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Introduction

Lung cancer is considered as the “leading killer” around the world. Its incidence rate ranks second in male and female patients, and the tumor-rela-ted mortality ranks first in all tumors1. Among, non-small cell lung cancer (NSCLC) accounts for nearly 85% of all types of lung cancers, and includes two pathological patterns: lung adeno-carcinoma (LAD) and lung squamous carcinoma. In recent years, despite of the rapid development of chemotherapy, radiotherapy and molecular tar-geted therapy in the comprehensive treatment of NSCLC, the 5-year survival rate (about 16.6%) is still unsatisfactory2. The important reason for the low 5-year survival rate is that invasion and me-tastasis occur easily in NSCLC. Therefore, it is important to understand the potential molecular mechanism of NSCLC metastasis and recurrence and find effective molecular markers.

About 98% of the transcripts of the human ge-nome have no protein translation capacity, so such kind of RNA is named non-coding RNA (ncR-NA)3,4. At first, the researchers believe that ncR-NA has no special biological characteristics and it is regarded as the “junk” of evolution. However, more and more research results show that ncR-NA, as an important regulatory factor, is invol-ved in cell metabolism, differentiation, stem cell induction and other biological processes5,6. And lncRNA is a kind of ncRNA with the length of more than 200bn, which, as an important mem-ber of the ncRNA family, is associated with the diabetes-related microvascular dysfunction, de-velopment, and progression of the tumor, etc. Qiu et al7 found that the knockdown of MEG3 (mater-nally expressed 3) expression can mediate the re-tinal vascular inflammation and accelerate vascu-lar deterioration in diabetic mice via mediating

Abstract. – OBJECTIVE: Long non-coding RNAs (lncRNAs) play an important role in vari-ous cellular biological processes. It is also in-volved in the occurrence and development of the tumor. BCAR4 is reported to be highly expressed in breast cancer and promotes cell proliferation. However, the biological effects of BCAR4 in non-small cell lung cancer (NSCLC) remains unclear.

PATIENTS AND METHODS: qRT-PCR was performed for detecting BCAR4 expression in 76 pairs of NSCLC tissues and corresponding cancer-adjacent tissues and 6 NSCLC cell lines. BCAR4 expression was knocked down, and its effects on NSCLC cell proliferation, cycle, apop-tosis, invasion and metastasis were studied via MTT, clone formation, flow cytometry, TUNEL and Transwell assay. Metastatic tumor model of nude mice was established to investigate its ef-fects on NSCLC cell metastasis. BCAR4 down-stream target gene protein expression was detected using Western blotting and immunoflu-orescence assay.

RESULTS: BCAR4 was higher in NSCLC tis-sues than that in cancer-adjacent tissues and was positively correlated with tumor size, clin-ical stage, and distant metastasis, suggesting that BCAR4 can be used as an independent pre-dictor of prognosis. Results also showed that BCAR4 knockdown could inhibit tumor cell inva-sion, metastasis, and proliferation, induce cell cycle arrest and increase cell apoptosis. BCAR4 knockdown inhibits the metastasis and invasion of tumor cells via regulating Vimentin, N-cad-herin and E-cadherin in Epithelial-Mesenchymal Transition (EMT).

CONCLUSIONS: BCAR4 promotes the inva-sion and metastasis of NSCLC via regulating EMT and BCAR4/EMT interaction can be used as a new target for the diagnosis and therapeutics of NSCLC.Key Words:

Carcinoma, Non-small-cell lung, Epithelial-mesen-chymal transition, Neoplasm invasiveness, Neoplasm metastasis, RNA, Long noncoding.

European Review for Medical and Pharmacological Sciences 2017; 21: 2075-2086

N. LI, W.-J. GAO, N.-S. LIU

Department of Pathology, Nanjing Medical University, Nanjing, Jiangsu, China

Corresponding Author: Ningsheng Liu, MD; e-mail: xiashi474959@163.com

LncRNA BCAR4 promotes proliferation, invasion and metastasis of non-small cell lung cancer cells by affecting epithelial-mesenchymal transition

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the activation of the PI3/AKT signaling pathway. Liu et al8,9 found that lncRNA HOTAIR (HOX transcript antisense RNA) is highly expressed in NSCLC, and the highly-expressed HOTAIR can enhance the metastasis and invasion of NSCLC cells. Though there are more and more studies on lncRNA, researchers still know little about its biological role in NSCLC.

