Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line

Summary

This study describes a protocol for exploring the effects of altered pH on oncogene expression via RNA-seq analysis of a pancreatic ductal cell line.

Abstract

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line.

Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs < ± 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the primecancers and ranks fourth among cancers in terms of number of related deaths¹. A higher mortality rate is evident among PDAC patients, and according to recent studies, the five-year survival rate is only 12%. Patients with documented chronic pancreatitis, a disease resulting in inflammation of the pancreas², are more likely (approximately 15-16-fold) to develop PDAC³. There are various types of CP with different etiologies, such as alcohol-related, hereditary, and idiopathic CP⁴. The conditions that exist during CP are known to lead to the formation of calcified stones, cancer cell development, and even diabetes. The prevalence of PDAC is high in patients with hereditary chronic pancreatitis⁵.

Some physiological conditions within the pancreas of CP patients might be beneficial for the initiation of other ailments; these include the inflammatory milieu, active digestive enzymes, and the acidic pH of the pancreatic juice⁶. A wide range of studies are being carried out in the area of inflammation, as inflammation creates chaos in the normal functioning of the organ^7,8,9. However, an imbalance in pH regulation is another phenomenon that might trigger the initiation of the uncontrolled growth of cells¹⁰. The pancreatic juice of healthy individuals is alkaline, which helps neutralize the acidic chyme produced by the stomach.In contrast, the pancreatic juice remains acidic in CP patients because of insufficient bicarbonate secretion. Low levels of bicarbonate cannot completely neutralize the juice, which results in a slightly acidic environment within the duct¹¹.

Earlier studies indicate that cancer cells adapt to and thrive in acidic environments¹². In such cases, conditions that exist during the process of chronic pancreatitis suggest a favorable environment for the proliferation and metastasis of these cells¹³. Hence, our study aimed to assess the morphological changes in human pancreatic ductal cells and assess oncogene expression under slightly acidic conditions.

Protocol

1. Revival of the cell line

1. Procure the human normal pancreatic ductal cell line (HPDEC/ H6C7) and revive the cells in fresh medium.
   1. Thaw the cells at 37 °C in a water bath.
   2. Freshly prepare complete medium and filter it using a syringe filter.
      NOTE: Complete medium contains Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
   3. Add 6 mL of the complete medium to a fresh 15 mL conical tube, add the thawed cell suspension, and mix it thoroughly.
   4. Centrifuge the suspension at 1,800 × g for 6 min. Discard the supernatant, add 1 mL of culture medium to the pellet, and mix gently.
      NOTE: Culture medium contains DMEM with 10% fetal bovine serum (FBS) and 1% pen/strep antibiotics.
   5. Add the cells drop-wise to a T-25 flask containing 4 mL of culture medium and incubate at 37 °C, 5% CO₂.

2. Changing the pH of culture medium

1. Prepare 100 mL of 0.5 M sodium phosphate buffer stock solutions with pH values of 6.0, 6.5, and 7.0 as mentioned in Table 1.
2. Check the pH with a pH meter and adjust it using 1 N HCl/1 M NaOH.
3. Filter-sterilize the buffers in a laminar air flow hood with a 0.45 µm syringe filter.
   NOTE: The buffer can be stored at 4 °C until further use.
4. Prepare culture media with pH values of 6.0, 6.5, and 7.0 by adding 20 mL of sterile 0.5 M sodium phosphate buffer with the respective pH to 80 mL of culture medium.
5. Add the media with the altered pH to 85-90% confluent human pancreatic ductal cells, and check for the morphological changes under a bright-field microscope at 1 h intervals for 6 h.

3. RNA isolation

NOTE: Sterile gloves must be used for RNA isolation from cells and the surface must be decontaminated using 100% ethanol.

1. Remove the culture medium and rinse the cells with phosphate-buffered saline (PBS).
2. Place the plate on ice, add PBS to the well,and then scrape the cells with a cell scraper.
   NOTE: The amount of PBS added should be enough to cover the cells for scraping. There is no need to add a high volume of PBS.
3. Collect the suspension in a fresh tube and centrifuge it at 8,000 ×g for 10 min. Discard the supernatant.
4. Add lysis buffer containing β-mercaptoethanol to the pellet and pipette it up and down for 5-10 min on ice.
5. Add an equal volume of 70% ethanol to the lysate and mix thoroughly. Do not vortex.
6. Add the solution to the silica membrane column, centrifuge at >8,000 ×g for 30 s, and discard the flow through.
7. Add wash buffer to the sample and centrifuge at >8,000 ×g for 30 s. Centrifuge the empty tube for 1 min at >8,000 ×g.
8. Add 30-50 µL of nuclease-free water to the column and leave it for 5 min at room temperature. Centrifuge the column at >8,000 ×g for 5 min.
   NOTE: RNA samples can be stored at -20 °C until further use.

