Knowing that we have so much to do at the end of the term, we tried to make our draft as finalized as possible. I think this was a very wise decision that we have made since we still had more work to do near the end of the final deadline.
Long noncoding RNA UCA1: Function in Cisplatin Resistance in Neuroblastoma Cell Lines
Background
Long non-coding RNAs (lncRNAs) are evolutionary conserved gene transcripts of over 200 nucleotides that do not encode proteins (Geisler and Coller, 2013). lncRNAs are involved in regulation of gene expression at transcriptional, post-transcriptional, and translational levels (Moran et al., 2012) either by directly interacting with its target gene (cis-acting) or by interacting with transcription factors (trans-acting) (Ponting et al., 2009). lncRNAs affect chromatin remodeling and transcript modification in the nucleus and interact with other RNAs and proteins in the cytoplasm (Schmitt and Chang, 2016). They regulate diverse cellular functions such as embryonic development and cell cycle progression, and when expressed in abnormal amounts, carcinogenesis (Corra et al., 2018).
In recent studies, a variety of lncRNAs such as HOTAIR, MALAT1, and UCA1 have been found to play an important role in carcinogenesis, metastasis, and prognosis. The function of lncRNA in oncogenesis may vary depending on the identity of lncRNA and the cellular context of cancers. lncRNA can be involved in promoting cancer but also prohibit oncogenic phenotypes such as metastasis (Malik et. al, 2014). LncRNA Urothelial carcinoma associated 1 (UCA1) is the most common isoform of the UCA1 gene in bladder cancer, breast cancer, and hepatocellular carcinoma (Wang et al., 2017). The biological role of other isoforms such as lncRNA CUDR is not well studied (Wang et al., 2017). The oncogenic role of lncRNA UCA1 has been identified in various cancers including bladder cancer, breast cancer, hepatocellular carcinoma, ovarian cancer, and tongue squamous cell carcinoma (Wang et al., 2017). Furthermore, studies have shown that lncRNA UCA1 regulates chemoresistance in various cancers (Wang et al., 2017).
In a study by Wang et al. (2008), targeted expression of lncRNA UCA1 induced the bladder TCC cell line to become highly proliferative and invasive as well as more resistant to cisplatin (Wang et al., 2017). They also revealed the differential gene expression resulting from UCA1 overexpression including WNT6 and AURKC as upregulated genes and MBD3 and SPRK1 as downregulated genes. Pan et al. (2016) suggested that lncRNA UCA1 induces cisplatin resistance by upregulating miR-196a-5p through activation transcription factor CREB.
Cisplatin, a platinum based cytotoxic drug, is one of the most commonly used chemotherapy agent to treat cancer including neuroblastoma, lung cancer and bladder cancer (Dasari and Tchounwou, 2014). Although cisplatin is effective in many patients, a large proportion of patients are resistant to cisplatin-based therapies (Galluzzi, 2012). Furthermore, a large faction of initially sensitive cancers become resistant after frequent treatment with cisplatin (Galluzzi, 2012). Mechanisms of cisplatin resistance include change in the signaling pathways, silencing of certain genes by miRNA, changes to the cell cycle, development of an efflux system, and DNA repair (Chen, 2017). In addition, several studies have revealed that long non-coding RNAs (lncRNAs) are involved in chemoresistance to cisplatin by interacting with histone modification tools and with other chromatin regulatory factors (Guttman et al., 2011).
Relevance and Importance
Neuroblastoma is a childhood malignancy in the sympathetic nervous system and accounts for 15% of all deaths in pediatric cancer patients (Piskareva et al, 2015). Treatment of neuroblastoma is most commonly performed using cisplatin, a platinum based anticancer drug that induces apoptosis by activating various signal transduction pathways (Dasari and Tchounwou, 2014). Development of drug resistance has made it hard to effectively treat neuroblastoma patients, and doctors often resort to multi drug treatments (Piskareva et al, 2015). Despite these efforts, drug resistance in neuroblastoma is still prevalent, and the cause of the resistance is not fully understood. In order to effectively treat cancer, it is important to understand the mechanism of cancer resistance and find ways to prevent their effect (Ayers and Vandesompele, 2017). Recent studies have shown that lncRNA plays a major role in the development of cisplatin resistance in various cancer types, including breast cancer, cervical cancer, and lung cancer (Wang et al, 2017). In particular, Urothelial cancer-associated 1 (UCA1), a lncRNA that plays a regulatory role in proliferation of cells, has been found to play a major role in development of cisplatin resistance (Wang et al, 2017). 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. Although UCA1 has been studied in other cancers such as bladder and lung cancer, we cannot conclude that the effect of UCA1 will be conserved across all cancer types including neuroblastoma. For example, lncRNA MALAT1 was suggested to function as a tumor suppressor gene in breast cancer (Eastlack, 2018), but as a promoter of tumor growth and metastasis in oral squamous cell carcinoma (Zhou, 2015).
