Analysis of lncRNA UCA1‐related downstream pathways and molecules of cisplatin resistance in lung adenocarcinoma

Abstract Background To analyze the lncRNA UCA1‐related downstream pathways and molecules of cisplatin resistance in lung adenocarcinoma. Methods We constructed overexpression and siRNA vectors targeting UCA1 and TXNIP and then used next‐generation sequencing to compare the UCA1 overexpression and negative control from A549 cell. Results It shown that 647 upregulated mRNAs and 633 downregulated differentially expressed mRNAs‐related UCA1, and the top ten upregulated mRNAs were CPD, AC007192.1, TGOLN2, LGR4, TFPI, CYP1B1, TOMM6, HLA‐B, SLC35F6, and TOP2A, and top ten downregulated mRNAs were TXNIP, SESN2, STC2, HSPA1A, MMP10, CHAC1, DNAJB1, AC004922.1, ATF3, and GABARAPL1. We found TXNIP mRNA expression level was the most significantly downexpressed mRNA. TXNIP mRNA expression level of LAD tissues was clearly lower than the adjacent tissues. UCA1 expression level of cisplatin insensitive group was markedly higher than that of cisplatin‐sensitive group, while TXNIP mRNA expression level of cisplatin insensitive group was clearly lower than that of cisplatin‐sensitive group. Compared to the BEAS‐2B, TXNIP mRNA expression level cut down in A549 and A549/DDP cell and that of A549/DDP cell was lower than A549 cell. After UCA1 overexpression, TXNIP mRNA obviously decreased, while proliferation ability and IC50 of A549 heightened. After knocking down UCA1, TXNIP mRNA was significantly increased, while proliferation ability and IC50 of A549/DDP lowered. PPI analysis result showed that TXNIP could interact with multiple proteins such as TXN, DDIT4, and NLRP3. Conclusion UCA1 promoted cisplatin resistance by downregulating TXNIP expression in LAD, and TXNIP could interact with multiple proteins. So, UCA1/TXNIP axis might affect cisplatin resistance in LAD.


| INTRODUC TI ON
Lung adenocarcinoma (LAD) is one of the most common types of NSCLC, accounting for 40% of lung cancer. It is on the rise for LAD had the biological characteristics of early distant metastasis, and most patients are diagnosed at an advanced stage. Therefore, cisplatin-based combination chemistry still played an important role in its comprehensive treatment program. 1 With the wide application of cisplatin, it would inevitably cause tumor cells to develop resistance to it, so that the chemotherapy effect was significantly reduced. 2 Studies had shown that 70%-80% of patients can temporarily relieve the disease in the early stage of chemotherapy, but long-term application would produce resistance to cisplatin, resulting in a recurrence rate of more than 60%, while drug resistance of recurrent lung cancer is significantly heightened, chemotherapy drug remission rate is less than 30%, 3 the current chemotherapy rate of patients with advanced LAD is only 30%-40%, and the 5-year survival rate is less than 15%. 4 According to the American Cancer Society survey, more than 90% of cancer patients' deaths are related to tumor drug resistance to varying degrees. Once cancer cells are resistant to cisplatin, they will be resistant to doxorubicin, vinblastine, fluorouracil, mitomycin, etc So, many first-line chemotherapeutic drugs become multi-drug resistance to cancer cells, which is particularly harmful. 5 UCA1 was first confirmed to be highly expressed in bladder cancer tissues 6 and shown to be highly expressed in other cancers. We had used high-throughput lncRNA microarray technology to compare LAD cisplatin-resistant A549/DDP cells and cisplatin-sensitive A549 cells and obtained differential lncRNA expression profiles of LAD cisplatin-resistant cell. Among them, we confirmed UCA1 was an upregulated lncRNA in these candidate lncRNAs by microarray and qPCR.
Subsequently, our qPCR showed that the expression of UCA1 in LAD was significantly higher than that in adjacent tissues, which was consistent with the related reports, 7,8 indicating that UCA1 might play an important role in the development of LAD. It had been reported in the literature that UCA1 was involved in the cisplatin resistance mechanism of ovarian cancer and bladder cancer. 9,10 Therefore, we speculated that UCA1 might be a novel lncRNA molecule that plays an important role in LAD resistance to cisplatin. How would UCA1 regulate the cisplatin resistance mechanism of LAD through downstream signaling pathways and molecules? These issues deserved our in-depth study, but no relevant literature reports had been reported so far. So, we used a high-throughput next-generation sequencing (NGS) to compare the UCA1 overexpression (A549 OE) and negative control from A549 cell (A549 OE NC) samples to analyze lncRNA UCA1-related downstream pathways and molecules of cisplatin resistance in LAD.

