Cell lines and reagents
Lung epithelial cells Beas-2B, lung large cell carcinoma cells H1299, and lung adenocarcinoma cells H1975 and H2228 were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's Modified Eagle Medium or RPMI 1640 Medium (Gibco, California, USA), containing 10% fetal bovine serum (Gibco) and 1% antibiotics (Corning, New York, USA). Cell culture was conducted in a 37 °C incubator with 5% CO2. AKT inhibitor MK2206 was purchased from Selleckchem (TX, USA). Ferrostatin-1 was obtained from Xcess Biosciences. Other drugs were from Sigma-Aldrich. Antibodies against p-AKT and T-AKT were obtained from Cell Signaling Technology (Danvers, USA). Slc7a11 primary antibody was from Abcam (Cambridge, United Kingdom). GAPDH primary antibody and all the secondary antibodies were from Proteintech (Chicago, USA).
Human lung cancer tissues and The Cancer Genome Atlas
Human lung cancer tissues and adjacent tissues were collected from the patients before any therapeutic intervention at Kunming Fourth People's Hospital, Seventh Affiliated Hospital of Dali University. Written informed consent was obtained from the patients. The experiments were approved by Ethics Committee of Kunming Fourth People's Hospital, Senventh Affiliated Hospital of Dali University (ky201620) and performed according to the World Medical Association Declaration of Helsinki. The mRNA and protein expression of CCT3 was analyzed in these tissues.
Transcript abundance of CCT3, overall survival, and disease-free survival of LUAD and LUSC were analyzed from http://gepia.cancer-pku.cn/. For overall and disease-free survival analysis, LUAD patients were divided into CCT3 high and low group. The patient number was 120 and 239 in each group when the data analysis was performed using Quartile and Median, respectively. LUSC were divided into CCT3 high and low group. The patient number was 121 and 241 in each group when the data analysis was performed using Quartile and Median, respectively.
CCT3 knockdown and overexpression
Lentivirus system was applied to knock down and overexpress CCT3. For CCT3 knockdown, short hairpin RNAs (shRNA) targeting CCT3 was cloned into pLKO1.1-puro vector. The shRNA targeting sequence of CCT3 was as follow: shCCT3-1: 5’-GCCAAGTCCATGATCGAAATT-3’ and shCCT3-2: 5’-GCTACTGCGAATTGATGACAT-3’. For CCT3 or slc7a11 overexpression, the coding sequence of CCT3 or slc7a11 was cloned into pLV105 plasmid. To package lentivirus, psPAX2 and pLV-VSVG vectors were co-transfected with the lentivirus vectors into 293FT cells. The virus were harvested 48 h later and used to infect H2228 and H1975 cells. Puromycin was used to construct stable cell lines with CCT3 knockdown and overexpression.
Quantitative real-time PCR (qRT-PCR)
Human lung cancer tissues or indicated cells were subjected to total RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocols. Then, 1 ug of the RNA was reversely transcribed into cDNA using RT-for-PCR kit (Clontech, Takara, Japan). Quantification of mRNA level was performed using SYBR Green II (Takara, Japan). The primer sequence was as follow: CCT3 forward, 5’-TCAGTCGGTGGTCATCTTTGG-3’ and reverse, 5’-CCTCCAGGTATCTTTTCCACTCT-3’; Ki-67 forward, 5’-AGAAGAAGTGGTGCTTCGGAA-3’ and reverse, 5’-AGTTTGCGTGGCCTGTACTAA-3’; PCNA forward, 5’-CCTGCTGGGATATTAGCTCCA-3’ and reverse, 5’-CAGCGGTAGGTGTCGAAGC-3’; Cyclin D1 forward, 5’-CAATGACCCCGCACGATTTC-3’ and reverse, 5’-CATGGAGGGCGGATTGGAA-3’; GPX4 forward, 5’-GAGGCAAGACCGAAGTAAACTAC-3’ and reverse, 5’-CCGAACTGGTTACACGGGAA-3’; GAPDH forward, 5’-TGTGGGCATCAATGGATTTGG-3’ and reverse, 5’-ACACCATGTATTCCGGGTCAAT-3’. The 2−ΔΔCt method was applied to determine mRNA expression and GAPDH acted as the internal control.
Total protein was extracted from human tissues and cells using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), which was supplemented with protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). 30–50 ug of the protein was separated on SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. After blocking with 5% non-fat milk for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight and with secondary antibodies at room temperature for 3 h. Protein abundance was detected by Chemiluminescent ECL reagent (Beyotime Biotechnology). Antibodies against CCT3 (60,264–1-Ig) and GAPDH (60,004–1-Ig) were obtained from Proteintech. Slc7a11 antibody (PA1-16,893) was from Invitrogen. p-AKT (#4060) and AKT (#4691) primary antibodies were purchased from Cell Signaling Technology. Mouse (SA00001-1) and rabbit (SA00001-2) secondary antibodies were from Proteintech.
Cell proliferation and ferroptosis detection
Cell counting kit-8 (CCK8) kit (YEASEN, Shanghai, China) was used to detect cell proliferation. Indicated lung cancer cells were seeded into 96-well plates in triplicate at the density of 2000 cells per well. 6, 30, 54 and 78 h later, the OD value at 450 nm was detected. Cell viability at 6 h after seeding was recognized as day 1. Cell proliferation was normalized to the OD450 value of day 1.
To determine ferroptosis, we treated the cells with DMSO, ferroptosis inducer erastin (5 uM) and apoptosis activator 2 (5 uM) in combination with or without ferroptosis inhibitor ferrostatin-1 (2 uM), apoptosis inhibitor Z-VAD-FMK (10 ug/ml) or 3-methylademine (2 mM). Then cell viability was examined by trypan blue staining.
Colony formation assay
For CCT3 knockdown, a total of 2000 shCtrl, shCCT3#1 and shCCT3#2 H2228 cells were seeded into 6-well plates. For CCT3 overexpression, a total of 800 Ctrl and CCT3 overexpressed H1975 cells were seeded. 10 days later, colonies were washed with PBS and stained with crystal violet solution. Images of colonies were photographed under the camera.
Apoptosis and cell cycle detection
Apoptosis of LUAD cells was measured using PI/Annexin V-FITC kit (Invitrogen). In brief, control, CCT3 silenced H2228 cells and CCT3 overexpressed H1975 cells in 6-well plates in triplicate were trypsinized and washed by PBS. After stained with PI and Annexin V-FITC, apoptosis was detected on the flow cytometer (BECKMAN COULTER, Minnesota, USA), according to the manufacturer's protocols.
Cell cycle of LUAD cells was measured using PI kit (YEASEN, Shanghai, China) and was detected on the flow cytometer (BECKMAN COULTER), according to the manufacturer's protocols.
Dual luciferase reporter assay
The promoter sequence was cloned into pGL3.basic vector. Coding sequence of CCT3 was inserted into pCDNA3.1. After transfecting pCDNA3.1 and shRNAs, accompanied with pGL3.basic and TK vectors to H1975 and H2228 cells, dual luciferase activity was assessed by using Dual-Luciferase Reporter Assay Kit (Promega, USA), according to manufacturers’ instructions. The luciferase activity was adjusted to Renilla luciferase expression vector, pCMV-RL-TK.
All the data were presented as mean ± standard error of the mean (SEM) and were analyzed using GraphPad Prism. Student’s t test was used to compare the difference between two groups. One-way ANOVA was applied when more than two groups. Statistical difference was considered significantly when P was less than 0.05.