RMP promotes venous metastases of hepatocellular carcinoma through promoting IL-6 transcription


Hepatocellular carcinoma (HCC) is believed to arise from tumor-initiating cells (T-ICs), which are responsible for tumor relapse and metastases. Portal vein tumor thrombus (PVTT) is raised from HCC and strongly correlated to a poor prognosis. However, the mechanism underling the formation of PVTT is largely unknown. Herein, we provide evidence that RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) was progressively upregulated in PVTT and overexpressed RMP appeared to increase T-ICs self-renewal. Moreover, RMP promoted metastases of PVTT cells and HCC cells in vitro and in vivo. Knockdown of RMP attenuated T-ICs self-renewal and reversed epithelial–mesenchymal transition (EMT) in HCC and PVTT cells. The neutralizing assays suggested that interleukin-6 (IL-6) had an indispensable role in RMP regulating metastases and self-renewal of HCC cells. Furthermore, the transcription of IL-6 was verified to be modulated by RMP via interaction with p65 and RPB5, through which expanding the T-IC/cancer stem cell populations, as well as inducing EMT was promoted. These results suggested that RMP may promote PVTT formation by promoting IL-6 transcription. Thus, RMP serves as a potent factor contributed to develop PVTT and a promising therapeutic target for HCC patients.


Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related mortality worldwide.1 Portal vein tumor thrombosis (PVTT) in patients with HCC is a major complication and is associated with poor survival.2 Approximately 50–80% of HCC has been reported to be accompanied by portal or hepatic vein invasion, as demonstrated by magnetic resonance imaging and ultrasonography.3 However, the mechanism underlying the formation of PVTT remains largely unknown. Tumor-initiating cells (T-ICs) are associated with metastases, recurrence and resistant to chemotherapy in variety of solid tumors, because of their capacity to self-renew and differentiate. On the basis of this hypothesis, cancer cells may be more dependent on the activation of oncogenic pathways.4 Therefore, targeting T-ICs in HCC is one novel therapeutic approach for intervention of metastatic recurrence of HCC.

Interleukin-6 (IL-6) is one of the most important nuclear factor (NF)-κB-dependent cytokines,5 and has been implicated as an autocrine and paracrine promoter of cancer growth for several human cancers. The production of IL-6 accounted for the striking male preference in HCC development in both human and mice.3,6 In addition, a highly metastatic cell line derived from HCC produces much higher levels of IL-6 than poorly metastatic cells derived from the same tumor in rat model.7 The IL-6/signal transducer and activator of transcription 3 (STAT3) pathway has been shown to be important for chemoresistance of EpCAM hepatic T-ICs.8 CD24 may also interact in this pathway to drive HCC tumor initiation through STAT3-mediated NANOG regulation and tumor initiation of hepatic T-ICs.9 However, what causes the aberrant expression of IL-6 remains elusive.

RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP), also known as unconventional prefoldin RPB5 interactor, is associated with the RNA polymerase II subunit, RPB5. The RMP gene was first isolated and cloned from a human HepG2 complementary DNA library in 1998 and counteracted transactivation by hepatitis B virus X protein, which has an important role in hepatitis B virus-associated HCC.10 RMP also is required to maintain genome stability by having an important function in controlling the cell cycle.11 Depletion of RMP increases cisplatin and rapamycin-induced apoptosis in ovarian cancer cells by activating mitochondrial S6K1-BAD signaling,12 and overexpression of RMP had an anti-apoptotic effect in the proliferation and growth of HCC cells.13,14 Nevertheless, the role of RMP in HCC invasion and metastases remains to be determined.

In this study, we showed that RMP was preferentially expressed in PVTT clinical samples and liver T-ICs and exerted roles in maintaining the stem cell population and metastases potential by promoting IL-6 production.


RMP expression was associated with venous metastases in HCC

The expression of RMP in HCC cell lines was examined and the results showed that RMP was moderately expressed in low metastatic SMMC7721, and was strongly expressed in highly metastatic PVTT cell CSQT-2 (Figures 1a and b), suggesting that RMP is involved in HCC aggressiveness and metastases. To text this hypothesis, we examined the expression of RMP in 40 human HCC patients with PVTT by quantitative reverse transcriptase–polymerase chain reaction (PCR) (Figure 1c) and found that RMP was overexpressed in PVTT clinical samples. Furthermore, immunohistochemical analysis showed that RMP was upregulated in 18/20 of PVTT and 174/269 of HCC tumor according to the German immunoreactive score, which has been described before (Figure 1d).15 On basis of the clinical and pathological parameters, RMP levels were found to be significantly associated with portal venous invasion (P=0.011) (Supplementary Table 1).

