Human tissue samples
Sixty pairs of the gastric cancer samples as well as the adjacent non-neoplastic tissues were acquired from the Department of Gastrointestinal Surgery of The First Afﬁliated Hospital, Wannan Medical College (Wuhu, Anhui, PR China) from March 2019 to October 2020. In accordance with the guidelines of World Health Organization (WHO), all of the specimens were assessed by two pathologists. All the tissues were given at the time of surgery, which were stored directly in a liquid nitrogen. No systemic or local treatment was implemented to the patients prior to operation. The experiments and investigation have been agreed by patients and authoritied by the Ethic Committee for Clinical Research of the First Affiliated Hospital of Wannan Medical College (No. 2020-52). The study was conducted in accordance with the Declaration of Helsinki (https://www.wma.net/what-we-do/medical-ethics/declaration-of-helsinki/). We did not provide any data that could help identify patients.
Cell line and cell culture
The cell lines of gastric cancer (BGC-823, AGS together with SGC-7901) and GES-1, the normal gastric mucosal epithelial cells were offered through the Chinese Academy of Sciences Cell Bank (Shanghai, China). Cell viability, DNA fingerprinting, Mycoplasma as well as isozyme were detected to characterize cell lines. The last identification of cell was implemented on September 2017. Five cell lines were cultivated in Dulbecco’s Modified Eagle medium with high glucose (DMEM; Invitrogen, Carlsbad, California, USA) containing antibiotics (penicillin (100 units/mL) and streptomycin (100 mg/mL)) together with fetal bovine serum (10%, FBS; Gibco, Bethesda, MD, USA). All of the cells were cultivated with 5% CO2 under a temperature of 37 °C.
The lentivirus establishing the TULP3 gene knockout was offered by genepharma (Shanghai, China). The cell lines of gastric cancer were inoculated in plates (6 wells) at 50 percent of confluence, which were then respectively infected by scramble control (called shCtrl) or TULP3 knockout lentivirus (called shTULP3) in BGC-823 and AGS cells. The stable transduction pools were produced via selecting with 4µg/ml puromycin for two weeks. The above cell transfection protocol shall comply with the instructions of manufacturer. After transfection, the cells were cultivated and gathered to conduct the subsequent studies.
Quantitative real-time polymerase chain reaction(qRT-PCR)
Trizol reagent (Sigma, Cat.No.T9424-100m) was utilized for the extraction of total RNA from the samples. qPCR (+gDNA wiper) (Vazyme, Cat. No. R123-01) was reverse transcribed through utilizing Hiscript QRT Supermix. In a VII7 Real-Time PCR System (Applied Biosystems, Weiterstadt, Germany), the products of cDNA could be amplified and then quantified through applying AceQ qPCR SYBR Green master mix (Vazyme, Cat. No. Q111-02). The TULP3 mRNA primers are composed of forward primer 5′-TTGAGGTGGAGTCTCGCTCTGTC-3′ and reverse primer 5′-GAGGCAGGAGAATGGCGTGAAC-3′. The forward primer 5′-AGC CAC ATC GCT CAG ACA-3′ together with reverse primer 5′-TGG ACT CCA CGA CGT ACT-3′were employed for the internal control of mRNA. The mRNA level was determined by the comparative Ct method (ΔΔCt).
RIPA (Thermo Fisher, MA, USA) lysis buffer involving phosphatase suppressor (1%), PMSF (1%) and protease suppressor (0.1%) was utilized to dissolve cells BCA Protein Assay Kit (Cat. # 23225, Thermo Fisher Scientific, MA, USA) was exploited for the assessment of total protein, and the same amount of proteins were transferred to PVDF membranes by SDS-PAGE Protein electrophoresis apparatus (Cat. # VE-180, TIANGEN, Shanghai, China). The membrane was blocked by skimmed milk (5%) in a Tris buffered saline involving Tween 20 (0.1%) for two hours and cultured overnight with the below primary antibodies under a temperature of 4 °C. The above primary antibodies contains: TULP3 (1:1000, ab254692, Abcam, Cambridge, MA, USA), PTEN(1:1000, #9188, Cell Signaling Technology, Danvers, MA, USA), p-PTEN(1:1000, #9554, Cell Signaling Technology, Danvers, MA, USA), Akt(1:1000, #2920, Cell Signaling Technology, Danvers, MA, USA), p-Akt(1:1000, #4060, Cell Signaling Technology, Danvers, MA, USA), Snail(1:1000, #3879, Cell Signaling Technology, Danvers, MA, USA), Alix(1:1000, #2171, Cell Signaling Technology, Danvers, MA, USA), HSP70(1:1000, #4876, Cell Signaling Technology, Danvers, MA, USA), GM130(1:1000, #553612480, Cell Signaling Technology, Danvers, MA, USA), Flotillin-1(1:1000, #18634, Cell Signaling Technology, Danvers, MA, USA), EpCAM(1:1000, #5532626, Cell Signaling Technology, Danvers, MA, USA), and β-Actin (1:3000, A1978, Sigma, Victoria, BC, Canada). The membrane was cleaned 3 times with TBST (0.1%) for five minutes each time, then cultured for two hours by anti-rabbit or anti-mouse secondary antibody combined with the horseradish peroxidase (Cell Signaling Technology, Danvers, MA, USA), and cleaned 3 times with TBST (0.1%) for five minutes each time. The reaction products were visualized with chemiluminescent ECL Plus reagents (Pierce, USA). And Tanon 5200 (Tanon, Shanghai, PR China) was employed to scan the membrane. Quantity One Software(Tanon, Shanghai, PR China) was applied for the measurement of band strength via densitometry. The levels of β-actin and protein were standardized. All of the experiments were conducted three times and reflected representative outcomes.
