Patient samples
Nine paired ESCC tumor tissues and corresponding adjacent normal tissues were collected from patients undergoing surgical resection at the Department of Thoracic Surgery, Shenzhen People’s Hospital, China. All patients were histologically confirmed by two pathologists. After surgical resection, all tissues were immediately frozen in liquid nitrogen and then stored at -80oC until use. None of these patients were treated with radiotherapy or chemotherapy prior to surgery.
Establishment of CTD-2017C7.1-overexpressing cell lines
To generate ESCC cells in which CTD-2017C7.1 was stably overexpressed, full-length human CTD-2017C7.1 (ENSG00000256705.3) was synthesized by Rochen Biotech (Shanghai, China) and was inserted into the EcoRI and NotI sites of pCDH-CMV-MCS-EF1-copGFP-T2A-Puro vector (pCDH, Supplementary Fig. 1A). Then, pCDH-CTD-2017C7.1 (12 µg) and packaging vectors (psPAX2, and pMD2.G) were co-transfected into 293T cells (ATCC) using the PEI reagents (Sigma, 408727). The supernatant containing the lentivirus particles was harvested 72h after transfection and concentrated using PEG8000 (Beyotime Biotech, Shanghai, China). TE-1 cells were seeded in 12-well plates with 1×105 cells per well. Lentivirus were added after TE-1 attachment. The infected cells were then subjected to selection with 5µg/ml puromycin. The stable transfected cells were harvested after 2 weeks screening. Cell lines with stably expressed CTD-2017C7.1 were confirmed by quantitative real-time PCR (Supplementary Fig. 1B).
Construction of CTD-2017C7.1 CRISPR/Cas9 knockout plasmids
To establish plasmids with silenced CTD-2017C7.1 expression, two guide RNA (gRNA) sequences which were predicted to have knockout effects on CTD-2017C7.1 (Supplementary Fig. 3A-D, 4A-D) were synthesized by GenePharma Co. (Shanghai, China). Then gRNAs were cloned into the pGE-4 (pU6gRNA1U6gRNA2Cas9puro, Supplementary Fig. 2A), using the BbsI digestive enzyme and the T4 DNA ligase. CTD-2017C7.1 knock-out sequences were verified by sequencing the genomic Cas9 guide-RNA binding site. The primers, gBlocks, and sequence of CTD-2017C7.1 can be found in the Supplementary materials (Supplementary Table 1, Supplementary Fig. 3–4). To construct the knockout cell lines, TE-1 cells were transfected with 10 µg CTD-2017C7.1 CRISPR-Cas9 knockout plasmids using the Lipofectamine 2000 kit (Invitrogen,11668-019). After incubation at 37oC for 48h, the knockout cells were further verified by PCR of genomic DNA showing CTD-2017C7.1 gene disruption (Supplementary Fig. 2B).
Subcellular fraction location
Nuclear/Cytosol Fractionation Kit (Phygene, PH1466) was used to separate cytoplasmic and nuclear fraction of TE-1 cells in accordance with supplier’s suggestions. Trizol reagent (Invitrogen, CA, USA) was applied to isolate total RNA, which was reverse transcribed in a final volume of 20 µl using random primers under conditions for RT reagent kit (TransGen Biotech, Beijing). QPCR was used to detect CTD-2017C7.1 levels in cytoplasm and nuclear fraction, using the SYBRGreen PCR kit (TransGen Biotech, Beijing). The expression levels of CTD-2017C7.1 in nuclear and cytoplasmic fraction were normalized to GAPDH, respectively.
Cell culture and transfection
ESCC cell lines (EC109, KYSE180, KYSE70, and TE-1) were obtained from the cell bank of Shanghai Biological Institute (Shanghai, China). Normal esophageal epithelium cells (NE3) were obtained from Pro. Fu L [20]. ESCC cells were cultured in RPMI 1640 (Gibco) with 10% FBS and NE3 cells were incubated in a 1:1 mixture of EpiLife and dKSFM (Gibco). All of these cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Cell transfections were performed using Lipofectamine 2000 kit (Invitrogen, CA, USA) according to the manufacturer’s instructions.
