Chapter 7 Exploring potential silencing by miRNA
RNAseq data from Zhao et al. 2018 highlighted several differentially expressed genes (DEG) between epithelium and fiber cells. In parallel or in concert with RBP, miRNA might be responsible for the differential expression (DE) of genes in epithelium and fiber cells. To explore this hypothesis, we used a list of the top expressed micro RNA (miRNA) expressed in whole lens at several stage of development according to the miRNA dataset published by Khan et al. 2015. So as to identify potential regulation through degradation of DEG’s transcripts we looked for negative correlation between pairs gene and miRNA.
7.1 Identification of DEG and top-expressed miRNA
7.1.1 Zhao et al RNAseq data
So as to identify interesting DEG in Zhao et al. dataset, we computed mean \(\overline{x}\), standard deviation \(\sigma\) and relative difference \(\Delta\) as: \(\frac{\overline{x}^{epithelium}-\overline{x}^{fibers}}{\overline{x}^{epithelium}+\overline{x}^{fibers}}\) of FPKM for one gene per tissue per stage. We filtered the table based on following parameters:
relative difference between epithelium and fibers: \(|\Delta| >\) 0.25
mean FPKM in at least one tissue (without considering stages): \(\overline{x} >\) 1000
With these parameters we retained 1219 genes differentially expressed between lens and fiber cells.
7.1.2 Kahn et al. miRNA dataset
This dataset does not discriminate between epithelium and fiber cells. Taking into consideration that highly expressed miRNA might be We focus on miRNA with FPKM above 500 at a least one stage.
7.2 Negative correlation between miRNA and DEG
We computed expression’s correlation between our list of 140 miRNA and 1219 DEG. miRNA:DEG pairs showing correlation inferior to -0.75 were conserved. This threshold aims to conserve pairs that might be involved in silencing regulation and that might explain the differential expression between epithelial and fiber cells.
We found 20788 miRNA:DEG pairs answering this correlation parameters. This consider a combination of 137 miRNas and 1093.
7.3 Highlight predicted or validated interactions
With this list of negatively correlated pairs, we will interrogate multiple databases of miRNA:gene interactions.
See below for known validated interactions:
| miRNA | gene | experiments |
|---|---|---|
| mmu-miR-107-3p | Hif1a | Western blot |
| mmu-miR-124-3p | Cd164 | Luciferase reporter assay, Microarray, qRT-PCR |
| mmu-miR-133a-3p | Prkacb | Igf1r | Pola1 | Flow, Immunofluorescence, Immunohistochemistry, Immunoprecipitaion, Luciferase reporter assay, qRT-PCR, Western blot | Luciferase reporter assay | Immunofluorescence, Luciferase reporter assay, Microarray, Western blot |
| mmu-miR-204-5p | Ntrk2 | Luciferase reporter assay, qRT-PCR, Western blot |