Figure 1

RMP expression was associated with venous metastases in HCC. (a, b) Expression of RMP in human HCC cell lines was examined by western blot assay and real-time PCR. Error bars represent s.e.m. from at least three independent experiments. (c) Real-time PCR analysis to quantify the endogenous levels of RMP in 40 HCC patients with PVTT. (PVTT, PVTT tumor tissues; tumor, HCC tumor tissues; para-tumor, adjacent normal-like tissue). Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01 (Student’s t-test). (d) Representative images of RMP staining in PVTT, HCC tumor and para-tumor. Bars, 100μm (upper panel). Statistics of the RMP immunohistochemistry (IHC) scores. P<0.0001 (Chi-square test) (low panel).

RMP regulated the aggressive behavior in HCC

PVTT arises from the HCC cells invasion into the portal vein that markedly deteriorates hepatic function.2 As shown in Supplementary Table 1, high RMP expression was positively correlated with portal venous invasion. This finding suggested that tumor cells with high RMP were endowed with improved metastatic abilities. To test this hypothesis, lentivirus delivered two different interfering sequences significantly inhibited the mRNA and protein levels of RMP (Supplementary Figure 1A). The migratory and invasive abilities of PVTT cell line, CSQT-2, and HCC cell line, MHCCLM3 were quantified. Knockdown RMP in CSQT-2 and MHCCLM3 cells generated a significant reduction in migration and invasion capability (Figures 2a and b,Supplementary Figure 1B). To test the metastatic role of RMP in vivo, CSQT-2-control cells and CSQT-2-shRMP cells were injected into the lateral tail vein of nude mice. Six weeks later, less and smaller micrometastatic lesions were microscopically detected in the lungs of nude mice inoculated with CSQT-2-shRMP cells compared with those inoculated with control cells (Figure 2c). At the same time, we also knocked down RMP in HCC cell lines MHCC97H cells, which possessed high metastases to lung tissue, and similar results were observed (Figure 2c). The CSQT-2-control cells and CSQT-2-shRMP cells were also inoculated intrasplenically into nude mice. As shown in Figure 2d, knockdown RMP significantly reduced metastatic growth (Figure 2d). The similar results were observed by SMMC7721 control/SMMC7721 shRMP cells in the same model (Figure 2d).

Figure 2

RMP enhanced metastatic potential of HCC cells. (a) The migration properties of CSQT-2 control/shRMP(shRMP1) and MHCCLM3 control/shRMP(shRMP1) cells were analyzed by way of the migration assay using a transwell chamber. Cell migration was determined with crystal violet staining. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05 calculated by Student’s t-test. Bars, 500 μm. (b) The invasive properties of the cells were analyzed by way of invasion assay using a Matrigel-coated Boyden chamber. Invasive cells were determined with crystal violet staining. Data are mean±s.e.m. and are representative of three independent experiments. **P<0.01 calculated by Student’s t-test. Bars, 500 μm. (c) Representative images of lung tissue sections from each group were shown (hematoxylin and eosin stain; bars, 100 μm) (left panel). RMP expression was analyzed by immunoblot (middle panel). The number of lung metastatic foci in each group (n=5) of CSQT-2 control or CSQT-2 shRMP and MHCC97H control and MHCC97H shRMP xenografted mice was calculated microscopically 6 weeks after tail vein injection. **P<0.01 calculated by Student’s t-test. (right panel). (d) Photographs of HCC tumors growing in the livers of nude mice given intrasplenic injections of CSQT-2 control or CSQT-2 shRMP and SMMC7721 control or shRMP. Bars, 5 mm. (upper panel) The number of tumors in each group (n=5) was counted after inoculation 8 weeks. ***P<0.001 calculated by Student’s t-test (low panel).

