Shannah and I finalized the topic of our project after talking with Pam and Evan. It was great that we decided on our topic early on so that we have a direction to work on. Not being able to find a direction was the most worried aspect of the final project for me, because I think I spend a lot of time on things that are not important. From this project I learned that it’s fine to reach for help at an early stage. I always thought that I should have a clear thought and specific questions to ask before approaching professors or TAs, but that’s not true. I wish I would have done that more during my undergrad, and I will in the future.

Topic chosen: long noncoding RNA and neuroblastoma cisplatin drug resistance

 

SPECIFIC QUESTION: What is the effect of lncRNA UCA1 in the development of cisplatin resistance in neuroblastoma?

 

HOW IS THIS QUESTION NOVEL AND ORIGINAL?

The effects of  long noncoding RNA (lncRNA) urothelial cancer‐associated 1 (UCA1) in the development of cisplatin resistance has been studied in many types of cancer, including cervical, bladder and ovarian cancer (REF). However, the effects of UCA1 on resistance to cisplatin treatment in neuroblastoma, a childhood malignancy in the sympathetic nervous system, has yet to be investigated. Studies have looked at the relationship between differential expression of proteins and development of cancer resistance.

 

Previous  research were done of the proteins that are expressed in resistant cancer cell lines. A study by _____ suggested the involvement of miRNA in silencing expression of proteins, thereby inducing chemoresistance. Recent studies have also demonstrated chemoresistance mechanisms beyond the protein level. For example, long non-coding RNA HOTAIR was shown to be involved in cancer metastasis.

 

POTENTIAL IMPACT OF THE PROPOSED QUESTION (WERE IT TO BE ANSWERED BY YOUR PROPOSED EXPERIMENT):

Neuroblastoma is a childhood malignancy in the sympathetic nervous system and accounts for 15% of all deaths in pediatric cancer patients (REF). Treatment of neuroblastoma is most commonly performed using cisplatin, a platinum based anticancer drug that induces apoptosis by activating various signal transduction pathways (REF). Development of drug resistance has made it hard to effectively treat neuroblastoma patients, and doctors often resort to multi drug treatments. Despite these efforts, drug resistance is still prevalent and the cause of the resistance is unknown. In order to effectively treat cancer it is important to understand the mechanism of cancer resistance and find ways to prevent their effect (REF). Recent studies have shown that long non-coding RNA plays a major role cisplatin drug capabilities in various cancer types, including breast cancer (REF), cervical cancer (REF), and lung cancer (REF). This includes urothelial cancer‐associated 1 (UCA1), a lncRNA that plays a regulatory role in the proliferation of cells and has been found to be upregulated in bladder cancer. However, UCA1 has never been studied in cisplatin resistance in neuroblastoma, posing a potential gap in knowledge that could be vital to finding effective treatment methods for patients who are resisting this drug. An investigation of the presence of UCA1 in cell lines that are resistant to drug treatment and identification of the changes in mRNA expression it promotes, will allow us to take a step further in understanding the mechanisms involved in resistance, a vital step in finding the best treatment methods.

 

HYPOTHESIS AND EVIDENCE ON WHICH THE HYPOTHESIS IS BASED (INCLUDE REFERENCES):

 

Experiment 1: If lncRNA UCA1 is crucial  in developing cancer resistance, then neuroblastoma cells without UCA1 expression should show little to no resistance. We predict that cisplatin sensitive neuroblastoma cells would have no or low levels or UCA1. Cisplatin resistant neuroblastoma cells will show reduced resistance after knockout of UCA1.

If high UCA1 is detected in cisplatin sensitive cells, then UCA1 may not be involved in cancer resistance or there are specific conditions required for UCA1 to be involved. If high levels of  resistance is shown even after knockout of UCA1 in cisplatin resistant neuroblastoma cells, then UCA1 may not be a crucial component of cisplatin resistance.

 

Study by Wang et al. (2008) has shown that UCA1 is correlated with cell proliferation and migration in bladder cancer. (rewording). Overexpression of UCA1 in bladder cancer cells resulted in invasion and migration of cells. The researchers identified that UCA1 may be  involved in activating the wnt signaling pathway. The wnt pathway is  known to contribute to chemoresistance development in various cancer cell lines including neuroblastoma, multiple myeloma, and hepatocellular carcinoma cells. Study by Fan et al. (2014) has demonstrated that UCA1 levels are higher in cisplatin resistant T24 cells, a type of bladder cancer cell line. Knock out of UCA 1 reduced cell proliferation

 

Experiment 2:

Studies looking at cisplatin resistance in numerous cancers have found a relationship between lncRNA UCA1 and changes in expression levels of genes that are involved in the cells susceptibility to treatment (REFS). This includes a study performed by …  looking at microarray mRNA expression analysis of transfected UCA1 cells compared to control. Expression levels of 42 genes were found to change by at least two-fold in the presence of UCA1 in bladder cancer, including an upregulation of Wnt signaling pathway member 6 (Wnt6), CYP1A1 (a cytochrome) and AURKC (kinase) and a downregulation of methyl‐CpG binding domain protein 3 (MBD3) and SR (serine/arginine‐rich) protein‐specific kinase 1 (SRPK1). These results are verified by several other studies looking further into the effects of UCA1 on gene expression in bladder cancer (REF’s). Similar results have been found in ovarian cancer, including a study by … that focuses on the change in expression of SRPK1 in the presence of UCA1 and cisplatin resistance. The gene expression analysis profiles continue to show similar changes in expression of genes in other cancers, including cervical, lung and bladder cancer [REFS]. Based on this continuing similarity between cancers, we hypothesize the expression levels of these genes will be similar in neuroblastoma cell lines [REF]. Among others, this includes an upregulation of Wnt6, CYP1A1, and AURKC and a downregulation of MBD3 and SRPK1.

PREDICTION(S):

Experiment 1:

	Cisplatin Resistant	Cisplatin Susceptible
UCA1 not knockout	Cells grow	Cells don’t grow
UCA1 knocked out	Cells don’t grow	Cells don’t grow

 

Experiment 2:

We predict RNA sequencing will show an increase in expression levels in Wnt6, CYP1A1, and AURKC and a decrease in expression levels in MBD3 and SRPK1 when UCA1 is present in the cisplatin resistant cells compared to UCA1 knockout cisplatin resistant cell lines and cisplatin susceptible cell lines.

 

EXPERIMENTAL APPROACH TO TEST PREDICTION (INCLUDE ANY DETAILS THAT YOU HAVE WORKED OUT SO FAR):

 

1. Collect neuroblastoma cisplatin resistant and susceptible cell lines
2. Knockout UCA1 using RNAi such that we have a control group and a knockout group
3. Cell proliferation assay (MTT) of control (cisplatin resistant with UCA1) and UCA1 knockout cells (from cisplatin resistant).
   1. Seed cells separately in two 96 well plates for 24 hours
   2. Treat both plates with cisplatin and allow them to proliferate for 3-4 days
   3. Analyze cell proliferation. If our hypothesis is correct, then we expect to see less cell proliferation in the UCA1 knockout cells
   4. Follow the same protocol with cisplatin susceptible cells as a negative control

 

1. Perform expression profile analysis using RNA sequencing of cell lines before and after knockout to compare their RNA expression levels. CONTROL: To ensure that RNAi sufficiently knocked out UCA1, check the expression levels of UCA1 in the knockout.

 

 

 

 

LIST OF RELEVANT PRIMARY AND REVIEW ARTICLES READ, AND SUMMARY OF RELEVANT INFORMATION FROM EACH (this is the start of the annotated bibliography that you will need to include in your portfolio)

 

Note to Pam: This will be a more fleshed out annotated bibliography in the future. Currently, we have point form for all of the

 

Wang, F., Zhou, J., Xie, X., Hu, J., Chen, L., Hu, Q., . . . Yu, C. (2015). Involvement of

SRPK1 in cisplatin resistance related to long non-coding RNA UCA1 in human ovarian cancer cells. Neoplasma,62(03), 432-438. doi:10.4149/neo_2015_051

 

This article investigates UCA1’s effects on SRPK1 in cisplatin resistance in ovarian cancer. They started by looking at UCA1 expression in cancer and non cancer cells and its corresponding resistance to cisplatin treatment followed by assessing the expression of SRPK1 and apoptosis pathway proteins to explore the mechanism. Lastly, they looked at the effects of knocking out SRPK1 on cisplatin resistance. Results found an increased expression of SRPK1 and anti-apoptosis proteins in transgenic UCA1 cells, and knocking down SRPK1 could partly rescue the effect of UCA1 expression on cell migration, invasion and cisplatin resistance in cells. Based on these findings, they suggested that SRPK1 and apoptosis pathway proteins may be involved in the effect of UCA1.

