Expression data sets
Seven human lung cancer cohorts and corresponding clinical information were extracted from the Gene Expression Omnibus (GEO; GSE19188, GSE40791, GSE75037, GSE41271, GSE50081 and GSE31210) and The Cancer Genome Atlas (TCGA) database to analyze the expression of TTI1.
Patients and following-up
A total of 160 NSCLC tissues and matched nontumor tissues were gathered from patients who underwent surgery at the Second Affiliated Hospital of Nanchang University. The pathological diagnosis was carried out by two pathologists respectively in accordance with the standards of the World Health Organization. Those tissues were constructed into a tissue microarray (TMA) that was used to perform immunohistochemistry (IHC) staining. Clinicopathological information was collected from 12 March 2009 to 28 February 2011. The last follow-up was in December 2019. All patients had written informed consent regarding the collection and use of their tissue samples. The ethical approval was authorized by the Second Affiliated Hospital of Nanchang University.
Tissue Microarray establishment and Immunohistochemistry
Tissue microarrays (TMAs) of 160 pairs of NSCLC tissues and matched nontumor tissues were built by Shanghai Biochip Co, Ltd (Shanghai, China). Rabbit polyclonal to human TTI1 antibody (1:200, ab234871, Abcam, USA) and Rabbit polyclonal to human KI67 antibody (1:200, ab16667, Abcam, USA) were used to detect the expression of TTI1 and KI67.The immunohistochemistry (IHC) assay was performed according to a standard protocol. In brief, after baking at 37℃ for 30 minutes, all paraffin sections of the human lung cancer tissues were first dewaxed and then rehydrated. 5% bovine serum albumin (BSA) (YESEN, Shanghai, China) was incubated for 1 h at room temperature to block nonspecific binding sites. Then, the sections were incubated with primary antibodies overnight at 4°C. Subsequently, endogenous peroxidase activity was blocked with incubation of the slides in 0.3% H2O2 at room temperature for 30 min, followed by incubation with secondary antibody for 1 h at room temperature. Next, the sections were stained with diaminobenzidine (DAB)-H2O2 (Gene Tech, Shanghai, China) under a microscope and counterstained with hematoxylin. Finally, neutral balsam (Yeasen, Shanghai, China) was used to seal the slides with a cover slip. All assays included Negative control slides without the primary antibodies. TTI1 staining was seen mainly in the cytoplasm of NSCLC and KI67 was usually seen in the nucleus. Histochemistry score(H-score) was defined as the score for staining intensity multiplying with the score for staining percentage. According to the average intensity,the intensity was scored as follows: score 0: negative (0-<25%),score 1: weak (≥25%-50%),score 2；moderate (≥50%-75%) and score 3: strong (≥75%). Based on the percentage of positive tumor cells, the percentage was scored as follows: score 1: negative (0-<25%), score 2: weak (≥25%-50%), score 3: moderate (≥50%-75%) and score 4: strong (≥75%).
The cell lines involved in this study included the human NSCLC cell lines A549, PC-9, NCI-H460, NCI-H1299 and the human bronchial epithelioid cell HBE, which were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultivated in DMEM or RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Yeasen, Shanghai, China). The environment conditions were 37°C and 5% CO2 in a humidified incubator.
Total RNA extraction and qRT-PCR detection
Total RNA from cultured cells was extracted using Trelief TM RNAprep FastPure Tissue＆Cell Kit(Tsingke, Beijing, China) and reverse transcribed into cDNA using a Hifair® III 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen, Shanghai, China) according to the manufacturer’s instructions. Subsequently, according to the manufacturer’s protocol, quantitative real-time polymerase chain reaction (qRT-PCR) was performed with Hieff® qPCR SYBR Green Master Mix (High Rox Plus) (Yeasen, Shanghai, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene for quantification of mRNA. The relative RNA level was calculated by using the 2-ΔΔCt method. The RT-qPCR primers used in this study are as follows, TTI1, Forward primer: AAGTCATGCTGCGGAACTCA, Reverse primer: TGGGAACCACTGGGCTAATG; mTOR, Forward primer: CCAGGCCGCATTGTCTCTAT, Reverse primer: AGTCTCTAGCGCTGCCTTTC; S6K1, Forward primer: CTGAGGACATGGCAGGAGTG, Reverse primer: ACAATGTTCCATGCCAAGTTCA. EIF4EBP1，Forward primer: CAAGGGATCTGCCCACCATT, Reverse primer: AACTGTGACTCTTCACCGCC. All experiments were done three times.
The lentiviral vectors of TTI1 and shTTI1 were obtained from Genomeditech (Shanghai, China) to construct stably transfected cell lines. The three shRNA target sequences respectively is 5′-GCAGAACAGGAGAAATCAAAG-3′,5′-GGTCACAGCATTGTCGTATCT-3′ and 5′-GCAAG CTGCCATGATCCTTAA-3′. The transfection efficiency was examined by western blotting and qRT-PCR.