Therefore, we propose a study that will determine if UCA1 is present in neuroblastoma cell lines that are resistant to cisplatin drug treatment and identify the changes in mRNA expression when UCA1 is present. If successful, this research would serve as a first step in understanding and finding better treatment methods for neuroblastoma patients who are resistant to cisplatin drug treatments. As a next step, the validation of UCA1 as a biomarker for drug resistance could serve as novel drug targets and could ultimately lead to the development of antagonists and/or mimics for adjunct therapy with traditional cisplatin treatment methods (Ayers and Vandesompele, 2017). Adjunct therapy methods have shown to increase susceptibility of the tumour, ultimately enhancing treatment effectiveness (Ayers and Vandesompele, 2017). In addition, use of RNA directed therapy could be used in patients who are highly resistant to the treatment due to dosage dependent resistance, reducing the discomfort of patients by permitting a lower treatment dosage (Ayers and Vandesompele, 2017). Lastly, use of UCA1 biomarker could be quantified in patients through RT-qPCR assays and provide pre-emptive knowledge to the oncologist on the best drug combination treatment for their patients (Ayers and Vandesompele, 2017).
Hypothesis
Several studies investigating the molecular mechanisms of lncRNA UCA1 in promoting cisplatin resistance have found that it is involved in silencing of tumor suppressor genes and inducing expression of multidrug resistant proteins (Want et. al, 2017). If the role of lncRNA UCA1 is conserved across different types of cancers that have been studied thus far, then we hypothesize that upregulated expression of lncRNA UCA1 causes increased resistance to cisplatin in neuroblastoma cell lines.
Experimental Plan
Level of cisplatin resistance after induced changes in lncRNA UCA1 expression
To assess the changes in cisplatin resistance after changes in expression of lncRNA UCA1, a cell viability assessment will be performed on both susceptible and resistant cells that were treated in various concentrations of cisplatin after either loss or further gain of expression of lncRNA UCA1. ()
Differential Gene Expression Analysis
To quantify changes in RNA expression levels between all conditions, an expression profile analysis will be performed on all RNA samples. RNA will be extracted in equal amounts from all conditions: lncRNA UCA1 KO, lncRNA UCA1 overexpressed, no change in lncRNA UCA1 (control) in both cisplatin resistant and cisplatin susceptible cell lines (control). All of the extracts will be sequenced and analysed. A summary of the steps and tools that will be used are outlined in Figure 2, with a detailed pipeline in Materials and Methods. To ensure that RNAi sufficiently knocked out UCA1, check the expression levels of UCA1 in the knockout.
Materials and Methods
Cell Lines
Cisplatin susceptible SK-N-AS and cisplatin resistant SK-N-ASrCDDP500 cells will be purchased from the Michaelis Lab, UK. The cisplatin resistant SK-N-ASrCDDP500 is a cell line that gained cisplatin resistance from its parental cell line SK-N-AS and therefore, is expected to exhibit characteristics similar to SK-N-AS compared to other cell lines with a different parental line.
Cisplatin treatment and Cell Viability Assay
Cisplatin treatment procedure will follow that outlined by Piskareva (2015). SK-N-AS neuroblastoma cells will be seeded at 10^4 cells/mL on a 96-well plate at 100µL medium per well. The plate will be incubated overnight at 37 °C in 5% CO2. Multiple concentrations of cisplatin was tested in serial dilution the following day. Cell proliferation will be monitored over 5 days. Cell viability will be assessed using the MTT assay as described by Wang et al (2008).
lncRNA UCA1 Knockdown via RNAi
The scrambled siRNA control (Si-NC) and siRNA that targets lncRNA UCA1 will be purchased from Thermo Fisher Scientific. si-UCA1 lentivirus will be constructed and infected into SK-N-AS cells, which will then be screened with puromycin over 7 days (Fang et. al, 2017). Successful knockdown of lncRNA UCA1 will be assessed using RT-PCR.
lncRNA UCA1 Overexpression
A recombinant plasmid containing pcDNA-UCA1 will be constructed by inserting UCA1 gene with BamHI and EcoRI restriction enzymes. The recombinant plasmid will be transfected into SK-N-AS cells using Lipofectamine 2000 from Thermo Fisher Scientific (2018). Transfected cells that stably express UCA1 will be selected by RT-PCR. A negative control of cells transfected with pcDNA3.1 will also be tested (Wang et. al, 2014).
