LncRNA WT1-AS downregulates lncRNA UCA1 to suppress non-small cell lung cancer and predicts poor survival

We selected 66 NSCLC patients (40 males and 26 females; 36 to 68 years old; mean age 52.7 ± 6.4 years old) selected from the 178 NSCLC patients admitted by Shenzhen Second People’s Hospital from June 2010 to June 2013. The 66 NSCLC patients included 30 cases of adenocarcinoma, 25 cases of squamous cell carcinoma and 11 cases of large cell carcinoma. All patients were smokers or had a previous history of smoking. No exposure to asbestos and other carcinogens was observed. A family history of lung cancer was not observed. Inclusion criteria: 1) patients received histopathological diagnosis; 2) all patients were newly diagnosed cases; 3) no initiated therapy. Exclusion criteria: 1) patients transferred from another hospital (45 cases excluded); 2) other clinical disorders were diagnosed (64 cases excluded, including 12 cases of heart disease, 26 cases of diabetes, 1 case of skin cancer, 17 cases of severe infection, 2 cases of bone fracture, 1 case of metal disorder, 2 cases of renal injury, 1 case of cystitis, and 2 cases of glaucoma); 3) patients with a history of malignancies (3 cases excluded). Based on clinical findings, the 66 patients included 12, 10, 22 and 22 cases at stage I-IV (AJCC), respectively. Written informed consent was signed by all patients. This study was approved by the Ethics Committee of the aforementioned hospital. Lung biopsy was performed to diagnose NSCLC. During lung biopsy, NSCLC and adjacent normal (non-cancer) tissues were obtained and stored in − 80 °C. All biopsies were completed under the guidance of MRI before the initiation of therapies. All tissue samples were subjected to histopathological exams and all NSCLC tissues contained more than 90% cancer cells and all non-tumor tissues contained less than 2% cancer cells.

After admission, the 66 NSCLC patients were monitored for 5 years through phone calls and/or outpatient visits every 1–2 months. Patients died of causes unrelated to NSCLC were excluded.

Two human NSCLC cell lines H1993 and H1581 (ATCC, USA) were used. RPMI-1640 medium (90%) was mixed with FBS (10%) to grow cells. Cells were grown at 37 °C in an incubator with 95% humidity and 5% CO2. WT1-AS or UCA1 overexpression vector was constructed with pcDNA3 vector (GenePharma, Shanghai, China). At the confluence of 70–90%, H1993, and H1581 cells were harvested and transfected with either 10 nM WT1-AS or UCA1 overexpression vector. Negative control (NC) experiment was performed by transfecting empty vector into the same number of cells. To perform Control (C) cells, cells without transfections were grown until the end of experiments. After cell transfections, cells were cultivated under normal conditions for another 24 h.

RNAzol (Sigma-Aldrich, USA) was used for RNA isolation from H1993 and H1581 cells (10⁶ cells) or tissues (about 0.025 g tissues in 0.5 ml RNAzol reagent). DNase I digestion was performed on all RNA samples. Following that, reverse transcriptions were performed to synthesize cDNAs. With cDNAs as a template, qPCRs were performed to detect the expression of WT1-AS and UCA with 18S rRNA as the endogenous control. Three technical replicates were included in each experiment and Ct values of targeted genes were normalized to 18S rRNA using the 2^-ΔΔCT method. The primer sequences were: WT1-AS Forward: 5′-GAGGACAGA GAGGCATGGAG-3′; Reverse: 5′-ACCCCTAGGCAAGGAGAAGA-3′; UCA1 Forward: 5′-ACGCTAACTGGCACCTTGTT-3′; Reverse: 5′-TGGGGATTACTGGGGTAGGG-3′; 18S rRNA Forward: 5′-CGGCTACCACATCCAAGGAA-3′; Reverse: 5′-TGTCACTACCTCCCCGTGTCA-3′.

H1993 and H1581 cells were transferred to the upper Transwell chamber (8 μm pore, Corning, Corning, NY) with 3000 cells in 0.1 ml non-serum medium per well. Each cell transfection group included three replicates. The lower Transwell chamber was filled with medium supplemented with 20% FBS. Matrigel (356,234, Millipore, USA)–coated membrane was used in invasion assay, while uncoated membranes were used in migration assay. Cells were cultured under the condition of 37 °C and 5% CO₂ for 12 h. After that, upper surface was cleaned, and 0.5% crystal violet (Sigma-Aldrich, USA) was used to stain lower surface at 25 °C for 30 min. An optical microscope was used to observe the stained cells. All groups were normalized to the C group, which was set to “100”.

