C -reactive protein promotes tumor progression in Hepatocellular Carcinom by 1 interacting with Ephrin type B receptor 3

31 C-reactive protein (CRP), an acute phase protein, has been increasingly implicated in 32 various tumors, and the role of CRP is positively correlated with invasion and 33 metastasis in hepatocellular carcinoma cells. However, the mechanism of CRP 34 affecting HCC progression remains poorly investigated. The present study 35 investigated the role of CRP in HCC and the underlying mechanisms. We first found 36 that CRP was significantly upregulated in HCC tissues and HCC cells, the expression 37 level correlated with the metastatic ability of HCC cells. Knockdown of CRP 38 significantly suppresses migration and invasion capacity in HCC cells. Through a 39 proteomic analysis of CRP co-immunoprecipitation complexes, the Eph receptor B3 40 was identified as a new CRP interactor. Then we found that the expression and 41 functions of EphB3 were consistent with CRP in HCC. In addition, 42 co-immunoprecipitation and immunofluorescence assays suggested that EphB3 was 43 able to interact with MAPK/ERK to activate MAPK/ERK signaling pathways. 44 Furthermore, we showed that CRP can induce the phosphorylation of MAPK/ERK by 45 binding EphB3. Our findings showed that CRP increased HCC cells migration and 46 invasion by binding EphB3 to activate MAPK/ERK signaling pathways. It suggested 47 that CRP may become a prognostic factor and a potential therapeutic target for liver 48 cancer.


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Hepatocellular carcinoma (HCC) is the sixth most common human malignancy and 54 the second leading cause of cancer-related death worldwide, with more than 50% of 55 HCC cancer cases and deaths occurred in China 1-3 . Despite great advancements in 56 HCC early diagnosis, intervention and prevention have been made, patients still have 57 a high recurrence rate and poor prognosis. These poor outcomes are primarily due to 58 metastases and recurrences 4,5 . Over the past decades, remarkable progress has been 59 made to illuminate the pathogenesis of HCC 6,7 . However, the underlying mechanisms 60 of recurrence and metastasis of HCC remain unclear. Therefore, it is necessary to 61 develop a more reliable biomarker for predicting recurrence and understanding the 62 mechanism of liver cancer metastasis as soon as possible.

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C-reactive protein (CRP), the first acute-phase protein described and an ancient and 64 highly conserved protein of the pentraxin family, is mainly synthesized by quantified by RT-PCR in 20 pairs of tumor and their matched normal liver tissues. 97 Figure 1A shows that CRP mRNA levels were significantly increased in the liver 98 tumor specimens. Results from Western blotting and immunohistochemistry further 99 confirmed the upregulation of CRP in liver cancer specimens (Fig.1B, C). migration and invasion ability of HCC cells was significantly reduced, the migration 119 ability was reduced by 50% and 55%, compared with the control group, and the 120 invasive ability was decreased by 40% and 50% (Fig. 2B, C, D). Similarly, the ability 121 of CRP-silenced liver cancer cells to close scratch wounds was also reduced 122 compared to control group (Fig. 2E, F). As CRP is a secreted protein, we used 123 CRP-specific antibodies to treat liver cancer cells, and then decreased the ability of To clarify the mechanism of CRP in the progression of liver cancer, we used was applied to all iTRAQ ratios to minimize false positives when determining 141 proteins as up-expressed. This cutoff value was widely employed in the iTRAQ 142 approach 21 . 52 unique proteins were successfully identified (Data not shown).

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Gene Ontology analysis with DAVID suggested that the main molecular biological 144 functions of these proteins were protein binding, cell adhesion, cytoskeletal 145 organization, and small molecule activation. , modification; and the main biological  Table S1).

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To determine the reliability of the iTRAQ analysis data, we selected several 149 interesting protein candidates and confirmed by co-immunoprecipitation (Co-IP) and  EphB3 is overexpressed in hepatocellular carcinoma 154 Since literature data and bioinformatic analysis suggest the functional involvement 155 of EphB3 in tumor metastasis 22 , we hypothesized that CRP may bind to EphB3 156 leading to HCC progression. The relationship between EphB3 and CRP was explored.
of HCC tissues (Fig.3 C), and EphB3 was mainly located in the cell membrane and 163 cytoplasm of HCC cells by Confocal assay (Fig.3 F).  analysis showed that the expression of EphB3 and CRP were consistent in HCC 165 tissues and cells.

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To study the role of EphB3 in HCC cell motility, the expression of EphB3 was 168 inhibited using siRNA transduction of SMMC7721 and Huh7 cells. Western blotting 169 was performed to confirm the successful knockdown of EphB3 in these two cell lines

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To examine whether EphB3 interacts with CRP in HCC cells, we performed 178 co-immunoprecipitation assay and western blotting in SMMC7721 and Huh7 cells.

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EphB3 interacting with CRP and CRP binding EphB3 were observed in SMMC7721  In summary, we found that CRP was overexpressed in HCC tissues and cells, and      38, 189-197 (2001  targeting HIF-1α (100nmol), and MMP-9 was detected by immunoblot analysis after