RNA Extraction
RNA will be extracted from each of the cell line conditions, with twelve replicates for each condition, as recommended by Mortazavi, et al (2008). Total RNA will be extracted with TRIzol reagent following the recommendations of the manufacturer. The purity of total RNA will be evaluated using the A260/A280 ratio of sample absorbance at 260 and 280 nm using NanoDrop ND-1000 (Thermal Fisher Scientific, 2011). Integrity of RNA samples will be measured using the 28S/18S ratio based on a densitometry plot using Agilent 2100 Bioanalyzer.
RNA Illumina Sequencing
RNA-seq will be performed using Illumina HiSeq™ 2000 Sequencing System with paired end sequences for improved accuracy. Standard library preparation procedure will occur using the manufacturer’s protocol for RNA library preparations. In order to have a high enough coverage, we will generate ~15-25 million reads per sample, as recommended by Mortazavi, et al (2008). Sequences will be open sourced on GenBank. Sequence read quality will be analyzed using FASTQC using the standard pass/fail metrics of the program.
Sequence Alignment
Sequence alignment will be performed using the program STAR and quality of alignment will be measured using RSeQC.
Gene-based Read Counting
Transcript quantification will be calculated using the R package, featureCounts, to generate integer-based read counts for each gene.
Differential Gene Expression Analysis
To analyze the differential gene expression between samples, we will use the R package, DESeq. Genes with a fold change > 2 and p-value < 0.05 will be considered to have a significant change in expression levels. To validate mRNA-seq data, 5 samples will be randomly chosen to run through qRT-PCR for analysis.
Predictions
Level of cisplatin resistance after induced changes in lncRNA UCA1 expression
The control experiment in which both cisplatin resistant and susceptible cells are treated with various concentrations of cisplatin will give us the concentration of cisplatin in which both types of neuroblastoma cells die. We predict that the IC50 of cisplatin in SK-N-AS cells will be extremely low at 0.4uM as provided by the supplier (Michaels Lab, UK). The IC50 in SK-N-ASrCDDP500 is predicted to be 9.6uM. If lncRNA UCA1 plays a significant role in inducing cisplatin resistance, then we expect the knockdown of lncRNA UCA1 to decrease percent cell viability and IC50in both cells. An overexpression of lncRNA UCA1 would increase the percent cell viability and IC50 in both cells.
| Type of Experiment |
SK-N-ASrCDDP500 cells (resistant) |
SK-N-AS cells (susceptible) |
| lncRNA UCA1 KD |
% viability decrease |
% viability remains the same or slightly lower |
| lncRNA UCA1 Overexpressed |
% viability higher than control resistant cells |
% viability higher than control susceptible cells but lower than resistant cells |
| Control (no change) |
% viability higher than susceptible ells |
Extremely to zero % viability in cisplatin treatment over IC50 |
Figure 3. Change in cisplatin resistance by knockdown or overexpression of lncRNA UCA1
Differential Gene Expression Analysis
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 (Wang et al, 2017). This includes a study performed by Wang et al, looking at microarray mRNA expression analysis of transfected UCA1 cells compared to control (2008). 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 (Fan et al, 2014). Similar results have been found in ovarian cancer, including a study by Wang et al that focuses on the change in expression of SRPK1 in the presence of UCA1 and cisplatin resistance (2015). The gene expression analysis profiles continue to show similar changes in expression of genes in other cancers, including cervical, lung and bladder cancer (Wang et al, 2017). If our hypothesis is correct, then we would expect to see similar changes in expression of genes in neuroblastoma cell lines.
Therefore, we predict expression analysis 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 knockdown cisplatin resistant cell lines and cisplatin susceptible cell lines, as shown in Figure 5.
