UCA1 was highly expressed in LUAD tissues and cells and was related to the prognosis
In the previous study, we screened the LncRNAs between A549/DDP cell and A549 cell through a high-throughput microarray. We found 984 LncRNAs were up-regulated more than two-fold in the A549/DDP group compared to A549 group, while 559 LncRNAs were down-regulated through the LncRNAs expression profiles and qRT-PCR. Among these, UCA1 was one of the significantly up-regulated genes. To further confirm this result, we detected the expression of UCA1 in A549 and A549/DDP cells, and found that UCA1 was high expressed in A549/DDP cells (t=10.35, P<0.001, Fig. 1A), which change trend was consistent with that of our previous study. Next, we investigated the UCA1 expression levels in LUAD tissues and normal adjacent tissues through TCGA database, and found that the expression of UCA1 was greatly higher in cancer tissues than in adjacent tissues (http://ualcan.path.uab.edu/cgibin/TCGAExResultNew2.pl?genenam=UCA1&ctype=LUAD, P<0.001, Fig. 1B).
In addition, to investigate the correlation between UCA1 expression and clinical characteristics of LUAD patients, the 433 patients from TCGA database were divided into two groups according to gene expression, and expression levels higher than the median were classified into the high expression group; otherwise, they were classified into the low expression group. The result showed that no correlations were noted between UCA1 expression and age, gender, T stage, N stage, smoking history, and tumor location. While UCA1 was significantly associated with M stage (P=0.019) and clinical stage (P=0.046, Table 2).Kaplan-Meier analysis showed that the overall survival time of patients with high UCA1 expression was significantly shorter than those with low expression level of UCA1(http://ualcan.path.uab.edu/cgi-bin/TCGA-survival1.pl?genenam=UCA1&ctype=LUAD, P=0.021, Figure 1C).
These results shown the role of UCA1 in LUAD cancer development and cisplatin resistance and the potential as a biomarker to predict poor prognosis and cisplatin resistance in LUAD patients.
UCA1 promoted cell proliferation and reduced sensitivity to cisplatin in LUAD cell line
RT-PCR showed that the RNA expression of UCA1 in overexpression group A549-Lenti-UCA1 was markedly elevated compared with the negative control group A549-Lenti-NC (t=54.71, P<0.001, Fig. 2A). The expression level of UCA1 decreased significantly in A549/DDP-Lenti-shUCA1 group than that in A549/DDP-Lenti-shNC group (t=95.10, P<0.001, Fig. 2B).
It was showed that the absorbance at 450 nm of A549-Lenti-UCA1 group was higher than that of A549-Lenti-NC cells at 24 h (t=15.03, P=0.0044), 48 h (t=7.248, P=0.0185), 72 h (t=3.390, P=0.0275), 96 h (t=4.477, P=0.0465), 120 h (t=12.00, P=0.0069) after overexpression of UCA1 (Fig. 2C). Under the effect of 2 μg/ml cisplatin, the absorbance values of A549-Lenti-UCA1 group were still higher than that of A549-Lenti-NC group(t=4.578, P=0.0102; t=10.33, P=0.0005; t=3.817, P=0.0316; t=5.391, P=0.0057; t=4.357, P=0.0121, Fig. 2C). In contrast, the absorbance of A549/DDP-Lenti-shUCA1 group at 450 nm at 48 h (t=5.358, P=0.059), 72 h (t=12.58, P=0.002), 96 h (t=10.21, P=0.0005) and 120 h (t=10.21, P=0.0005) were lower than those in A549/DDP-Lenti-shNC group (Fig. 2D). After 4 μg/ml cisplatin treatment, except for 24 h the absorbance, the proliferation levels of A549/DDP-Lenti-shNC cells were still stronger than that of UCA1 knockdown cell lines (t=4.165, P=0.0141; t=8.800, P=0.0009; t=4.935, P=0.0078; t=3.756, P=0.0198; Fig. 2D). Proliferating cell nuclear antigen (PCNA) was an auxiliary protein involved in DNA replication and has been confirmed to be an indicator to evaluate the proliferation status of tumor cells[23-25]. PCNA protein expression was significantly increased after UCA1 overexpression (t=2.990, P=0.0403, Fig. 2E) and was decreased after UCA1 knockdown (t=5.847, P=0.0043, Fig. 2F). These results demonstrated that UCA1 could promote the proliferation of LUAD cells and reduce sensitivity to cisplatin in LUAD cell lines.
