Long noncoding RNA UCA1: Function in Cisplatin Resistance in Neuroblastoma Cell Lines
Shannah Fisher and Joanne Lim
Background
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). Cisplatin, a platinum based cytotoxic drug, is one of the most common chemotherapy agents used to treat neuroblastoma (Dasari and Tchounwou, 2014). However, a large proportion of patients are resistant to cisplatin-based therapies either at the beginning of treatment or after ongoing exposure to treatment (Galluzzi, 2012). 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).
Cisplatin resistance is not only found in neuroblastoma but also in other cancers, including breast, cervical, lung and bladder cancer (Dasari and Tchounwou, 2014). 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). Several studies have also 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).
Long non-coding RNAs (lncRNAs) are evolutionary conserved gene transcripts of over 200 nucleotides that do not encode proteins (Ponting et al., 2009). They are involved in regulation of gene expression at the transcriptional, post-transcriptional, and translational levels (Moran et al., 2012) either by directly interacting with a target gene (cis-acting) or by interacting with transcription factors (trans-acting) (Ponting et al., 2009). A variety of lncRNAs such as HOTAIR, MALAT1, and UCA1 have been found to play an important role in carcinogenesis, metastasis, prognosis and treatment. In particular, Urothelial cancer-associated 1 (UCA1), a lncRNA that has a regulatory function in proliferation of cells, has been found to play a major role in development of cisplatin resistance in bladder cancer, breast cancer, hepatocellular carcinoma, ovarian cancer, and tongue squamous cell carcinoma (Wang et al., 2017).
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, where 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 and ovarian cancer (Fan et al, 2014; Wang et al, 2015).
Relevance
The function of lncRNA UCA1 in cisplatin resistance has never been studied neuroblastoma, posing a 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, we cannot assume that the effect of UCA1 will be conserved across all cancer types, including neuroblastoma. For example, lncRNA MALAT1 functions 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, it is possible that lncRNA UCA1 may take a unique function depending on the cancer cell type.
Therefore, we propose a study that will determine the function of lncRNA UCA1 in cisplatin resistance specific to neuroblastoma cell lines and identify the changes in expression levels of various genes at high levels and in absence of lncRNA UCA1. 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). In addition, RNA directed therapy could be used in patients who are highly resistant to the treatment, reducing the discomfort of patients by permitting a lower treatment dosage (Ayers and Vandesompele, 2017). Lastly, a UCA1 biomarker could be quantified in patients through RT-qPCR assays and provide pre-emptive knowledge to oncologists on the best drug combination treatment for each patient (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 (Wang et. al, 2017). We hypothesize that upregulated expression of lncRNA UCA1 is associated with increased resistance to cisplatin in neuroblastoma cell lines, accompanied by changes in the expression of other genes. This will further determine if the role of lncRNA UCA1 is conserved in neuroblastoma compared to other cancer types that have already been studied.
Experimental Plan
Figure 1: Overview of Experimental Plan
Cisplatin resistance after induced changes in lncRNA UCA1 expression
In order to assess whether upregulated expression of lncRNA UCA1 is associated with resistance to cisplatin in neuroblastoma, we will manipulate the levels of UCA1 via knockdown and overexpression in neuroblastoma cell lines. A cell viability assessment will be performed on susceptible SK-N-AS cell line that will be overexpressed with UCA1 and resistant SK-N-ASrCDDP500 cell line that will knockdown expression of UCA1.
Other experiments investigating the function of lncRNA have mapped lncRNA binding sites to the genome after manipulating its expression level (Luo, 2016). However, this technique only gives information on how lncRNA regulates gene expression in cis. Alternatively, the system we propose that manipulates UCA1 levels, will allow us to study the changes in resistance to cisplatin by UCA1, and identify the genes that are potentially affected by UCA1 expression that may lead to resistance. The challenge to our experiment is that when we overexpress lncRNA UCA1 via UCA1 gene transfection, isoforms of the UCA1 gene other than lncRNA UCA1 can be formed. These isoforms may have an effect on cisplatin resistance mechanisms and expression of genes. As a strategy to overcome this challenge, we will silence the isoforms.
