Role of Bile-Derived Extracellular Vesicles in Hepatocellular Proliferation after Partial Hepatectomy in Rats
Abstract
:1. Introduction
2. Results
2.1. Extraction of Rat Bile-Derived EVs
2.2. Examination of the Number of EVs
2.3. Verification of Bile EV Migration to Hepatocytes
2.4. Transcriptome Analysis
2.5. Examination of Cell Proliferative Ability
3. Discussion
4. Materials and Methods
4.1. Preparation of the Bile Duct Cannulated Rat Model
4.2. Bile Collection 70% Partial Hepatectomy
4.3. EV Extraction from Bile
4.4. Evaluation of EVs
4.4.1. Transmission Electron Microscope (TEM)
4.4.2. Nano Tracking Analysis (NTA)
4.5. Verification of Bile EV Migration to Hepatocytes
4.6. Transcriptome Analysis of Hepatocytes after Bile EV Addition
4.6.1. Addition of EVs to the Hepatocyte Cell Line
4.6.2. RNA Extraction of Cultured Cells
4.6.3. RNA Fragmentation and Purification
4.6.4. Construction of Whole Transcriptome Library and Reverse Transcription Reaction
4.6.5. cDNA Purification
4.6.6. cDNA Amplification, Purification and Library Quantification
- Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, USA: 4475936);
- IonXpress RNA-Seq Barcode 1–16 Kit (Thermo Fisher Scientific, USA: 4475485);
- D1000 ScreenTape (Agilent Technologies, USA: 5067–5582);
- D1000 Reagents (Agilent Technologies, USA: 5067–5583).
4.6.7. Sequencing
4.6.8. Expression Analysis
4.7. Examination of Cell Proliferative Ability
4.8. Statistical Methods
- ontology = Biological Process;
- p-value cutoff = 0.5;
- p.adjust.method = FDR.
4.9. Ethical Considerations
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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ID | Term | GeneRatio | p Value | p Adjusted | Count |
---|---|---|---|---|---|
GO:0044772 | mitotic cell cycle phase transition | 122/4596 | 0.0012 ** | 0.0755 | 122 |
GO:0044770 | cell cycle phase transition | 133/4596 | 0.0015 ** | 0.0823 | 133 |
GO:0044843 | cell cycle G1/S phase transition | 67/4596 | 0.0041 ** | 0.1334 | 67 |
Up Regulated | Down Regulated | ||||||
---|---|---|---|---|---|---|---|
Gene Symbol | Log2 Fold Change | p Value | Gene Symbol | Log2 Fold Change | p Value | ||
PH (24 h) | Sham | PH (24 h) | Sham | ||||
Hacd1 | 1.0618 | −0.5067 | <0.0001 *** | Pidd1 | −7.2416 | 0.0024 | <0.0001 *** |
Rptor | 1.2947 | 0.1998 | 0.0003 *** | Rcc1 | −1.1955 | −0.1498 | 0.0001 *** |
Rpl17 | 1.6784 | −0.3205 | 0.0008 *** | Cirbp | −1.0556 | −0.0983 | 0.0015 ** |
Tmod3 | 1.3730 | 0.1943 | 0.0009 *** | Plcb1 | −6.8962 | 0.2994 | 0.0032 ** |
Chmp4c | 2.4938 | 1.0174 | 0.0010 ** | Camk2b | −5.5836 | 0.6711 | 0.0055 ** |
E2f3 | 3.6683 | 1.1763 | 0.0010 ** | LOC102555319 | −6.4815 | −0.0902 | 0.0081 ** |
Bcl2 | 1.0480 | 0.1521 | 0.0010 ** | Ccnj | −7.3298 | 0.0335 | 0.0099 ** |
Ccni | 2.3697 | 0.9410 | 0.0013 ** | Inhba | −6.4521 | 0.4295 | 0.0139 * |
Vps4a | 1.1111 | 0.1982 | 0.0016 ** | Eps8 | −6.2677 | −0.2896 | 0.0204 * |
Ctdsp2 | 1.3427 | 0.4218 | 0.0016 ** | Hus1 | −6.4833 | −0.0456 | 0.0210 * |
Anxa1 | 1.4453 | 0.5099 | 0.0028 ** | Trim39 | −6.7484 | −0.2955 | 0.0217 * |
Brsk1 | 3.3370 | 1.0763 | 0.0031 ** | Fbxl6 | −1.3573 | −0.3980 | 0.0281 * |
Cit | 2.1396 | 0.6820 | 0.0031 ** | Haspin | −1.0681 | −0.3645 | 0.0318 * |
Chek2 | 1.8543 | 0.5042 | 0.0034 ** | Eif4e | −1.0115 | −0.3972 | 0.0355 * |
Dbf4 | 2.0209 | 0.8112 | 0.0035 ** | Appl2 | −6.8909 | −0.3617 | 0.0366 * |
Ppm1d | 2.0957 | 0.7411 | 0.0041 ** | Xrcc3 | −6.7349 | −0.4998 | 0.0371 * |
Mad2l1 | 1.4244 | 0.2698 | 0.0060 ** | Cdk5rap3 | −1.0748 | −0.3954 | 0.0395 * |
Donson | 1.8242 | 0.6657 | 0.0123 * | Foxm1 | −1.5041 | −0.2065 | 0.0461 * |
Ddx3x | 1.8009 | 0.9050 | 0.0146 * | Pdpn | −1.7263 | −0.5369 | 0.0473 * |
Pias1 | 1.4560 | 0.5660 | 0.0163 * | ||||
Sox4 | 1.6782 | 0.7718 | 0.0217 * | ||||
Adamts1 | 1.2637 | 0.2707 | 0.0225 * | ||||
Pten | 1.0504 | 0.5068 | 0.0278 * | ||||
Gen1 | 1.4655 | 0.5676 | 0.0300 * | ||||
Nek6 | 1.3541 | 0.6354 | 0.0316 * | ||||
Mnat1 | 1.1033 | 0.6977 | 0.0347 * | ||||
Mastl | 1.9228 | 1.1178 | 0.0449 * | ||||
Dcun1d3 | 1.1699 | 0.1316 | 0.0476 * |
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Share and Cite
Sugahara, S.; Haga, H.; Ikeda, C.; Makino, N.; Matsuda, A.; Kakizaki, Y.; Hoshikawa, K.; Katsumi, T.; Ishizawa, T.; Kobayashi, T.; et al. Role of Bile-Derived Extracellular Vesicles in Hepatocellular Proliferation after Partial Hepatectomy in Rats. Int. J. Mol. Sci. 2023, 24, 9230. https://doi.org/10.3390/ijms24119230
Sugahara S, Haga H, Ikeda C, Makino N, Matsuda A, Kakizaki Y, Hoshikawa K, Katsumi T, Ishizawa T, Kobayashi T, et al. Role of Bile-Derived Extracellular Vesicles in Hepatocellular Proliferation after Partial Hepatectomy in Rats. International Journal of Molecular Sciences. 2023; 24(11):9230. https://doi.org/10.3390/ijms24119230
Chicago/Turabian StyleSugahara, Shinpei, Hiroaki Haga, Chisaki Ikeda, Naohiko Makino, Akiko Matsuda, Yasuharu Kakizaki, Kyoko Hoshikawa, Tomohiro Katsumi, Tetsuya Ishizawa, Toshikazu Kobayashi, and et al. 2023. "Role of Bile-Derived Extracellular Vesicles in Hepatocellular Proliferation after Partial Hepatectomy in Rats" International Journal of Molecular Sciences 24, no. 11: 9230. https://doi.org/10.3390/ijms24119230