RMP knockdown reduced stem/progenitor characteristics of HCC cells

More and more studies indicated that human metastases-initiating cells may be found within sub-populations of cancer stem cells (CSCs).16 CSQT-2 and MHCCLM3 cells, derived from suspension-cultured spheroids, exhibited a much higher expression of RMP, compared with cells from a monolayer culture (Figure 3a). Furthermore, we found CSQT-2-shRMP and MHCCLM3-shRMP cells formed much fewer spheroids in suspension culture compared with control cells (Figure 3b, Supplementary Figure 2A). Moreover, limiting dilution assay revealed that knockdown RMP in MHCCLM3 decreased the proportion of T-ICs (Supplementary Figure 2B). Overexpressed RMP in MHCCLM3 cells formed more spheroids in suspension culture (Supplementary Figure 2C). We then compared the expression of stemness-associated genes between RMP knockdown clones and controls (Figure 3c,Supplementary Figure 2D). Consistently, the downregulation of the stem cell-associated genes, including KLF4, NANOG, C-MYC, BMI1, NOTCH1, OCT4 and SOX2, was observed in RMP knockdown cells. The specific hepatic T-ICs biomarkers, CD24, CD90 and EpCAM, were also significantly decreased in knockdown cells (Figure 3d, Supplementary Figures 2E and F). Forced RMP expression in MHCCLM3 increased stem cell-associated genes, KLF4, NANOG, BMI1, OCT4 and SOX2, and hepatic T-ICs biomarkers, CD24, CD90 and EpCAM (Supplementary Figures 2G and H).

Figure 3

RMP knockdown reduced characteristics of liver T-ICs. (a) Spheroid assay of CSQT-2 and MHCCLM3 cells was performed, and the mRNA of spheroids was extracted and subjected to real-time PCR. Error bars represent s.e.m. from at least three independent experiments. *P<0.05 (Student’s t-test). (b) Spheroid assay of CSQT-2 control/CSQT-2 shRMP and MHCCLM3 control/shRMP cells were performed. Bars, 500 μm Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01 (Student’s t-test). (c) The mRNA expression of genes related to stemness in CSQT-2 control/shRMP and MHCCLM3 control/shRMP cells by real-time reverse transcriptase (RT)–PCR. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (Student’s t-test). (d) The mRNA expression of genes related to hepatic T-ICs biomarkers in CSQT-2 control/shRMP and MHCCLM3 control/shRMP cells by real-time RT–PCR. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01 (Student’s t-test). (e) The mRNA expression of genes related to EMT in CSQT-2 control/shRMP and MHCCLM3 control/shRMP cells by real-time RT–PCR. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (Student’s t-test). (f) Knockdown of RMP in CSQT-2 and MHCCLM3 cells resulted in upregulation of E-cadherin as determined by immunoblot.

As epithelial–mesenchymal transition (EMT) has been linked to the CSCs phenotype, we examined whether the decrease in RMP resulted in shift of EMT markers. The results showed that the expression of EMT markers, Snail and Fibronectin, was downregulated, but epithelial-associated genes including E-cadherin was increased in CSQT-2-shRMP and MHCCLM3-shRMP cells (Figures 3e and f, Supplementary Figure 2I). Moreover, upregulation of RMP in MHCCLM3 cells resulted in induction of EMT markers (Supplementary Figure 2J).

As tumorigenicity is one of the characteristics of T-ICs/CSCs, we examined the tumorigenicity of hepatoma without RMP in vivo. MHCCLM3-shRMP had a significant inhibition of tumor formation in nude mice xenograft tumors compared with control mice. Knockdown of RMP in SMMC7721 cells resulted in no tumor formation in any mice (Supplementary Figure 2K). Importantly, MHCCLM3 RMP cells were more tumorigenic in nude mice compared with the control cells (Supplementary Figure 2L).

RMP maintained T-ICs self-renewal and drove tumor initiation by promoting IL-6 production

Although the key mechanism in regulating hepatic T-ICs/CSCs remains elusive, several signaling pathways, such as Wnt, Akt, transforming growth factor-beta and STAT3 have been found to be frequently deregulated in hepatic CSCs.17 Transforming growth factor-beta, Akt and Wnt/β-catenin pathways were slightly altered in the RMP knockdown cells (Supplementary Figures 3A and B). Interestingly, we found that phosphorylation of STAT3 was robustly decreased in CSQT-2-shRMP and MHCCLM3-shRMP cells compared with control cells (Figure 4a, Supplementary Figure 3C). As a major STAT3 activator, IL-6 was also decreased in CSQT-2 and MHCCLM3 cells with RMP depletion (Figure 4b, Supplementary Figure 3D). Alternatively, overexpression of RMP increased IL-6 expression in MHCCLM3 cells (Figure 4b). Luciferase reporter assay indicated that downregulation of IL-6 by knockdown RMP was achieved through inhibiting the activity of the IL-6 promoter activity and upregulation of IL-6 was achieved through the enhancement of the IL-6 promoter (Figure 4c). Meanwhile, secretion of IL-6 in cell culture supernatant increased in a similar manner as quantified through enzyme-linked immunosorbent assay (Figure 4d, Supplementary Figure 3E). Consistently, tumors derived from MHCCLM3 cells also displayed higher IL-6 and p-STAT3 immunostaining than MHCCLM3-shRMP (Supplementary Figure 3F). In addition, we determined the expression of RMP, IL-6, CD24 and EpCAM in clinical HCC samples. The results showed that the RMP mRNA level is positively associated with the IL-6, CD24 and EpCAM level in primary tumors and PVTT tumors when the values for each individual patient were plotted (Supplementary Figure 3G). These results were also confirmed by western blot and immunohistochemical assays (Supplementary Figures 3H and I). Again, these clinical data provide strong support for our hypothesis that RMP was likely the reason for elevating IL-6 in the development of PVTT.

