Transcriptomics Analysis of Lens from Patients with Posterior Subcapsular Congenital Cataract
Abstract
:1. Introduction
2. Materials and Methods
2.1. Subjects
2.2. RNA Extracting, Library Construction and RNA-seq
2.3. Gene Expression Data Analysis
2.4. Real-time Quantitative PCR (RT-qPCR)
2.5. Immunohistochemical Staining
2.6. Statistical Analysis
3. Results
3.1. Differential Expression and Functional Enrichment Analysis in the Anterior Lens Epithelial Cells
3.2. Differential Expression and Functional Enrichment Analysis in the Fibre Cells
3.3. PPI Network/Strings Network
3.4. RT-qPCR Validates the Results of RNA-seq
3.5. Lens-Associated Differential Gene Expression to Identify High-Priority Candidate Genes
3.6. A Protein Marker of Cell Growth and Epithelial Cell Proliferation Confirms RNA-seq Transcriptome Findings
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
cDNA | complementary DNA |
GO | gene ontology |
KEGG | Kyoto Encyclopaedia of Genes and Genomes |
FPKM | per million fragments mapped |
DEG | differentially expressed genes |
PPI | protein–protein interaction |
RT-qPCR | real-time quantitative PCR |
RIPA | radio-immunoprecipitation assay |
SDS-PAGE | sodium dodecylsulphate-polyacrylamide gel electrophoresis |
PVDF | transferred to a polyvinylidene difluoride |
GRIFIN | galectin-related inter-fibre protein |
TGF-β | transforming growth factor-β |
ECM | extracellular matrix |
α-SMA | deposition of α-smooth muscle actin |
FDR | false discovery rate |
PBS | phosphate-buffered saline |
RNA-seq | RNA sequencing |
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Target | F/R | Primer Sequence (5′→3′) | Amplicon Size (BP) |
---|---|---|---|
CRYGC | Forward | TCCCAACTACCAAGGTCAACA | 232 |
Reverse | GTGGAGGGAACGGATCTCG | ||
CRYGB | Forward | CACCTCACTGAAATTCACTCCC | 125 |
Reverse | CCCCAATCAAGAAACCTCCTGT | ||
CRYGA | Forward | GGTCCAATCCTGCCGTATAATTC | 109 |
Reverse | ACAGGCGCAGTCATCAGTG | ||
CRYBB1 | Forward | GTGCTCAAATCTGGCAGACC | 92 |
Reverse | GGAAGTTGGACTGCTCAAAGG | ||
CRYBA1 | Forward | GGGCAAGAGGATGGAGTTCAC | 122 |
Reverse | CACAGAAGCTGGTATGCTCATAA | ||
HBA2 | Forward | CAAGACCTACTTCCCGCACTTCG | 341 |
Reverse | GGGCAGGAGGAACGGCTACC | ||
HBA1 | Forward | TCAACTTCAAGCTCCTAAGCCACTG | 110 |
Reverse | CACAGAAGCCAGGAACTTGTCCAG | ||
HBB | Forward | AGGAGAAGTCTGCCGTTACTG | 190 |
Reverse | CCGAGCACTTTCTTGCCATGA | ||
S100A9 | Forward | CTGTGTGGCTCCTCGGCTTTG | 98 |
Reverse | TGGTGGAAGGTGTTGATGATGGTC | ||
HBG2 | Forward | GTGGAAGATGCTGGAGGAGAAACC | 106 |
Reverse | TGATGGCAGAGGCAGAGGACAG | ||
APOE | Forward | GTTGCTGGTCACATTCCTGG | 146 |