As a preliminary experiment, the level of UCA1 expression in cisplatin susceptible SK-N-AS cell line and cisplatin resistant SK-N-AsrCDDP500 cell line will be measured. If lncRNA UCA1 is involved in inducing cisplatin resistance in neuroblastoma, we would expect higher levels of UCA1 in SK-N-AsrCDDP500 cells than in SK-N-AS cells. For the purpose of this project, we design our manipulative experiments assuming that higher levels of UCA1 is observed in the resistant SK-N-ASrCDDP500. If we observe higher UCA1 in susceptible cells, then we should knockdown UCA1 in susceptible cells instead.
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 knockdown in cisplatin resistant cell lines and lncRNA UCA1 overexpression in cisplatin susceptible cell lines. RNA will also be extracted from susceptible and resistant cell lines with no change in lncRNA UCA1, which serves as a control. All of the extracts will be sequenced and analysed. In comparison to microarrays and qPCR, RNA-seq would allow us to look at differential expression of a broader dynamic range of genes. A summary of the steps and tools that will be used are outlined in Figure 2, with a detailed pipeline in Materials and Methods.
Materials and Methods
Cell lines: Susceptible and Resistant
Both cell lines will be purchased from the Michaelis Lab, UK. SK-N-AS cells are parental cells of SK-N-ASrCPPD500, which have gained cisplatin resistance. We chose the same lineage of neuroblastoma with the effort to keep the cells to be as genetically similar as possible.
lncRNA UCA1 Overexpression
lncRNA UCA1 will be overexpressed in the susceptible SK-N-AS cell line via plasmid DNA transfection of the UCA1 gene. Both plasmid DNA transfection and RNA transfection are valid methods for overexpression of RNA. However, plasmid DNA transfection is a better method because lncRNA UCA1 will be produced for multiple rounds of replication from the transfected plasmid (Hayashi et al., 2010). In an RNA based transfection, the overexpression of lncRNA UCA1 will rapidly decrease as RNA degrades. A recombinant plasmid of pcDNA-UCA1 will be constructed by inserting UCA1 gene into pcDNA3.1. 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. Cells transfected with pcDNA3.1 will also be tested as a negative control to ensure that the transfection procedure does not alter the expression of UCA1 (Wang et. al, 2014).
lncRNA UCA1 Knockdown
Short hairpin RNA (ShRNA) will be used to knockdown lncRNA UCA1 because it is a powerful silencing technique that imitates the endogenous RNA interference mechanism and allows for long term knockdown (Luo, 2016). The scrambled shRNA control (Si-NC) and siRNA that targets lncRNA UCA1 will be purchased from Thermo Fisher Scientific to show that the shRNA protocol does not alter the expression of UCA1. sh-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. To prevent UCA1 isoforms from affecting our results, shRNA against isoform RNAs will be transfected as well.
Cisplatin treatment and cell viability assessment
The cisplatin treatment procedure outlined by Piskareva (2015) will be adapted. SK-N-AS cells and SK-N-ASrCDDP500 cells will be seeded at 10^4 cells/mL on two separate 96-well plates with 100µL medium per well. The plate will be incubated overnight and will be treated with cisplatin at multiple concentrations the following day. Cell proliferation will be monitored over 5 days. In order to assess the degree to which the cells gain cisplatin resistance, we will perform the MTT assay as described by Wang et al (2008) and therefore measure cell viability.
RNA Extraction
RNA will be extracted from each of the conditions with TRIzol reagent, with twelve replicates for each condition, as recommended by Mortazavi, et al (2008). 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 (Thermo 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. In order to have a high enough coverage, we will generate ~15-25 million reads per sample, as recommended by Mortazavi, et al (2008). Sequence read quality will be analyzed with the pass/fail metrics of the program FASTQC.
