Cell culture
The 92.1 choroidal melanoma cell line was provided by the Eye and Ear Research Institute of the University of Pittsburgh Medical Center. 92.1 cell line was maintained in 37℃,5%CO2 incubator in RPMI1640 (Corning, USA) containing10% fetal bovine serum (FBS，Corning, USA) .
Animals
Four-to-six-week-old male and female EGFP transgenic nude mice at an average weight of 25 g were provided by our group [10]. All the animals were bred and maintained in the Specific Pathogen Free Animal Care Facility, Nasal1000 grade. The National Institutes of Health guidelines for the care and use of laboratory animals were followed in all animal procedures.
Tumor cells inoculation
Orthotopic transplantation model of human uveal melanoma in nude mice.
After the cell suspension was vortexed, cell suspension with 2.5 x 105 cells in 25 µL of the cell suspension was drawn into the inoculation needle for subsequent use. After narcotizing the experimental mice with 1% pentobarbital sodium at the dose of 50mg/kg through intraperitoneal injection, the skin around the right orbit was disinfected using iodophor. The inoculation 27G needle was used to puncture the right eye of the animal 3 mm inside the nasal side orbital rim at an angle of approximately 45 degrees; when the needle reached the retro-ocular soft tissue, the pre-collected cells in the inoculation injector were injected into the eye. The injector was pushed slowly, and the needle was allowed to remain in the eye for a brief period.
Transplantation model of human uveal melanoma of brain ventricle in nude mice
As previously described [11], after narcotizing the experimental mice, a mini-sized cranial drill was used to drill a hole at the position 0.22 mm posterior to the bregma and 1 mm left of the sagittal suture under the guidance of the stereotactic apparatus. Then, a microinjection needle was inserted into the hole at the depth of 2.5 mm, Fifteen microliters of cell suspension（2 x 105 cells） was injected under the control of a micropump in 10 min. The needle was then withdrawn after being retained in the hole for 5 min. As required by the experiment, the tumor-bearing mice were sacrificed in due time, and the whole brains were taken out.
H & E staining.
Mice were sacrificed, then eyeballs, orbits, brain, heart, lung, and liver were also observed using the naked eye. Tissues were fixed with 10% paraformaldehyde, the specimens were embedded and sliced (5 μm). Staining was performed.
Immunofluorescence labeling
Tumor tissues were fixed with 4% phosphate-buffered paraformaldehyde, dehydrated with 20% sucrose solution, and cut into 30 μm coronal sections with a cryostat. All the sections were washed 3 times with PBS, 10 min each. The primary antibody HBM45 (1:100) (diluted in 0.3% Triton X-100/PBS) was applied overnight at 4 °C. The sections were washed 3 times with PBS, 10 min each before the second antibody was applied. Sections were exposed to secondary antibody CY3(1:800) at room temperature. Finally, sections were mounted with Vectashield medium (Vector laboratories) and coverslipped. Results were viewed using a confocal laser scanning microscope (Lecai TCS SP2, German).
Monoclonal culture of 92.1-A cells
Tumor-bearing mice were sacrificed. Fresh tumor tissues were gently aspirated using a pipette and placed in 6-well culture plates filled with 5 mL of 1640 culture medium. The culture medium was replaced every 3 days. After 2 weeks, the cells were transferred to a culture flask, and monoclonal subculture was repeatedly performed using the limiting dilution method [12].
Cell cycle detection using flow cytometer
After trypsin digestion, cells in the logarithmic growth period was centrifuged at 1000 rpm for 5 min; the supernatant was discarded before adding the medium; after being measured by counting chamber, the solution was diluted into single cell suspension with 106 cells and then tiled in a 6-well plate. The plate was placed in the 5% CO2 incubator at 37 °C for 24 h of adherent growth. After incubation for 24 h, trypsin digestion and centrifugation, the cells were washed by PBS twice before addition of 70% ice ethanol and placement at 4 °C overnight. Fixed cells were centrifuged and washed by PBS again, followed by action by RNAaes at 37 °C for 1 h, addition of PI and staining at 4 °C for 1 h. At last, cell cycle was detected by flow cytometer.