| mmu-miR-20a-5p | App | Luciferase reporter assay |
| mmu-miR-23a-3p | Smad5 | qRT-PCR, Western blot |
| mmu-miR-23b-3p | Smad5 | Luciferase reporter assay | In situ hybridization, Luciferase reporter assay, Microarray, Northern blot, qRT-PCR |
| mmu-miR-24-3p | Smad5 | In situ hybridization, Luciferase reporter assay, Microarray, Northern blot, qRT-PCR |
| mmu-miR-27a-3p | Lrpprc | qRT-PCR, Luciferase reporter assay, Western blot |
| mmu-miR-34c-5p | Smad5 | Luciferase reporter assay |
| mmu-miR-375-3p | Yap1 | Pdk1 | Immunohistochemistry, qRT-PCR, Western blot | Immunofluorescence, In situ hybridization, Luciferase reporter assay, qRT-PCR, Western blot | Luciferase reporter assay, qRT-PCR, Western blot |
| mmu-miR-99b-5p | Mfge8 | Luciferase reporter assay, qRT-PCR, Western blot |
7.4 Regulation of Crystallin’s genes by miRNA
miRWalk database was interrogated for crystallin predicted miRNA in both human and mice. We filtered based on expression data from Khan et al. and looked for prediction in both human and mouse:
| mirnaid | humantargets | mousetargets | shared | E15 | E18 | P0 |
|---|---|---|---|---|---|---|
| let-7e-5p | CRYAA, CRYBA4, CRYGB, CRYGC, CRYGS | Cryaa, Crybb1, Crybb2, Crygb, Crygc, Crygd, Cryge, Crygf, Crygn | CRYAA, CRYGB, CRYGC | 2721.095 | 2351.445 | 2327.900 |
| miR-744-5p | CRYAA, CRYAB, CRYBA1, CRYBA2, CRYBB1, CRYBB2, CRYGA | Cryaa, Cryab, Cryba2, Cryba4, Crybb2, Crygs | CRYAA, CRYAB, CRYBA2, CRYBB2 | 1340.890 | 889.680 | 724.640 |
| miR-423-5p | CRYAA, CRYBB3, CRYGC, CRYGS | Cryba1, Cryba4, Crybb2, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf | CRYGC | 759.835 | 445.640 | 391.545 |
| miR-181b-5p | CRYAA, CRYAB, CRYGB, CRYGC | Cryba4, Crybb1, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf | CRYGB, CRYGC | 13609.130 | 14627.655 | 12768.755 |
| miR-92a-1-5p | CRYAB, CRYBB3, CRYGA, CRYGC, CRYGD, CRYGS | Cryab, Crybb2, Crybb3, Cryga, Crygb | CRYAB, CRYBB3, CRYGA | 651.705 | 341.905 | 223.840 |
| miR-541-5p | CRYAA, CRYBB1, CRYGC | Cryba1, Cryba2, Cryba4, Crybb1, Crygf, Crygs | CRYBB1 | 4414.160 | 4063.510 | 4570.285 |
| miR-181d-5p | CRYAB, CRYGC | Cryba1, Cryba4, Cryga, Crygb, Crygc, Crygd, Crygf | CRYGC | 3351.590 | 2355.050 | 1745.020 |
| miR-210-3p | CRYAB, CRYBA2, CRYGA, CRYGB, CRYGS | Cryab, Crybb2, Crygd, Cryge | CRYAB | 576.235 | 645.760 | 876.680 |
| miR-431-5p | CRYAB, CRYBB3 | Cryba4, Crybb1, Crybb2, Cryga, Crygc, Crygd, Crygs | 559.865 | 256.145 | 238.090 | |
| miR-331-3p | CRYBA1, CRYBA4, CRYBB1 | Cryab, Cryba4, Crybb1, Crybb2, Crybb3, Crygd | CRYBA4, CRYBB1 | 301.120 | 321.080 | 606.365 |
| miR-133a-3p | CRYBA2, CRYBB1, CRYGD | Cryba2, Crybb1, Crybb2, Crybb3, Crygs | CRYBA2, CRYBB1 | 4566.355 | 3689.930 | 7141.340 |
| miR-423-3p | CRYBA1, CRYGA, CRYGD | Cryba2, Cryga, Crygb, Crygc, Crygs | CRYGA | 1829.670 | 1202.975 | 765.820 |