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
We predict that knocking down lncRNA UCA1 in SK-N-ASrCDDP500 resistant cell line will result in a rapid decrease in percent cell viability over increasing concentrations of cisplatin and the overexpression of lncRNA UCA1 in SK-N-AS cell lines will result in an increase in percent cell viability.
| Type of Experiment |
SK-N-ASrCDDP500 cells (resistant) |
SK-N-AS cells (susceptible) |
| lncRNA UCA1 KD |
% viability rapid decrease |
|
| lncRNA UCA1 OX |
|
% viability higher than control susceptible cells |
| Control (no manipulation) |
% viability higher than susceptible cells |
Extremely low to zero % viability |
Figure 3. Change in cisplatin resistance by knockdown or overexpression of lncRNA UCA1
Differential Gene Expression Analysis
Experiments performing gene expression analysis after manipulating levels of lncRNA UCA1 in the presence and absence of cisplatin continue to show similar changes in RNA expression profiles across cancers that have been studied (Wang et al, 2017). In neuroblastoma, we predict expression analysis to follow suit with other cancers and show a decrease in expression levels in Wnt6, CYP1A1, and AURKC and an increase in expression levels in MBD3 and SRPK1 in the cisplatin resistant knockout lncRNA UCA1 cell line compared to its resistant cell line control (Figure 5). We predict the opposite pattern in UCA1 overexpressed cisplatin susceptible cell line compared to its control, as shown in figure 5.
Results
Result 1: Hypothesis is not rejected and predictions are confirmed
We would see a higher percent cell viability when UCA1 is knocked down in the resistant cell line as concentration of cisplatin increases compared to its control, and a higher percent cell viability when UCA1 is overexpressed in the susceptible cell line compared to its control (Figure 4). We would also see changes in expression levels of RNA from the expression analysis in both the knockdown and overexpression of lncRNA UCA1 relative to controls of their respective cell lines (Figure 5). The expression profile of resistant cell line control and susceptible cell line with overexpression would be similar. We can conclude that high levels of lncRNA UCA1 is both sufficient and necessary to increase the percent viability of neuroblastoma cells with increasing doses of cisplatin and to change the expression levels of RNA in the cell lines studied.
SK-N-ASrCDDP500 (resistant) SK-N-AS (susceptible)
Figure 4: Percent cell viability of neuroblastoma cells measured in increasing concentration of cisplatin. Percent viability is higher in control SK-N-ASrCDDP500 than knockdown of UCA1. Percent viability is higher in SK-N-AS overexpressed with UCA1 than the control SK-N-AS.
| Type of Experiment |
Expression levels compared to resistant SK-N-ASrCDDP500 control |
Expression levels compared to susceptible SK-N-AS control |
| lncRNA UCA1 Knocked down in SK-N-ASrCDDP500 (resistant) |
Change in RNA expression
For instance:
Downregulated:
Wnt6, CYP1A1, AURKC
Upregulated:
MBD3, SRPK1 |
may have similar RNA expression patterns |
| lncRNA UCA1 Overexpressed in SK-N-AS (susceptible) |
Similar RNA expression |
Change in expression
For instance:
Upregulated:
Wnt6, CYP1A1, AURKC
Downregulated:
MBD3, SRPK1 |
Figure 5: Table of possible result 1 for the expression profile analysis
Remarks
We may infer that lncRNA UCA1 regulates a molecular mechanism that induces cisplatin resistance such as silencing of a tumor suppressor gene or inducing expression of multidrug resistant proteins (Wang et al., 2017). Further, if we observe similar changes in RNA expression profiles of neuroblastoma to those in other cancers, we could infer that lncRNA is involved in cisplatin resistance in neuroblastoma in the same pathway found in other cancers. For example, if we observe a similar upregulation of Wnt6 mRNA in neuroblastoma to that of lung cancer, we may infer that lncRNA UCA1 is involved in cisplatin resistance in neuroblastoma by interactions with Wnt6 in the Wnt pathway (Wang et al., 2008). This is under the assumption that the genes that change in expression are involved in cisplatin resistance rather than unrelated cellular functions. For further investigation into the function of UCA1 and the pathway it is involved in, we can use RNA-TRAP to assess the degree to which lncRNA UCA1 interacts with specific genes and postulate its role in regulating expression of various genes in cis. In addition, we can identify the proteins that lncRNA UCA1 interacts with via crosslinking protein to lncRNA followed by mass spectrometry.
Result 2: Prediction confirmed for overexpression of lncRNA UCA1 in susceptible neuroblastoma cells
We would observe no change in percent cell viability when UCA1 is knocked down in SK-N-ASrCDDP500 compared to its control, but an increase in percent cell viability when UCA1 is overexpressed in SK-N-AS compared to its control. 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 high expression of lncRNA UCA1 is sufficient to increase percent cell viability and change the expression levels of RNA of cisplatin susceptible SK-N-AS, but not necessary to maintain percent cell viability and to change the expression levels of RNA in an already resistant SK-ASrCDDP500.