Invasion metastasis is an important cause of NSCLC treatment failure. The molecular mecha-nisms of NSCLC invasion and metastasis mainly include extracellular matrix degradation, tumor microenvironment changes, activation or inhibi-tion of invasion and metastasis-related genes10,11. The epithelial-mesenchymal transition (EMT), as one of the important molecular mechanisms, is closely related to tumor invasion and metastasis. Its main features include decreased E-cadherin expression, a marker for cell adhesion, and increa-sed N-cadherin expression, and the transformation from keratin-based cytoskeleton to vimentin-ba-sed cytoskeleton12. However, NSCLC invasion and metastasis are the results of multi-factor network regulation, and lncRNA-mediated EMT is confir-med by more research results. It is reported in the literature that lncRNA TUG1 (taurine-up-regula-ted gene 1) can promote the metastasis and inva-sion of colon cancer, and its molecular mechanism is the regulation of EMT pathway13. Li et al14 found that lncRNA HULC is highly expressed in liver cancer and can promote the invasion and metasta-sis of hepatocellular carcinoma cells. Further me-chanism research has shown that lncRNA HULC can competitively absorb miRNA-200a-3p, and the decrease of miR-200a-3p promotes the increa-se of ZEB1 protein expression, which induces the occurrence of EMT in hepatocellular carcinoma cells15. LncRNA-mediated EMT provides a good idea for researchers to study the potential molecu-lar mechanisms of NSCLC invasion and metasta-sis. LncRNA BCAR4 (breast cancer anti-estrogen resistance 4) was the first gene that was found to be related to hormone resistance in breast cancer16. Godinho et al17,18 reported that BCAR4 plays a role similar to “oncogene” in breast cancer and can promote the breast cancer metastasis and prolife-ration by regulating Hedgehog/GLI2 pathway, and promote its resistance to estrogen. In breast can-cer patients, highly-expressed BCAR4, as a new biological marker, suggests that patients are prone to invasion and metastasis19, but there has been no report on relative expression quantity of BCAR4 in NSCLC and its biological effects. In this study, the biological function of BCAR4 in NSCLC was

studied by in vivo/in vitro experimental studies, and the correlation between its relative expression quantity and the clinical and pathological characte-ristics of NSCLC patients was analyzed. Results showed that BCAR4 was relatively highly expres-sed in NSCLC, which is positively correlated with tumor size, clinical stage, invasion and metastasis. Highly-expressed BCAR4 can be used as a predi-ctive factor of prognosis of NSCLC patients. Both in vivo and in vitro studies showed that the knock-down of BCAR4 in NSCLC cells can promote cell apoptosis, inhibit proliferation and inhibit cell in-vasion and metastasis by affecting EMT.

Patients and Methods

Tissue CollectionFresh and formalin-fixed, paraffin-embedded

NSCLC tumor tissue samples were obtained from patients diagnosed with NSCLC. These patients received the elective surgery at the First Affiliated Hospital of Nanjing Medical University. A total 76 pairs of fresh NSCLC and adjacent non-tumor tis-sues (more than 5 cm away from the tumor) were freshly frozen in liquid nitrogen and stored at −80°C until further use. The archived, formalin-fixed and paraffin-embedded NSCLC tissue samples from 76 cases, were used for clinicopathological and progno-stic investigation of lncRNA BCAR4. Ethics Com-mittee of the First Affiliated Hospital of Nanjing Medical University has approved the use of tissues for this study. Before using these clinical materials for research purposes, informed consent has been signed by all the patients. No patient received any preoperative chemotherapy or radiotherapy.

Cell Culture and TransfectionNSCLC cell lines (A549, SPC-A-1, PC-9, H157

and SK-MES-1) and the 16HBE cell lines were from the Institute of Biochemistry and Cell Bio-logy of the Chinese Academy of Sciences (Shan-ghai, China). RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum was used to culture cells in 100 U/ml penicillin, and 100 mg/ml streptomycin (Gi-bco, Grand Island, NY, USA) at 37°C with 5% CO2. si-BCAR4, were purchased from Invitrogen (Carlsbad, CA, USA). Cells were culture using complete medium without antibiotics at least 24 hours prior to transfection. 1× phosphate buffer saline (PBS) (pH7.4) was adopted to wash cells and, then, cells were transiently transfected with 50 nmol/l si-NC, si-BCAR4, using Lipofectamine