Figure 4

RMP maintain tumor cell self-renewal and metastases though promoting IL-6 production. (a) Decreased level of phosphorylated STAT3 (Y705) but not its parental form was observed after RMP knockdown in CSQT-2 and MHCCLM3 cells. (b) Knockdown of RMP in CSQT-2 and MHCCLM3 cells resulted in down-regulation of IL-6 and overexpressed of RMP resulted in upregulation of IL-6 by real-time PCR. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; ***P<0.001 (Student’s t-test). (c) Luciferase reporter assay of IL-6 promoter was assessed in MHCCLM3 control/MHCCLM3 shRMP and MHCCLM3 CMV/MHCCLM3 RMP cells. Data are mean ±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01 (Student’s t-test). (d) Knockdown of RMP in CSQT-2 and MHCCLM3 cells resulted in down-regulation of IL-6 and overexpressed of RMP resulted in upregulation of IL-6 by enzyme-linked immunosorbent assay (ELISA). Data are mean ± s.e.m. and are representative of three independent experiments. *P<0.05; ***P<0.001 (Student’s t-test). (e) Representative images showing the formation of tumor metastases in the mouse liver. Arrows indicate metastatic tumors in the mouse liver. Bars, 1 cm. (f) The number of tumors in each group (n=5) was counted after inoculation 8 weeks. **P<0.01 calculated by Student’s t-test. (g) Representative images of lung tissue sections from each group were shown (hematoxylin and eosin stain; bars, 100 μm). (h) The number of lung metastatic foci in each group (n=5) calculated microscopically 6 weeks after tail vein injection. **P<0.01 calculated by Student’s t-test.

To determine whether RMP drives tumor initiation and T-ICs self-renewal by IL-6, we neutralized the IL-6 effect by antibody and IL-6 short hairpin RNA. The major stem cell-associated markers were decreased by IL-6 antibody or lentivirus delivered short hairpin RNA (Supplementary Figure 4A). Spheroids formation assay revealed that neutralizing antibody could counteract more spheroids formation by RMP overexpressed (Supplementary Figure 4B). On the contrary, IL-6 treatment largely eliminated the effect of RMP knockdown on spheroid formation (Supplementary Figure 4C). Furthermore, we also found that IL-6 neutralizing antibody can attenuate the enhanced ability of migration by RMP overexpressed (Supplementary Figure 4D). Consistently, IL-6 treatment restored shRMP-mediated migration reduction (Supplementary Figure 4E). In vivo assay overexpressed RMP significantly, promoted metastatic growth (Figures 4e and f). In contrast, knockdown IL-6 had a potent effect in block the metastases of MHCCLM3 cells, completely overcoming promoting effect by RMP (Figures 4e and f). In another metastases model, a similar result was observed. Knockdown IL-6 expression nearly completely blocked the formation of lung metastases by MHCCLM3 RMP and control cells (Figures 4g and h). As the main functions of IL-6 were likely mediated by activation of STAT3,5 we activated MHCCLM3 control and shRMP cells by infecting cells with an adenovirus that constitutively expresses active STAT3 (Ad-caSTAT3). Spheroids formation assay revealed that both RMP knockdown and control cells generated more hepatospheres upon constitutive STAT3 activity than Ad-GFP-treated cells (Supplementary Figure 4F). Furthermore, no difference was observed between RMP knockdown and control cells by Ad-caSTAT3 stimulation. In contrast, a STAT3 inhibitor inhibited spheroids formation (Supplementary Figure 4F). These results provided direct evidence that RMP mediated tumor cell self-renewal and metastases mainly through IL-6 production and STAT3 activation.

