Hsc 2

In this study, the type strain of SAA, NCTC10713^T, showing SLS-dependent β-hemolysis, and a non-hemolytic SAA strain, NCTC11169, were used. In addition to these strains, we tested certain genetically modified strains derived from NCTC10713^T, an erythromycin (erm) resistant strain carrying the inserted erm cassette near the upstream region of the intact sag operon, a non-hemolytic erm resistant strain lacking both the sagA1 and sagA2 genes (ΔsagAs), and a sagA1-gene trans-complemented strain of ΔsagAs (psagA1). The tested strains were inoculated into BHI broth (Becton Dickinson and Company, Franklin Lakes, NJ) and incubated at 37°C overnight under 5% CO₂. The human oral squamous cell carcinoma cell line HSC-2 (RCB1945, RIKEN BRC, Ibaraki, Japan) and Eagle’s Minimum Essential Medium (EMEM, FUJIFILM Wako, Osaka, Japan) containing 10% (v/v) of heat-inactivated fetal bovine serum (FBS), 10% (v/v) of BHI, and 50 mM HEPES (pH 7.4) were used for the co-cultivation. The human acute monocytic leukemia cell line THP-1 (RCB1189, RIKEN BRC), and RPMI1640 (FUJIFILM Wako) containing 10% (v/v) of heat-inactivated FBS, 10% (v/v) of BHI, and 50 mM HEPES (pH 7.4) were also used for the co-cultivation.

Eight human OSCC cell lines (Ca922, HO-1-u-1, HSC2, HSC3, HSC4, SAS, HSQ89, and Sa-3) were purchased from the RIKEN BioResource Center (Ibaraki, Japan). The human keratinocyte HaCaT cell line was obtained from the Cell Line Service (Eppelheim, Germany) as a control. All cell lines were maintained in appropriate media (RPMI-1640 or Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin.

Eleven human OSCC-derived cell lines (HSC-2, HSC-3, HSC-3-M3, HSC-4, Sa3, Ca9-22, KOSC-2, SAS, SAS-H1, Ho-1-u-1, and Ho-1-N-1) were purchased from RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) and the Japanese Collection of Research Bioresources Cell Bank (Ibaraki, Osaka, Japan) 11 (link)-13 (link). We obtained human normal oral keratinocyte (HNOKs) from young healthy patients and cultured the cells as described previously 14 -17 . The cells used in this study were within 10 passages from thawing. They also were routinely tested using an EZ-PCR Mycoplasma Test Kit (Biological Industries, Kibbutz Beit Haemek, Israel).

Six specimens (three HNSCC tissues and three normal oral epithelial tissues) were analyzed by RNA sequencing to determine the HNSCC miRNA signature. The clinical features of HNSCC patients are summarized in Table S2.
All specimens used were obtained by surgical resection at Chiba University Hospital. All patients provided written informed consent for the use of their specimens. This study was approved by the Bioethics Committee of Chiba University (approval number: 28–65, 10 February 2015).
Two human HNSCC cell lines (HSC-2 and SAS) were obtained from the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) and used in this study.

The human oral squamous cell carcinoma cell line HSC-2 (RCB1945; RIKEN BRC, Ibaraki, Tsukuba, Japan) was cultured in EMEM cell culture medium (FUJIFILM Wako) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin G and 100 U/mL streptomycin; FUJIFILM Wako) under cell culture conditions (37 °C, 5% CO₂ atmosphere).

HSC-2, a well-differentiated OSCC cell line, and HSC-3, a poorly differentiated OSCC cell line, developed from the metastatic lymph nodes of the floor of mouth and tongue cancer, respectively, were obtained from the Riken BRC Cell Bank (Tsukuba, Japan). The cells were maintained in minimum essential medium (MEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere with 5% CO₂/95% air at 37 °C.