Colony formation and Cell Counting Kit-8 (CCK-8) assay
The colony formation and the CCK-8 assay were employed to determine the cellular proliferation. For colony formation assay,1000 cells per well were cultured in a 6-well plate (Corning, NY, USA) for 10 days. Then, all wells were rinsed with phosphate-buffered saline (PBS) three times, fixed with 4% paraformaldehyde for 10 minutes, and stained with 0.4% crystal violet for 15 minutes. After rinsing with water and drying, all wells were photographed, and the total number of colonies was observed and counted. The results of three independent experiments were shown as mean ± standard deviation. For the CCK-8 assay, the Cell Counting Kit-8 (CCK-8, Yeasen, Shanghai, China) was purchased and performed according to the manufacturer’s protocol. Firstly, seeded cells in a 96-well plate (Corning, NY, USA) approximately at 1000 cells/well, Then, 10 µl of CCK-8 reagent was added to each well after 24, 48, and 72h; the plates were incubated for 2h; and the absorbance of all plates was measured at 450 nm.
Wound healing, migration and transwell invasion assays
The wound healing assay and transwell assay (with Matrigel coating) were performed to detected cell invasion. For wound healing assay, seeded cells in a 6-well plate (Corning, NY, USA) and cultured until confluent. Then, using a (yellow) pipette tip made a straight scratch, simulating a wound. Subsequently, the cells were photographed at 0,24 and 48h.For transwell assay (with Matrigel coating) (BD Biosciences, Franklin Lakes, NJ), first, placed Matrigel coating into the upper chamber and then, 1×106 cells in serum-free medium were seeded into it.500ul culture medium containing 10% FBS was added into under chamber. After incubation for 36 h at 37 °C in a humidified atmosphere of 5% CO2, the transwell chambers was fixed with 4% paraformaldehyde for 10 minutes and stained with 0.4% crystal violet for 15 minutes. Finally, the transwell chambers was rinsed and the upper cell layer from the filter was wiped with a cotton swab, and the cells were photographed under a microscope and counted.
Western blot analysis
The procedures of the experiments were performed according to previous studies[10, 11]. In brief, cells were lysed with RIPA buffer (Beyotime, China) containing PMSF (Beyotime, China). The cell lysates were collected and centrifuged for 15 min at 12,000g (4°C). The supernatants were transferred to clean tubes, and SDS-PAGE sample loading buffer (Beyotime,China) was added. Then, they were boiled for 10 min and cooled down to room temperature. Proteins were subsequently separated on SDS-PAGE electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with protein free rapid blocking buffer (Epizyme, China) and incubated overnight at 4°C with primary antibodies. At the following day, the membranes were incubated with an HRP‐conjugated secondary antibody for 1h at room temperature and visualized by super ECL detection reagent (Yeasen, Shanghai, China) according to the manufacturer’s instructions. The TTI1 (1:5000, ab176696, Abcam, USA), p-mTOR(1:1000, ab109268, Abcam, USA), mTOR(1:5000, ab134903, Abcam, USA), p-S6K1 (1:1000, ab228513, Abcam, USA), S6K1(1:1000, ab32359, Abcam, USA),p-4EBPI (1:1000, ab27792, Abcam, USA), 4EBP1(1:2000, ab32024, Abcam, USA), Tublin (1:1000, AF0001, Beyotime, China) and GAPDH (1:1000, AF0006, Beyotime, China) was used as primary antibodies.
In vivo tumor growth
Immunodeficient nude mice (4-6 weeks of age) were purchased from Jiesijie (Shanghai, China) and fed in a pathogen-free environment. The experiment was approved by the Animal Experimentation Ethics Committee of the Second Affiliated Hospital of Nanchang University. About 5 × 106 cells were resuspended in 150 µl DMEM and injected subcutaneously into the right side of the flank area of nude mice. Tumor volumes were calculated (length × width2)/2 every four days.
The SPSS 23.0 software program (IBM SPSS, Chicago, IL, USA) was used for statistical calculations. The values were showed as the mean ± standard deviation. The chi-squared or Fisher’s exact tests was used to compare the categorical variables and the Student’s t test was chosen to compare difference of measurement data between two groups. Spearman correlation analysis was used to detect correlation between the expression of TTI1 and KI67. The best cut-off value of TTI1 and KI67 expression was divided by X-tile software. The overall survival (OS) and disease-free survival (DFS) analyses were estimated by the Kaplan-Meier method, and the comparison was evaluated by the log-rank test. The univariate and multivariate analyses were conducted using a model of Cox’s proportional hazards regression. P < 0.05 was regarded as statistically significant.