Detection of cell proliferation via CCK-8 assay
Both 92.1 and 92.1-A cells were cultured in RPMI1640 containing 10% fetal bovine serum and placed in a 5% CO2 incubator at 37 °C. The cells in the logarithmic growth period were inoculated to the 96-well plate (2000 cells/well) with 6 duplicate wells for each group; From day 1 to day 10, CCK-8 reagent was added to each well in the dark, followed by culture in the incubator for 2 h and detection of OD value at 450 nm using the microplate reader. The cell survival rate was calculated. SPSS 22 software was adopted to analyze the OD value of two cell lines and GraphPad Prism 5 was used for plotting to demonstrate the differences between groups.
In vitro invasiveness test
Scratch assay as described in the following steps. Horizontal lines spaced approximately 0.5-1.0 cm apart were evenly marked across the wells on the back of the 6well plates using a color pen. At least 5 lines passed through each well. Approximately 5 x10 5 cells were injected into each well; The cells were incubated in serum containing solution and transferred to stem cell conditions 2 h later. The next day, a tip was positioned with a ruler to ensure, to every possible extent, that it was placed perpendicular to the horizontal lines drawn on the back of each plate. The cells were rinsed with PBS three times. The detached cells were discarded and the remaining cells were cultured in media containing serum. Samples were obtained and photographed 0, 24, and 48 h after incubation.
Transwell assay
Transwell invasion assay were conducted with a Corning Inc. transwell chamber. Melted Matrigel was mixed with serum-free medium in a 1:8 rate. the upper compartment was precoated with 100 μl of Matrigel. After the glue is solidified, the remaining liquid in the plate was discarded, 200 μ l of warm serum-free medium was added, and retained at room temperature for 30 min after which the remaining culture medium was removed. 1 × 105 cells suspended in 200 μl serum-free medium were seeded in the upper compartment of the chamber and 500 μl RPMI1640 with 10% FBS were added to the lower compartment of the chamber. The cells were incubated for another 24 h. After that, the cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet. The non-migrating cells in the upper chamber were removed carefully using a cotton swab. The migrated cells were cells on the lower surface were photographed with microscope in five randomly selected visual fields and the migrated cells were quantified using SPSS.
Key protein screening and protein interaction diagram
Protein chip making and differential proteins of 92.1-Aand 92.1 cells were completed by Hangzhou Jingjie Biotechnology Co., Ltd. The protein relationship was analyzed by STRING 10.5. Proteins were imported into string 10.55 protein database for analysis, and preliminary protein interaction diagrams were generated. The results mentioned above were imported into Cytoscape 3.6.1 for further protein screening Finally, proteins of which the up-regulation difference was less than 1.2-fold, and the down-regulation difference was more than 0.83-fold were deleted. The Gene Card gene library was used to query the function of the related protein which may induce 92.1 malignant progression.
Plasmid extraction
A single transformed colony was selected, and cultured in 5 ml lysozyme broth culture medium containing ampicillin (50 ug / ml), and then shaked at 37 ℃ (250 rpm) for overnight cultivation. After centrifuging for 5 minutes (4000 rpm), the supernatant was discarded and precooled buffer P1 250ul was added into the bacterial precipitation. The bacteria was shaken by shaking table until completely suspended
Next, 250 UL buffer P2 was added, the centrifuge tube was turned gently to mix the suspension, for 4 minutes at room temperature. Then 350 UL buffer P3 was added and the suspension was mixed again. The supernatant was collected after centrifuged for 10 minutes, 12000 × g ,at 4 ℃ and transferred to the adsorption column. The supernatant in adsorption column was centrifuged again, 9000 × g, for 30s and the filtrate was discarded. 500 UL deproteinized buffer dw1 was added into adsorption column, and centrifuged, 9000 × g ,for 30 seconds. Finally 500 UL wash solution was added after the filtrate was discarded and centrifuged at 9000 × g for 30 seconds, which was repeated once. The empty adsorption column and collection tube were put into the centrifuge, and centrifuged at 9000 × g for 60 seconds.50 UL Elusion buffer was added to the center of the adsorption membrane and centrifuged at 9000 × g for 1 min at room temperature. The obtained plasmids were stored in refrigerator
p62 knockdown of 92.1-A
The cells in logarithmic growth phase were inoculated into 6-well plates. The cells were transfected by Lipofectamine 2000, when cells grown to about 60% confluence. The plasmid（Fig 1，Applied Biological Materials abm, Canada ） was mixed with Lipofectamine 2000 at room temperature for 15 min. Mixture was added to the cells culture medium and cultured for 6 h after which medium was changed to complete medium. After 24 hours, the plasmid can be used in the following experiments.
