In clinical treatment of Glioma, TMZ is necessary, but it always causes resistance and decreases the survival rates of patients. So, it’s really important to screen for new target genes to conquer this problem. In this project, we developed a bioinformatics analysis protocol to screen TMZ resistance-related genes.
In order to carry forward the spirit of iGEM, we specially searched the iGEM Biological Parts library for related research. However, there is no related research. Our team provided a bioinformatics analysis protocol and successfully pick up a target gene, PDRG1, adding data for TMZ resistance-related genes. This information can be a good reference for future iGEM teams working on TMZ resistance.

The aim of this project is to screen for target genes that can reverse TMZ drug resistance during the treatment of gliomas. Therefore, the public dataset GSE199689 related to TMZ resistance was obtained by screening in the GEO database. This dataset includes two cell lines, U87 (TMZ-sensitive) and U87TR (TMZ-resistant), each with three biological repeats. the biological repeats of U87 are the biological replicates of U87TR are GSM5981756, GSM5981757, and GSM5981758. Using this dataset, the differences between the two cell lines were analyzed and the target genes associated with TMZ resistance were expected to be found. We download the probe and annotated the genes with AnnoProbe. We analyzed the data with R.
The genes that were significantly up- and down-regulated in the differential genes were screened separately and the expression amounts were observed in each sample. We can see the difference in the expression of differential genes between the two groups (Figure 1).

Based on the mechanism of TMZ drug resistance, we put our emphasis on DNA damage repair. Here, we listed 10 GO terms. We found that the downregulated genes are remarkably enriched in terms that are related to DNA damage repair, which points out that the downregulation of certain genes affects DNA damage repair (Figure 2). The differential genes that are enriched in the GO terms are potential target genes that could reverse TMZ drug resistance, which is one of our focuses.

Through extensive literature review, we found that PDRG1, the down-regulated differential gene, was up-regulated in various tumor tissues, such as colorectal cancer, ovarian cancer, lung cancer, breast cancer, and endometrial cancer. At the same time, PDRG1 down-regulation can enhance the radiosensitivity of nasopharyngeal cancer. Therefore, PDRG1 was preliminarily identified as a potential target gene, and the function of PDRG1 was verified by using glioma-related data from TCGA and GTEx databases. Firstly, the expression of PDRG1 in glioma tumor samples and normal samples was analyzed, and the expression of PDGR1 in normal samples was lower than that in tumor samples (Figure 3A). Secondly, whether PDRG1 affects the prognosis of glioma was analyzed, and patients with low expression of PDRG1 had a better prognosis and longer survival time (Figure 3B).

We observed that the expression of PDRG1 in GBM and LGG was lower in normal samples than in tumor samples, which verified our hypothesis. What’s more, the survival time of samples with low PDRG1 expression was significantly higher than that of samples with high PDRG1 expression (Figure 3C). Both OS and RFS can reach the same conclusion. So again, it verifies our hypothesis. In conclusion, PDRG1 was taken as the target gene for further verification by cell assay.

The gene p53 and DNA-damage regulated 1 (PDRG1) was first cloned in 2003 as a small molecule that affects the cellular response to genotoxic stress. An increasing number of studies have confirmed PDRG1 as a tumor marker; the expression of PDRG1 is elevated in a variety of tumor tissues, such as colorectal, ovarian, lung, breast, and endometrial cancers.

GV248-vector is one of the most commonly used siRNA expression vectors, which uses the hU6 promoter to regulate the expression of siRNA. The vector contains an EGFP gene to verify if the plasmid was transfected into the host. We inserted the siRNA1 (BBa_K4251003) which targeted PDRG1, into the AgeI and EcoRI sites and regulated by the hU6 promoter. When expressed in the prokaryotic system, the Amp+ resistance can be used to screen the right colony, when transfected into mammalian cells, the puromycin resistance could be used to screen the transfected cells.

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.

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
4. Project Design: Prof. Jing Ren. He has been participating in our project ideation, especially experiment design.