RMP upregulated IL-6 expression through an interaction with p65 and RPB5

To investigate whether NF-κB activation accounts for the stimulatory effect of RMP, we utilized inhibitors of NF-κB to determine their effects on IL-6 production in RMP knockdown and control liver cancer cells. Inhibition of NF-κB using Bay11 nearly fully abolished the RMP-induced production of IL-6 (Figure 5a and Supplementary Figure 5A). A dominant inhibitor of NF-κB (IkBaDN), which was co-expressed with RMP in SMMC7721 cells, also attenuated the increased expression of IL-6 (Supplementary Figure 5B). In order to assess the effect of RMP on NF-κB, we measured NF-κB activity through a luciferase reporter assay. The results indicated that knockdown of RMP significantly attenuated the activity of NF-κB in hepatoma cells (Figure 5b and Supplementary Figure 5C). Furthermore, RMP could enhance p65 transcriptional activity, and knockdown of RMP decreased p65-induced IL-6 production (Figure 5c and Supplementary Figure 5D). The coimmunoprecipitation assay revealed that RMP could associate with p65 and RPB5 with treatment of tumor necrosis factor-α, which were components of the transcriptional PIC of IL-6 (Figure 5d). Meanwhile, we also performed coimmunoprecipitation assay between RMP and STAT3 or cAMP-response element binding protein which are important transcription factors for IL-6 expression.18, 19, 20 However, no interactions were observed between RMP and the two transcriptional factors (Supplementary Figure 5E). RPB5, one of the polII subunits that provide a major anchoring site for the other factors,21 had been reported as one of the targets of gene transcription. Therefore, we used small interfering RNA to knockdown RPB5 expression, and found that the interaction between RMP and p65 was reduced compared with control cells (Figure 5e). To further examine the different potential binding between p65 and the IL-6 promoter, a chromatin immunoprecipitation (ChIP)-quantitative PCR assay was performed. After RMP knockdown, a decrease of p65 binding to IL-6 promoters in CSQT-2 and MHCCLM3 cells was observed (Figure 5f and Supplementary Figure 5F).

Figure 5

RMP upregulated IL-6 expression via interaction with p65 and RPB5. (a) Secretion of IL-6 by RMP in MHCCLM3 cells were completely inhibited by the NF-κB inhibitor (5 mM Bay11–7082). Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; ***P<0.001 (Student’s t-test). (b) Luciferase reporter assay of NF-κB was assessed in MHCCLM3 RMP, MHCCLM3-CMV, MHCCLM3-shRMP cells. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01 (Student’s t-test). (c) MHCCLM3 cells co-transfected with pFlag-p65, pFlag-RMP, pGPU6-shRMP and pFlag-CMV. Data are mean±s.e.m. and are representative of three independent experiments. *P<0.05; **P<0.01; ***P<0.001 (Student’s t-test). (d) HEK293T cells were co-transfected with Flag-RMP, Flag-p65 and green fluorescent protein (GFP)-RPB5. At 48 h after transfection, cells were treated with tumor necrosis factor-α (TNFα) at a concentration of 10 ng/ml for 20 min before harvesting. Whole-cell lysates were immunoprecipitated with RMP, p65, GFP antibody respective or control immunoglobulin G and analyzed by western blotting. Cell lysates were also subjected to immunoblotting (lower panel). (e) HEK293T cells were transfected with small interfering RNA (siRNA) of RPB5 or control siRNA. At 12 h after transfection, Flag-RMP was transfected. 48 h later, cells were treated with TNFα at a concentration of 10 ng/ml for 20 min before harvesting. Whole-cell lysates were immunoprecipitated with Flag-tag antibody or control immunoglobulin G. Endogenous p65 was analyzed by western blotting. (f) A marked decrease of p65 bound on the upstream of transcriptional start site of IL-6 was found upon RMP knockdown MHCCLM3 and CSQT-2 cells as compared with the corresponding vector control in ChIP assay. Normal rabbit antibody added served as a negative control. Error bars represent s.e.m. from at least three independent experiments.*P<0.05; **P<0.01 (Student’s t-test).


Great efforts have been made to elucidate the molecular mechanisms underlying the formation of PVTT, in order to identify potential biomarkers for prediction and intervention. Here, we found that RMP was highly expressed in PVTT clinical samples, which was a special type of HCC metastases leading to deteriorating hepatic function and determined the significance and underlying mechanism in PVTT formation by RMP overexpression. We believed that RMP was an attractive candidate gene for risk prognosis and therapy of HCC.

