S100A4 regulates migration and invasion in hepatocellular carcinoma HepG2 cells via NF-κB-dependent MMP-9 signal.
OBJECTIVE
We previously showed that the calcium-binding protein S100A4 is overexpressed and related to metastasis in hepatocellular carcinoma (HCC). However, whether S100A4 participates in the regulation of metastasis and its mechanisms in HCC is mostly unknown. Given the associations of S100A4, nuclear factor-kB (NF-kB/RelA) and MMP-9 with metastasis in a variety of malignancies, we explored a potential role of S100A4 in HCC metastasis and its mechanism.


METHODS
20 patients with HCC invasion (Lymph node metastasis, microvascular invasion, major portal vein invasion and intrahepatic metastasis) and 20 patients without HCC invasion were included. These tissues were detected for the expression of S100A4, NF-kB/RelA and MMP-9 by immunohistochemistry and quantitative real time polymerase chain reaction (Q-PCR). Correlation between the expressions of S100A4, NF-kB/RelA and MMP-9 with the invasion was analysed. The expressions of S100A4, nuclear factor-kB and MMP-9 was evaluated in HepG2 cells by western blot and immunohistochemistry. HepG2 cells were stably transfected with S100A4-specific small interfering RNA (S100A4 siRNA) to knockdown of S100A4, then transiently transfected with S100A4 cDNA to rescure the S100A4 level and evaluated for effects on invasion and expression analysis for molecules involved in invasion. After the HepG2 cells recurred the S100A4 levels, the HepG2 cells was treated with 5 µM Pyrrolidine Dithiocarbamate (PDTC) (a selective NF κ B inhibitor) to inhibit the NF-kB activity, or treated with Batimast (BB94: a MMPs inhibitor) to inhibit the MMP-9 activity. The expression analysis for molecules involved in invasion was analyzed.


RESULTS
A significant increase of S100A4, NF-kB/RelA and MMP-9 expression in HCC tissues with invasion than that of without invasion. A positive correlation was observed between S100A4, NF-kB/RelA, MMP-9 and invasion, respectively. In addition, S100A4 was positively correlated with NF-kB and MMP-9. S100A4 siRNA mediated knockdown of S100A4 in HepG2 cells resulted in significant reduction in the NF-kB activity and MMP-9 expression, and dramatically decreased its invasion. Moreover, the HepG2 cell metastatic potential was rescued by overexpression of S100A4 completely, at the same time, the NF-kB activity and MMP-9 expression was also increased. Pretreatment with PDTC or BB94 was observed to significantly reduce NF-kB activity and MMP-9 expression and dramatically decreased S100A4 -induced invasion.


CONCLUSIONS
Our findings indicate that S100A4 contributes to HCC metastasis by activation of NF-kB dependent MMP-9 expression, suggesting S100A4 as a novel diagnostic biomarker and therapeutic target in HCC.
Department of Surgery, the Affiliated Hospital of Medical College, Qingdao University, Qingdao, China. qyfyzhang@126.com.
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D014408:Biomarkers, Tumor / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis / Q000235:genetics / Q000473:pathology*; D002465:Cell Movement; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000175:diagnosis / Q000235:genetics / Q000473:pathology*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D011759:Pyrrolidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000008:administration & dosage; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000235:genetics* / Q000378:metabolism; D013859:Thiocarbamates / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D014408:Biomarkers, Tumor; D016328:NF-kappa B; D011759:Pyrrolidines; D034741:RNA, Small Interfering; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D013859:Thiocarbamates; D013876:Thiophenes; D051996:Transcription Factor RelA; C077591:S100A4 protein, human; C020972:pyrrolidine dithiocarbamic acid; D010649:Phenylalanine; C080985:batimastat; D020780:Matrix Metalloproteinase 9