Long Non-coding RNA X-Inactive Specific Transcript Promotes Esophageal Squamous Cell Carcinoma Progression via the MicroRNA 34a/Zinc Finger E-box–Binding Homeobox 1 Pathway

Samples from 66 patients were used in this study. All patients were surgically treated at The Fourth Hospital of Hebei Medical University (Hebei, China) between January 1, 2014 and December 31, 2015. Patient selection was as previously described [26]. Inclusion criteria for patient samples were as follows: 1, pathological diagnosis on initial gastroscopic bite: esophageal squamous cell carcinoma; 2, no preoperative history of radiotherapy; 3, preoperative assessment capable of R0 resection. Exclusion criteria for patient samples were as follows: 1, postoperative paraffin pathology was not esophageal squamous cell carcinoma; 2, patients who died within one month after surgery. All specimens were obtained and pathologically confirmed to be ESCC. Surgical samples were obtained within 30 min post-operative and immediately stored in liquid nitrogen.

The patients (52 males and 14 females) ranged in age from 50 to 87 years. The patients’ general data (gender, age, occupation, and family history) and data on tumor (pathological type, TNM stage and surgical situation) were extracted from medical records. Follow-up was performed according to the standard follow-up system of the hospital every 6 months after patients were discharged from the hospital. The deadline for the follow-up was December 31, 2019. The survival period was measured from the date of admission to the date of death or to the date of the follow-up deadline. During the follow-up period, 35 patients died due to ESCC, with no other underlying causes implicated. The clinical characteristics and outcomes of all participants are summarized in Table 1.

Paraffin sections (4 mm thick) were deparaffinized and rehydrated, followed by treatment with 0.02 M EDTA buffer (pH 9.0, Gene Tech, USA). Then, the sections were immersed in 3% H₂O₂ and blocked with 5% normal goat serum in proper sequence, followed by incubation with monoclonal anti-ZEB1 antibody (1:500; Sigma, USA) overnight at 4 °C. The antibody was diluted in PBS buffer containing 5% normal goat serum. The negative control for each slide was incubated with 5% normal goat serum without the anti-ZEB1 antibody. The sections were then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (ZSGB-BIO, China) for 45 min at 37 °C and revealed with diaminobenzidine tetrahydrochloride. The stained slides were scored by three pathologists who were unaware of the clinical diagnosis. The indexes of ZEB1 labeling were implemented so that samples were scored according to the percentage and intensity scores of positively stained tumor cells.

The ESCC cell lines were obtained from The Chinese National Infrastructure of Cell Line Resources (NICR; Beijing, China). Cells were routinely cultured in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10% Fetal Bovine Serum (FBS; Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA) in the 37 °C incubator with 5% CO₂.

KYSE150 cells were planted in plates about 16 h before transfection. The plasmids expressing control scramble shRNA and sh-XIST were prepared by Genehem (Shanghai, China). The sequences are listed below: scramble control shRNA, 5ʹ-TGGATCCAAGGTCGGGCAGGAAGAG-3ʹ; sh-XIST, 5ʹ-TCTCTGTCATTGCTTCTGTAGTCACAGTC-3ʹ. Transfection of KYSE150 cells was conducted with lipofectamine 2000 (Invitrogen, CA, USA) following the manufacturer’s instructions. Lentivirus for knocking down of miR-34a was ordered from Genehem (Shanghai, China), and the sequence cloned into the vectors is as follows: 5ʹ-TGCTGTTAGC TAATACTCAC AACAACGTTT TGGCCACTGA CTGACGTTGT TGTGTATTAG CTAA-3ʹ.

Cell proliferation was measured by the CCK-8 assay kit (Dojindo Molecular Technologies, Inc.; Japan). Briefly, ESCC cells with the indicated gene expression manipulation were seeded in 96-well flat-bottom microplates with a density of approximately 5 × 10³ cells/well, and incubated for 12, 24, 36, and 48 h, respectively. Then, 20 µL CCK-8 reagent was added into each well. Cells were incubated at 37℃ subsequently for another 2 h. The cell viability in each well was determined by measuring the optical absorbance at 450 nm on a microplate reader (Promega™; Madison, WI, USA).