| Human LAD tissue specimen
Human LAD tissue specimens were collected from the First Affiliated Hospital of Wenzhou Medical University from 2010 to 2015.
Specimens were obtained through lung puncture biopsy, surgical resection, and metastatic lymph node biopsy and were strictly identified by the pathology department. All tissue specimens must meet the following conditions: Patients with primary LAD and clinical stage are IIIB to IV. First-line chemotherapy was cisplatin 25 mg/m 2 on the first 1-3 days, combined with gemcitabine 1000 mg/m 2 or paclitaxel 80 mg/ m 2 on days 1 and 8. The 21 days were one cycle, and each patient was treated for 3-4 cycles. According to medical imaging examinations such as CT and MRI and detection of serum tumor markers and the RECIST standard, they were divided into "cisplatin-sensitive group" (complete response + partial response) and "cisplatin insensitive group" (progression), and a total of sensitive groups were collected. There were 25 specimens and 32 insensitive specimens. Tissue specimens were stored in liquid nitrogen before use.

| Cell culture of LAD cell line and cisplatinresistant strain
A549 cells and cisplatin-resistant cell line A549/DDP were added with appropriate amount of RPMI1640 medium containing 10% calf serum and gently pipetted into a single-cell suspension with a pipette, and the cell suspension was transferred to a cell culture flask with a pipette. The cells were cultured in a 5% CO 2 incubator at 37°C, and A549/DDP cells were required to add cisplatin at a concentration of 2 μg/mL to maintain drug resistance. The medium was changed every 2-3 days. The cell growth state was observed to be good, and the cell passage was performed at a density of 70%-90%.
Transfections were performed by seeding 2 × 10 5 cells in 6-well plate. After 24 hours, these mediums were replaced, and the cells

| Cell proliferation assay
Cell Counting Kit-8 (CCK-8, Corning Corporation) was evaluated to cell proliferation assay comply with the manufacturer's standard operating procedure. 11 In short, 3000 cells were resuspended and inoculated in 96-well plates in the presence of 10% FBS for 1 week.
On the second day, the cells were incubated with 10 μL CCK-8 for 1 hour, and the absorbance was measured at 450 nm with TECAN (Germany) on 0, 24, 48, and 72 hours.

| Cisplatin sensitivity test
The cell inoculation density was 2000/well. After 24 hours of cell attachment, 100 μL of complete medium (including cisplatin) was added to each well. The concentration of cisplatin was 1, 2, 4, 10, and 25 μg/mL, and only the zeroing hole of the medium and the control hole of the single-cell suspension were set to zero. After 48 hours of culture, the complete medium containing 10% CCK8 was used instead of the medium and kept training for 45 minutes. The microplate reader detects the absorbance at 450 nm wavelength, cell viability % = (A plus-A blank)/(A0 plus drug-A blank) × 100%, using the SPSS18.0 software profit regression model to calculate the IC50 of the cell. Each group was provided with three sets of repeat holes.

| Sequencing and data analysis
Through NanoDrop ND-1000 concentration measurement, 1-2 µg of total RNA was selected from each sample for the construction of an   Figure 1). The overexpression level of UCA1 in A549 UCA1 siRNA group (8013.33 ± 234.45) was clearly lower than that in the A549 UCA1 siRNA NC groups (t = 13.45, P = .0002). ***P < .001, ###P < .001 and calculated the expression differences between the gene level and transcript level, respectively. Differentially expressed genes.
New genes/transcript predictions were assembled, merged, and compared with the official annotation information by StringTie for each sample separately. They were obtained through Ballgown calculations, and the new transcripts were predicted by CPAT. 15,16 We also used GO function significance enrichment analysis, pathway significance enrichment analysis, and other data mining analysis.

| qPCR
Take 0.1 g of tissue, ground it thoroughly with liquid nitrogen (to powder) or discard the medium with 1-5×10 7 cells, and rinse with prechilled PBS twice. Total RNA was extracted from cell or tissues samples using TRIzol reagent (Invitrogen life technologies) and then extracted into cDNA according to the manufacturer's instructions using Thermo Scientific RT Kit (Thermo Scientific). These different mRNA expressions were measured by qPCR on the ABI 7500 analyzer. The primers of these genes are shown in Table 1. PCR was carried out in a total volume of 20 μL, including 10 μL SYBR premix (2×), 2 μL cDNA template,

| Statistical methods
Statistical analysis was performed for the comparison of two groups using Student's t test and one-way ANOVA test for differences in variables between the groups from normal distribution data. The threshold value we used to designate differentially expressed mRNAs and mRNAs was a fold change of ≥2.0 or ≤0.5 (P < .05). Differences with P < .05 were considered statistically significant in both cases.

| Construction of lentivirus-mediated UCA1 overexpression and siRNA vector
The overexpression level of UCA1 in A549 UCA1 OE group (8013.33 ± 234.45) was significantly higher than that in the A549

F I G U R E 3 GO analysis of downregulated mRNAs-related UCA1.
The genes corresponding to the downregulated mRNAs involved in biological processes, such as response to organic substance, response to stress, cellular response to organic substance, system development, regulation of cell death, and so on. Some genes involved in cellular components, such as extracellular space, extracellular region, extracellular matrix, cytoplasm, actin cytoskeleton, and so on. Some different genes involved in molecular functions, such as protein binding, peptide binding, amide binding, molecular function regulator, transcription factor activity, RNA polymerase II proximal promoter sequence-specific DNA binding, and so on UCA1 OE NC groups (t = 15.26, P = .0001, Figure 1). The overexpression level of UCA1 in A549 UCA1 siRNA group (8013.33 ± 234.45) was clearly lower than that in the A549 UCA1 siRNA NC groups (t = 13.45, P = .0002, Figure 1). These results showed that we had successfully established UCA1 overexpressing and siRNA cell lines.

