2.1 Bioinformatic Analysis
The expression and prognosis of OSBPL10 was calculated with the help of GEPIA and TCGA database. JASPAR online tool has been used for the prediction on binding sites between HIF-1α and OSBPL10.
2.2 Patients and Specimens
Pancreatic cancer tissues and their adjoining normal tissues were collected from Ruijin Hospital Affiliated with Shanghai Jiaotong University School of Medicine. It’s necessary for these paticipants to satisfy the following standards:they were diagnosed pancreatic cancer pathologically; having not received any preoperative chemotherapy; there are medical history data completely. At the same time, we obtained the agreement of these patients to join in our study and the ethics committee of Ruijin Hospital has approved this study protocol.
2.3 Immunohistochemistry (IHC)
To perform IHC assay, the tumor tissues collected from mice and patients were embedded in paraffin and sliced into 4 or 5 μm sections. These paraffin slides were then laid at 65℃ until they melt completely. After the treatment of xylene, ethanol and antigen retrieval solution, the antigen has been recovered. Next, the corresponding primary and secondary antibodies were selected for incubating these samples. Having finished the processes above, we used DAB color solution and hematoxylin for staining these tissues to detect the expression of OSBPL10. Finally, different concentrations of xylene and ethanol were applied again for dehydration and transparency and the pictures for IHC analysis were captured by scanner.
2.4 Cell culture and Tranfection
Cells in the study including human normal pancreatic duct epithelial (HPNE) cell, 293T , Pan02 and PDAC cell lines (PATU8988,Panc1,Bxpc-3,Miapaca2,Aspc-1,CFPAC,Capan2) were purecured from Cell Bank of the Chinese Academy of Sciences. All cell lines were maintained in Modified Eagle Medium (DMEM,Invitrogen) with the supplementation of 1% penicillin/sreptomycin and 10% fetal bovine serum,in a 37℃incubator with the atmosphere of 5% CO2. For tranfection experiments, Hilymax reagent as well as HEK-293T cells at appropriate confluence were used as mediators to transfect siRNA and target plasmids into pancreatic cancer cells. The siRNAs sequences are described in Table 1.
2.5 Western Blotting
In the study, the protein samples from cells and tissues were extracted with the application of RIPA buffer containing protease inhibitor and the BCA protein quantification kit determined concentrations of them. Subsequently, these protein samples were loaded and seperated by a 10% SDS-polyacrylamide gel electrophoresis separation gel, followed by being transferred onto a polyvinylidene difluoride membrane. The membrane was then incubated with primary and secondary antibodies.The protein expression levels were assessed by western blot imaging analyzer (Thermo Fisher Scientific , USA). Antibodies are listed in Table 2.
2.6 qt-PCR
The pancreatic frozen tissue and cell lines’ total RNA was isolated under the use of SteadyPure Quick RNA Extraction kit(ACCURATE BIOLOGY). The SYBR qPCR Master Mix was used to perform RT-PCR for detecting the expression of OSBPL10, CNBP, HIF-1α, and GAPDH played a role as the normalized gene . The primer sequences can be found in Table3. The data was evaluated using the 2-ΔΔCT method.
2.7 Colony Formation and Cell Counting Kit-8 (CCK-8)
The colony formation and CCK-8 assays were used for detecting the biological effects of OSBPL10 and CNBP on pancreatic cancer cell survival and proliferation. The cancer cells after transfection (1000 cells/per well) were plated into six-well plates for colony formation. After 14 days, the colonies were treated with 4% paraformldehyde for 30 minutes and crystal violet for 20 minutes. We choosed Image J software for counting cell colonies. And cell viability was evaluated by Cell Counting Kit-8, the transfected PC cells were placed in 96-well plates at 3000 cells/well for 24h. The absorbance was calculated under the treatment of cck-8 solution(Dojindo, China).
2.8 TRANSWELL
The transfected cancer cells were cultured in the 24-well Transwell plates with precoated Matrigel. 40000 cells under treatment were incubated in the upper chamber with the DMEM medium, while the lower chamber was supplemented with DMEM medium and 10% FBS. The membranes were collected and stained with the Crystal Violet Solution after 48 hours’cultivation. Cell invasion ability was calculated and counted by an inverted microscope.