SK-N-ASrCDDP500 (resistant) SK-N-AS (susceptible)
Figure 6: percent cell viability of neuroblastoma cells measured in increasing concentration of cisplatin.
| Type of Experiment |
Expression levels compared to resistant SK-N-ASrCDDP500 control |
Expression levels compared to susceptible SK-N-AS control |
| lncRNA UCA1 Knocked down in SK-N-ASrCDDP500 (resistant) |
Similar RNA expression |
may have similar RNA expression |
| lncRNA UCA1 Overexpressed in SK-N-AS (susceptible) |
Similar RNA expression |
Change in expression
For instance:
Upregulated:
Wnt6, CYP1A1, AURKC
Downregulated:
MBD3, SRPK1 |
Figure 7: Table of possible results for the expression profile analysis of the second potential result.
Remarks
This result supports the hypothesis that upregulated levels of lncRNA UCA1 may be associated with increased cisplatin resistance, but RNA therapy against only UCA1 would not be sufficient to treat neuroblastoma. We infer that lncRNA UCA1’s function is redundant to other lncRNAs that induce cisplatin resistance. This is assuming other lncRNAs could have similar to identical functions in regulating gene expression and that the expression of these other lncRNAs are high and saturated in the control resistant SK-N-ASrCPPD500 cells. For example, the alteration of the wnt pathway is a common way of inducing cisplatin resistance and several lncRNA such as HOTTIP and MALAT have been identified to affect this pathway (Hu et. al, 2018). As a next step, we could compare the expression profiles resulting from manipulation of putative lncRNAs with that of lncRNA UCA1. In addition, we could study the proteins that lncRNA UCA1 interacts with via CHART technique and compare with other lncRNAs.
Result 3: Hypothesis rejected with no change in cell viability and expression profile
In the case where there is no change in percent cell viability and RNA expression profile after manipulation of UCA1 levels, our hypothesis will be rejected. We conclude that lncRNA UCA1 is neither sufficient nor necessary to increase percent cell viability and change the RNA expression profile in the neuroblastoma cell lines of study treated under multiple concentrations of cisplatin. We cannot conclude that lncRNA UCA1 has no function in neuroblastoma, because it is still possible that it is involved in other ways.
SK-N-ASrCDDP500 (resistant) SK-N-AS (susceptible)
Figure 8: percent cell viability of neuroblastoma cells measured in increasing concentration of cisplatin.
| Type of Experiment |
Expression levels compared to resistant SK-N-ASrCDDP500 control |
Expression levels compared to susceptible SK-N-AS control |
| lncRNA UCA1 Knocked down in SK-N-ASrCDDP500 (resistant) |
Similar RNA expression |
Different expression
- This will be the same differences in expression that were found when comparing the resistant and susceptible controls
|
| lncRNA UCA1 Overexpressed in SK-N-AS (susceptible) |
Different expression
- This will be the same differences in expression that were found when comparing the resistant and susceptible controls
|
Similar RNA expression |
Figure 9: Table of results if both the hypothesis and prediction are rejected.
Assuming that lncRNA UCA1 has all of the requirements to perform its normal functions in the in vitro experiment, we can infer that lncRNA UCA1 does not have a function in the pathways that induce or reduce cisplatin resistance in our neuroblastoma cell lines. Previous studies have found other lncRNAs apart from UCA1 can induce cisplatin resistance, so it is possible that the UCA1 knockdown resistant cell line is resistant to cisplatin because of other lncRNAs and proteins. To investigate these, RNAs expressed differently in SK-N-ASrCDDP500 control compared to SK-N-AS control could be investigated using a similar method to this experiment. Once confirmed of their potential involvement in cisplatin resistance, we can further study their function by assessing the proteins they interact with and the genes that they regulate expression of, as described in result 1 and 2. Additionally, if we observed higher levels of UCA1 in the resistant cell line control compared to the susceptible control, we might infer that upregulated UCA1 is a consequence of cisplatin resistance rather than the cause.
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