The shTULP3 and shCtrl cells were inoculated into the plates (96-well) (Cat. # 3599, Cornning, NY, USA) overnight at 2×103 cells/well density. Before adding DMSO (150 μl, Sigma-Aldrich), each well was added with the MTT (20 μl, 5 mg/mL, Genview Inc., Santa Clara, California, U.S.), and then cultured for four hours, after which Microplate Reader was conducted to determine the optical density (Cat. # M2009PR, Tecan infinite, Shanghai, China) at 490 nm. All of the studies were implemented for three times.
Colony formation assay
The transfected BGC-823 together with AGS cells were added to the plates (6 well) and kept in the medium under a standard condition. After incubation for 2 weeks under a temperature of 37 ℃, rinse the above plates through using ice-cold phosphate buffer saline (PBS; Sigma-Aldrich, St. Louis, MO, USA). Next, the colony cells were fixed with paraformaldehyde (4%, Sigma-Aldrich, St. Louis, MO, USA) for fifteen minutes, subsequently stained by 0.1 precent crystal violet (0.1%, Sigma-Aldrich, St. Louis, MO, USA) at RT. The optical microscope (Nikon, Japan) was used for imaging and calculating the colony cells number.
Transwell chambers (BD Biosciences, USA) were used for the invasion and migration of cell. After two days of related treatment, in each group, BGC-823 along with AGS cells were cultured into upper Transwell chamber at 4×104 cells each well density (100μL medium involving 5 percent fetal bovine serum). In addition, the culture plate (24 well) in lower chamber was added with 100μL medium involving 10 precent of fetal bovine serum. After one day of the conventional culture, take out the chamber and wipe the cells on the upper microporous membrane layer through exploiting the cotton swab. Then, the cells were fixed in 4% solution of paraformaldehyde for ten minutes at RT and stained by crystal violet solution for fifteen minutes (0.5%, Sigma-Aldrich; Merck KGaA). In the end, five visual fields could be randomly chose and under the optical microscope (Nikon, Japan), they were subsequently observed to calculate the cells number invading the chamber microporous membrane sublayer.
Wound healing assay
Wound healing experiment was implemented for the observation of the cell migration. In short, when the BGC-823 and AGS cells after transfection remained in the 6-well plate and reached a confluence from 90 to 95%, the tip of a micropipette was utilized to create scratches. After scratching, X71 inverted microscope (Olympus, Tokyo, Japan) was applied for the observation of wound state at 0 and 24 hours.
In accordance with the instructions of manufacturer, ExoQuick precipitation solution (System Biosciences, Mountain View, CA, USA) was applied for acquiring exosomes from serum. Shortly, it will be ExoQuick solution (63μL) and serum (250μL) was mixed and incubated under a temperature of 4 °C overnight. After centrifugation for half an hour at 1500×g, suspending the pellets in PBS (50μL) and filtrated by 0.22μm of filter (EMD Millipore, Billerica, MA, USA). The separated ectoplasts were maintained under a temperature of −80 °C prior to use.
Transmission electron microscopy
A pipette was used to transfer 20 μL of produced exosomes onto the copper grid coated with Formvar carbon, allow it to adsorb for ten minutes, and then discharge the excess liquid. The exosomes after absorbed were subsequently negatively stained by 2% (w/v) phospphotungstic acid (with a pH of 6.8) for five minutes, and electric incandescent lamp was subsequently employed to dry them, and transmission electron microscope (Fei TECNAI 12, Philips) was exploited to analyze them, bar = 200 nm.
Size analyses of exosomes
The separated exosomes were diluted in the PBS, NanoSight LM 10-HSBFT 14 Instrument (NanoSight, Malvern, UK) was employed for analyzing them based on the protocol. The exosomes size were subsequently analyzed studied with the software of Nanoparticle Tracking Analysis 2.0 (NTA 2.0).
All data were produced from three separated studies and they were described as mean ± standard deviation. GraphPad Prism 8.0.1 for Windows (Graphpad Software Inc., La Jolla, California USA, www.graphpad.com) was utilized to conduct the statistical calculations. Between the two groups, the difference was calculated via proper unpaired Chi-square test or using Student’s t-test, and there exist a statistical significance when P < 0.05.