In our study, we have found that RMP was closely associated with PVTT. In addition, PVTT, as one special type metastases of HCC, was endowed with high metastases capacity. The mechanism associated with the pathogenesis of PVTT remains largely unclear. Previously, study found that the whole population of CSQT-2 cells come from PVTT in the cell line was EpCAM positive,22 indicating that PVTT was raising from sub-populations of T-ICs/CSCs in HCC because of the tumor-initiating capacity and EMT phenotype. Meanwhile, we showed that RMP had an indispensable role in maintaining T-ICs. That may partly explain high level RMP expression in PVTT. We also provided the evidences that clinical PVTT samples highly expressed CD24 and EpCAM, which were specific cell surface markers enriching HCC T-ICs.8,9 However, the definite mechanism of RMP high expression in PVTT was still unknown, which needed further investigation.

Knockdown of RMP notably attenuated IL-6 production. Furthermore, a significant correlation between RMP expression and IL-6 expression was found in HCC clinical samples. IL-6 has been reported to be capable of expanding the T-IC/CSC populations, as well as inducing EMT, both of which are implicated in tumor metastases and therapeutic resistance.23, 24, 25 Reports had also shown that IL-6 is stimulated and secreted not only in Kupffer cells but also in parenchymal liver cells.6,26 Furthermore, autocrine IL-6 from parenchymal cells is important for T-ICs to HCC progression and for tumorigenic growth.27 Therefore, the effect of RMP on tumor invasion and metastases in vitro and in vivo was mainly dependent on the IL-6 secreted from parenchymal cells.

It has been reported STAT3, cAMP-response element binding protein and p65 were the major transcription factors of IL-6.18, 19, 20 However, we did not find an interaction between RMP and STAT3 or cAMP-response element binding protein. Nevertheless, RMP was observed in association with p65 and RPB5, which are assembled in the PIC of IL-6. Consequently, we detected other target genes of p65 in both CSQT-2 and MHCCLM3 cells (Supplementary Figure 5G). IL-6, IL-1β and CCL5 were dropped in absent of RMP in both cell lines. Thus, RMP may promote PVTT forming by NF-κB. However, IL-6 was the most significant downregulated target genes in both cells. Owing to the impact of IL-6 on expanding the CSC population displaying an EMT phenotype, IL-6 was considered as the major mechanism to promote PVTT forming in HCC. In addition, the results indicated that RMP selectively modulated the transcription of p65 in HCC and PVTT cells. However, the exact mechanisms of RMP on NF-κB need further studies to illustrate. Together with above data, expression of RMP may account for IL-6 elevating in HCC to facilitate hepatocytes malignancies and PVTT formation.

Taken together, RMP has an important role in HCC PVTT formation by maintaining tumor self-renewal and metastases capacity via promoting IL-6 production. As the significant role of IL-6 inducing transformation from viral hepatitis to HCC and predicting poor prognosis,28, 29, 30 targeting RMP in HCC may markedly lower IL-6 production, which is an attractive therapeutic strategy against this disease.

Materials and methods

Cell culture, transfection and lentivirus infection

The HCC cell lines SMMC7721 and MHCCLM3 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (shanghai, China). Human embryonic kidney cell line HEK293T was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). PVTT cell line CSQT-2 has been described previously.22 MHCC97H cells were obtained from Dr Z Tang. All cells were cultured in Dulbecco’s modified Eagle’s media supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution (Sigma-Aldrich, St Louis, MO, USA), and maintained at 37 °C in an atmosphere of humidified air containing 5% CO2.

HCC cells were seeded into well. After incubating for 24 h, these cells were transfected with using jet-PEI (Polyplus, New York, NY, USA) according to the manufacturer’s protocol. The RMP expression plasmid was constructed by cloning human RMP opening reading frame into pFLAG-CMV. For depletion of RMP, plasmids containing pGPU6-shRMP and two different lentivirus-shRMP/lentivirus-shRMP2, lentivirus-shIL-6 and control plasmids and virus were purchased from Genechem Co., Shanghai, China. Plasmids complementary DNA3.1-IKbα ΔDN was conserved in our lab.