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2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Western Blot AnalysisAccording to the protocol described previou-

sly, Western blot was performed16. The primary antibodies were used as follows: rabbit anti-E-ca-dherin (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-N-cadherin (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-vimentin (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-GAPDH (Sig-ma-Aldrich, St. Louis, MO, USA).

siRNA Knockdown ExperimentsStealth siRNA oligonucleotides were synthesi-

zed by Invitrogen (Carlsbad, CA, USA). The siRNA sequences were used as follows for knockdown of lncRNA BCAR4: 1# 5’-GCUGCGAGGGUAGA-CAUCUCUGUUU-3’; 2# 5’-CAGCUGGGCUU-GACAUUCCUGGGAA-3’; 3# 5’-UGGACC-GUCUGUGGAUGGGUGGGAA-3’. Cells were transfected with 50 pmol of siRNA and the scram-bled control oligo with RNAimax Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. Quantitative PCR was performed to determine knockdown efficiency at the time of plating for assay.

RNA Isolation and qRT-PCRTRIZOL reagent was used for extraction of total

RNA from tissue samples or cells in accordance to the manufacturer’s instructions. Random pri-mers were used to reversely transcribe total RNA (500 ng) in a final volume of 10 mL under stan-dard conditions with the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). To measure PVT1 expression levels, SYBR Premix Ex Taq (TaKa-Ra, Dalian, China) was used according to the manufacturer’s instructions. GAPDH expression was used for normalization. GAPDH(F): 5’-GG-GAGCCAAAAGGGTCAT-3’, GAPDH(R): 5’-GAGTCCTTCCACGATACCAA-3’ Real-time PCR was performed in triplicate on an ABI 7500, and comparative cycle threshold (CT) (2-∆∆CT method) was adopted for data calculation.

Proliferation AssayCell viability was determined by cell coun-

ting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Briefly, cells (2.5*103 cells per well) were seeded on the plate (96-well) and allowed for adhesion. CCK-8 solution (10 µL) was added into each well at different time points and then the cells were in-cubated for 3 h and measured at 450 nm.

Colony Formation AssayFor colony formation assay, a total of 800 cel-

ls were seeded in a six-well plate to allow colony formation for 2 weeks. Methanol was used to fix colonies and Giemsa was used to stain colonies for 20 min, and the visible colonies were manual-ly counted.

Cell Cycle AnalysisAfter being trypsinized, cells were washed

with PBS and then fixed in cold ethanol (10 ml) for 2 h. After being washed with PBS, cells were incubated with 1mL RNase (0.25 mg/mL) at 37°C for 1 h. After being pelleted and resuspended in propidium iodide (PI) (50 µg/mL), cells were in-cubated in the dark at 4°C for 30 min. Finally, PI signal was examined using flow cytometry.

Apoptosis Assay by Flow CytometryPhosphatidylserine externalization was de-

tected via flow cytometry with 7-aminoacti-nomycin (7-AAD) and Annexin V (FITC-labeled) (BD Pharmingen, San Diego, CA, USA), and ta-ken as an endpoint indicator of apoptosis accor-ding to the manufacturer’s instructions.

Immunofluorescence Cells were grown on glass coverslips, fixed

with 4% formaldehyde for 10 min, and permeabi-lized with Triton X-100 (0.2%) for 10 min at room temperature. After being washed with PBS, cells were incubated at 4°C overnight with anti-E-ca-dherin (Sigma-Aldrich, St. Louis, MO, USA), anti-N-cadherin (Sigma-Aldrich, St. Louis, MO, USA), anti-vimentin (Sigma-Aldrich, St. Louis, MO, USA). The Alexa Fluor 488® IgG (Invitro-gen, Carlsbad, CA, USA) was used as secondary antibody. Lamellipodia was observed via Alexa Fluor568® phalloidin (Invitrogen, Carlsbad, CA, USA). 1 mg/mL DAPI (Sigma-Aldrich, St. Louis, MO, USA) was used to stain nuclei at 37°C. Final-ly, coverslips and SlowFade® Gold confocal mi-croscope (Olympus, Tokyo, Japan) were mounted.

