RESULT OF INVESTIGATION
After reading through all the literature based on the disease Leptospirosis, we observed that while our complement immune system is compromised; complement factor H (CFH), Complement component 3 (CPAMD8), and Toll-Like Receptor 2 (TLR2) are particularly downregulated via microRNAs in response to bacterial Lipopolysaccharides (LPS) of Leptospira. After further data mining, we designed our project to study the LPS-Complement components-miRNA axis.
Using Online tools TargetScans, miRBase, BLAST, miRDB, and NCBI; 3’UTR of TLR2, 3’UTR of CPAMD8, and 3’UTR of CFH have been identified as for the target gene sequence. hsa-miR-144-3p, hsa-miR630, hsa-miR3646 and hsa-miR4427 have been selected as regulatory microRNA based on their relevance in disease Leptospirosis (Table 1). Using iDT tools the modified anti-miRNAs or inhibitors were designed (Table 2).
Table 1. The target gene and regulatory miRNAs selected for the wet lab work.
| Target gene | Regulatory microRNAs |
|---|---|
| TLR2 | hsa-miR-144-3p |
| CPAMD8 (C3) | hsa-miR-630 |
| CFH | hsa-miR-3646 |
| CFH | hsa-miR-4427 |
Table 2. The sequence of microRNAs and corresponding inhibitors.
| Name | Sequence |
|---|---|
| miR-144-3p mature | 5’ UAC AGU AUA GAU GAU GUA CU 3’ |
| miR-144-3p inhibitors | 5’ mA/ZEN/mGmU mAmCmA mUmCmA mUmCmU mAmUmA mCmUmG mU/3ZEN/ 3’ |
| miR-144-3p inhibitors | 5’ AGU AUU CUG UAC CAG GGA AGG U 3’ |
| miR630 inhibitors | 5’ mA/ZEN/mCmC mUmUmC mCmCmU mGmGmU mAmCmA mGmAmA mUmAmC/3ZEN/ 3’ |
| miR3646 mature | 5’ AAA AUG AAA UGA GCC CAG CCC A 3’ |
| miR3646 inhibitors | 5’ mU/ZEN/mGmG mGmCmU mGmGmG mCmUmC mAmUmU mUmCmA mUmUmU/3ZEN/ 3’ |
| miR4427 mature | 5’ UCU GAA UAG AGU CUG AAG AGU 3’ |
| miR4427 inhibitors | 5’ mA/ZEN/mCmU mCmUmU mCmAmG mAmCmU mCmUmA mUmUmC mAmG/3ZEN/ 3’ |
The oligos were ordered from iDT along with gene fragments from GenScript. The plasmids were cloned (Picture 1, Picture 2, Picture 3). Primers were designed to introduce Restriction sites Xba1 and Sal1 In 3’UTR-TLR2, Xho1 and Xba1 in both the sequence 3’UTR-C3 and 3’UTR-CFH. Insert sequences were PCR amplified (Picture 4, Picture 5), Luciferase Vectors were double digested (Picture 6 , Picture 7). Insert sequences were then subcloned into the pMIR-Dual-Glo luciferase vector (Picture 8, Picture 9, Picture 10).
The positive colonies were confirmed by Colony PCR respectively for the target sequence of 3’UTR-TLR2 (Picture 11), 3’UTR-CPAMD8 (Picture 12) and 3’UTR-CFH (Picture 13).
Positive colonies were then inoculated in LB broth overnight. Plasmids were extracted via QiaGen miniprep and taken to Hong Kong University (HKU) for downstream experiments.
Prof Chan, Chi Wai Michael, the Division of Public Health Laboratory Science, in HKU provided us with the mammalian secondary cell line A549. He is also generous in facilitating the requisite training and lab space for our work.
Here we subcultured the cell line every 3 days. After the cells had attained 80%-90 % confluency (Picture 14). The cells were counted using the trypan blue staining method (Picture 15, Picture 16 ). The cell culture was then diluted. 5.0 X 10*5 cells per well been seeded on 24 wells plates.