The authentication of cell lines
TE-1, EC109, KYSE180, and KYSE70 cells were obtained in January, 2018. All these cell lines were tested and authenticated by STR. Genomic DNAs of TE-1, EC109, KYSE180, and KYSE70 cells were extracted and amplified respectively using GenePrint System (Promega). Amplified products were processed using the ABI3730xl Genetic Analyzer. Data were analyzed using GeneMapper4.0 software and then compared with the ATCC, DSMZ or JCRB databases for reference matching. The STR results showed that there were no four alleles on the main nine locus, it is certainly no cross contamination of human cells in these cell lines. TE-1, EC109, KYSE180, and KYSE70 cells were tested on Jan.19, 2018 (Supplementary 1–4).
RNA extraction, quantitative realtime PCR (qRT-PCR)
Total RNA was isolated from tissues or cultured cells using Trizol reagent (Invitrogen, CA, USA). Total RNA (1µg) was reverse transcribed to cDNA using random primers with the SuperScript™ III RT Reagent kit (Invitrogen, CA, USA) following the manufacturer’s protocol. QRT-PCR was performed in the CFX96TM Real-Time System (Bio Rad, Hercules, CA, USA). GAPDH was employed as an endogenous control. Results were normalized to the expression of GAPDH. The relative expression of RNAs was calculated using the 2−ΔΔCt method. All experiments were performed in triplicate. Sequences of specific primers are listed in Supplementary Table 1.
Cell proliferation assay
Cell proliferation was determined by the cell counting kit-8 (CCK-8) (TransGen Biotech, FC101-02) according to the manufacturer’s instructions. Briefly, cells were cultured in a 96-well plate at 37oC for 24h. Subsequently, 10µl CCK-8 reagent was added into each well and incubated with cells for 2h. Finally, cell viability was measured every 24h (0, 24, 48, 72h) using the microplate reader MK3 (Thermo Fisher Scientific, Shanghai, China) at the absorbance of 450 nm. Five replicates were performed for each treatment group.
Cell invasion and migration assays
For the invasion assay, 5×104 cells in serum-free RPMI-1640 medium were placed into the upper chamber (Corning Falcon, USA), and 500 µL of RPMI-1640 with 10 % FBS was added to the lower chamber. After 24 h of incubation, the cells on the upper chamber were removed with cotton swabs, and the cells on the lower membrane surface of the membrane that had migrated or invaded from the upper chamber were fixed with 4 % paraformaldehyde for 15 min and then stained with 0.1 % crystal violet for 10 min. The number of invaded cells was counted under a microscope. All fields were counted randomly in each well.
Cell migration was evaluated by wound healing assay. In brief, cells in 6-well plates were allowed to reach 80–90 % confluence, and wounds were scratched in the cell monolayers using a sterile micropipette tip. Wound closure was recorded using a microscope. The distance across the wound was measured at baseline and at 24h, and the wound closure was calculated using the following formula: distance of wound closure = 1 − length of wound (24h)/length of wound (0h).
In vivo animal study
BALB/cA nude mice (female, 4–6 weeks old) were purchased from the Experimental Animal Center of Guangdong, China; and were maintained under pathogen-free conditions. The mice were randomly divided into two treatment groups (n = 5 for each group): one group was injected with cells (1×106) of NC control cells; another group was injected with cells (1×106) of stably expressing CTD-2017C7.1 (in 0.1ml PBS). Cells were injected subcutaneously into the right flanks of mice. Tumor size was measured every 3 days using a Vernier caliper. Tumor volumes were calculated using the following formula: 0.5×L×W2 (L refers to long and W refers to short diameters of the tumor). The tumor weight was measured at the endpoint of the study. At 21 days post-injection, mice were euthanized, and tumor were resected and preserved in formaldehyde for HE or IHC staining. The animal study was approved by the Animal Ethical Committee of Shenzhen University Health Science Center. All experimental procedures were manipulated following the approved protocols.