| let-7c-5p | CRYBB1, CRYGB, CRYGC | Cryaa, Crygb, Crygd, Crygf | CRYGB | 1341.270 | 1455.530 | 1405.790 |
| miR-24-3p | CRYAA, CRYBA2 | Cryaa, Crybb1, Cryga, Crygf, Crygn | CRYAA | 377.475 | 542.340 | 837.105 |
| miR-31-5p | CRYGA, CRYGS | Crybb3, Crygc, Crygd, Crygs | CRYGS | 7317.845 | 5700.630 | 9052.220 |
| miR-93-5p | CRYAB | Cryaa, Crygb, Crygc, Crygd, Crygn | 4402.715 | 3161.065 | 2552.605 | |
| miR-127-3p | CRYAB, CRYGC, CRYGN | Crybb1, Crybb2 | 19788.955 | 15686.235 | 18170.400 | |
| let-7g-5p | CRYGA | Cryba1, Crygb, Crygd, Cryge | 1437.645 | 2161.855 | 1262.400 | |
| miR-183-5p | CRYAB, CRYGN | Cryga, Crygb, Crygc | 1381.615 | 3431.745 | 1078.885 | |
| miR-125b-2-3p | CRYBA4, CRYBB1, CRYBB2 | Cryaa, Crybb2 | CRYBB2 | 204.040 | 261.860 | 472.260 |
| miR-181a-5p | CRYGS | Crybb1, Crybb3, Crygs | 45584.200 | 65691.180 | 53448.200 | |
| miR-92b-3p | CRYGB, CRYGN | Cryab, Crygb | 27766.170 | 14352.145 | 9378.940 | |
| miR-25-3p | CRYAA, CRYAB, CRYGN | Cryaa | CRYAA | 10070.005 | 5884.985 | 4215.970 |
| let-7a-5p | CRYAA, CRYGB | Cryaa, Crygb | CRYAA, CRYGB | 3892.035 | 5208.760 | 4621.925 |
| miR-378a-3p | CRYBA4, CRYBB2, CRYGA | Crygs | 2798.740 | 2866.830 | 4092.610 | |
| miR-19b-3p | CRYGB, CRYGC | Cryba1, Cryga | 2130.480 | 1273.455 | 1739.070 | |
| miR-34c-5p | CRYAA, CRYGS | Cryaa, Cryba2 | CRYAA | 2067.450 | 1868.765 | 2646.510 |
| miR-125b-5p | CRYAA | Cryaa, Cryga, Crygf | CRYAA | 2026.495 | 2733.485 | 2883.550 |
| miR-20a-5p | CRYAB | Crygd, Cryge, Crygf | 1662.725 | 1421.255 | 1169.450 | |
| miR-484 | CRYGB | Cryba1, Crybb3, Crygs | 1070.745 | 958.280 | 799.570 | |
| let-7d-5p | CRYAA, CRYGA, CRYGB | Cryaa | CRYAA | 351.040 | 490.605 | 305.245 |
| miR-1251-5p | CRYBB1, CRYGB, CRYGC | Crybb3 | 146.385 | 200.170 | 411.100 | |
| miR-193b-3p | CRYGD, CRYGN | Crybb3, Crygn | CRYGN | 42.495 | 32.265 | 97.995 |
| miR-92a-3p | CRYAB | Cryab, Crygd | CRYAB | 78424.960 | 40102.225 | 25677.025 |
| miR-204-5p | CRYBA4, CRYGN | Cryba4 | CRYBA4 | 74195.665 | 64165.925 | 85982.685 |
| miR-99b-5p | CRYAB | Cryaa, Cryga | 38034.775 | 44824.330 | 39097.940 | |
| miR-106b-5p | CRYAA, CRYGC | Cryba1 | 664.570 | 538.765 | 665.655 | |
| miR-23a-3p | CRYGD | Cryba4, Crygb | 220.985 | 290.395 | 500.590 | |
| miR-124-3p | CRYAB, CRYGC | Cryba2 | 162.970 | 291.235 | 83.165 | |
| miR-96-5p | CRYAB, CRYBB2 | Cryaa | 52.725 | 183.410 | 59.185 | |
| miR-27b-3p | CRYBA4 | Crybb3 | 8015.050 | 10459.260 | 12127.460 | |
| let-7f-5p | CRYBB1 | Crygb | 4164.680 | 5778.100 | 5275.050 | |
| miR-532-5p | CRYGA | Cryba1 | 811.490 | 661.110 | 657.065 | |
| miR-136-3p | CRYGD | Crygc | 760.280 | 599.885 | 784.450 | |
| miR-19a-3p | CRYGB | Cryga | 572.765 | 281.610 | 313.440 | |
| miR-27a-3p | CRYBA4 | Crybb3 | 389.905 | 495.750 | 1006.845 |
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