 

Pan, J., Li, X., Wu, W., Xue, M., Hou, H., Zhai, W., & Chen, W. (2016). Long non-coding

RNA UCA1 promotes cisplatin/gemcitabine resistance through CREB modulating miR-196a-5p in bladder cancer cells. Cancer Letters,382(1), 64-76. doi:10.1016/j.canlet.2016.08.015

 

Fan, Y., Shen, B., Tan, M., Mu, X., Qin, Y., Zhang, F., & Liu, Y. (2014). Long non-coding RNA UCA1 increases chemoresistance of bladder cancer cells by regulating Wnt signaling. FEBS Journal,281(7), 1750-1758. doi:10.1111/febs.12737

Investigated the role of UCA1 lncRNA in cisplatin resistance during chemotherapy for bladder cancer. We showed that cisplatin‐based chemotherapy results in up‐regulation of UCA1 expression in patients with bladder cancer. Similarly, UCA1 levels are increased in cisplatin‐resistant bladder cancer cells. Over‐expression of UCA1 significantly increases the cell viability during cisplatin treatment, whereas UCA1 knockdown reduces the cell viability during cisplatin treatment. We finally demonstrate that UCA1 increases the cisplatin resistance of bladder cancer cells by enhancing the expression of Wnt6, and thus represents a potential target to overcome chemoresistance in bladder cancer.

 

Wang, F., Li, X., Xie, X., Zhao, L., & Chen, W. (2008). UCA1, a non-protein-coding RNA up-regulated in bladder carcinoma and embryo, influencing cell growth and promoting invasion. FEBS Letters,582(13), 1919-1927. doi:10.1016/j.febslet.2008.05.012

In addition to other experiments, did an expression analysis of genes to find potential targets of UCA1 in drug resistance

1. 1. Results [this is copied from the paper but will be changed later]: The changes of expression of the several representative genes were confirmed through real time PCR (Fig. 4C3–C5), the up‐regulated genes including
      1. wingless‐type MMTV integration site family, member 6 (WNT6) 15
      2. CYP1A1 (cytochrome P450, 1A1) 16,
      3. AURKC (a urora kinase C) 17,
      4. and the down‐regulated genes including
      5. methyl‐CpG binding domain protein 3 (MBD3) 18–20, and
      6. SR (serine/arginine‐rich) protein‐specific kinase 1 (SRPK1) 21, 22, which were identical with the microarray results (Tables 2 and 3).

 

Casinelli, G., Larosa, J., Sharma, M., Cherok, E., Banerjee, S., Branca, M., . . . Graves, J. A. (2016). N-Myc overexpression increases cisplatin resistance in neuroblastoma via deregulation of mitochondrial dynamics. Cell Death Discovery,2(1). doi:10.1038/cddiscovery.2016.82

 

Wang, B., Huang, Z., Gao, R., Zeng, Z., Yang, W., Sun, Y., . . . Zhou, S. (2017). Expression of Long Noncoding RNA Urothelial Cancer Associated 1 Promotes Cisplatin Resistance in Cervical Cancer. Cancer Biotherapy and Radiopharmaceuticals,32(3), 101-110. doi:10.1089/cbr.2016.2156

 

Xia, Y., He, Z., Liu, B., Wang, P., & Chen, Y. (2015). Downregulation of Meg3 enhances cisplatin resistance of lung cancer cells through activation of the WNT/β-catenin signaling pathway. Molecular Medicine Reports,12(3), 4530-4537. doi:10.3892/mmr.2015.3897

 

Piskareva, O., Harvey, H., Nolan, J., Conlon, R., Alcock, L., Buckley, P., . . . Stallings, R. L. (2015). Corrigendum to “The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro” [Cancer Lett 364 (2015) 142–155]. Cancer Letters,369(2), 428. doi:10.1016/j.canlet.2015.09.010

 

https://www.sciencedirect.com/science/article/pii/S0304383515003250?via%3Dihub#bib0025

Identified the proteins that are involved in cisplatin resistant cell lines in neuroblastoma through proteomic profiling, a method to identify the proteins that are present. They showed that proteins that are differentially expressed in resistant cell lines are involved in cancer promoting mechanisms such as cell migration and proliferation. Proteins that were absent (or expressed in lower levels) in resistant cells were predicted to be due to miRNA that target specific genes.

 

 

 

POTENTIAL WAYS TO MAKE YOUR QUESTION KNOWN TO THE PUBLIC AT LARGE (e.g. TO YOUR NON-BIOLOGIST FAMILY AND FRIENDS):

• Infographic of our findings and share on social media
• Make a video that is easy for a non-biologist to understand and share on social media
• Present at events that are intended for the public, for example: https://vancouver.nerdnite.com/

 

ANY OTHER PARTS OF THE PROJECT COMPLETED SO FAR:

 

 

ANYTHING YOU WOULD LIKE SPECIFIC FEEDBACK ON:

• How much detail should we provide for the hypothesis, possible results and discussion of the expression analysis. There are a TON of genes that UCA1 could affect, as found in previous studies. Is it ok to mention a few and dive deeper into these? Or should we try to include all that we can find and not go into too much detail? Or some sort of combination of both?
• Is our question novel enough? Is our evidence for our hypothesis valid and sufficient? How can we improve on this?