Reverse | GCAGGTAATCCCAAAAGCGAC | ||
TSPAN12 | Forward | CCAGAGAAGATTCCGTGAAGTG | 107 |
Reverse | GTCCCTCATCCAAGCAGAAAC | ||
PRSS35 | Forward | CATCGAATGCCAGAAAGAACTCC | 135 |
Reverse | GTCGGCTCAAGAACCAAATCT | ||
ASS1 | Forward | TCCGTGGTTCTGGCCTACA | 126 |
Reverse | GGCTTCCTCGAAGTCTTCCTT | ||
SULF1 | Forward | GATCCCCGAGGTTCAGAGGA | 178 |
Reverse | GGTGTAGTCACAAAGGCATTGA | ||
METRN | Forward | CACAGACACCGCCAGGCAAG | 103 |
Reverse | CCACTCCTCTCCCGTCCACAC | ||
PRAP1 | Forward | ACAGCCTGTACCACCCTCC | 82 |
Reverse | AGCACCTGGTGATTTGGCATC | ||
RAMP1 | Forward | CCAGGAGGCTAACTACGGTG | 297 |
Reverse | GGGACCACGATGAAGGGGTA | ||
TMEM54 | Forward | TGGGCCATGTGAGCTTCATC | 176 |
Reverse | GAGGTAGCGTGACAACACGAT | ||
GAPDH | Forward | GGAGCGAGATCCCTCCAAAAT | 197 |
Reverse | GGCTGTTGTCATACTTCTCATGG |
Sample | Tissue | Raw Reads | Clean Reads | Clean Read-Pairs | Average Length | Mapped Ratio (%) |
---|---|---|---|---|---|---|
PSC patient 1 | Capsules | 79,150,688 | 23,475,256 | 11,737,628 | 138.3 | 88.80% |
PSC patient 2 | Capsules | 92,790,046 | 36,750,674 | 18,375,337 | 141.9 | 93.40% |
PSC patient 3 | Capsules | 102,532,016 | 47,760,460 | 23,880,230 | 142.0 | 91.70% |
PSC patient 4 | Capsules | 100,012,388 | 32,018,416 | 16,009,208 | 142.4 | 92.60% |
PSC patient 5 | Capsules | 98,193,322 | 36,350,310 | 18,175,155 | 134.7 | 79.10% |
PSC patient 6 | Capsules | 100,226,088 | 40,541,834 | 20,270,917 | 134.1 | 79.90% |
NC 1 | Capsules | 127,355,192 | 60,141,116 | 30,070,558 | 144.6 | 95.60% |
NC 2 | Capsules | 100,869,818 | 37,378,548 | 18,689,274 | 138.0 | 86.40% |
NC 3 | Capsules | 111,902,712 | 47,398,990 | 23,699,495 | 141.5 | 92.20% |
NC 4 | Capsules | 127,358,610 | 48,182,942 | 24,091,471 | 144.2 | 94% |
NC 5 | Capsules | 120,259,640 | 52,969,554 | 26,484,777 | 134.6 | 90.60% |
NC 6 | Capsules | 91,405,500 | 31,710,346 | 15,855,173 | 133.6 | 84.20% |
NC 7 | Capsules | 125,882,600 | 56,962,562 | 28,481,281 | 136.5 | 93% |
NC 8 | Capsules | 131,778,762 | 54,214,656 | 27,107,328 | 135.5 | 91.70% |
PSC patient 1 | Cortex | 123,977,234 | 45,747,380 | 22,873,690 | 141.6 | 93.40% |
PSC patient 3 | Cortex | 91,869,478 | 25,684,820 | 12,842,410 | 137.9 | 85.90% |
PSC patient 4 | Cortex | 110,104,320 | 109,339,070 | 54,669,535 | 150.0 | 97.60% |
PSC patient 5 | Cortex | 128,652,528 | 48,532,586 | 24,266,293 | 138.7 | 89.40% |
PSC patient 6 | Cortex | 116,294,432 | 47,393,278 | 23,696,639 | 136.5 | 84.80% |
NC 1 | Cortex | 98,977,182 | 32,642,774 | 16,321,387 | 141.8 | 92.40% |
NC 4 | Cortex | 167,149,114 | 67,734,538 | 33,867,269 | 142.4 | 95.10% |
