Glioma, the most common malignant tumor of the adult central nervous system, has a high mortality rate. In clinical treatment, radiotherapy and TMZ are always used. However, the patients always get resistance to TMZ which causes the mean survival rate for GBM patients to be only 15 months. To further study the molecular mechanism of its resistance to TMZ, we need to construct a TMZ-resistance cell line.
In order to carry forward the spirit of iGEM, we specially searched the iGEM Biological Parts library for related projects, but there are almost no related parts. So we provided a new TMZ-resistance T98G cell line to start the research.
In this project, our team carried out a T98G cell line for this part in the laboratory, adding data for TMZ resistance research. What’s more, we also screened out a TMZ resistance-related gene, PDRG1, and verified its role in this process. This information can be a good reference for future iGEM teams working on TMZ resistance.
In order to verify if the new composite part PDRG1 we provided, we knockdown the PDRG1 gene of the T98G TMZ resistance cell line and measured the abundance of tits transcripts, and test its resistance of TMZ. As a result, we set up a T98G TMZ resistance cell line and verified the role of PDRG1 in the TMZ resistance process.

T98G is a fibroblast-like cell that was isolated from the brain of a glioblastoma multiforme White, 61-year-old, male patient. T98G expresses a unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells become arrested in the G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality.

To construct the TMZ resistance cell line, we set up a dose escalation method, and cultured the T98G cell with TMZ containing medium, the same concentration of TMZ is maintained for two weeks. Then calculate the new IC50, and increase the TMZ concentration until IC50 increases to 500 μM. After the successful preparation of TMZ-resistant cells, the culture is maintained at a low concentration of TMZ (different cell concentrations vary).
The CCK8 cell viability assay was used to examine the TMZ tolerance of the cells. Both the control group (transfected with NC plasmid) and the experimental group (TMZ-resistance cell line) of T98G cell lines were tested. Raw data from the cell viability assay was normalized and listed below in Table 1.

We can observe that TMZ-treated cells have significantly higher viability than the control group (Figure 1). This illustrates that we successfully screened TMZ-tolerance cell lines.

In order to knock down the PDRG1 gene in the T98G TMZ resistance cell line, we used a siRNA method. We designed two different siRNA targeting PRDG1 and give them to the company to construct the plasmids and lentivirus packaging. After transfecting the T98G TMZ resistance with the lentivirus, we measured the abundance of PDRG1 transcripts through qPCR. As shown below, the mRNA expression level of PDRG-1 is significantly lower in shRNA treated group (Figure 2).

The control group and the PDRG-1 knockdown group are treated with TMZ. The relative cell viability was recorded over 72 hours for T98G (Figure 3). As shown in the figure, the PDRG1 knockdown cell line showed lower cell viability over time and lower TMZ tolerance. What’s more, different siRNA showed different efficiency of inhibition, which may be caused by the different secondary structures of siRNA et.al.
In conclusion, knockdown PDRG1 can significantly increase the susceptibility to TMZ.