4. RNA-seq/Transcriptome

1. RNA integrity assessment
   1. Check the integrity of RNA in a bioanalyzer by taking 1 µL (25-500 ng) and mixing it with 5 µL of dye buffer.
   2. Vortex the solution at 500 ×g for 1 min and spin briefly.
   3. Run a one-step polymerase chain reaction (PCR) for each sample at 72 °C for 3 min and then, take the tubes and incubate them on ice for 2 min before analysis with a bioanalyzer.
2. rRNA-depleted total RNA
   1. Add 5 µg of total RNA, 50 µL of hybridization buffer, and 1-3 µL of probe mixture to a fresh PCR tube.
   2. Perform the following steps: first step, denaturation at 70 °C for 10 min; second step, hybridization at 37 °C for 20 min; and incubation at 37 °C until use.
   3. In a fresh tube, add 500 µL of magnetic beads and the sample; place the mixture on a magnetic stand for 1 min until the solution becomes clear.
   4. Carefully discard the supernatant. Remove the tube, wash the beads by adding 500 µL of nuclease-free water, and place the mixture back on the magnetic stand for 1 min. Remove the supernatant once the solution becomes clear.
   5. Repeat the process 5-7x. Add 200 µL of hybridization buffer and mix properly. Keep the tubes at 37 °C for 5 min.
   6. Add 100 µL of RNA probe to the above mixture, mix gently and incubate the mixture at 37 °C for 15 min.
   7. Briefly spin the tube before placing it on the magnetic stand for 1 min.
   8. Collect the clear supernatant containing the rRNA-depleted RNA in a fresh tube.
3. RNA fragmentation
   1. In a fresh PCR tube, add 8-10 µL of rRNA-depleted total RNA (500 ng/g), 1 µL of 10x buffer, and 1 µL of RNase III.
   2. Mix the solution by tapping and spinning briefly.
   3. Place the tubes in a thermal cycler for 10 min at 37 °C.
   4. In a fresh tube, add 5 µL of nucleic acid-binding beads and then, add 90 µL of binding solution concentrate.
   5. Add 30 µL of the RNA fragments and 150 µL of 100% ethanol to the above mixture. Mix the sample by pipetting 10x, and incubate it for 5 min at room temperature.
   6. Place the tubes on the magnetic stand until the solution becomes clear. Remove and discard the supernatant.
   7. Wash the beads with 150 µL of ethanol while the tubes are on a magnetic stand; let the mixture stand for 30 sec and then remove the supernatant.
   8. Leave the tubes on a magnetic stand for 1-2 min to allow the remaining ethanol to evaporate.
   9. Add 12 µL of prewarmed nuclease-free water to the beads, and mix the sample by pipetting 10 times.
   10. Incubate the sample for 1 min at room temperature. Place the tubes on a magnetic stand and collect the supernatant once it is clear.
4. Library preparation and cDNA preparation
   1. Add 3 µL of the fragmented RNA sample to a PCR tube and then add 5 µL of hybridization mixture. Mix the sample by pipetting a few times and then briefly spin the sample.
   2. Keep the tubes in a thermal cycler for 10 min at 65 °C, followed by 5 min at 30 °C.
   3. Add 10 µL of ligation buffer and 2 µL of ligation enzyme mix to the above solution. Incubate the reaction in a thermal cycler for 1 h at 30 °C.
   4. Add reverse transcriptase master mix to the ligation mixture and incubate at 70 °C for 10 min, followed by incubation on ice.
5. Amplification of cDNA
   1. Add 6 µL of cDNA (500 ng) to a fresh PCR tube and then add 46 µL of barcoded PCR mix from a tube containing 46 µL of high-fidelity polymerase enzyme and 1 µL of 3' barcode primers.
   2. Set the PCR conditions as follows: first stage, hold at 94 °C for 2 min; second stage, cycles at 94 °C for 30 s and 50 °C for 30 s; third stage, 16 cycles at 94 °C for 30 s, 62 °C for 30 s, and 68 °C for 30 s. Hold the reaction at 68 °C for 5 min.
   3. Purify the amplified cDNA by repeating steps 4.3.5 to 4.3.10 and replace the fragmented RNA sample with the PCR product obtained in the previous step.
   4. Analyze the purified RNA sample on a sequencer (NGS) to generate data in the form of FASTq files.

