Correlation with Apoptosis Process through RNA-Seq Data Analysis of Hep3B Hepatocellular Carcinoma Cells Treated with Glehnia littoralis Extract (GLE)
Abstract
:1. Introduction
2. Results
2.1. Glehnia littoralis Extract (GLE) Induces Cell Death of Hep3B Cells
2.2. Identification of Genes and Differentially Expressed Genes (DEGs)
2.3. Gene Ontology Term Enrichment Analysis of DEGs in GLE-Treated Hep3B Cells
2.4. KEGG (Kyoto Encyclopedia of Genes and Genomes) Enrichment Pathway and Protein–Protein Interaction (PPI)
2.5. GLE Induces Apoptotic Cell Death in Hep3B Cells
2.6. Increase or Decrease in Genes Related to the Apoptotic Process
3. Discussion
4. Material and Methods
4.1. Reagents and Chemicals
4.2. Cell Culture and Viability Assay
4.3. Annexin V/Propidium Iodide Apoptosis Detection
4.4. RNA Isolation
4.5. Library Preparation and Sequencing
4.6. Data Analysis
4.7. Source Code
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
GLE | Glehnia littoralis extract |
HCC | Hepatocellular carcinoma |
DEG | Differentially expressed gene |
GO | Gene Ontology |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
PPI | Protein–protein interaction |
BP | Biological process |
MF | Molecular function |
CC | Cellular component |
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Name | Type | Reads | Bases | Bases (Gb) | GC | N | Q30 |
---|---|---|---|---|---|---|---|
getC | Raw | 22,236,783 (100%) | 1,689,995,508 (100%) | 1.69 | 722,506,852 (42.75%) | 122,257 (0.0%) | 1,546,777,236 (91.53%) |
Clean | 21,935,961 (98.65%) | 1,563,188,663 (92.50%) | 1.56 | 685,071,168 (43.83%) | 17,858 (0.0%) | 1,471,970,080 (94.16%) | |
getT | Raw | 16,504,954 (100%) | 1,254,376,504 (100%) | 1.25 | 525,542,567 (41.90%) | 91,010 (0.0%) | 1,138,775,855 (90.78%) |
Clean | 16,279,626 (98.63%) | 1,148,658,184 (91.57%) | 1.15 | 496,909,263 (43.26%) | 13,046 (0.0%) | 1,075,923,116 (93.67%) |
Biological Process | ||||
ID | Description | Gene Ratio | BgRatio | p Value |
GO:0042594 | response to starvation | 29/994 | 206/18,866 | 1.31859 × 10−6 |
GO:0001558 | regulation of cell growth | 38/994 | 420/18,866 | 0.000847655 |
GO:0006984 | ER-nucleus signaling pathway | 14/994 | 51/18,866 | 2.40565 × 10−7 |
Molecular Function | ||||
ID | Description | Gene Ratio | BgRatio | p Value |
GO:0001217 | DNA-binding transcription repressor activity | 39/1015 | 336/18,352 | 9.92372 × 10−6 |
GO:0003712 | transcription coregulator activity | 48/1015 | 498/18,352 | 0.000130717 |
GO:0004402 | histone acetyltransferase activity | 8/1015 | 55/18,352 | 0.010272488 |
Cellular Component | ||||
ID | Description | Gene Ratio | BgRatio | p Value |
GO:0016607 | nuclear speck | 41/1029 | 401/19,559 | 3.81569 × 10−5 |
GO:0005635 | nuclear envelope | 41/1029 | 473/19,559 | 0.001216517 |
GO:0019908 | nuclear cyclin-dependent protein kinase holoenzyme complex | 3/1029 | 12/19,559 | 0.022350144 |
ID | Description | Gene Ratio | Bg Ratio | p Value | p.Adjust | q Value | GeneID | Count |
---|---|---|---|---|---|---|---|---|
hsa04722 | Neurotrophin signaling pathway | 14/480 | 119/8223 | 0.00923833 | 0.22409163 | 0.21320458 | ATF4/FOXO3/FRS2/IRAK2/IRS1/JUN/MAP3K3/ MAPK12/NFKBIA/NFKBIB/NFKBIE/RHOA/RPS6KA5/SOS2 | 14 |
hsa04152 | AMPK signaling pathway | 12/480 | 121/8223 | 0.04947161 | 0.50476492 | 0.48024193 | ADIPOR2/FOXO3/HNF4A/IRS1/IRS2/PRKAG1/ RHEB/RPTOR/SIRT1/STRADB/TBC1D1/ULK1 | 12 |
hsa04110 | Cell cycle | 13/480 | 127/8223 | 0.03374595 | 0.48461782 | 0.46107364 | CCNE1/CDC6/CDK7/CDKN1C/CDKN2D/DBF4B/GADD45A/ GADD45B/GADD45G/ORC1/ORC3/ORC6/TTK | 13 |
hsa04068 | FoxO signaling pathway | 16/480 | 131/8223 | 0.00387417 | 0.13927125 | 0.13250504 | BCL2L11/CDKN2D/FOXO3/GABARAPL1/GADD45A/GADD45B/ GADD45G/IRS1/IRS2/MAPK12/PRKAG1/SGK1/SGK2/SIRT1/SOD2/SOS2 | 16 |