Results
Potential Results 1: Hypothesis is not rejected and predictions are confirmed
Cell Viability
Expected cell viability results for potential results 1: relative % cell viability is in accordance with our prediction
Figure 4: percent cell viability of neuroblastoma cells measured in increasing concentration of cisplatin. Figures on the left show percent viability when NS-K-ASrCDDP500 cells are treated with si-UCA1 or overexpressed with lncRNA UCA1. Figures on the right show percent viability when NS-K-AS cells are treated with si-UCA1 or overexpressed with lncRNA UCA1.
We can conclude that high levels of expression of UCA1 is necessary and sufficient to increase the percent viability of both cisplatin resistant and susceptible cells when treated with various doses of cisplatin. We can also conclude that lncRNA UCA1 is not necessary for cellular functions other than resistance mechanism to cisplatin if there is no significant change in percent cell viability between control NS-K-AS cells and NS-K-AS with knockdown of UCA1. We may infer that lncRNA UCA1 regulates a molecular mechanism that induces cisplatin resistance such that higher levels of expression of lncRNA UCA1 results in resistant cells to become more resistant and susceptible cells to become resistant.
Our results support the conclusion made by Fan et al. (2014) that lncRNA UCA1 increases chemoresistance in cancer cells. We cannot conclude that the significance of UCA1 expression will be the same in vivo. An experiment could be done in vivo by performing lncRNA UCA1 knockdown and overexpression in neuroblastoma mouse models.
Differential Gene Expression Analysis
In the case where the hypothesis is confirmed with an upregulated expression of lncRNA UCA1 causes increased resistance to cisplatin in neuroblastoma cell lines, we would expect to see changes in expression levels of RNA in both the knockdown and overexpression cases relative to control (Figure 5). From this, we can conclude that lncRNA UCA1 is both sufficient and necessary to change the expression levels of RNA in the cell lines studied. If we observe similar changes in RNA compared to expression profiles performed on other cancers (REFS), then we can infer that the molecular mechanism is the same. For example, if we observe an upregulation of Wnt6 mRNA, we could infer that lncRNA UCA1 is involved in cisplatin resistance through interactions with the Wnt6 in the Wnt pathway (REF). However, we cannot make conclusions about this pathway without further experimentation. One potential experiment includes knocking down and over expressing the genes of interest (genes found in molecular pathway of UCA1 inducing cisplatin resistance in other cancers) and then performing a 3C experiment to see the gene interaction.
| Type of Experiment |
Genes with fold change > 2 in cisplatin resistant cell lines |
Genes with fold change > 2 in cisplatin susceptible cell lines |
| lncRNA UCA1 Knocked down |
No change in gene expression |
No change in gene expression |
| lncRNA UCA1 Overexpressed |
Compared to both control and cisplatin resistant + no change in UCA1:
Highly Upregulated:
Wnt6, CYP1A1, AURKC
Highly Downregulated:
MBD3, SRPK1 |
Upregulated:
Wnt6, CYP1A1, AURKC
Downregulated:
MBD3, SRPK1
This may be in similar amounts to cisplatin resistant cell lines with no change to UCA1 |
| No change
in lncRNA UCA1 |
Upregulated:
Wnt6, CYP1A1, AURKC
Downregulated compared to susceptible cell lines control:
MBD3, SRPK1 |
CONTROL for the normal expression of all genes
(including: Wnt6, CYP1A1, AURKC,
MBD3, SRPK1) |
Figure 5: Table of possible results if the hypothesis and prediction is confirmed. All results are relative to the control for the normal expression of RNA with cisplatin susceptible cell lines. The genes described are a subset of the genes that were found to have >2 fold change in past experiments (REF), however, is not a complete set.
Result 2: Hypothesis rejected but prediction confirmed for overexpression of lncRNA UCA1 in susceptible neuroblastoma cells
Cell Viability
Induced lncRNA UCA1 expression increases cell viability in susceptible cells, but change in percent viability in other conditions is not significant.
Figure 6: percent cell viability of neuroblastoma cells measured in increasing concentration of cisplatin. Figures on the left show percent viability when NS-K-ASrCDDP500 cells are treated with si-UCA1 or overexpressed with lncRNA UCA1. Figures on the right show percent viability when NS-K-AS cells are treated with si-UCA1 or overexpressed with lncRNA UCA1.
