Long-Term Helicobacter pylori Infection Switches Gastric Epithelium Reprogramming towards Cancer Stem Cell-Related Differentiation Program in Hp-Activated Gastric Fibroblast-TGFβ Dependent Manner
Abstract
1. Introduction
2. Materials and Methods
2.1. Bacterial Hp Strain
2.2. Technique of Rat Gastric Fibroblasts Isolation and Their Activation towards Fibroblasts Possessing CAFs Characteristic
2.3. Isolation of Hp-AGF and Normal Gastric Fibroblast (GF)-Conditioned Media
2.4. Long-Term Influence of Supernatants Collected from Hp-AGFs and GFs on Epithelial RGM1 Cells
2.5. RT-PCR Technique
2.6. Influence of TGFβR1 Kinase Activity Receptor Inhibition on Phenotype of Long-Term RGM1 Cells
2.7. Cell Proliferation
2.8. Western Blot
2.9. Determination of the Cells Ability to Release TGFβ1
2.10. Image Acquisition and Immunofluorescence
2.11. Statistical Analyses
3. Results
3.1. Long-Term Influence of Hp-AGF Secretome Induces Phenotypical Changes in Pro-Invasive s.t.EMT+RGM1 Cells
3.2. The Phenotypic Plasticity of l.t.EMT+RGM1 Cells
3.3. TGFβR1 Activation Participates in Both Pro-Pluripotent and Pro-Fibrotic RGM1 Reprogramming
3.4. Hp-AGF Secretome Induces Potentially Pro-Proliferative Phenotype of l.t.EMT+RGM1 Cells
3.5. Hp-AGF Secretome Prompts TGFβ-Dependent Proliferation of l.t.EMT+RGM1 Cells
3.6. Interrelations between TGFβR1 and TGFβR2 Activity Underlie Differential Microevolution Pattern of l.t.EMT+RGM1 and l.t.EMT−RGM1 Cells
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Sequence | Size of PCR Product (bp) | Annealing Temp. (°C) |
---|---|---|---|
18S | Forward 5′-GTTGGTTTTGATCTGATAAATGC-3′ Reverse 5′-CATTAAATCAGTTATGGTTCCTTTG-3′ | 143 | 60 |
Bax | Forward 5′-CTGCCAACCCACCCTGGTCT-3′ Reverse 5′-TGGCAGCTGACATGTTTTCTG-3′ | 195 | 55 |
BCL2 | Forward 5′-ACTGAGTACCTGAACCGGCATC-3′ Reverse 5′-GGAGAAATCAAACAGAGGTCGC-3′ | 108 | 60 |
c-Myc | Forward 5′-CCACACAGCCCACTGGTCCT-3′ Reverse 5′-GGCTGGAGCATTTGCGGTTGTT-3′ | 163 | 60 |
Cyclin D1 | Forward 5′-TGCTTGGGAAGTTGTGTTGG-3′ Reverse 5′-AATGCCATCACGGTCCCTAC-3′ | 126 | 60 |
c-Myc | Forward 5′-CCACACAGCCCACTGGTCCT-3′ Reverse 5′-GGCTGGAGCATTTGCGGTTGTT-3′ | 163 | 60 |
Ki-67 | Forward 5′-AACCAGGACTTTGTGCTCTGTAA-3′ Reverse 5′-CTCTTTTGGCTTCCATTTCTTC-3′ | 209 | 60 |
Klf4 | Forward 5′-TTCTCCACGTTCGCGTCCGG-3′ Reverse 5′-TCTCGCCAACGGTTAGTCGGGG-3′ | 80 | 60 |
Oct4 | Forward 5′-GGAGGGATGGCATACTGTGGACCT-3′ Reverse 5′-TCCTGGGACTCCTCGGGACTAGG-3′ | 197 | 60 |
p53 | Forward 5′-AGTGAAGGGACTAGCATTGTC-3′ Reverse 5′-GGATGCCCGTGCTGCCGAGGAG-3′ | 243 | 60 |
Sox2 | Forward 5′-AGAACCCCAAGATGCACAAC-3′ Reverse 5′-CTCCGGGAAGCGTGTACTTA-3′ | 204 | 60 |
TGFβ rec 2 | Forward 5′-TGTGGCAGAGCGCTTCAGT-3′ Reverse 5′-TGTTCAGGGAGCCGTCTTCT-3′ | 95 | 60 |
TGFβ rec 1 | Forward 5′-GCAGACTGGACCAGCAATGAC-3′ Reverse 5′-CTGCAATCAGGATCACTGCAA-3′ | 118 | 60 |
Hp 16S | Forward 5′-GTCAAGAGATCAGCCTATGTCC-3′ Reverse 5′-TGGCAATCAGCGTCAGGTAATG-3′ | 522 | 54 |
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Krzysiek-Maczka, G.; Targosz, A.; Szczyrk, U.; Wrobel, T.; Strzalka, M.; Brzozowski, T.; Czyz, J.; Ptak-Belowska, A. Long-Term Helicobacter pylori Infection Switches Gastric Epithelium Reprogramming towards Cancer Stem Cell-Related Differentiation Program in Hp-Activated Gastric Fibroblast-TGFβ Dependent Manner. Microorganisms 2020, 8, 1519. https://doi.org/10.3390/microorganisms8101519
Krzysiek-Maczka G, Targosz A, Szczyrk U, Wrobel T, Strzalka M, Brzozowski T, Czyz J, Ptak-Belowska A. Long-Term Helicobacter pylori Infection Switches Gastric Epithelium Reprogramming towards Cancer Stem Cell-Related Differentiation Program in Hp-Activated Gastric Fibroblast-TGFβ Dependent Manner. Microorganisms. 2020; 8(10):1519. https://doi.org/10.3390/microorganisms8101519
Chicago/Turabian StyleKrzysiek-Maczka, Gracjana, Aneta Targosz, Urszula Szczyrk, Tomasz Wrobel, Malgorzata Strzalka, Tomasz Brzozowski, Jaroslaw Czyz, and Agata Ptak-Belowska. 2020. "Long-Term Helicobacter pylori Infection Switches Gastric Epithelium Reprogramming towards Cancer Stem Cell-Related Differentiation Program in Hp-Activated Gastric Fibroblast-TGFβ Dependent Manner" Microorganisms 8, no. 10: 1519. https://doi.org/10.3390/microorganisms8101519
APA StyleKrzysiek-Maczka, G., Targosz, A., Szczyrk, U., Wrobel, T., Strzalka, M., Brzozowski, T., Czyz, J., & Ptak-Belowska, A. (2020). Long-Term Helicobacter pylori Infection Switches Gastric Epithelium Reprogramming towards Cancer Stem Cell-Related Differentiation Program in Hp-Activated Gastric Fibroblast-TGFβ Dependent Manner. Microorganisms, 8(10), 1519. https://doi.org/10.3390/microorganisms8101519