Results

BCAR4 Was Up-Regulated in NSCLC Tissues and Correlated with the Pathological Characteristics of Patients

To explore the effects of BCAR4 on NSCLC, the qRT-PCR assay was used to detect 76 cases of NSCLC tissues and cancer-adjacent tissues. The results showed that BCAR4 was highly expressed

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for 8.62 times on average in 76 cases of NSCLC tissues compared with that in cancer-adjacent tis-sues (Figure 1A). Then, the chi-squared test was used for correlation analysis between the relati-ve expression quantity of BCAR4 and the clini-cal and pathological characteristics of NSCLC patients. First, the median of multiple of BCAR4 differential expression was taken as the cut-off point, and patients with NSCLC were divided into two groups: BCAR4 high-expression group (n=38) and BCAR4 low-expression group (n=38). The results of the chi-squared test showed that highly-expressed BCAR4 was closely associated with tumor size (p<0.01), clinical stage (p<0.001) and lymph node metastasis (p<0.001), but uncor-

related with age, gender and pathological type (Table I).

Research on Biological Function of BCAR4 in NSCLC cells via in vitro Experiment

The results of our preliminary study showed that BCAR4 expression was relatively higher in NSCLC tissues, but its relative expression quanti-ty in NSCLC cells and its biological function re-main unknown. First, qRT-PCR was employed to detect BCAR4 expression in 5 kinds of NSCLC cell lines (A549, SPC-A-1, PC-9, H157 and SK-MES-1) and human normal bronchial epithe-lial cells (16HBE). Results revealed that relative

Figure 1. Relative PVT1 expression in NSCLC tissues and cells. A, relative expression of BCAR4 in NSCLC tissues (n=78) and in paired adjacent normal tissues (n=78). BCAR4 expression was examined by qPCR assays and normalized to GAPDH expression. B, BCAR4 expression levels of NSCLC cell lines (A549, PC-9, SK-MES-1, H157, and SPC-A1) compared with that in normal human bronchial epithelial cells (16HBE). C, A549 and SPC-A1 cells were transfected with si-BCAR4.

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expression level of BCAR4 was higher than that of 16HBE in 4 kinds of NSCLC cell lines, but the relative expression level in H157 was not statisti-cally significant (Figure 1B). 2 kinds of cell lines with the highest relative expression level, A549 and SPC-A-1, were selected as the model cell to design and synthesize BCAR4-specific interfe-rence sequence that was transiently transfected into A549 and SPC-A-1 cells using lip3000. After 48h, RNA was extracted from the two groups and the interference efficiency of Si-BCAR4 was de-tected (Figure 1C); 1# and 2# sequences were se-lected for subsequent experiments.

Then, MTT assay was used to study the effect of BCAR4 knockdown on the proliferation capa-city of A549 and SPC-A-1 cells, and 0h, 24h, 48h, 72h and 96h were selected as the observation poin-ts. The results showed that the proliferation capa-city of the experimental group was significantly decreased compared with control group (Figure 2A). And the clone formation results were consi-stent with MTT assay (Figure 2B). To investigate the effect of BCAR4 on the cell cycle progression and apoptosis of NSCLC cells, lip3000 was used to transiently transfect si-BCAR4 to A549 and SPC-A-1 cell lines. After 48h, cells were collected and flow cytometry was used to detect the chan-ge in cell cycle and apoptosis. The results showed that compared with the si-NC group, the cell cycle

progression of the si-BCAR4 group was blocked in G1 phase and the apoptosis rate was also in-creased (Figure 3A, 3B). Then, Transwell assay was used to investigate whether BCAR4 knock-down affected the invasion and metastasis of NSCLC cells. The cells of si-BCAR4 and si-NC groups were obtained by the same method. The cells were planted in transwell chamber, and after 48h, the cells crossing the chamber were counted. The results showed that the cell migration and in-vasion in the si-BCAR4 group were significantly decreased (Figure 3C, 3D).

Research on Influences of BCAR4 on NSCLC cell Invasion and Metastasis Via in vivo Experiment

Then the metastatic tumor model of nude mice was established, and effects of BCAR4 knock-down on metastasis and invasion capacities of NSCLC cells was studied via in vivo experiments. First, A549 cell lines with the highest transfection efficiency were screened as the model cells. Cel-ls in the transfected sh-BCAR4 group and empty plasmid group were injected into the nude mice via caudal vein. The growth situation of nude mice was observed and they were weighed every four days. At 28d after the caudal vein injection of cells, mice were executed, and the lungs of nude mice were taken out, followed by a photograph.

