Nanodrop 2000

We selected seven candidate SNPs of ApoB according to previous published papers which demonstrated association with lipid metabolism abnormality related diseases and minor allele frequencies >5% in the HapMap Chinese Han Beijing population. We used the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd. Xi’an City, China) extracted from whole blood. Using a NanoDrop 2000 (Gene Company Limited) were measured DNA concentrations. We used SequenomMassARRAY Assay Design 3.0 Software to design a Multiplexed SNP MassEXTEND assay [15 ]. SequenomMassARRAY RS1000 was used for genotyping, and the related data were managed using SequenomTyper 4.0 Software [15 , 16 (link)]. Laboratory personnel were blinded to the genotyping results of all samples.

SNPs were selected based on published studies demonstrating an association with cancer [27 (link)–28 (link), 31 (link)–33 (link)], and by using search of HapMap and dbSNP (Chinese population). Only SNPs, which may impact miRNA:LncRNA interaction according to the lncRNASNP database and limited the minimum allele frequency of SNPs in the Chinese population(CHB) > 5%, were selected. Finally, 16 SNPs were selected as candidate SNPs. GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd. Xi'an City, China) was used to extract DNA from the whole blood. DNA concentration was measured by NanoDrop 2000 (Gene Company Limited). Sequenom MassARRAY Assay Design 3.0 Software was used to design a Multiplexed SNP MassEXTEND assay [34 (link)]. Sequenom MassARRAY RS1000 was used for genotyping, and the data were managed using Sequenom Typer 4.0 Software [34 (link), 35 (link)]. Primers used for this study are listed in Supplementary Table 1. Laboratory personnel were blinded to the genotyping results of all samples.

The Human Signal Transduction PathwayFinder^™ RT2 Profiler^™ PCR Array (PAHS-014Z, Qiagen, Frederick, MD, USA) was used to evaluate the expression of a panel of 84 genes representative of ten different signal transduction pathways, in FaDu cells treated with pepsin. Total RNA was isolated from pepsin treated Fadu cells and control cells using the Qiagen RNeasy Mini Kit, following manufacturer’s protocol. RNA was quantified using the Nanodrop 2000 (Gene Company Limited, Hong Kong, China), and quality was assessed based on the integrity of 18 S and 28 S ribosomal RNA bands in 1% agarose gels. First-strand cDNA was mixed with 2 × RT2 SYBR Green qPCR Master Mix and ddH₂O. qPCR was performed in the Applied Biosystems (ABI) 7500 system using the following conditions: 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Each array contained five independent housekeeping genes (Actb, B2m, Hprt1, Ldha and Rplp1) that were used for data normalization.

Among the 14 SNPs were selected, rs11125529 was chosen from previously published polymorphisms associated with telomere length [13 (link)], others were randomly chosen from the published gene ACYP2 associated with telomere length, with minor allele frequencies >5% in the HapMap Chinese Han Beijing population. We used the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd. Xi’an City, China) extracted from whole blood. Using a NanoDrop 2000 (Gene Company Limited) were measured DNA concentrations. We used Sequenom MassARRAY Assay Design 3.0 Software to design a Multiplexed SNP MassEXTEND assay [19 ]. Sequenom MassARRAY RS1000 was used for genotyping, and the related data were managed using Sequenom Typer 4.0 Software [19 , 20 (link)]. Laboratory personnel were blinded to the genotyping results of all samples.

We selected the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd. Xi’an City, China) to extract the DNA from the 5 ml peripheral venous blood; and Nanodrop 2000 (Gene Company Limited) was used to detect the concentration and purity of samples, DNA to ensure that the samples could be used for subsequent experiments. Same as previously published articles [14 (link), 15 (link)]. rs2277698, rs2009196, rs7342880, rs11654470, rs2003241, and rs4789936 Six SNPs were selected in our study based on minor allele frequency data more than 0.05 in the global population [16 (link)]. Primer design and SNP typing were performed in the same way as previously published articles [14 (link), 15 (link)]. The genotyping primers were designed with the Agena MassARRAY Assay Design 3.0 Software [17 ]. The Agena MassARRAY RS1000 was used for genotyping, and the related data were managed using Agena Typer 4.0 Software [13 (link), 17 , 18 (link)].

Total RNA was extracted from muscle samples using TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, US) according to the manufacturer's instructions. The concentration and integrity of the RNA samples were determined using a NanoDrop 2000 instrument (Gene Company Limited, HK, China) with an OD260:OD280 ratio ranging from 1.8 to 2.0. The reverse transcription system was performed using random primers and a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) according to the instructions. The primers for myosin heavy chain (MyHC) I, IIa, IIx, and IIb were described in Han et al. (13 (link)), while primers for PFKM, PKM, HK2, and GAPDH were designed using Primer 5.0 software (Premier Biosoft International, Palo Alto, CA). The 2-ΔΔCt method was used to analyze the relative changes in gene expression after normalization to that of GAPDH as the internal control (Supplementary Table S2).