Part:BBa_K2440030

miR-152 target sequence

It is the target sequence of miR-152, a modularized DNA part from a set of chemically synthetic oligo DNA library.

Usage and Biology

MiRNA locker assembled by using this modularized DNA part was able to bind miR-152 in an Ago2 dependent manner, that is, knockdown of miR-152 was achieved by transfecting cells with miRNA locker.

Dre-miR-152 is a novel miRNA founded in zebrafish.¹

It was found that miR-152 was underexpressed in human osteosarcoma (OS) tissues and cell lines. Decreased miR-152 was inversely correlated with lymph-node metastasis and advanced clinical stage. Overexpression of miR-152 significantly inhibited cell proliferation, colony formation, migration and invasion of OS cells. Bioinformatics analyses showed that miR-152 directly targeted E2F transcription factor 3 (E2F3), as further confirmed by a dual-luciferase reporter assay. Dre-miR-152 may be a therapeutic target for OS.²

Moreover, MiR-152 regulated cell proliferation and apoptosis of glioma mediated by Runx2, while the mechanism of down regulated miR-152 in glioma tissues and cells was its hypermethylation.³

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Experimental Validation

This part is sequenced as correct after construction.

Reference

1.The developmental miRNA profiles of zebrafish as determined by small RNA cloning. Chen PY, Manninga H, Slanchev K, Chien M, Russo JJ, Ju J, Sheridan R, John B, Marks DS, Gaidatzis D, Sander C, Zavolan M, Tuschl T Genes Dev. 19:1288-1293(2005).

2.MicroRNA-152 Suppresses Human Osteosarcoma Cell Proliferation and Invasion by Targeting E2F Transcription Factor 3. Ma C, Han J, Dong D, Wang N.

3.MiR-152 regulated glioma cell proliferation and apoptosis via Runx2 mediated by DNMT1. Zhang P, Sun H, Yang B, Luo W, Liu Z, Wang J, Zuo Y.

Improvement by 2022 SubCat_Shanghai

RDRG1-SiRNA-1

Profile

Name: PRDG1-siRNA

Base Pairs: 21bp

Origin: Company synthetic

Properties: Silencing RNA of PDRG1

Contribution

siRNA is a double-stranded non-coding RNA molecule, it usually between 20 and 24 base pairs, and it is also known as silencing RNA and short interfering RNA. siRNAs have very tight target specificity, this anti-sense strand binds to the target mRNA, and then the target mRNA cleavage is induced. PRDG1-siRNA is designed according to the DNA sequence of PRDG1, and it’s 21bp long. We insert the siRNA into AgeI and EcoRI sites of the GV248 plasmid, and the correct plasmid was sent to the company for lentivirus packaging. The lentivirus was then transfected to T98G and U118 TMZ resistance cell line to knock down the PRDG1 transcripts.

Engineering Success

1. PDRG-1 knockdown cell-line construction

In order to verify if knockdown of the PDRG1 gene could decrease the TMZ-resistance cells’ lifespan, we transfected T98G cells and U118 cells with Lentivirus packaged plasmids containing siRNAs and screened them using purine. The fluorescence microscope can be used to check if the plasmids have been transfected successfully by detecting the green fluorescence (Figure 1).

Figure 1. Detection of green fluorescent protein expression in the stable knockdown cell using fluorescence microscopy. The green fluorescence proves successful plasmid transfection.

To get more reliable and quantitative results, fluorescence real-time quantitative PCR was done to test the mRNA expression of PDRG1 in the transfection cell line (Figure 2). As shown below, the mRNA expression level of PDRG-1 is significantly lower in shRNA treated group (Table 2).

Figure 2. Bar graph of mRNA level of the control group and PDRG-1 knockdown group.

Table 1. Data of relative expression of PDRG-1 from qPCR.

4. PDRG-1 knockdown cell line PDRG1 protein expression level measurement

In addition, the Western blot was done to further confirm PDRG-1 expression on the protein level in each group. The thickness of the protein band indicates the amount of the expressed protein. GAPDH was used as a control group, as it is a common housekeeper gene. It can be easily observed that in the PDRG-1 group, the protein band of Lv-sh1 and Lv-sh2 are thinner than the Lv-shNC, showing the lowered expression of PDRG-1 in the experimental group (Figure 3).

Figure 3. Western blot result of PDRG-1 and GAPDH.

Proof of function

5. TMZ tolerance test

The control group and the PDRG-1 knockdown group are treated with TMZ. The relative cell viability was recorded over 72 hours for U118 (Figure 4A) as well as T98G (Figure 4B). In both groups, there are significant differences between the control group and the PDRG-1 knockdown group. Comparatively, the PDRG-1 knockdown groups show lower cell viability over time and lower TMZ tolerance, which is our expected result.

Figure 4. TMZ tolerance of different glioma cell lines over time. The significance is indicated near the data point using the same abbreviation as listed above. A. the result of the U118 cell line, B. the result of the T98G cell line

Future plan

We have already collected the figures from our experiments. PDRG1 is a small oncogenic protein related to TMZ resistance. However, TMZ resistance may cause chemotherapy failure which significantly decreases the survival rates of GBM patients. To conquer this problem, we screened the databases to search for related genes that could be used as therapeutic targets, so PDRG1 was chosen for our project.

We constructed a PDRG1 knockdown cell line that has been shown to have a higher potential to counter TMZ tolerance. This cell line can be used to further investigate the PDRG signaling pathway to gain a deeper understanding of the molecular mechanisms of TMZ tolerance development and thereby inspire new research directions seeking to eliminate or moderate TMZ tolerance. We believe that if we can fully understand the mechanism of PDRG1 in the TMZ-resistance process, we could provide new ways to treat GBM.

Reference

1. Lee, S. Y. (2016, May 11). Temozolomide resistance in glioblastoma multiforme. Genes & Diseases. Retrieved July 26, 2022, from https://www.sciencedirect.com/science/article/pii/S2352304216300162

2. Jiapaer, S., Furuta, T., Tanaka, S., Kitabayashi, T., & Nakada, M. (2018, October 15). Potential strategies overcoming the temozolomide resistance for glioblastoma. Neurologia medico-chirurgica. Retrieved July 26, 2022, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6186761/

3. Kato, T., Natsume, A., Toda, H., Iwamizu, H., Sugita, T., Hachisu, R., Watanabe, R., Yuki, K., Motomura, K., Bankiewicz, K., & Wakabayashi, T. (2010, June 3). Efficient delivery of liposome-mediated MGMT-Sirna reinforces the cytotoxity of temozolomide in GBM-initiating cells. Nature News. Retrieved July 26, 2022, from https://www.nature.com/articles/gt201088