Table I. LncRNA-BCAR4 expression and clinicopathologic charateristics in non-small cell lung cancer (NSCLC).

lncRNA-BCAR4 expression

Characteristics Case number low high p-value

Gender male 33 15 18 0.488 female 43 23 20 Age (year) <60 48 27 21 0.154 ≥60 28 11 17 Site of tumor left lung 29 14 15 0.813right lung 47 24 23 pathological type squamous carcinoma 36 15 21 0.168 adenocarcinoma 40 23 17 Tumor stage I/II 45 30 15 <0.001* III/IV 31 8 23 Lymph node metastasis negative 20 17 3 <0.001* positive 56 21 35 Tumor size T1/T2 38 25 13 0.006* T3/T4 38 13 25

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As shown in Figure 4A, compared with the con-trol group, the number of metastatic tumor in the lung of nude mice in the sh-BCAR4 group was significantly reduced. Then the metastatic tumor in the lung of nude mice was taken out and total RNA was extracted from tumor tissue. The rela-tive expression quantity of BCAR4 in metastatic tumor tissue was detected by qRT-PCR. The re-sults showed that compared with control group, the expression quantity of BCAR4 in the tran-sfected sh-BCAR4-derived metastatic tumor was significantly decreased (Figure 4B).

BCAR4 Affected the Invasion and Metastasis of NSCLC Cells by Acting on EMT Pathway

The preliminary results showed that inter-ference on BCAR4 expression in NSCLC cells could significantly inhibit the invasion and me-tastasis capacities of NSCLC cells. But there is no report on how BCAR4 regulates down-stream molecular pathways and plays its role in NSCLC. Recent researches have shown that epithelial-mesenchymal transition is closely re-lated to tumor invasion and metastasis. Howe-

Figure 2. Effects of knockdown of BCAR4 on NSCLC cell viability in vitro. A, MTT assays were conducted to determine the cell proliferation ability for si-BCAR4-transfected A549 and SPC-A1 cells. Values indicate the mean (SD from three in-dependent experiments). B, Colony -forming assays were performed to determine the proliferation of si-BCAR4-transfected A549 and SPC-A1 cells.

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ver, Sun et al20 showed that lncRNA SPRY4-IT1 inhibits NSCLC cell invasion and metastasis by inhibiting EMT. Western blot assay was used to observe whether the expressions of EMT

molecular markers (E-cadherin, Vimentin, and N-cadherin) change after the interference on BCAR4. The results showed that compared with control group, the expression of E-cadherin was

Figure 3. The effect of BCAR4 on NSCLC cell cycle, apoptosis, metastasis in vitro. A, Cell cycle of A549 and SPC-A1 cells was analyzed by flow cytometry. The bar chart represents the percentage of cells in G0-G1, S, or G2-M phase, as indicated. B, A549 and SPC-A1 cells were stained and analyzed by flow cytometry. C, Transwell assays were used to investigate the changes in migratory and invasive abilities of si-BCAR4-transfected A549 and SPC-A1 cells.

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up-regulated and expressions of N-cadherin and Vimentin were down-regulated after the knock-down of BCAR4 expression (Figure 5A). Then immunohistochemistry was used to detect the protein expressions in E-cadherin, Vimentin, and N-cadherin in the above metastatic tumor. The results were consistent with those of We-stern blot (Figure 5B). It was concluded from the above in vivo/in vitro experiments that the knockdown of BCAR4 can inhibit the invasion and metastasis of NSCLC cells by acting on EMT pathway.

Discussion

At present, the prognosis of NSCLC patients still remains to be unsatisfactory, and the 5-year survival rate is about 16.6%2. The main reason for treatment failure and death of NSCLC patien-

ts is the invasion and metastasis. The micro-en-vironment changes around tumor cells and the interaction between tumor cells make cells lose the established biological habits, so metastasis occurs easily. Tumor cells colonize in other or-gans through the blood, lymph nodes and direct spreading, which leads to malignant prolifera-tion and organ dysfunction20,21. Therefore, the in-depth understanding of genes and proteins involved in NSCLC invasion and metastasis can provide a good idea for searching the accurate biological markers and developing effective tre-atment strategies.

With the rapid development of molecular biolo-gy, it has been found that lncRNA has a strong bio-logical function in the tumor, so it attracts the at-tention of researchers around the world and studies on lcnRNA is also becoming a forward-looking hot spot. LncRNA has a role in maintaining normal cell physiology, and its expression imbalance will

Figure 4. Effects of BCAR4 on tumor metastasis in vivo. A, Analysis of an experimental metastasis in animal model was performed by injecting si-BCAR4 A549 cells into nude mice. Lungs from mice in each experimental group. B, The numbers of tumor nodules on lung surfaces were shown.

