Subject characteristics
The study population was composed of 20 lung cancer patients (14 men, 6 women) with a median age of 49.9 years (range 32–65 years) at the time of diagnosis in our hospital between May 2016 and June 2018 (Table 1). All patients were diagnosed with NSCLC confirmed by pathological tissue examination. 8 (40%) patients were classified as TNM stage grade I or II, while 12 (60%) were stage III or IV. All patients underwent surgical resection and we obtained tissues from the surgical specimens. Both cancerous and noncancerous tissues were histologically confirmed. Written informed consent was signed by all the participants. This study was approved by the ethics committee of Huai'an Second People's Hospital, The Affiliated Huai'an Hospital of Xuzhou Medical University.
Table 1
Clinical characteristics of the 20 study participants.
Variables
|
N
|
Percent (%)
|
Age at diagnosis
|
|
|
≤ 50
|
16
|
80.00%
|
> 50
|
4
|
20.00%
|
Sex
|
|
|
Male
|
14
|
70.00%
|
Female
|
6
|
30.00%
|
Histology
|
|
|
Adenocarcinoma
|
17
|
85.00%
|
Squamous cell
|
3
|
15.00%
|
TNM stage
|
|
|
I-II
|
8
|
40.00%
|
III-IV
|
12
|
60.00%
|
Total
|
20
|
100.00%
|
Cell culture
A549, H1299, H1650, H292 and 293T cells were purchased at American Type Culture Collection (ATCC). A549 H1299, H1650 and H292 were grown in RPMI-1640 (Hyclone). 293T were grown in DMEM. The cell medium were added with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were cultured under 37 °C with 5% CO2.
Transfection
Lentiviruses plasmid for knockdown and overexpression of PIK3R1 were provided by Genepharma (Shanghai, China). 293T cells were used to produce lentiviruses. The supernatants were collected 48 hours after transfection, filtered by a 0.45-um filter, and obtained at -80℃ until use. sh-NC, sh-PIK3R1 were transfected into A549 and H1299 cells and LV-NC, LV-PIK3R1 were transfected into H1650 and H292 cells according to the manufacturer’s protocol.
Cell viability assay
CCK-8 assay was used to detect the cell viability. 5000 cells/well cells were cultured in a 96-well plate. After transfection, each well was replaced with 100 ul mixture of 10 ul CCK8 (Dojindo, Japan) and 90 ul medium and kept at 37°C with 5% CO2. Absorbance at 450 nm as valibiliy value was determined using an enzyme microplate reader (LabSystems) 2 hours later. A cell growth curve was plotted every 24 h for five days.
Quantitative real-time PCR
We used TRIzol (Invitrogen Corp) to extract total RNA. Reverse Transcriptase cDNA Synthesis Kit (Cat. no. RR037A; Taraka) was utilized to reverse transcribe total RNA. After that, SYBR Premix Ex Taq II (TaKaRaBio Technology) were used for quantitative real-time PCR (qPCR). Fianlly, the qPCR run on an Applied Biosystems 7500 Fast Real-Time PCR System (ABI, Foster, USA). The primers used were as follows: PIK3R1 Forward: 5’-CATCCCAGGACCTCTCT-3’, Reverse: 5’-CGGGGGACTGGCGA-3’; GAPDH Forward: . 5’-CAAGGTCATCCATGACAACTTTG-3’, Reverse: 5’-GTCCACCACCCTGTTGCTGTAG-3’.
Western blot
Proteins were run on a 10% SDS-PAGE. We used polyvinylidene fluoride (PVDF) membranes to transfer protein. The primary antibodies for anti-PIK3R1, anti-p-PI3K, anti-PI3K, anti-p-AKT, anti-AKT, anti-p-mTOR, anti-mTOR and anti-GAPDH (Abcam, Cambridge, UK) and secondary antibodies conjugated with horse radish peroxidase were used for analysis. The enhanced chemiluminescence assay was used to visualize the protein bands.
Statistical analysis
Student paired t-tests were used for the comparison between primary tumors and adjacent noncancerous tissues. Student’s unpaired t-test were used to measure the differences between two groups. Data were shown as the standard deviation of the mean (mean±standard deviation (SD)). Differences were considered significant with a P value <0.05.