RNA collection, complementary DNA synthesis and real-time PCR analysis

Total RNA was extracted from fresh-frozen tumor specimens, healthy control tissues and cell lines in Trizol (Invitrogen, Carlsbad, CA, USA). Reverse transcription of total RNA was performed using random hexamers (Roche Diagnostics, Penzberg, Germany) and SuperScriptII reverse transcriptase (Invitrogen). PCR amplifications of the respective genes were carried out with 40 ng complementary DNA, 500 nM forward and reverse primer, and iTaqSYBRGreen Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in a final volume of 10 μl. The primer sequences and microRNA targeting sequences are provided in Supplementary Table 2.

Immunoblotting,coimmunoprecipitation assays, antibodies and chemicals

Whole-cell extracts or HCC tumor specimens were prepared in RIPA buffer and centrifuged at 12 000 g for 15 min. Protein concentrations were measured using the bicinchoninic acid assay. Immunoblotting was performed using specific primary antibodies, and immune-complexes were incubated with the fluorescein-conjugated secondary antibody and then detected using Odyssey fluorescence scanner (Li-Cor, Lincoln, NE, USA). For coimmunoprecipitation experiments, cell lysates were prepared in RIPA buffer and protein concentrations were measured and incubated with 2 μg anti-RMP, anti-p65 and anti-green fluorescent protein or normal mouse immunoglobulin G (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 8 h at 4 °C, followed by addition of Protein A/G Plus-Agarose (Santa Cruz Biotechnology Inc.) for another 2 h. The samples from these assays were analyzed by western blotting. Anti-P-STAT3, anti-P-p65, anti-p65, Anti-vimentin and anti-green fluorescent protein were from Cell Signalling Technology, Beverly, MA, USA. Anti-β-actin and anti-glyceraldehyde 3-phosphate dehydrogenase were purchased from Santa Cruz Biotechnology Inc. The anti-RMP and anti-RPB5 were from ProteinTech Group, Chicago, IL, USA. Anti-E-cadherin was from BD Biosciences, Franklin Lakes, NJ, USA. Anti-IL-6 and anti-pol II was from Abcam Ltd, Cambridge, UK. Anti-EpCAM was from Abgent, San Diego, CA, USA. Anti-CD24 was from ABclonal Biotech (Woburn, MA, USA). Flow cytometry antibody of anti-EpCAM-APC and anti-CD24-APC were purchased from eBioscience, San Diego, CA, USA. The cytokine of tumor necrosis factor-α and IL-6 were purchased from PeproTech, Rocky Hill, NJ, USA and used at indicated concentrations. STAT3 Inhibitor VII was purchased from EMD Chemicals, Billerica, MA, USA. NF-κB inhibitor, Bay11–7082 was purchased from Beyotime, Shanghai, China.

Patients and follow-up

All tissue samples were obtained from consenting patients registered at the Eastern Hepatobiliary Surgery Hospital, Shanghai, China, from January 2002 to June 2005 and approved by the ethics committee. Forty paired HCC patients with PVTT were recruited to testing set for PCR assays and 20 PVTT samples were recruited to testing for immunohistochemistry. Details on the characterization of the clinical data can be found in Supplementary Table 1.


After screening hematoxylin and eosin-stained slides for optimal tumor content, we constructed tissue microarray slides (Shanghai Biochip Company, Ltd, Shanghai, China). Two cores were taken from each formalin-fixed, paraffin-embedded HCC samples and normal liver samples by using punch cores that measured 0.8 mm in greatest dimension from the center of tumor foci. Immunohistochemistry was performed as described before.31

Spheroid assay

Single cell of hepatoma cells were plated at 2 × 103/ml in Corning 3471 ultra-low attachment culture dishes (Corning, NY, USA) and cultured for 2 weeks. Spheroids from three independent experiments were counted and the representative view was photographed. To inhibit IL-6 signaling, HCC cells was treated with an IL-6 signal-neutralizing Ab (cat# ab6672, Abcam) or control rabbit immunoglobulin G (cat# SC-2027, Santa Cruz Biotechnology Inc.), respectively, at a concentration of 0.4 mg/ml for 2 weeks.

In vitro limiting dilution assay

MHCCLM3 control or shRMP cells were seeded into 96-well ultra-low attachment culture dishes at various cell numbers and incubated under spheroid condition for 14 days. Colony formation was assessed by visual inspection. On the basis of the frequency of wells without colony, proportion of T-ICs was determined using Poisson distribution statistics and the L-Calc Version 1.1 software program (Stem Cell Technologies, Inc., Vancouver, BC, Canada).

