Molecular networks regulated by tumor suppressive microRNA-375 in head and neck squamous cell carcinoma Abstract #137 Takashi Kinoshita 1,2, Toyoyuki Hanazawa 1, Nijiro Nohata 1,2, Naoko Kikkawa 1, Miki Fuse 2, Takeshi Chiyomaru 3, Hirofumi Yoshino 3, Hideki Enokida 3, Masayuki Nakagawa 3, Yoshitaka Okamoto 1, Naohiko Seki 2 1 Department of Otorhinolaryngology / Head and Neck Surgery, Graduate School of Medicine, Chiba University, Chiba Japan 2 Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan 3 Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan MicroRNAs (miRNAs) constitute a class of small non-protein coding RNA molecules, 19 – 22 nucleotides in length. They negatively regulate multiple genes by mRNA cleavage or translational repression. Down-regulated miRNAs in cancer cells could function as a tumor suppressors by negatively regulating oncogenes. Our miRNA expression signatures of hypopharyngeal squamous cell carcinoma (HSCC), maxillary sinus squamous cell carcinoma (MSSCC) and esophageal squamous cell carcinoma (ESCC) revealed that microRNA-375 (miR-375) was significantly down-regulated in cancer tissues compared to normal epithelium. The aim of this study was to clarify the functional significance of miR-375 and to identify the gene targets miR-375 regulates in head and neck squamous cell carcinoma (HNSCC). Background and Aims 1. qRT-PCR revealed that miR-375 was down-regulated in HNSCC tissues compared with adjacent normal epithelium in 20 patients with HNSCC. 2. Gain-of-function analysis revealed that cancer cell proliferation was inhibited and apoptosis was induced in miR-375 transfected HNSCC cells. 3. Genome-wide molecular targets search and TargetScan database indicated that LDHB (lactate dehydrogenase B) and MTDH (metadherin) were candidate genes of miR-375 target. 4. qRT-PCR, Western blots, and luciferase assays revealed that miR-375 directly inhibits LDHB and MTDH expression. 5.LDHB, which is the glycolytic enzyme that catalyze the formation of lactic acid from pyruvate, promotes cell proliferation in HNSCC. 6.MTDH, which promotes tumorigenesis by modulating multiple signal transduction pathways such as NF- κB, PI3K/AKT, and Wnt pathway, promotes cell proliferation in HNSCC. Key Findings Conclusions miR-375 functions as a tumor suppressor in HNSCC. LDHB and MTDH are directly regulated by miR-375. These genes may function as oncogenes and contributed to cell proliferation in HNSCC. Tumor suppressive miR-375 and its target oncogenes may provide new insights into the molecular networks of HNSCC. References 1.Tumor suppressive microRNA-375 regulates oncogene AEG-1 /MTDH in head and neck squamous cell carcinoma (HNSCC) Journal of Human Genetics, : Tumor suppressive microRNA-375 regulates lactate dehydrogenase B in maxillary sinus squamous cell carcinoma International Journal of Oncology, : Top 10 down-regulated miRNAs from TaqMan LDA in MSSCC No.miRNA Fold Change P-value 1miR E-02 2miR E-05 3miR-139-5p E-04 4miR E-02 5miR-125b E-04 6miR-199b E-03 7miR E-03 8miR E-04 9let-7c E-03 10miR-30a* E-02 No.miRNA Fold Change P-value 1miR E-02 2miR-133a E-03 3miR E-02 4miR E-03 5miR E-02 6miR-139-5p E-03 7miR E-02 8miR E-02 9miR-486-3p E-02 10miR-146a E-03 Top 10 down-regulated miRNAs from TaqMan LDA in HSCC NormalTumor P=0.008 miR-375 expression (Normalized to RNU48 ) mock control Early apoptosis cell (relative to mock) miR * mock control Cell proliferation (% of mock) miR * SAS No. Gene Gene Name log2 ratiomiR-375 SymbolSASFaDuAveragetarget site 1HERPUD1 homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member PDIA4protein disulfide isomerase family A, member HSPA5heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) LDHBlactate dehydrogenase B NUCB2nucleobindin PSIP1PC4 and SFRS1 interacting protein CXCR7chemokine (C-X-C motif) receptor MTDHmetadherin SLC7A11 solute carrier family 7, (cationic amino acid transporter, y+ system) member KRT5keratin YWHAZ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide DNAJB11DnaJ (Hsp40) homolog, subfamily B, member KRT8keratin HSP90B3Pheat shock protein 90kDa beta (Grp94), member 3 (pseudogene) EBPLemopamil binding protein-like C6orf129chromosome 6 open reading frame TTC7Btetratricopeptide repeat domain 7B HSP90B1heat shock protein 90kDa beta (Grp94), member C4orf3chromosome 4 open reading frame SDF2L1stromal cell-derived factor 2-like NormalTumor P=0.014 Normalized to GAPDH LDHB miR-375 expression in 20 pairs of HNSCC samples miR-375 inhibited cancer cell proliferation and induced apoptosis Top 20 down-regulated genes by transfection of miR-375 in HNSCC cell lines MTDH NormalTumor P=0.015 Normalized to GAPDH HERPUD Normal Tumor P=0.12 Normalized to GAPDH PSIP NormalTumor P=0.16 Normalized to GAPDH SLC7A11 0 NormalTumor P=0.38 Normalized to GAPDH The mRNA expression of candidate genes for miR-375 target in 20 pairs of HNSCC samples * P< Cell proliferation (% of mock) * SAS * mock si-control si-MTDH_1 si-MTDH_2 WT-3’UTR Luminescence (normalized) * DEL-3’UTR MTDH (NM_178812) 3’UTR length:1637 miR-375 target site Position of MTDH 3’UTR 5‘...ACUAGGAAAGCUAAACGAACAAA... ||| ||||||| 3’ AGUGCGCUCGGCUUGCUUGUUU 5‘...ACUAGGAAAGCUAAA A... ||| ||||||| 3’ AGUGCGCUCGGCUUGCUUGUUU Position deletion MTDH mRNA expression (% of mock) * 120 MTDH normalized to β-Actin MTDH β-Actin SAS mock control miR-375 mockcontrolmiR-375 miR-375 directly regulates MTDH expression both in mRNA and protein levels Knocking down of MTDH inhibited cancer cell proliferation * P<0.0001