Over expression of p62 in 92.1 cells
92.1 cells were placed in a 6-well plate. After 24 hours, when cells grown to 40%- 60% confluence, the cells were infected with lentiviral overexpressed of p62 (Applied Biological Materials abm, Canada). 5 ng / μ l Polybrene was added to improve the efficiency of virus infection.6 hours later, cell culture medium was changed completely, and the transfection efficiency was observed under fluorescence microscope. After 24-48 hours, the cells were collected for protein extraction
Western blot
Cells from each group were collected and placed in a 1.5 mL EP tube, followed by addition of appropriate amount of lysate for completely lysis and ultrasonic testing in the ice bath sing ultrasonic processor; after centrifugation at 2000 rpm for 15 min at 4 °C, the supernatant was extracted and stored at − 20 °C for further use. The oncentration of proteins extracted above was quantified sing BCA Protein Quantification Kit; 5× loading buffer as added to the sample, followed by boiling at 100 °C or 5 min to denature the proteins. After processing by DS-PAGE, the proteins were transferred to the NC embrane using protein transfer device and then locked by 5% skimmed milk powder for 1 h. After ddition of IL-6, p-STAT3 and NF-κB antibodies and incubation vernight at 4 °C, the proteins were washed by BST three times before 1 h of fluorescent secondary ntibody incubation and another three times of TBST ashing; Western Blot imaging software Odyssey nalyzer was used to detect and produce images.
Animal experiment
Both 92.1 and 92.1-A cells in logarithmic growth phase were inoculated to 36 nude mice totally at the concentration of 1 × 106 cells/animal. After two weeks, when the tumor grew to the extent that vernier caliper could be used for measuring, the animals were divided into A) control group; B) chloroquine (CQ) treatment group; C) dacarbazine (DTIC) treatment group; and D) CQ + DTIC treatment group. Both drugs were intraperitoneally injected. The dosage of chloroquine was 50 mg / kg, intraperitoneal injection every day; dacarbazine was 50 mg / kg, o intraperitoneal injection every three days. The drug treatment lasted for three weeks. The body weight and tumor size of modeled mice were measured every 3 days. At the end of the experiment, as previously described, mice were anesthetized and sacrificed. The tumor tissue was removed and weighed in the aseptic condition. Paraffin section and immunohistochemical staining were conducted to analyze the traditional pathological and molecular pathological features of xenografts.
Immunohistochemical staining
Immunohistochemical staining was performed for the paraffin sections of the transplanted tumor tissue according to the antibody instructions. The steps were as follows in briefly: 1) add in goat serum to block heterogenetic antigens after PBS hydration and endogenous peroxidase blocking with 0.3% hydrogen peroxide; 2) add in the detection antibody (p62 Cell Signaling Technology); 3) incubate them overnight at 4℃; 4) add in the corresponding biotin-labeled second antibodies; and 5) seal the sections with neutral resins after DAB color development and hematoxylin counterstaining.
Statistical analysis
The experiment was duplicated at least 3 times. Graph-Pad Prism 5 software was used for imaging analysis; one-way ANOVA and Student’s t-test were employed for data statistical processing, where the data were represented by mean ± standard deviation (x ± SD). P < 0.05 was defined as statistical significance.