Telomerase

The newly constructed expression cassettes are shown in Fig. 1a. The promoters, RU5′, BGH (bovine growth hormone) polyadenylation (polyA) signal, and a sequence for multiple cloning sites, were synthesized by IDT Inc. (Coralville, IA) and inserted into pDNR-1r promoter-less vector (Clontech, Mountain View, CA) or pIDT-SMART promoter-less vector (IDT Inc.). The RU5′ sequence (269 bp: Accession No. J02029 (374–642)) is derived from the R segment and a part of the U5 sequence of HTLV Type 1 long terminal repeat and used to enhance transcription efficiency [9 (link)]. Sequences of the promoter elements were as follows: hTERT (189 bp: Accession No. DQ264729 (1618–1806)), SV40 (319 bp: Accession No. AY864928 (2156–2474)), and CMV (479 bp: Accession No. AJ318513 (159–637)). The CAG promoter was obtained from the pCAGGS vector (a kind gift from Dr. Jun-ichi Miyazaki; Osaka University, Japan). pTracer-EF/V5-His-A and pEF6/Myc-His-A were purchased from Invitrogen. Full-length cDNAs of human S100A11, REIC/Dkk-3, CD133, LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5), telomerase, erythropoietin (EPO), and green fluorescence protein (GFP) were amplified by RT-PCR.Fig. 1

Schematic diagram of modified gene expression systems and their capabilities for gene expressions. a A series of indicated plasmids were constructed on the basis of the promoter-less pDNR-1r vector. b Expression of KLF16 protein was assessed by Western blot analysis after transfecting the indicated plasmids carrying KLF16 cDNA in HEK293, MCF7, PC-3, HeLa, and HepG2 cells. c Plasmid vectors carrying various cDNAs were constructed using the same series of vectors as those shown in (A). The vectors were transfected to HEK293 cells, and the level of each protein was determined by Western blot analysis. Lane numbers in b and c correspond to the vector numbers shown in (a)

The activity of telomerase was tested based on the classical TRAP method (Kim and Wu, 1997 (link)) with minor modification. Briefly, cultured cells were washed with PBS and the cell numbers were counted. A total of 1 × 10⁶ cells were lysed with CHAPS lysis buffer for 30 min on ice. After centrifugation at 12,000 ×g for 20 min, the supernatant was removed into a new prechilled tubes and the concentration was determined by the Pierce BCA Protein Assay Kit (Thermo Scientific). Two steps were applied for TRAP assay. The first reaction system contained 1 μl cell lysate, 1 μl dNTP (2.5 mM), 1 μmol/l TS primer (5‘-AATCCGTCGAGCAGAGTT-3’), 1 μl 10 × TRAP buffer (200 mM Tris–HCL, 15 mM MgCL₂, 630 mM KCL, 0.5% Tween 20, 10 mM EGTA, and 0.1% BSA, pH 8.3), and 6.5 μl ddH₂O. The mixture were incubated at 30°C for 40 min, inactivated at 95°C 5 min, and stored at 4°C. The second step was PCR reaction, each 20 μl reaction system contained 2 μl of products from the first step, 2 μl 10 × TRAP buffer, 3 μl dNTP (2.5 mM), 0.5 μmol/l TS primer, 0.5 μmol/l ACX primer (5‘-GCGCGGCTTACCCTTACCCTTACCCTAACC-3’), 0.5 μmol/l NT primer (5‘-ATCGCTTCTCGGCCTTTT-3′), 0.5 amol/μl TSNT (5‘-AATCCGTCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-3′), and 11 μl ddH₂O. PCR was performed at 95°C for 3 min, 30 cycles of 94°C for 30 s, 59°C for 30 s, 72°C for 1 min, and 72°C for 5 min. PCR products were separated on a 15% native-PAGE gel in TBE buffer. After electrophoresis, the gel was stained with GelRed for 30 min and the images were captured under UV light with reverse processing.

Human telomerase-immortalized retinal pigmented epithelial cells (hTERT-RPE1) were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (FUJIFILM Wako), supplemented with 2 mM L-glutamine (FUJIFILM Wako), 10% fetal bovine serum (FBS, Thermo Fisher Scientific), and 0.375% sodium bicarbonate. NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. All cells were incubated at 37 °C in a humidified 5% CO₂/95% air atmosphere.

The workflow of this study is presented in Figure 1. The expression data including mRNA and lncRNA of PTC patients were got from TCGA database (https://www.cbioportal.org) [24 (link)]. This dataset consisted of 507 subjects, 510 tumor samples, and 58 adjacent normal tissue samples. Only the patients with clinical data and follow-up time/PFI ≥ 30 days were included. Overall, 498 cases were finally included and were randomly split into a training cohort (n = 299) and a validation cohort (n = 199). A total 222 autophagy-related genes (ARGs) were obtained from the Human Autophagy Database (HADb, http://autophagy.lu/clustering/index.html), which collected those genes from the literature. LncRNAs and ARGs mRNAs expression were extracted according to GENCODE annotations (https://www.gencodegenes.org). LncRNAs with zero expression levels in more than 50% of samples were excluded. Clinical variables including age, gender, race, cancer history, thyroid gland disorder history, histological types, TNM stages, T stage, N stage, M stage, tumor location, residual tumor, American Thyroid Association (ATA) risk stratification, the distant metastasis, patient age, completeness of resection, local invasion, and tumor size (MACIS) scores were collected. The methods of assessing the tumors with ATA risk stratification and MACIS scores have been described in the previous report [24 (link)] and are introduced in the Supplementary data. We also re-evaluated the tumors based on the methods. Additionally, BRAF mutation and telomerase reverse transcriptase (TERT) promoter mutation status information were extracted, which have been reported to be associated with prognosis in TC [25 (link)]. Patients’ progression-free survival (PFS) and overall survival (OS) were also extracted.