NSCLC specimens’ collection
87 pairs of NSCSC tissues and adjacent normal lung tissues were obtained from the Affiliated Hospital of Yangzhou University,Yangzhou University(Yangzhou, China) from January 2018 to December 2019. NSCSC was confirmed by two pathologists. The age of NSCLC patients ranged from 39 to 76 years old, including 56 males and 31 females. All fresh specimens were stored in liquid nitrogen before use. The use of specimens and the collection of clinical data were approved by the ethics committee of the Affiliated Hospital of Yangzhou University(Approval No. 2018-08). Written informed consent was also provided for the patients. All the clinicopathological parameters are summarized in Table 1, including age, gender,smoking, tumor size,grade, lymphatic metastasis and TNM stage.
Cell culture
NSCLC cell lines (A549, H1650 and H460) and normal Lung epithelial cell (BEAC-2B) were purchased from the Chinese Peking Union Medical College Cell Bank (Beijing, China). These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (HyClone, Australia), 120 IU/ml penicillin, and 120 mg/ml streptomycin at 37°C in humidified incubators with 5% CO2.
Plasmid and Transfection
Plasmid of piR-1366 mimics/inhibitor and piRNA negative control (NC) were all purchased from Sangon Biotech (Shanghai) Co., Ltd.(Shanghai, China). Plasmids were transfected into cultured cells using Lipofectamine 2000 Reagent (Invitrogen) according to the product protocol, Then tumor cells were incubated for 36 hour at 37°C with 5% CO2 before next experiments.
qRT-PCR
Total RNAs were isolated using TRIzol reagent (Invitrogen). The RNAs were set at an optical density (OD) A260/280 ratio between 1.8 and 2.1 and an OD A260/230 ratio >1.8. RNAs were reverse-transcribed to cDNA using the PrimeScript First Strand cDNA synthesis kit (Takara) according to the instructions(13). qRT-PCR was performed with fluorescent quantitative Kit (2 × SYBR Green qPCR mastermix) on an Applied Biosystems 7500 Real Time PCR system. The related primers were in table S1. The expression of piRNAs and mRNAs was normalized to U6 and GAPDH, respectively.
Arraystar piRNA array
The extracted total RNAs were submitted to AKSOMICS (Shanghai) Biotechnology Co., Ltd. (Shanghai, China) for The Human Arraystar piRNA array, which is designed for profiling 23000 human piRNAs (ArrayStar, Rockville, MD). Data analysis of expression profiling and image acquisition were provided by AKSOMICS.
RNA in situ hybridization (RISH)
Paraffin sections were dewaxed to water. Endogenous enzymes were eliminated by hydrogen peroxide treatment at room temperature. The exposed tissues were digested with pepsin. Tissues were incubated in an incubator with pre-hybridizing solution, and then hybridizing solution was added and incubated 12 hours. Add sealing solution after washing. Biotinylated digoxin was added. The tissues were added with SABC. Biotinylated peroxidase was added. The tissues were stained with DAB, hematoxylin re-dyeing,washing,dehydration, transparency, sealing. The hybridization solution containing the probe was replaced by pre-hybridization solution as blank control.
Nude mice lung metastasis models
For metastasis models, LC cells (5 × 106) suspended by PBS were injected subcutaneously into the tail veins of null mice (BABL/c, nu/nu, 23–27 g, 5–7 weeks of age, 10 mice/group) from Animal Center of Yangzhou University. At the end of the arrays, the mice were scarified and the tumors and their lungs were removed, quantified and frozen for further assay. All nude mouse studies were approved in the animal facility at Ethics Committee on Animal Experimentation of Yangzhou University accordance with institutional guidelines.
Western blot assay
Western blotting has been described in the previous article (14). The simple steps are as follows: Total protein extraction, Protein content determination, SDS-PAGE electrophoresis, Transfer membrane, Immune reaction, Final gel image analysis. The primary antibodies included WT1(#ab180840), CDH1(E-cadherin,#ab231303)), MGE3(#ab223162) and GAPDH(#2118), the second antibody was anti-rabbit immunoglobulin G antibody (which had been combined with horseradish peroxidase). CD9 (#ab2215), CD81(#ab109201), WT1 (#ab180840), CDH1(E-cadherin, #ab231303)), MGE3(#ab223162) and anti rabbit immunoglobulin G antibody (#ab6721) were purchased from Abcam Biotechnology company, GAPDH antibody (#2118) were purchased from CST Biotechnology company.
Dual-luciferase reporter assay
Bioinformatics method was used to predict the piR-1366 binding sites in 3'UTR of WT1. The length 368 nt of WT1 3'UTR was amplified and inserted into the luciferase reporter gene plasmid pGL3-BS. Wild type (WT) gene plasmid and mutation type(Mut) gene plasmid were co-transfected into A549 cells. The protein was extracted and used for luciferase detection.The activity of luciferase was determined by adding substrate. The relative fluorescence intensity was calculated, Firefly luciferase signal was used for normalization. The primers of WT1 3′-UTR are shown in table S1.
Transwell migration
In 180 µl culture media of the upper wells, ten thousand of lung cancer cells without serum were seeded on a fibronectin-coated polycarbonate membrane. In the lower wells, culture solutions contain 10% FBS. After 12 hours, the tumor cells migrated to the membrane’ bottom surface and were fixed with methanol for 20 minutes, then stained with 0.1% crystal violet for 15 minutes, observed and photographed under a light microscope (Leica, Germany) at 10×20or 10×400 magnification.
Statistical analysis
SPSS V.16 software and GraphPad Prism V5.0 software were used for statistical analysis and all graphs. Students’ T test was used to evaluate the differences between groups. The variables of count data were analyzed by chi square test (χ2-test). P < 0.05 as a significant difference between the two groups.