Penter vector

Plasmids pEnter-MMP1-Flag and pEnter-vector were obtained from Vigene Bioscience (Rockville, MD, USA). Short interfering RNA (siRNA) oligonucleotides targeting MMP1 were purchased from GenePharma (Shanghai China); the siRNA sequences are shown in Supplementary Table S1. Plasmid (2 μg) and siRNA oligonucleotide (100 nmol/L) transfections were carried out using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. The cells were used for further studies at 48 h after transfection.

To construct a plasmid for overexpressing TMEM98, the coding sequence and 3'-UTR of TMEM98 was inserted into the p-Enter vector (Vigene Bio.) according to the manufacturer’s instructions. FaDu and HSC-3 cells were transfected with miR-29c-5p mimic, an miR-29c-5p inhibitor, a negative control (RiboBio), and a TMEM98-expression vector in the presence of Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s instructions. FaDu and HSC-3 cells were harvested at least 24 h after transfection.

The ETS1 overexpression construct generated from the ETS1 cDNA sequence and the pcDNA3.1 vector was kindly provided by Professor G. Huang from the Children’s Hospital of Fudan University. The cDNA sequences of NKX2.5 and JUN, which were cloned into the pENTER vector (Vigenebio, China), were synthesized by Shanghai Sunny Biotechnology Co., Ltd. The zebrafish and human DNA sequence of the enhancer was downloaded from the ECR Browser. DNA regions of the candidate enhancers were amplified by PCR, cut with XhoI and BglII and cloned into pCNE7.04-E1b-GFP-T2KXIGQ, the enhancer activity detection vector (Li et al., 2010 (link)) (Fig. 1A). To construct a luciferase reporter plasmid (Fig. 1B), enhancer fragments were cut with KpnI and XhoI, and the original SV40 promoter of the luciferase reporter vector pGL3-promoter (Promega; USA) was replaced by E1b, which is a widely used basic promoter. We termed this new vector pGL3-E1b. In the mutated enhancer constructs, a single base in the TFBSs was mutated but was not introduced a new heart-related TFBS. The primer sequences used for PCR amplification of the enhancer activity detection and mutation analysis constructs are listed in Table 1.

Surgical tissue specimens were obtained from patients with OSCC diagnosed from 2016 to 2018 at the Affiliated Hospital of Qingdao University, China. Specimens were obtained from the surgical resection of OSCC primary tumors, along with paired samples of adjacent normal tissues. All samples were immediately frozen at −196°C (liquid nitrogen) and placed at −80°C for further storage. Ethical approval was ratified by the local Ethics Committee, and all patients provided written informed consent for the utilization of tissue samples in research.
The human OSCC cell lines, SCC15 and SCC25, were kindly provided by the Chinese Academy of Medical Sciences (Beijing, China). For the construction of the PIP overexpression plasmid, whole sequences of PIP gene were amplified from a human complementary DNA (cDNA) library, AsiSI and Mlu I restriction sites were inserted, and the construct cloned into a pEnter vector by Vigene Biosciences (Shandong, China). Small interfering RNAs for PIP (siRNA‐PIP) were designed by GenePharma (Shanghai, China). Primers were supplied by Thermo Fisher Scientific (Carlsbad, CA). The AKT inhibitor MK‐2206 and ERK inhibitor SCH772984 were obtained from ApexBio Technology (TX) and dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer's instructions.

Short-interfering RNAs (siRNAs) targeting MTX1 or CISD1 were synthesized by Shanghai Genepharma Co., Ltd, Shanghai, China. The sequences were as follows: siNC: 5′-UUCUCCGAACGUGUCACGU-3′; siMTX1: 5'-CCCACAUUCUCAGUCUCUA-3'; siCISD1-1: 5'-CAGCUGCAAUUGGUUAUCU-3'; siCISD1-2: 5'-CGUGAAGUUACCUGAUUGU-3'.
For gene overexpression, FLAG-tagged MTX1 plasmid was constructed through cloning MTX1 cDNA (GeneBank Accession Number: NM_002455.5) into pcDNA3.1 vector. CISD1 overexpression plasmid (also FLAG tagged) was constructed based on pENTER vector, which was obtained from Vigene Biosciences, Shandong, China. MTX1-overexpressing lentivirus was prepared by Shanghai Genepharma Co., Ltd, Shanghai, China.

The expression plasmids, pENTER-NRF2, pENTER-NQO1, and the pENTER-vector, were obtained from Vigenebio Inc. (MD, USA). The siRNAs (small interfering RNAs), siNQO1, siNRF2, and siNC (negative control), and miRNAs, miR-450b-5p mimic, miR-450b-5p inhibitor, and their respective negative controls, were all purchased from Ribobio Inc. (Guangzhou, China). The plasmids, miRNAs and siRNAs, were transfected alone or co-transfected into cells as mentioned in the result part using Lipofectamine 2000 (Thermo Fisher, CA, USA) according to the manufacturer’s instructions. The sequences of siRNAs and miRNAs were listed in the Supplementary Table S2.