We designed our 2 plates for all the 3 constructs as in Fig 1 and Fig 2.
The wells transfected with only plasmid vectors were taken as negative control. Then wells transfected with plasmids and miRNAs were the experimental ones, which reflects the strength of their interaction. The stronger the strength, its luciferase activity will be quenched strongly. Wells transfected with plasmids, miRNA mimics, and their corresponding inhibitors were taken as another negative control, which reflects the restored activity of plasmids in the presence of sequestered regulatory microRNAs. All transfections were done in triplicates.
Following the transfection, we carried the luciferase assay for all the 3 constructs. The readings are recorded (Picture 17, Picture 18, Picture 19, Picture 20) and calculated as in table 3, table 4 and table 5. We calculated the relative luminance and plotted the graph. Fig 3, Fig 4 and Fig 5.
Our recorded readings of luciferase activity show that designed anti-miRNAs or inhibitors successfully bind with their respective miRNAs, restoring the luminescence to 100%.
Also, our initial findings confirm the interaction between the target sequence of 3’UTR-TLR2, 3’-UTR CPAMD8 and 3’UTR-CFH with their respective miRNAs.
The observed interaction between 3’UTR-TLR2 and hsa-miR144-3p were 88%, between 3’UTR-CPAMD8 and hsa-miR630 were 80 % and between 3’UTR-CFH and hsa-miR4427 were 87%. However, this percentage is higher than expected suggesting the mismatched sequence or mutated sequence.
Due to time limitations, we could do only 1 trial for each construct and to get any conclusive results we were suggested to record readings from 6 plates or trials.
Future Prospective.
5 more trials will be carried out to confirm the results.
The inhibitors will be evaluated for drug design.
The pharmacokinetic/pharmacodynamic (pk/pd) modeling will be studied for its delivery to the infected organs.
Picture 1. showing cloning of pUC19-3’UTR-TLR2 in E.coli Top10
Picture 2. showing cloning of pUC19-3’UTR-C3 in E.coli Top10
Picture 3. showing cloning of pUC19-3’UTR-CFH in E.coli Top10
Picture 4. Gel picture showing PCR amplification of 3’UTR-TLR2..
Picture 5. Gel picture showing PCR amplification of 3’UTR-C3 and 3’UTR-CFH..
Picture 6. Gel picture showing double digested vector with Xba1 and Sal1.
Picture 7. Gel picture showing double digested vector with Xba1 and Xho1.
Picture 8. Subcloning of 3’UTR-TLR2 into pMIR-Dual-glo-Luciferase vector in E.coli Top10.
Picture 9. Subcloning of 3’UTR-C3 (CPAMD8) into pMIR-Dual-glo-Luciferase vector in E.coli Top10.
Picture 10. Subcloning of 3’UTR-CFH into pMIR-Dual-glo-Luciferase vector in E.coli Top10.
Picture 11. Gel picture showing positive clones of 3’UTR-TLR2 into pMIR-Dual-Glo-Luciferase vector by colony PCR
Picture 12. Gel picture showing 2 positive clones (220 bp) of 3’UTR-CPAMD8 (C3) into pMIR-Dual-Glo-Luciferase vector by colony PCR
Picture 13. Gel picture showing positive clones of 3’UTR-CFH into pMIR-Dual-Glo-Luciferase vector by colony PCR
Picture 14. Microscopic image of Mammalian cell line A549 before seeding into 24 well plates.