hsa04210 | Apoptosis | 14/480 | 136/8223 | 0.02708404 | 0.47755381 | 0.45435282 | APAF1/ATF4/BCL2L11/BIRC3/DDIT3/ENDOG/FADD/GADD45A/ GADD45B/GADD45G/ITPR2/JUN/NFKBIA/TUBA4A | 14 |
Number of nodes: 64 | Avg. local clustering coefficient: 0.678 |
Number of edges: 197 | Expected number of edges: 47 |
Average node degree: 6.16 | PPI enrichment p-value: <1.0 × 10−16 |
Biological Process | ||||
GO-Term | Description | Count in Network | Strength | False Discovery Rate |
GO:0006915 | Apoptotic process | 64 of 1041 | 1.28 | 1.87 × 10−77 |
GO:0050794 | Regulation of cellular process | 61 of 11,025 | 0.23 | 5.23 × 10−10 |
GO:0050896 | Response to stimulus | 54 of 7835 | 0.33 | 9.81 × 10−11 |
GO:0051716 | Cellular response to stimulus | 52 of 6357 | 0.4 | 6.42 × 10−13 |
GO:0048518 | Positive regulation of the biological process | 51 of 6207 | 0.4 | 1.61 × 10−12 |
Molecular Function | ||||
GO-Term | Description | Count in Network | Strength | False Discovery Rate |
GO:0005515 | Protein binding | 47 of 7242 | 0.3 | 7.04 × 10−6 |
GO:0019899 | Enzyme binding | 28 of 2084 | 0.62 | 6.33 × 10−8 |
GO:0042802 | Identical protein binding | 20 of 2144 | 0.46 | 0.0093 |
GO:0098772 | Molecular function regulator activity | 19 of 1960 | 0.47 | 0.0093 |
GO:0044877 | Protein-containing complex binding | 15 of 1261 | 0.56 | 0.0093 |
KEGG Pathways | ||||
Pathway | Description | Count in Network | Strength | False Discovery Rate |
hsa05200 | Pathways in cancer | 13 of 515 | 0.89 | 1.29 × 10−6 |
hsa04210 | Apoptosis | 12 of 131 | 1.45 | 1.03 × 10−11 |
hsa04621 | NOD-like receptor signaling pathway | 9 of 173 | 1.2 | 1.17 × 10−6 |
hsa05169 | Epstein–Barr virus infection | 9 of 192 | 1.16 | 1.29 × 10−6 |
hsa04068 | FoxO signaling pathway | 8 of 126 | 1.29 | 1.29 × 10−6 |
Reactome Pathways | ||||
Pathway | Description | Count in Network | Strength | False Discovery Rate |
HSA-162582 | Signal transduction | 25 of 2540 | 0.48 | 4.14 × 10−5 |
HSA-74160 | Gene expression (Transcription) | 17 of 1476 | 0.55 | 0.00080 |
HSA-168256 | Immune system | 17 of 1979 | 0.42 | 0.0136 |
HSA-2262752 | Cellular responses to stress | 15 of 747 | 0.79 | 1.69 × 10−5 |
HSA-212436 | Generic transcription pathway | 15 of 1215 | 0.58 | 0.0014 |
Wiki Pathways | ||||
Pathway | Description | Count in Network | Strength | False Discovery Rate |
WP1772 | Apoptosis modulation and signaling | 10 of 89 | 1.54 | 5.91 × 10−10 |
WP3888 | VEGFA-VEGFR2 signaling | 10 of 428 | 0.86 | 0.00012 |
WP254 | Apoptosis | 7 of 83 | 1.41 | 6.30 × 10−6 |
WP4754 | IL-18 signaling pathway | 7 of 271 | 0.9 | 0.0015 |
WP2882 | Nuclear receptors meta-pathway | 7 of 312 | 0.84 | 0.0024 |
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Park, M.-Y.; Lee, S.; Kim, H.-H.; Jeong, S.-H.; Abusaliya, A.; Bhosale, P.B.; Seong, J.-K.; Park, K.-I.; Heo, J.-D.; Ahn, M.; et al. Correlation with Apoptosis Process through RNA-Seq Data Analysis of Hep3B Hepatocellular Carcinoma Cells Treated with Glehnia littoralis Extract (GLE). Int. J. Mol. Sci. 2024, 25, 9462. https://doi.org/10.3390/ijms25179462
Park M-Y, Lee S, Kim H-H, Jeong S-H, Abusaliya A, Bhosale PB, Seong J-K, Park K-I, Heo J-D, Ahn M, et al. Correlation with Apoptosis Process through RNA-Seq Data Analysis of Hep3B Hepatocellular Carcinoma Cells Treated with Glehnia littoralis Extract (GLE). International Journal of Molecular Sciences. 2024; 25(17):9462. https://doi.org/10.3390/ijms25179462
Chicago/Turabian StylePark, Min-Yeong, Sujin Lee, Hun-Hwan Kim, Se-Hyo Jeong, Abuyaseer Abusaliya, Pritam Bhangwan Bhosale, Je-Kyung Seong, Kwang-Il Park, Jeong-Doo Heo, Meejung Ahn, and et al. 2024. "Correlation with Apoptosis Process through RNA-Seq Data Analysis of Hep3B Hepatocellular Carcinoma Cells Treated with Glehnia littoralis Extract (GLE)" International Journal of Molecular Sciences 25, no. 17: 9462. https://doi.org/10.3390/ijms25179462