Migration and invasion assays

The migration assay was performed as described.32 The cell migration assay was performed with a transwell chamber. In all, 1 × 106 Cells were seeded in upper surface of a transwell chamber for 24 h. To inhibit IL-6 signaling, HCC cells was treated with an IL-6 signal-neutralizing Ab (cat# ab6672, Abcam) or control rabbit immunoglobulin G (cat# SC-2027, Santa Cruz Biotechnology Inc.), respectively, at a concentration of 0.4 mg/ml for 2 days before the experiment. HCC cells were treated with IL-6 (cat# 200–06, PeproTech) at the concentration of 20 ng/ml for 2 days before the experiment.

The invasive cells of 5 × 106 that had invaded through the extracellular matrix layer to the lower surface of the membrane for 24 h were fixed with methanol and stained with crystal violet. Photographs of three randomly selected fields of the fixed cells were captured and cells were counted. The experiments were repeated independently three times.

In vivo metastases assay

Six-week-old male BALB/c nude mice were randomized into two groups (n=5) and inoculated with CSQT-2 control/shRMP, MHCC97H control/shRMP or MHCCLM3 RMP/CMV/RMP+shIL-6/CMV+shRMP (1 × 106) into the tail vein. All of the metastatic foci in lung were calculated microscopically to evaluate the development of pulmonary metastases.

Six-week-old male BALB/c nude mice were randomized into two groups (n=5) and inoculated with CSQT-2 control/shRMP, SMMC7721 control/shRMP or MHCCLM3 RMP/CMV/RMP+shIL-6/CMV+shRMP (2 × 106) in spleen. All mice were killed 8 weeks after inoculation, and metastatic tumor colonies in the liver were measured.

Enzyme-linked immunosorbent assay

Enzyme-linked immunosorbent assay was performed using the conditioned medium collected from two day cultures of cells seeded at 2 × 103cells per plate, and then analyzed by human IL-6 kit purchasing from Dakewei Biotech Company (shanghai, China).

ChIP assay

Cells (5 × 106) were fixed in 0.5% formaldehyde for 10 min and lysed. Chromatin was sonicated to an average length of 0.2–0.5 kb. Chromatin was then immunoprecipitated with p65 antibody, which recognizes transcriptionally active chromatin regions, or beads-only for the negative control. After washing, crosslinking was reversed and DNA was purified with phenol/chloroform and ethanol precipitation. Isolated DNA was analyzed by reverse transcriptase–PCR. The following primers were used for amplification of the IL-6 promoter and β-actin control region: IL-6-ChIP-fw: 5′-IndexTermCTAGTTGTGTCTTGCCATGC-3′, IL-6-ChIP-rv: 5′-IndexTermCAGAATGAGCCTCAGACATC-3′; β-actin-ChIP-fw: 5′-IndexTermTCCACCTTCCAGCAGATGTG-3′, β-actin-ChIP-rv: 5′-IndexTermGCAACTAAGTCATAGTCCGCCTAGA-3′.

Statistical analysis

The Pearson χ2 test or Fisher’s exact test was used to analyze the relationship RMP expression and the clinicopathologic features. The data shown represent mean values of at least three independent experiments and expressed as mean±s.e.m. Statistical analysis was performed by the Student’s t-test, using the statistical software GraphPad Prism 4 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was set at a level of P<0.05. SPSS 15.0 software (SPSS Inc., Chicago, IL, USA) was also used for statistical analyses and a P-value <0.05 was considered significant.


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We thank Liang Tang, Dong-Ping Hu, Shan-Na Huang, Dan-Dan Huang, Shan-Hua Tang, Lin-Na Guo and Huan-Lin Sun for technical assistance. We thank Dr Xiao-Ni Kong for providing luciferase reporter constructs and Ad-caSTAT3 virus. This work was supported by grants from National Natural Science Foundation of China (81370066, 81372355, 81001075, 91229205, BWS11J036), the Funds for Creative Research Groups of China (81221061) and the State Key Project for Liver Cancer (2012ZX10002–009, 2013ZX10002–010), Stem Cell and Medicine Research Center’s Innovation Research Program of the Second Military Medical University (SCMRC1306).

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Correspondence to L-W Dong or H-Y Wang.

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Zhang, J., Pan, Y., Ding, Z. et al. RMP promotes venous metastases of hepatocellular carcinoma through promoting IL-6 transcription. Oncogene 34, 1575–1583 (2015). https://doi.org/10.1038/onc.2014.84

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