Cell count Kit-8 (CCK-8, Sigma-Aldrich) was used to evaluate the effect of overexpression of WT1-AS and UCA1 on proliferation of H1993 and H1581 cells. Briefly, 3× 10⁴ cells were mixed with 1 mL medium. Under the above conditions, cells were grown with a cell culture plate (96 Wells, 0.1 mL per well) at 4 h before the end of cell culture, cck-8 solution (10 μL) was added to each well. Then 10 μL DMSO was added, and OD values at 450 nm were measured.

H1993 and H1581 cells were mixed with RIPA solution (Beyotime) to extract total proteins. The protein concentration was detected by the BCA assay kit (Beyotime). All protein samples were denatured by incubating the samples in boiling water for 15 min. After that, electrophoresis was performed using 10% SDS-PAGE gel to separate protein molecules. After that, proteins were transferred to PVDF membranes, followed by blocking in PBS containing 5% non-fat milk at room temperature for 2 h. After that, membranes were incubated with rabbit primary antibodies of E-cadherin (ab40772, Abcam), vimentin (ab92547, Abcam), p53 (ab131442, Abcam) and β-actin (ab8226, Abcam) at 4 °C for 18 h. After that, membranes were further incubated with IgG-HRP goat anti rabbit (MyBioSource) at 24 °C for 2 h. Signals were developed using ECL (Sigma-Aldrich) and quantity one was used to normalize the signals.

The expression of WT1-AS in two types of tissues (NSCLC and non-cancer) from NSCLC patients (n = 66) was detected by RT-qPCR. It was observed that the expression levels of WT1-AS were significantly lower in NSCLC tissues compared to that in non-cancer tissues (Fig. 1, p< 0.05). Based on clinical findings, the 66 patients included 12, 10, 22 and 22 cases at stage I-IV (AJCC), respectively. The chi-squared test showed that the expression levels of WT1-AS and UCA1 in were not significantly correlated with the clinical stage (Tables 1 and 2, p< 0.05). Moreover, no significant differences in the expression levels of WT1-AS and UCA1in either non-tumor tissues or NSCLC tissues were observed among different subtypes of NSCLC (data shown in Table 1 and Table 2).

Using the expression data of WT1-AS in NSCLC tissues before therapies, the above-average group was defined as high and sub-average group was defined as low. NSCLC patients were grouped into low (< 3.9, n = 34) and high (≥ 3.9, n = 32) WT1-AS level groups. Survival curves were plotted and compared using the methods aforementioned. Compared to patients in high in WT1-AS level group, low WT1-AS level group exhibited significantly lower overall survival rate (Fig. 2).

The expression of UCA1 in paired tissues was also determined. Compared to non-tumor tissues, NSCLC tissues exhibited significant higher expression levels of UCA1 in non-cancer tissues (Fig. 3a, p < 0.05). Correlation analysis showed that the expression of UCA1 and WT1-AS were inversely and significantly correlated in both NSCNC tissues (Fig. 3b) and non-cancer tissues (Fig. 3c).

WT1-AS or UCA1 expression vector was transfected into H1993 and H1581 cells. Transfections were confirmed at 24h post-transfection (Fig. 4a, p <0.05). UCA1 overexpression failed to affect WT1-AS, while WT1-AS overexpression led to inhibited UCA1 expression (Fig. 4b, p <0.05). Cell proliferation assay was performed to assess the effects of overexpression of WT1-AS and UCA1 on the proliferation of NSCLC cells. Compared to C and NC groups, cell proliferation assay showed that overexpression of WT1-AS led to decreased rates of cell proliferation, while overexpression of UCA1 led to increased proliferation of NSCLC cells. In addition, overexpression of UCA1 attenuated the effects of overexpression of WT1-AS. Moreover, silencing of UCAI decreased cell proliferation rate of the NSCLC cells (Fig. 5a p < 0.05).

Transwell assay data showed that overexpression of UCA1 resulted in increased, while overexpression of WT1-AS resulted in decreased invasion (Fig. 5b) and migration (Fig. 5c) of NSCLC cells (p < 0.05). In addition, the role of overexpression of WT1-AS was inhibited by the overexpression of UCA1 (p < 0.05). Moreover, we also explored the function of WT1-AS and UCA1 in the level of EMT, the expression of E-cadherin and vimentin indicated that the decreased influence of UCA1 overexpression in EMT was counteracted by synchronous introduction of WT1-AS overexpression in H1993 cell (Fig. 6a) and H1581 cell (Fig. 6b). Meanwhile, we detected the expression of p53 to further demonstrate that WT1-AS downregulated UCA1 to inhibit NSCLC cell. And we found that overexpression of WT1-AS overexpression could promote the expression of p53 and when UCA1 was silenced, the expression levels of p53 were increased (Fig. 6c and d), the original, uncropped blots are shown in Supplementary figure-1. In summary, WT1-AS downregulated UCA1 to inhibit NSCLC cell invasion and migration by suppressing EMT and promoting the expression of p53.