NC 5 | Cortex | 94,542,668 | 93,252,442 | 46,626,221 | 148.9 | 96.90% |
NC 6 | Cortex | 114,900,736 | 113,734,102 | 56,867,051 | 148.8 | 97.90% |
NC 7 | Cortex | 92,822,214 | 92,085,706 | 46,042,853 | 149.5 | 97.90% |
DEG | Symbol | Description | Log2 Fold Change | p-Value | False Discovery Rate (FDR) Value |
---|---|---|---|---|---|
Downregulated | CRYGC | Crystallin gamma C | −8.117 | 3.04 × 10−15 | 6.00 × 10−12 |
CRYGB | Crystallin gamma B | −7.952 | 4.24 × 10−14 | 6.30 × 10−11 | |
CRYGA | Crystallin gamma A | −7.818 | 4.67 × 10−18 | 1.61 × 10−14 | |
CRYBB1 | Crystallin beta B1 | −7.695 | 3.07 × 10−22 | 2.11 × 10−18 | |
CRYBA1 | Crystallin beta A1 | −7.667 | 7.63 × 10−24 | 7.00 × 10−20 | |
Upregulated | HBA2 | Hemoglobin subunit alpha 2 | 10.182 | 1.20 × 10−41 | 2.21 × 10−37 |
HBA1 | Hemoglobin subunit alpha 1 | 9.838 | 1.27 × 10−44 | 3.48 × 10−40 | |
HBB | Hemoglobin subunit beta | 9.414 | 4.16 × 10−49 | 2.29 × 10−44 | |
S100A9 | S100 calcium binding protein A9 | 5.087 | 3.54 × 10−15 | 6.71 × 10−12 | |
HBG2 | Hemoglobin subunit gamma 2 | 4.863 | 2.18 × 10−6 | 4.59 × 10−4 |
DEGs | Symbol | Description | Log2 Fold Change | p-Value | FDR Value |
---|---|---|---|---|---|
Downregulated | APOE | Apolipoprotein E | −5.889 | 1.47 × 10−11 | 9.71 × 10−8 |
TSPAN12 | Tetraspanin 12 | −4.435 | 3.64 × 10−7 | 0.001 | |
PRSS35 | Protease, serine 35 | −4.249 | 2.21 × 10−6 | 0.004 | |
ASS1 | Argininosuccinate Synthase 1 | −3.786 | 6.89 × 10−7 | 0.002 | |
SULF1 | Sulfatase 1 | −3.338 | 3.63 × 10−5 | 0.028 | |
Upregulated | METRN | Meteorin, glial cell differentiation regulator | 2.872 | 5.07 × 10−6 | 0.007 |
PRAP1 | Proline rich acidic Protein 1 | 2.852 | 2.85 × 10−5 | 0.024 | |
RAMP1 | Receptor activity-modifying protein 1 | 2.311 | 1.56 × 10−5 | 0.016 | |
TMEM54 | Transmembrane protein 54 | 1.921 | 1.37 × 10−6 | 0.003 |
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Lin, X.; Li, H.; Yang, T.; Liu, X.; Fan, F.; Zhou, X.; Luo, Y. Transcriptomics Analysis of Lens from Patients with Posterior Subcapsular Congenital Cataract. Genes 2021, 12, 1904. https://doi.org/10.3390/genes12121904
Lin X, Li H, Yang T, Liu X, Fan F, Zhou X, Luo Y. Transcriptomics Analysis of Lens from Patients with Posterior Subcapsular Congenital Cataract. Genes. 2021; 12(12):1904. https://doi.org/10.3390/genes12121904
Chicago/Turabian StyleLin, Xiaolei, Hongzhe Li, Tianke Yang, Xin Liu, Fan Fan, Xiyue Zhou, and Yi Luo. 2021. "Transcriptomics Analysis of Lens from Patients with Posterior Subcapsular Congenital Cataract" Genes 12, no. 12: 1904. https://doi.org/10.3390/genes12121904