotting showed an obviously decrease of PCNA protein expression, which indirectly suggested the role of Sam68 in the proliferation of HCC cells. The molecular mechanisms underlying the possible role of Sam68 in human cancer development and progression, however, remain largely unknown. The key discovery presented in this report are that suppressing the expression
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of endogenous Sam68 inhibited the proliferation and tumourigenicity of HCC cell lines. Our finding has linked Sam68 to dysregulation of cell cycle control by identifying the inhibitory role of Sam68 silencing on G1/S transitional phase entry. Mechanistic basis for such an anti-proliferative effect of Sam68 consumption might be linked to suppression of Akt phosphorylation and subsequent activation of FOXOs, which may consequently promote the enhancement of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. We found that siRNA knockdown of endogenous Sam68 inhibited cell proliferation through blocking the G1 to S phase transition. Moreover, we demonstrated that the anti-proliferative effect of silencing Sam68 on HCC cancer cells was associated with up-regulation of cyclin-dependent kinase inhibitor p21Cip1 and p27Kip1, enhanced transactivation of FOXO factors, and attenuation of Akt/GSK-3b signalling. Thus, our study suggests a potential usefulness of Sam68-targeting strategies to deliver an anti-proliferative therapeutic effect through activating the FOXO/p21/p27 pathway and deactivating the Akt/GSK-3b signaling. Recent studies have shown that FOXO4 mediates tumor-suppressive function in part through transcriptional regulation of the cell cycle arrest p27kip1 gene. Meanwhile, FOXO4 interacts with some molecules like Ku70 and inhibited FOXO4-mediated p27kip1 transcription and cell cycle arrest. (Brenkman et al. 2010). Hence, we put forward a hypothesis that Sam68 could interact with FOXO4 in HCC cell lines, then regulate the level of p27kip1 protein. Despite these findings, further investigations must be conducted to clarify the explicit role of Sam68 in HCC development. Accumulating evidence indicates that Sam68 plays a prominent role in tumor survival and progression. Future investigation will be aimed at determining whether aberrant Sam68 up-regulation could be accelerate the progress of HCC through other signal channel. Thus, the identification of such proteins would be valuable for further understanding the molecular mechanism underlying the progression of HCC cancer and might represent an approach to developing new therapeutic targets and intervention strategies against cancer. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81272708). Compliance with ethical standards Conflict of interest
None.
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