Immunohistochemical (IHC) analysis
IHC assay was conducted to quantify the expression levels of Ki67 protein in the tumor tissues, using Ki67 antibody (Abcam, ab16667) as the primary antibody and HRP-conjugated antibody as the secondary antibody. The detailed procedures for IHC assay have been described previously[21].
RNA pulldown and MS assays
The RNA pull-down assays were performed using the PierceTM Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific). Briefly, full-length sense and antisense CTD-2017C7.1 sequences were transcribed into TE-1 cells, using the RNA 3’ End Desthiobiotinylation Kit (Thermo Fisher Scientific) and RNA Production System-T7 (Promega, Madison, WI, USA), respectively. Cells were isolated and lysed using a protein lysis buffer. The biotin-labeled probe was captured using the streptavidin magnetic beads. Then the lysates of TE-1 cells were incubated with biotinylated nucleic acid compounds. The retrieved proteins were purified and detected by silver staining and the eluted RNA-binding proteins complex were subjected to mass spectrometry analysis. For LC-MS, proteins were digested by trypsin and separated by liquid chromatography, then detected by Q-Exactive MS (Thermo Fisher Scientific, Hudson, NH).
RNA immunoprecipitation (RIP)
RNA immunoprecipitation (RIP) analysis was performed using the Imprint® RNA Immunoprecipitation kit (Sigma, RIP-12Rxn) in accordance with the user manual. In brief, TE-1 cells (1×107) were treated with RIP lysis buffer to obtain the cell extract. The cell lysis was centrifuged at 13,000g at 4℃ for 10 min. Then the collected supernatant was incubated with RIP buffer containing magnetic beads conjugated to specific antibody or normal anti-rabbit IgG at 4℃ overnight. IgG was considered as the negative control. Subsequently, beads were incubated with proteinase K Buffer and RIP wash buffer to remove proteins. RNA was purified by phenol and chloroform. The purified RNAs were furthered subjected to qRT-PCR analysis to assess the expression of precipitated CTD-2017C7.1.
RNA Sequencing (RNA-seq)
To investigate the target genes regulated by CTD-2017c7.1, total RNA was extracted from TE-1 cells stably expressed CTD-2017C7.1 or NC control cells, using Trizol reagent (Invitrogen, CA, USA). RNA quality was assessed using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) was used for generating the sequencing libraries. In brief, magnetic beads attached with poly-T oligo were used to purifying mRNA from total RNA. First strand cDNA synthesis was performed using M-MuLV Reverse Transcriptase (RNase H-), then second strand cDNA synthesis was synthesized using DNA polymerase I and RNase H. The library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). After PCR amplification, the PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. TruSeq PE Cluster Kit v3-cBot-HS (Illumina) was used to perform the index-coded samples clustering on a cBot Cluster Generation System based on the manufacturer’s instructions. Then the index-coded samples were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Differential expression and normalized reads counts (FPKM, fragments per kilobase of gene/transcript model per million mapped fragments) were calculated using the DESeq2 R package (1.16.1).
Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
Statistical analysis
Experiments in vitro and in vivo were performed at least 3 times. Results are expressed as the mean ± standard deviation. Differences between two groups were assessed using Student t-test (two-tailed), one-way-ANOVA, or the Mann-Whitney U test, where appropriate. Kaplan-Meier survival analysis was used to compare esophageal cancer patient survival based on dichotomized CTD2017C7.1 expression levels retrieved from the TCGA database. Cox regression was applied to estimate prognosis, adjusting for multiple covariates, including age, gender, and stages. All statistical analyses were performed using the SAS 9.3 program (SAS Corp., NC, USA) and GraphPad Prism 7.0 (GraphPad software, Inc., USA). Statistically significant differences are presented as *P < 0.05 and **P < 0.01. In all cases, P < 0.05 was considered statistically significant.