Picture 15. Showing Cell counting of cells by hemocytometer using trypan blue staining method before seeding cells for construct pMIR-Glo-3’UTR-TLR2
Picture 16. Showing Cell counting of cells by hemocytometer using trypan blue staining method before seeding cells for construct pMIR-Glo-3’UTR-C3 and construct pMIR-Glo-3’UTR-CFH
Fig 1. Experimental set-up for the cell transfection with reporter plasmid Tlr2 and corresponding miRNA in 24 wells plates.
| 1 | 2 | 3 | 4 | 5 | 6 | |
|---|---|---|---|---|---|---|
| A | TT1 Reporter plasmid pmir-Glo-TLR2 | TT1 Reporter plasmid pmir-Glo-TLR2 | TT1 Reporter plasmid pmir-Glo-TLR2 | .|||
| B | TT2 pmir-Glo-TLR2 + hsa-miR144-3p | TT2 pmir-Glo-TLR2 + hsa-miR144-3p | TT2 pmir-Glo-TLR2 + hsa-miR144-3p | .|||
| C | TT3 pmir-Glo-TLR2 + has-miR144-3p+ anti-hsa-miR144-3p | TT3 pmir-Glo-TLR2 + has-miR144-3p+ anti-hsa-miR144-3p | TT3 pmir-Glo-TLR2 + has-miR144-3p+ anti-hsa-miR144-3p | .|||
| D | Negative control (No Transfection) | Negative control (No Transfection) | Negative control (No Transfection) | .
Fig 2. Experimental set-up for the cell transfection with reporter plasmid C3 and CFH and their respective miRNA in 24 wells plates.
| 1 | 2 | 3 | 4 | 5 | 6 | |
|---|---|---|---|---|---|---|
| A | TT1 Reporter plasmid pmir-Glo-C3 | TT1 Reporter plasmid pmir-Glo-C3 | TT1 Reporter plasmid pmir-Glo-C3 | .TT1 Reporter plasmid pmir-Glo-CFH | TT1 Reporter plasmid pmir-Glo-CFH | TT1 Reporter plasmid pmir-Glo-CFH |
| B | TT2 pmir-Glo-C3 + has -miR630 | TT2 pmir-Glo-C3 + has -miR630 | TT2 pmir-Glo-C3 + has -miR630 | .TT2 pmir-Glo-CFH + hsa-miR4427 | TT2 pmir-Glo-CFH + hsa-miR4427 | TT2 pmir-Glo-CFH + hsa-miR4427 |
| C | TT3 pmir-Glo-C3 + has-miR630+ anti-hsa-miR630 | TT3 pmir-Glo-C3 + has-miR630+ anti-hsa-miR630 | TT3 pmir-Glo-C3 + has-miR630+ anti-hsa-miR630 | .TT3 pmir-Glo-CFH + has-miR4427+ anti-hsa-miR4427 | TT3 pmir-Glo-CFH + has-miR4427+ anti-hsa-miR4427 | TT3 pmir-Glo-CFH + has-miR4427+ anti-hsa-miR4427 |
| D | No Transfection | No Transfection | No Transfection | .No Transfection | No Transfection | No Transfection |
Picture 17. FireFly Luciferase readings recorded on Sep 13, 2024 to study interaction between pMIR-Glo-3’UTR-TLR2 and hsa-miR-144-3p.
Picture 18. Renilla Luciferase reading 2 (Renilla as internal control) recorded on Sep 13, 2024 to study interaction between pMIR-Glo-3’UTR-TLR2 and hsa-miR-144-3p.
Picture 19. FireFly Luciferase reading 1 as recorded on Sep 20, 2024 to study interaction between pMIR-Glo-3’UTR-CPAMD8 with hsa-miR-630 and pMIR-Glo-3’UTR-CFH with hsa-miR-4427.
Picture 20. Renilla Luciferase reading 2 as recorded on Sep 20, 2024 to study interaction between pMIR-Glo-3’UTR-CPAMD8 with hsa-miR-630 and pMIR-Glo-3’UTR-CFH with hsa-miR-4427.
LUCIFERASE ASSAY RESULTS AND GRAPH
Fig 3 Graph illustrating the interaction between 3'UTR-TLR2 with hsa-miR144-3p
Fig 4. 3'UTR-CPAMD8 (C3) interaction with hsa-miR630
Fig 5. 3'UTR-CFH interaction with hsa-miR4427