UCA1 promoted migration and invasion of LUAD cells
We found that the number of A549-Lenti-UCA1 migrating cells passing through Transwell chamber was significantly higher than that in control group (t=2.596, P=0.0318, Fig. 3A). After treated with 2 μg/ml cisplatin, this trend was still obvious (t=8.352, P<0.0001, Fig. 3A). On the contrary, A549/DDP-Lenti-shUCA1 group had fewer migration cells than the A549/DDP-Lenti-shNC group (t=5.754, P=0.0004, Fig. 3B). The knockdown cells were treated with 4 μg/ml cisplatin, and the result was the same as without cisplatin treatment group (t=3.307, P=0.0107, Fig. 3B).
The results of the Matrigel invasion assays also showed that UCA1 significantly enhanced the cell invasion capability in A549 cells (t=7.537, P<0.0001; t=3.173, P=0.0131, Fig. 3C). However, the results were reversed after the knockdownnce of UCA1 (t=5.568, P=0.0005; t=3.325, P=0.0105, Fig. 3D). These results suggested that overexpression of UCA1 promoted the migration and invasion of LUAD cells, whether under the influence of cisplatin drug.
UCA1 enhanced the clonogenic capability of LUAD cells
Figure 4A showed that UCA1 overexpression group owed the higher colony forming ability than the negative control group (t=8.766, P=0.0009; t=5.935, P=0.0040; Fig. 4A). This result was reversed in UCA1 knockdown group (t=6.649, P=0.0027; t=5.375, P=0.0058; Fig. 4B). These results demonstrated that UCA1 markedly enhanced the colony forming ability of LUAD cells.
UCA1 enhanced cisplatin resistance of LUAD cells by ERCC1
Cell IC50 assay showed that the IC50 value to cisplatin of A549 cells increased from 0.865±0.071 μg/ml to 1.878±0.037 μg/ml (t=21.92, P<0.001, Fig. 5A). After UCA1 knockdown, the IC50 value of A549/DDP cells decreased from 5.135±0.472 μg/ml to 4.021±0.377 μg/ml (t=3.193, P=0.0331, Fig. 5B).
Excision repair cross complementing gene 1 (ERCC1) protein have been confirmed to be highly correlated with cisplatin resistance in non-small cell lung cancer. We detected the expression of ERCC1 in the cells, and found that ERCC1 increased with the increase of UCA1 expression (t=4.010, P=0.016, Fig. 5C) and decreased with the decrease of UCA1 expression (t=6.911, P=0.0023, Fig. 5D). According to the results, as ERCC1 involved in DNA repair pathway, we inferred that UCA1 might improve cisplatin induced DNA damage by activating the DNA repair pathway to enhance cell cisplatin resistance of LUAD cells.