We can conclude that high expression of lncRNA UCA1 is sufficient to increase percent cell viability of cisplatin susceptible NS-K-AS cells, but not in already resistant NS-K-ASrCDDP500 cells. Knockdown of UCA1 is not sufficient to reduce the percent cell viability in resistant cells.
Differential Gene Expression Analysis
In differential gene expression analysis, where the predictions are satisfied in the cases where genes are overexpressed but not in the knockdown, we would expect to see changes in expression outlined in figure 7. We can conclude that overexpressing lncRNA UCA1 is sufficient and knocking down lncRNA UCA1 is not sufficient, to change expression levels of RNA in cisplatin resistant cell lines compared to cisplatin susceptible cell lines.
| Type of Experiment |
Genes with fold change > 2 in cisplatin resistant cell lines |
Genes with fold change > 2 in cisplatin susceptible cell lines |
| lncRNA UCA1 KD |
No change in expression |
No change in expression |
| lncRNA UCA1 OX |
Gene expression changes (upregulated or downregulated) |
Most likely will see no change in expression, similar to control for cisplatin resistant cell lines. Might see slight changes in gene expression. |
| No change
in lncRNA UCA1 |
No change in expression of one or more genes |
CONTROL for the normal expression of all genes
(including: Wnt6, CYP1A1, AURKC,
MBD3, SRPK1) |
Figure 7: Table of possible results if the hypothesis is rejected but prediction is confirmed for changes in expression in lncRNA UCA1 overexpressed conditions. All results are relative to the control for the normal expression of RNA with cisplatin susceptible cell lines. The genes described are a subset of the genes that were found to have >2 fold change in past experiments (REF), however, is not a complete set.
Remarks
These results would suggest that there are mechanisms other than lncRNA UCA1 in cisplatin resistance such that the function of UCA1 is redundant. We cannot conclude, however, that lncRNA UCA1 has no effect in cisplatin resistance in control NS-K-ASr. It is possible that there are other lncRNA that have similar effects that are highly expressed in resistant cells. For example, the alteration of the wnt pathway is a common way of inducing cisplatin resistance and several lncRNA such as HOTTIP, MALAT, and MEG3 have been identified to affect this pathway (Hu et. al, 2018). Further investigation can be done by studying the pathways that lncRNA UCA1 may function in and compare with other lncRNAs.
Result 3: Hypothesis rejected with no change in cell viability and expression profile
Cell Viability
Percentages of cell viability of neuroblastoma cells before and after induced changes in UCA1 expression are the same.
Figure 8: percent cell viability of neuroblastoma cells measured in increasing concentration of cisplatin. Figures on the left show percent viability when NS-K-ASrCDDP500 cells are treated with si-UCA1 or overexpressed with lncRNA UCA1. Figures on the right show percent viability when NS-K-AS cells are treated with si-UCA1 or overexpressed with lncRNA UCA1.
From these results, we could conclude that lncRNA UCA1 is not necessary or sufficient for percent cell viability of cells treated with cisplatin.
Differential Gene Expression Analysis
| Type of Experiment |
Genes with fold change > 2 in cisplatin resistant cell lines |
Genes with fold change > 2 in cisplatin susceptible cell lines |
| lncRNA UCA1 KD |
No change in expression |
No change in expression |
| lncRNA UCA1 OX |
No change in expression |
No change in expression |
| No change
in lncRNA UCA1 |
No change in expression |
CONTROL for the normal expression of all RNA
(including: Wnt6, CYP1A1, AURKC,
MBD3, SRPK1) |
Figure 9: Table of possible results if both the hypothesis and prediction are rejected. All results are relative to the control for the normal expression of RNA with cisplatin susceptible cell lines. The genes described are a subset of the genes that were found to have >2 fold change in past experiments, however, is not a complete set.
From these results, we could conclude that lncRNA UCA1 is not necessary or sufficient to change expression levels in the cell lines.
Remarks
We infer that lncRNA UCA1 is not involved in the pathways that induce or reduce cisplatin resistance in our neuroblastoma cell lines. We cannot conclude, however, that lncRNA UCA1 has no function in neuroblastoma, because it is still possible that it is involved in other ways, apart from cisplatin resistance. Further experiments could be done to test cisplatin resistance in other neuroblastoma cell lines as well as other potential functions of lncRNA UCA1 in neuroblastoma.
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