| Quality assurance of RNA and quality control results of mRNA sequencing
According to the OD of A260/A280 ratio should be close to 2.0 for pure RNA (ratios between 1.8 and 2.1 were acceptable). The OD A260/A230 ratio should be more than 1.8 and Table 2, so our RNA quantification was passed. Generally, Q30 ≥ 80% indicates that the sequencing quality is extremely high. Our findings showed that the results of Q30 were higher than 90%, and hinted sequencing quality was qualified (shown in Table 3).

| Overview of mRNA profiles
The results showed that the expression profiles of mRNAs indicated that they were differentially expressed (fold change ≥ 2.0 or ≤0.5; P < .05) between A549 OE and A549 OE NC group. Among these, 647 mRNAs were found to be upregulated more than two-fold in the A549 OE group compared to the A549 NC group, while 633 mRNAs were downregulated more than two-fold (P < .05, Figure 2A Table 4).

| GO analysis
The genes corresponding to the downregulated mRNAs were involved in biological processes, such as response to organic substance, response to stress, cellular response to organic substance, system development, regulation of cell death, and so on. Some genes were involved in cellular components, such as extracellular space, extracellular region, extracellular matrix, cytoplasm, actin cytoskeleton, and so on. Some different genes were involved in molecular functions, such as protein binding, peptide binding, amide binding, molecular function regulator, transcription factor activity, RNA polymerase II proximal promoter sequencespecific DNA binding, and so on (shown in Figure 3).
The genes corresponding to the upregulated mRNAs were involved in biological processes, such as regulated exocytosis, exocytosis, neutrophil degranulation, neutrophil activation involved in immune response, neutrophil mediated immunity, and so on. Some genes were involved in cellular components, such as vesicle, membrane-bounded organelle, extracellular exosome, extracellular vesicle, endomembrane system, and so on. Some different genes were involved in molecular functions, such as catalytic activity, hydrolase activity, nucleoside-triphosphatase activity, protein binding, pyrophosphatase activity, and so on (shown in Figure 4).
F I G U R E 4 GO analysis of upregulated mRNAs-related UCA1. The genes corresponding to the upregulated mRNAs involved in biological processes, such as regulated exocytosis, exocytosis, neutrophil degranulation, neutrophil activation involved in immune response, neutrophil mediated immunity, and so on. Some genes involved in cellular components, such as vesicle, membranebounded organelle, extracellular exosome, extracellular vesicle, endomembrane system, and so on. Some different genes involved in molecular functions, such as catalytic activity, hydrolase activity, nucleoside-triphosphatase activity, protein binding, pyrophosphatase activity, and so on

| Pathway analysis
These different genes involved into 22 upregulated pathways were identified, including lysosome, axon guidance, steroid biosynthesis, cell cycle, fatty acid metabolism, and so on. These different genes involved into 55 down-regulated pathways were identified, including IL-17 signaling pathway, legionellosis, cytokine-cytokine receptor interaction, rheumatoid arthritis, TNF signaling pathway, and so on (shown in Figure 5A,B).

| Real-time quantitative PCR validation
According to fold difference, gene locus, nearby encoding gene, and so on, we initially identified several interesting candidate mRNAs (including TGOLN2, GR4, TFPI, CYP1B1, TOMM6, TXNIP, SESN2, STC2, HSPA1A, and MMP10) and found that qPCR results of most candidate mRNAs were relative to NGS; see Figure 6A. The NGS shown that the TXNIP mRNA expression level was the most significantly downexpressed changed mRNA of these mRNAs (fold change = 15.27070, P = .005815).TXNIP mRNA expression level of LAD tissues was distinctly lower than the adjacent tissues (fold change = −6.623, t = −6.220, P = 1.53E−6); see Figure 6B. QPCR shown that UCA1 expression level in cisplatin insensitive group was markedly higher than that in cisplatin-sensitive group (t = 9.019, P < .0001), while TXNIP mRNA expression level in cisplatin insensitive group was clearly lower than that in cisplatin-sensitive group

| Interaction prediction between TXNIP and protein
The analysis results from https://strin g-db.org/ shows that TXNIP could interact with multiple proteins such as TXN, DDIT4, and NLRP3 ( Figure 8). These results showed that TXNIP might play a key role in cisplatin resistance of LAD by interacting with some proteins.

| D ISCUSS I ON
It was extremely complex about the mechanism of cisplatin resistance and involved into multiple genes, proteins, and several pathways. It was currently believed to be achieved mainly through these mechanisms, 18

| CON CLUS IONS
Our study ascertained UCA1 could regulate a lot of downstream pathways and target genes. UCA1 promoted cisplatin resistance by downregulating TXNIP expression in LAD, and TXNIP could interact with multiple proteins. So, UCA1/TXNIP axis might affect cisplatin resistance in LAD.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.