For transwell assays, 2 × 10⁴ cells were plated as scheduled in the top chamber and covered with or without membrane. Cells were seeded in the preset serum-free medium. In the meanwhile, serum-supplemented medium was used as an effective chemoattractant and put in the lower chamber. The cells were incubated for 24 h, and cells that had not migrated or invaded through the hole were gently eliminated with a cotton swab. After being fixed, the cells located on the lower surface of the membrane were then stained with Giemsa crystal violet solution and counted with the help of an optical microscope (TS100-F, Nikon, Japan).

Total RNA was extracted from tissue samples according to the instructions in the manual of iRNAVana™ PARISTM (Ambion, TX, USA). The levels of miRNAs were analyzed using qPCR with TaqMan, miRNA reverse transcription assays, and the appropriate primers according to the manufacturer's instructions. Briefly, 10 ng of total RNA was used as the template for 15 µL reverse transcription reactions. Probes were specially designed for specific mature miRNAs. For each miRNA, reactions were performed in triplicate using the 7500 RT-PCR system (Applied Biosystems), and RNU66 (Applied Biosystems, cat. no. 4373382) was used as the normalization control.

The sequences of primers used for qPCR assays are as follows: XIST, Forward, 5ʹ-TGGCTCTTCTTTCACGCTTT-3ʹ, Reverse, 5ʹ-TGGTGTCGTGGAGTCG-3ʹ;

miR-34a, Forward, 5ʹ-ACACTCCAGCTGTGACTGGTTGACCAGA-3ʹ, Reverse, 5ʹ-TCAACTGGTGTCGTGGA-3ʹ, RT, 5ʹ-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCCCCTCTG-3ʹ;

U6, Forward, 5ʹ-CTCGCTTCGGCAGCACA-3ʹ, Reverse, 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ; ZEB1, Forward, 5ʹ-TTCGAGCCATCATTAAAATCAC-3ʹ, Reverse, 5ʹ-CTGGGTGGTTCAGACTCACA-3ʹ; E-cadherin, Forward, 5ʹ-TGCCCAGAAAATGAAAAAGG-3ʹ, Reverse, 5ʹ-GTGTATGTGGCAATGCGTTC-3ʹ; GAPDH, Forward, 5ʹ-CGCTGAGTACGTCGTGGAGTC-3ʹ, Reverse, 5ʹ-GCTGATGATCTTGAGGCTGTTGTC-3ʹ.

Cells were lysed with ice-cold lysis buffer (Keygen Biotech, China) for 30 min on ice. Cell lysates were then collected after centrifugation at 12,000 rpm for 5 min at 4 °C. The lysate protein at an amount of 60 µg was loaded, and the total cellular protein was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transblotted overnight at 4 °C onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with antibody (anti-ZEB1, 1:1,000 dilution, Cell Signaling Technology (CST), USA; anti-E-cadherin, 1:2,000 dilution, CST, USA) at 4 °C overnight, washed three times with tris-buffered saline supplemented with 0.1% tween (TBST), and incubated with the IRDye® 800CW Conjugated Goat anti-Rabbit IgG (Catalog Number 926-32211, LI-COR Biosciences, USA) secondary antibody (1:2000 dilution) for 1 h at room temperature. The membranes were washed three times with TBST, and the immunoreactive bands were detected using the kit and the infrared (IR) laser-based instrumentation from LI-COR Biosciences, USA.

The miRNA targets of XIST and the binding sites of miR-34a and XIST, as well as miR-34a and 3' untranslated region (UTR) of ZEB1 mRNA were predicted using the online tools starBase v 2.0 (http://​starbase.​sysu.​edu.​cn/​starbase2/​index.​php). The DNA sequences with the predicted binding site regions and the corresponding mutated regions, were cloned into a luciferase-expressing vector psiCHECK2 (Shanghai Genechem, China). The luciferase reporter vectors containing the full length XIST or 3' untranslated region (UTR) of ZEB1 were constructed. Esophageal cancer cells were co-transfected with the indicated vectors or the control luciferase reporter vector together with miR-34a-mimic. At 48 h after transfection, cells were harvested and subjected to luciferase activity analysis using the Dual-Glo luciferase assay system (Shanghai Genechem, China) in accordance with the manufacturer’s instructions.