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lead to many pathological and physiological phe-nomena in cells. For example, MALAT-1 was the first lncRNA that was confirmed to be associated with NSCLC cell invasion and metastasis22, and subsequent studies found that the highly-expressed MALTA-1 can also promote tumor cell invasion and metastasis in gastric cancer, ovarian cancer, and other tumors23,24. Also, Yang et al25,26 reported that lncRNA also plays an important role in the process of the cell cycle, differentiation and pro-grammed apoptosis.

BCAR4 was first discovered by the Meijer et al16 in breast cancer and might have a role in the resistance to tamoxifen, a chemotherapic drug in breast cancer. And Chen et al27 found that BCAR4 can promote the occurrence and deve-lopment of osteosarcoma by promoting the tran-scription of target gene GLI2 (GLI family zinc finger 2). Here, we found for the first time that lncRNA BCAR4 is highly expressed in NSCLC tissues and cells and can be used as a predictive factor of prognosis of NSCLC patients. In vivo/

Figure 5. BCAR4 inhibiton suppresses NSCLC cell invasion and metastasis by affecting EMT. A, Analysis of E-cadherin, N-cadherin, Vimentin expression in A549 and SPC-A1 cells treated with si-BCAR4 by Western blot. B, Analysis of E-cadhe-rin, N-cadherin, Vimentin expression in lungs from mice by immunohistochemical.

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in vitro experiments have proved that the knock-down of BCAR4 expression can inhibit the cell proliferation, invasion, and metastasis, and pro-mote the cell apoptosis. The above experiments showed that BCAR4 plays a role similar to “on-cogene”, which is closely related to the occurren-ce and development of NSCLC. Moreover, the study on BCAR4-specific interference sequence also provides a theoretical basis for clinical tre-atment of NSCLC patients.

Although BCAR4 has been proved to play a role similar to “oncogene” in a variety of tumors, the underlying molecular mechanisms that affect the metastasis and invasion of tumor cells are still unclear, and there has been no report about it so far. Epithelial-mesenchymal transition plays an important role in epithelial-derived tumors (such as esophageal cancer, non-small cell lung cancer)28-30. Epithelial cells lose cell polarity and lose connection to the basement membrane, but obtain higher migration and invasion via EMT31. In recent years, several studies have shown that lncRNA-mediated EMT is confirmed in a variety of tumors13,14. Western blot and immu-nofluorescence assay showed that knockdown of BCAR4 expression in NSCLC cells can increase the expression of E-cadherin (EMT molecular marker) and inhibit the expressions of N-cadhe-rin and vimentin.

Methylation of gene promoter region inclu-des DNA methylation and histone methylation32. DNMT1 (DNA (cytosine-5)-methyltransferase 1) is an important protein for maintaining high methylation of CPG island in gene promoter re-gion33,34, and enhancer of zeste homolog 2 (EZH2) is an important enzyme mediating the histone H3K27-site methylation. If there is methylation in gene promoter region, the expression of this gene will be silent. And such a phenomenon exists in E-cadherin gene promoter region35. Wu et al36 confirmed through RIP and CHIP experiments that lncRNA HNF1A-AS1 and linRNA 01133 can bind DNMT1 and EZH2 to the E-cadherin pro-moter region, and inhibit their expressions at the epigenetic level37. And there is no report on the relation of direct regulation and control between lncRNA and N-cadherin and Vimentin, and may-be the expressions of them are regulated by other mechanisms at the transcription or post-tran-scription levels. However, this paper did not fur-ther investigate whether there is a direct relation between BCAR4 and E-cadherin, N-cadherin and Vimentin, which is also the research direction in the future.

Conclusions

We observed for the first time that the knock-down of BCAR4 expression in NSCLC cells can inhibit the proliferation, invasion, and metasta-sis and promote apoptosis of tumor cells. And in vitro and in vivo experiments, our study fur-ther proved that BCAR4 promotes cell invasion and metastasis by influencing the EMT process. BCAR4 can be used as a predictive factor of prognosis of NSCLC patients. And blocking the interaction between BCAR4 and EMT can also provide an important theoretical basis for clinical reversal of NSCLC recurrence and me-tastasis.

Conflict of interestThe authors declare no conflicts of interest.

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