T98G and U118 cells were selected. The drug concentration of the starting TMZ is set with each cell line IC50, the culture medium containing TMZ is changed every two days, and the same concentration of TMZ is maintained for two weeks. Then calculate the new IC50, increase the TMZ concentration, and TMZ is maintained for two weeks, using the same method to change the culture medium. Repeat this 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 control cells were treated with the same concentration of DMSO, and the processing time and procedure were the same plus TMZ. The cell line is established and expanded, including breed conservation.
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 and U118 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.

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 (Figure 2A).
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. 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 2B). What’s more, we observed that the expression of PDRG1 in GBM and LGG was lower in normal samples than in tumor samples. And in GBM and LGG, the survival time of samples with low PDRG1 expression was significantly higher than that of samples with high PDRG1 expression (Figure 2C). 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.

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 3).

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 4). As shown below, the mRNA expression level of PDRG-1 is significantly lower in shRNA treated group (Table 2).

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 5).

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 6A) as well as T98G (Figure 6B). 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.