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GV248-PDRG1-SiRNA-2

GV248-PDRG1-SiRNA-2

Usage and Biology

Introduction

Glioma, the most common malignant tumor of the adult central nervous system, has a high mortality rate. The combination of temozolomide (TMZ) chemotherapy and radiotherapy is the current standard for curing gliomas, but most patients eventually develop resistance to TMZ. To better individualize and precision therapy, finding new molecular targets that increase TM therapeutic sensitivity is really important.

In our project, we screened out the gene PDGR1 (p53 and DNA damage regulated 1) which is related to TMZ resistance through bioinformatics analysis. To verify its role in the TMZ resistance process, we developed a U118 TMZ-resistance cell line and knocked down the PDRG1 gene by siRNA. Then we measured the transfected cell line’s lifespan in different concentrations of TMZ. Our experimental data indicated that PDRG1 could be a target gene to surmount TMZ resistance. How we construct the TMZ-resistance cell line We chose the U118 cell line as the Initial cell line to carry out the project. 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 U118 cell lines were tested. Raw data from the cell viability assay was normalized and listed below in Table 1.

Table 1. The left column lists different shRNA concentrations, varying from 0-3200 μM. U118 represents the control group. U118-R are the experimental groups.

We can observe that TMZ-treated cells have significantly higher viability than the control group (Figure 1). This illustrates that we successfully screened U118 TMZ-tolerance cell lines.

How we test PDRG1

a) U118 PDRG1 gene knockdown cell line construction

In order to verify if knockdown of the PDRG1 gene could decrease the TMZ-resistance cells’ lifespan, we transfected 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 2).

b) Measure the mRNA abundance and protein expression level of PDRG1 knockdown U118 cell line

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 3). As shown in figure 3, the abundance of the PDRG1 gene in the transfected cell line, the siRNA successfully inhibited the PDRG1 transcription.

Figure 3. the result of mRNA abundance of the control group and PDRG-1 knockdown group

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 4). 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 4).

c) TMZ-tolerance of the U118 PDRG1 knockdown cell line

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 5). 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 5. Measure the resistance of PDRG1 knockdown U118 cell line and the control cell line

How we learn from our project

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.

Sequence and Features