BALB/c nude mice (4–5 weeks old, male) were purchased from Beijing Vital River Laboratory Animal Technologies Co. Ltd (Beijing, China). All animals were kept in a pathogen-free environment in the Animal Center of the Fourth Hospital of Hebei Medical University. and fed with food and water ad lib. Twelve nude mice aged 6 weeks were divided into two groups for cancer cells inoculation: siXIST and control (6 mice in each group). Stable XIST-knockdown KYSE150 cells were generated after infection of shXIST-expression lentivirus. A total of 5 × 10⁶ stable XIST-knockdown KYSE150 cells (shXIST) or control KYSE150 cells (control) in 0.2 mL of phosphate buffered saline (PBS) (Corning, Manassas, V.A., USA) were subcutaneously injected into the right shoulder flank of nude mice. The xenograft size in each group was measured at 5, 10, 15, 20, 25, and 30 days after inoculation using a microcaliper, and the tumor volume was calculated according to the following formula: volume = 1/2 length × width². At 30 days after tumor inoculation, mice were euthanized with CO₂ inhalation and tumor tissues were harvested for additional analyses.

Comparisons of the results with OS of patients were carried out using Kaplan–Meier log-rank testing. The correlation between the expression levels of XIST and ZEB in tumor tissues was evaluated using the COX regression analysis. Correlations between XIST level and patient characteristics were assessed using the χ² statistical test. Data were analyzed by using SPSS 21.0 (SPSS Inc., Chicago, IL, USA) and are displayed as mean ± standard deviation (SD). A P < 0.05 was considered to be statistically significant.

We evaluated the expression levels of XIST and ZEB1 in 66 paired ESCC and adjacent non-tumor tissues. We found that the XIST levels were significantly higher in tumor tissues (2.69 ± 1.71) than that in the paired adjacent non-tumor tissues (1.84 ± 1.67) (P < 0.001) (Fig. 1A). In addition, the ZEB1 protein expression levels as revealed by IHC staining were higher in tumor tissues (3.24 ± 2.79) than that in adjacent non-tumor tissues (1.06 ± 1.31) (P < 0.001) (Fig. 1B). We then analyzed XIST expression level in ESCC tissues and its effect on patient characteristics and survival time. Patients were categorized into two groups (high and low) based on the median level. The results showed that XIST expression was associated with postoperative metastasis (χ² = 17.580, P < 0.001): a high level of XIST was correlated with postoperative metastasis (Table 2). Moreover, the ESCC patients with a high level of XIST had a significantly shorter overall survival (OS) and patients with a low XIST expression had a longer OS (Fig. 1C and Table 2). Additionally, patients were categorized into two groups (negative and positive) based on the IHC scores. We found that the expression of ZEB1 protein was associated with postoperative metastasis (χ² = 6.110, P = 0.013) (Table 2). As expected, ESCC patients with ZEB1 positive expression had significantly shorter OS than the patients with negative ZEB1 expression (Fig. 1D and Table 2). Furthermore, the relative expression level of XIST in all examined tissues was positively correlated with the ZEB1 protein level by regression analysis (F = 58.529, P < 0.001) (Fig. 1E). Collectively, our results suggest that XIST and ZEB1 are overexpressed in ESCC and their high expressions predict worse prognosis of ESCC patients.

