Evaluating The Function of Genes Implicating Glioblastoma Multiforme (GBM) Formation Using C. Elegans
The purpose of this project was to determine if genes implicating Glioblastoma Multiforme (GBM) formation play a role in cell division in Caenorhabditis elegans (C.elegans). To be able to detect genes predisposing to GBM, it is important to understand the role of cell division. Mutations in cell division cause the proliferation of cancerous cells. In order for cell division to produce malignant cell growth, genes responsible for proper cell division have to be altered. Gene regulation is usually affected making some cells do things that they should not be doing, like overproducing proteins or growth factors that may be useful for the destructive cancerous cells. To detect GBM genes, it is important to know what genes have increased variance in GBM affected patients. Genetic variation is important in cancer, because it causes evolutionary genetic susceptibility to cellular malfunctions, such as damage in DNA repair and replication. Using the Backes et al. (2012) study, we used the data published on GBM patients, which yielded multiple genes that had increased or abnormal genetic variance. These genes were not declared as markers of GBM, although with their suspicious presence in the studied GBM patients implicates that those genes may have a role in brain cancer development. There were some genes in the publication’s dataset that had high variance, but those genes were ignored because they have been already declared as cancerous genes and predisposition markers from other research studies.
To understand whether these genes have a role in GBM, we used C.elegans as a model organism to understand the morphological and cellular significance of the suspected genes. C.elegans is a nematode that possesses a nervous system. Its nervous system comprises of 302 neurons and 56 glial cells. (Oikonomou and Shaham, 2011) These cells have an influence on sensory and motor functions. We implicated in this study that if we knockout a suspected GBM gene from C.elegans, and observed changes in glial cell development using behavioral assay and advanced microscopy, then will be able to confirm the suspected genes association with GBM.
Using several online databases, including Wormbase, Ensembl and Ortholist, we were able to find 10 genes that had orthologous genes with C.elegans. We had to research the background of the 10 genes to pinpoint which ortholog had any association with glial cells or the nervous or neuromuscular system in C.elegans. One gene, Lev-9, a levamisole resistant gene, was one of the few genes from the bunch that had a connection to the nervous system in the nematode. Utilizing the services of a company that specializes in knocking out selected genes in organisms, we were able to order two strains of Lev-9: RB1717 and ZZ16. The two strains with similar genotypes allowed us to investigate the effects of the Lev-9 gene knockout.
Figure 1: Above graphs the median progeny count for two strains of Lev-9 C.elegans: RB1717 and ZZ16; and control, N2 (wild-type) strain. Median progeny count is taken from several tests performed using the Lev-9 strains and control. Blue indicated the number of unhatched eggs found on the plate. Orange indicates the amount of early larval stage (L1-L3) organisms inhabit a plate. Purple indicated the total amount of progeny present on the plate when counted after 48 hours of incubation in 20 C.
According to the data, it is clear that there is some statistical significance in the results we have collected. To determine significance, we conducted four tests on the N2 control and three tests each for both RB1717 and ZZ16 Lev-9 strain C.elegans. In Figure 1, the total progeny count is substantially lower in ZZ16 than in RB1717 in comparison to the control. We expected ZZ16 and RB1717 to have less progeny, since this would indicate that there are problems with cell division. In each test, ZZ16 still remained in its L4 stage, and unlike the other strains, it appeared that it needed a longer period of incubation time in order to develop into an adult. This suggests that ZZ16 may have developmental issues and a harder time laying eggs at the same pace as a wild-type. On the contrary, RB1717 appeared to develop at an increased rate and yielded an even higher progeny count than N2. This result was surprising, because we expected RB1717 to behave more closely as ZZ16 since they are both mutants from the same gene.
From the data we have collected, we can start discussing how the progeny count is affecting the nematode and whether this data is relevant to glial cells. We predicted that the organisms would have a deteriorated nervous system and that the neural tube would also be affected by the KO of the Lev-9 gene. The organisms did exhibit abnormal behavior and uncoordinated movement when observed under a 10X microscope. Body curling and slowed movements were two of the most obvious observations, when compared to a normal organism (control). This raised question whether glial cells have been affected. To determine whether glia are directly affected, we will need to follow up on our behavioral results with a much more advanced experiment.
Keeping in mind that there is no research adequate enough to understand to what extent GBM is hereditary, and the details of it being linked to cell division, this study has the potential to help provide significant insight for the gap of knowledge in genetic inheritance and variability in GBM. GBM has significant molecular characterization for its category as a tumor, and not all therapies like chemotherapy or radiotherapy have not been found to be completely effective. This study may contribute to biomedical and bioengineering professionals that are working on solutions for cancer development prevention and those who are finding simpler ways for disease prognosis. Additionally, this study may add information to new studies concerning C.elegans and cell reproductive health. It is also greatly anticipated that the contributions from this research can provide more direction for the development of both better drugs and therapies for life-threatening mutations in cell division. it is anticipated that the present and future studies may help reshape the current predisposition testing for cancer. For a bigger picture, it is hoped that the small, but significant information from this research and other studies can redesign treatment options, and revolutionize education on how cancer and may other diseases work. With the help of my mentor, this project has definitely enriched and enhanced my education in the fundamental knowledge of the reproductive biology and cellular biology. I hope that my research experience can be a motivation to others interested in the scientific field and that determination and commitment has the potential to create great things.
Backes C, Harz C, Fischer U, et al. New insights into the genetics of glioblastoma multiforme by familial exome sequencing. Oncotarget. 2015;6(8):5918-5931.
Oikonomou G, Shaham S. The glia of Caenorhabditis elegans. Glia. 2011 Sep; 59(9):1253-63. Doi:10.1002/glia.21084.