UCA1 promoted proliferation and cisplatin resistance of LUAD cells in vivo
The tumor formation ability of A549/DDP-Lenti-shNC and A549/DDP-Lenti-shUCA1 cells was checked by nude mice inoculation experiment. As shown in Fig. 6A, after 8 days, the transplanted xenograft tumors were constructed. There was no significant difference in the volume of transplanted tumor except the 10th day. At other time points, such as Day 8 (t=2.704, P=0.0221), Day 14 (t=3.581, P=0.0050), Day 16 (t=3.566, P=0.0051), Day 18 (t=3.711, P=0.005), Day 22(t=4.110, P=0.0021), Day 22 (t=4.315, P=0.0015), Day 27 (t=4.670, P<0.001), Day 32 (t=6.207, P<0.001), Day 35 (t=8.725, P<0.001) and Day 38 (t=6.295, P<0.001), the tumor volume of A549/DDP-Lenti-shNC group was significantly higher than that of and A549/DDP-Lenti-shUCA1 cells. The results showed that the weight of nude mice in A549/DDP-Lenti-shNC group (20.300±2.326 g) was slightly lower than that in A549/DDP-Lenti-shUCA1 group (21.533±2.482 g), but the difference was not statistically significant (Fig.6B). While, the weight of transplanted tumor in A549/DDP-Lenti-shUCA1 group (0.074±0.042 g) was significantly lower than that in A549/DDP-Lenti-shNC group (0.310±0.066g) (t=7.354, P<0.001, Fig. 6CD). The tumor growth was most significantly inhibited in mice following UCA1 knockdownd compared with the NC groups, which indicated that UCA1 played an important role in regulating the growth of LUAD cells in vivo.
With cisplatin treatment, the mice body weight of A549/DDP-Lenti-shUCA1+DDP group decreased from 19.417±1.137g to 17.067±0.784 g and that of A549/DDP-Lenti-shNC+DDP group changed from 20.383±0.947g to 18.000±1.643 g, but there were no significant difference between the two groups both before and after chemotherapy (Fig 7A). After intraperitoneal injection of cisplatin, the inhibition degree of transplanted tumor in A549/DDP-Lenti-shUCA1+DDP group was significantly higher than that in the control group. On the day 1 of chemotherapy(t=2.992, P=0.0281), day 3 (t=7.976, P<0.001), day 6 (t=4.886, P=0.0038), day 11 (t=10.42, P<0.001), day 14 (t=9.197, P<0.001), day 17 (t=10.53, P<0.001), day 21 (t=12.26, P<0.001), day 24 (t=8.150, P<0.001), day 27 (t=9.027, P<0.001), day 29 (t=7.794, P<0.001), the tumor volume of A549/DDP-Lenti-shUCA1+DDP group was significantly lower than that of A549/DDP-Lenti-shNC+DDP group (Fig.7B). After chemotherapy, the tumor weight of A549/DDP-Lenti-shUCA1+DDP group was 0.025±0.009 g, which was significantly lower than that of A549/DDP-Lenti-shNC+DDP group (0.285±0.071g, t=0.885, P<0.001, FIG. 7CD). These results demostrated that Knockdownnce with UCA1 restored the sensitivity of A549/DDP cells to cisplatin. Taken together, UCA1 promoted proliferation and cisplatin resistance of LUAD cells in vivo.
The RNA binding protein ENO1 of UCA1 was obtained
Through RNA pulldown and protein mass spectrometry (MS), we analyzed the proteins of RNA pulldown products of the UCA1_sense and UCA1_antisense. A total of 441 proteins were identified, among which 219 proteins were identified in both 2 samples, while 75 proteins were identified unique in UCA1_sense and 147 unique proteins in UCA1_antisense (Fig. 8A). Detail information of some protein of the MS results were shown in Table 3.
According to the results of protein mass spectrometry, was selected for subsequent experiments. Enolase 1 (ENO1), known as coding enolization enzyme 1, played a key role in in glucose metabolism and tumor development. It was reported that ENO1 was highly expressed in lung cancer tissues and promoted LUAD progression by regulating the glycolytic pathway. We explored whether ENO1 was bound to UCA1 by RIP experiment. The results confirmed that UCA1 was highly expressed in ENO1 immunoprecipitated (t=6.859, P=0.0024, Fig. 8B), indicating that ENO1 possibly was one of the RNA binding proteins of UCA1. It was quite possible that UCA1 might co-engage with ENO1 in regulating cisplatin resistance mechanisms in LUAD.