2.9 WOUND Healing Assay
PANC1, Patu8988, MiaPaCa2 cells were set into 6-well plates respectively (untreated or treated by small interfering RNAs) for 24 hour’s cultivation until 100% confluence of cells. Then, we created cells wound and washed these cells with PBS gently. After that, these cells were continued to be incubated in serum-free medium. 24 hours later, the different-group cells migrated from the wound for different distances. The distances were measured under the microscope. These results showed the different abilities of cells migration.
2.10 Cell Apoptosis
Transfected cells in this experiment were laid in 6-well plates and received 24 hours’ incubation.They were centrifuged and suspended by PBS twice after digestion of EDTA. Next,the sample was resuspended with binding buffer and stained by Annexin-V/7-AAD apoptosis kit,this process lasted 15minitues.
2.11 Tumor Xenograft Model
We selected male 5-to-6-week-old BALB/c-nu nude mice(16-18g) for our research. Stably transfected PANC1, PATU8988, and MiaPaCa-2 cells were injected into subcutaneous axilla and spleen of mice(5×10^6 cells/site) after being resuspended by PBS. Since the injection, we measured the longest longitudinal diameter (a) and longest transverse diameter (b) every 4 days and every tumor volume=1/2(a×b^6). 4 weeks later, the tumors were collected and weighed for the further use and the livers were removed to check the situation of metastases.
2.12 Co-immunoprecipitation(Co-IP)
Protein G, immunoprecipitation kit played key roles in performing Co-IP asssay.PBS and NP40 buffer were applied to washing and lysing PC cells respectively.Then,the supernatant after centrifugation were incubated by antibodies and protein G agarose.
As for the next process,wash buffer 1,2,3 were used for washing protein G agarose step by step. At last, loading buffer washed the agarose, which was collected for western blot assays later.
2.13 Chromatin Immunoprecipitation (ChIP) Assay
ChIP analysis were operated according to the protocol of Pierce Magnetic ChIP Kit (Thermo Fisher Scientific, USA) . The pancreatic cancer cells Panc1, Patu-8988 and MiaPaCa2 cells were crosslinked with 4% paraformaldehyde and terminated with glycine respectively. Nuclear lysate was disintegrated under the use of ultrasound and incubated with HIF-1α antibody. Rabbit IgG antibody was required as control. Primers used in CHIP assay are listed in Table4.
2.14 Dual-luciferase Reporter Assays
Dual-Luciferase reporter assay was as described previously.The HEK-293T cells were kept in 6-well plates and growing until a proper confluence(70-80%).After that,we transfected 293T cells prepared above with target plasmids for 48 hours.For the next step,they received further treatment after transfection.In the end,the absorbance was measured by the Dual-Luciferase Reporter Assay System .
2.15 Flow-cytometry Analysis
We selected male 5-to-6-week-old C57 mice(16-18g) and Pan02 cells for our research.The process of mouse tumorigenesis is as mentioned above. Macrophages from animal models’ tumor tissues were isolated under the digestion of enzyme, followed by being filterated, centrifuged and resuspended with staining buffer. After the processes above, we selected antibodies against CD45, CD11b, F4/80, CD206, CD163(Biolegend) to stain these cells for monitoring the alteration of M1 and M2 ratio between different groups.
2.16 ELISA
In order to compare different levels of inflammatory marker IL-6 in two groups (with and without treatment), ELISA assay was performed according to guides of manufacturer. We collected supernatant from cells after 20 minutes’ centrifugation for determining IL-6 expression by ELISA kit.
2.17 Statistical Analysis
Finally, we operated SPSS and GraphPad prism9.0 to analyze these results above. Paired t-test was selected to see whether there existed any differences between two groups and Kaplan Meier curve took part in survival analysis . Cox regression showed levels of expression among related genes and their influences on PC patients. P<0.05 is the criterion for statistical significance.