To investigate the effects of XIST expression on ESCC disease progression, we sought to examine the impacts of manipulated expressions of XIST on cell proliferation and metastasis ability of human ESCC cells. We first evaluated the expression levels of XIST in several common human ESCC cell lines. Compared with the normal immortalized esophageal epithelial cell line NE1, the tested ESCC cell lines including ECA109, KYSE150, KYSE170, and TE1 displayed remarkably increased levels of XIST transcript. Particularly, KYSE150 cells had the highest expression level of XIST (Fig. 2A). Therefore, we selected the KYSE150 cell line as model cancer cells to further decipher the potential roles of XIST in the disease progression of ESCC. We confirmed successful knockdown of XIST, and the relative level of siXIST transfected cells is 0.38 in comparison to the control cells which had a relative level of 1 (Fig. 2B). Moreover, our proliferation assays demonstrated that KYSE150 cells with XIST knockdown had significantly decreased proliferation when compared with the control cells during the 3-day culturing, while a reduced proliferation by 39% was observed on Day 3 (Fig. 2C). miR-34a was reported as a sponge target of XIST [22]. Thus, we co-transfected miR-34-targeting siRNA into XIST knockdown cells, and found that these cells only showed a slightly decreased proliferation in comparison to the control cells on Day 3 (Fig. 2C). Furthermore, transwell assays showed that XIST knockdown significantly decreased the migration and invasion abilities of KYSE150 cells by 58% and 50%, respectively, while additional miR34a silencing remarkably restored the abilities of migration and invasion of XIST-knockdown KYSE150 cells (Fig. 2D). Therefore, XIST knockdown compromised the proliferation, migration, and invasion of KYSE150 ESCC cells, which could be attenuated by additional miR-34a silencing.

Knockdown of XIST reduced the migration and invasion of esophageal cancer cells, and E-cadherin is the key player in EMT that promotes tumor migration [9]. Therefore, we hypothesized that XIST may promote tumor migration by regulating E-cadherin expression. Quantitative assays showed that the level of E-cadherin mRNA was up-regulated by 62% (Fig. 3A) and expression of E-cadherin protein was increased by 360% upon knocking down of XIST (Fig. 3B). Additionally, we speculated that XIST might regulate E-cadherin expression through the miR-34a/ZEB1 axis based on previous study [27]. We thus verified the signal pathway and found that the level of miR-34a was significantly up-regulated while the level of ZEB1 mRNA was significantly decreased upon knocking down of XIST (Fig. 3A). A similar trend in changes of the protein levels for ZEB1 and E-cadherin was also identified in our western blot assays (Fig. 3B).

Next, luciferase reporter assays were performed to determine whether miR-34a specifically interacted with XIST and ZEB1 by binding to the predicted target sites.

Compared with the control group, miR-34a mimic significantly suppressed the luciferase activity of luciferase reporter vectors containing XIST (Fig. 3C) or 3’ UTR of ZEB1 (Fig. 3D), respectively. Whereas, the co-transfections of miR-34a mimic and mutated XIST or mutated 3’ UTR of ZEB1 resulted in largely unchanged luciferase activities, compared to the negative control (NC) co-transfections (Fig. 3C, D). These results indicate that miR-34a directly binds to the predicted target sites, and XIST can indirectly regulate ZEB1 expression by combining with miR-34a as a sponge.

To test the potential for clinical translation of our in vitro findings, we set out to investigate whether silencing of XIST can facilitate in vivo tumor rejection in an ESCC xenograft animal model. We inoculated the control KYSE150 cells (Control) and the stable KYSE150 cells after infection of shXIST-expression lentivirus (siXIST group) into the right shoulder of nude mice, measured tumor growth, and euthanized the mice at 30 days after tumor inoculation. After 5 days, xenografted tumors grew to be palpable in all mice. As shown in Fig. 4A, the siXIST group at Day 30 had evidently smaller tumors in size. The volume of tumors in the siXIST group was significantly smaller than that in the Control group at the time of sacrifice (P = 0.012) (Fig. 4B). Protein quantitation assays showed that the relative expression level of the E-cadherin protein in the siXIST group was 3.12 folds of that in the Control group; while the relative expression of ZEB1 in the siXIST group was 6% of that in the control group (Fig. 4C). Taking together, these results are consistent with what we observed in the in vitro study: XIST silencing suppressed ESCC tumor growth.