5. Bioinformatics

1. Open the galaxy tool available online by copy pasting the link: https://usegalaxy.org into the search tab of the browser.
2. On the first visit to this site, register for a profile by clicking on the log-in or the register option on the top right corner.
3. After creating an account, log into the account by filling the public name/username and password.
4. Upload the FASTq files obtained after RNA-seq analysis by clicking on the upload option on the left-hand side panel.
5. Select the cutadapt tool from the tools section on the left side panel. This tool will trim low-quality sequences.
   NOTE: This file can now be used for mapping.
6. Click on the tool button on the left side panel and look for the RNA star tool. Fill in the details as follows: Use the output file of cutadapt, select the reference genome as human reference genome (hg38), and change the length of the genomic sequence around annotated junctions to 36. Select the output as gene count and click the run tool option on the top right corner.
   NOTE: This tool will generate a BAM file.
7. Select the featurecounts tool from the tool options and upload the BAM files obtained after RNA star analysis. Set the minimum mapping quality per read option in the read filtering options to 10, and click the run tool option in the top right corner.
8. Open the featurecount output file from the right panel and copy the data into a spreadsheet. Calculate the fold change relative to the control by dividing the number of read counts obtained in the test with the number of read counts obtained in the control with the command =log (fold change, 2).
   NOTE: A fold change <-2.0 is considered to indicate downregulation and a fold change > 2.0 is considered upregulation.

Results

Morphological changes
The incubation of human pancreatic ductal cells in an acidic medium altered the morphology of the cells. A confluent and tightly packed cell pattern resulted in dispersed cells under acidic conditions, and cell death becomes prominent with time. Compared with normal medium-containing cells, ductal cells shrank and decreased in size. A drastic change in the death rate was observed after 6 h of incubation. After 24 h, the cell number decreased, and the cells seemed to be in a st...

Discussion

This method was developed to determine the effects of acidic conditions on noncancerous human pancreatic ductal epithelial cells in biological repeats. A change in the pH of the medium caused the cells to be in a stressed state, and a change in the morphology was observed. The cells were cultured at pH 6.0, 6.5, or 7.0 for 24 h and monitored every hour to optimize the incubation period for further experiments. We observed a change in morphology after 4 h of incubation, and the cell mortality rate increased after 6 h of i...

Materials

Name	Company	Catalog Number	Comments
15 mL graduated centrifuge tubes (Falcon)	Tarsons	500031	used for sample preparation
50 mL graduated centrifuge tubes (Falcon)	Tarsons	500041	used for sample preparation
Agilent 2100 Bioanalyzer instrument	Agilent	Agilent G2938A	Instrment for RNA quality assessment
Antibiotic Antimycotic Solution 100x liquid	Himedia	A002-100 mL	Used to prevent contamination
Cell Scraper with rotatable blade	Himedia	TCP223	Scraping and collecting the cells
CO2 incubator	New Brunswick	Galaxy 170 S	Used for incubating the culture
Dulbecco's Modified Eagle Medium (DMEM), high glucose	Himedia	AL007S-500 mL	Medium used for culturing the cells
Dulbecco's Phosphate Buffered saline 1x	Himedia	TL1006-500mL	cell washing
Galaxy	(https://usegalaxy.org)		Online tool for processing NSG Data
HI FBS (origin: Australia)	Gibco	10100-147	Used for the growth of cells
Human Pancreatic Ductal Epithelial Cell Line (HPDEC/ H6C7)	Addex Bio	T0018001	Pancreatic ductal cell line
Ion Total RNA-Seq Kit v2	Thermo fisher scientific	4475936	RNA sample preparation kit
Laminar Air Flow
Na2HPO4 Diabasic	Sigma Aldrich	S3264-250G	Sodium phosphate buffer preparation
NaH2PO4 Monobasic	Sigma Aldrich	S3139-250G	Sodium phosphate buffer preparation
NovaSeq 6000	Illumina	3376672	Sequencer
Nunclon Delta Surface (6-well plate)	Thermo Scientific	140675	Culturing the cells
Refrigerated benchtop Centrifuge	Thermo Scientific	Sorvall ST 8R	Used for centrifugation
RiboMinus Eukaryote System v2	Thermo fisher scientific	A15026	rRNA depletion kit
Rneasy Mini kit	Qiagen	74104	Kit for isolating Total RNA from cells
Thermal cycler	Eppendorf	E950040025	PCR reaction
Water Bath	Equitron	#8406M	Thawing of sample