Background
Esophageal carcinoma ranks ninth in the new diagnosed cancer cases and occupies sixth in cancer deaths worldwide [
1]. Esophageal squamous cell carcinoma (ESCC), the most common histological subtype of esophageal carcinoma, accounts for approximately 90% of all cases globally [
2]. ESCC is highly prevalent in East Asia, South/East Africa and South Europe [
3,
4]. Multimodal treatment encompassing surgery, radiation and chemotherapy is currently the main therapeutic option for ESCC [
5]. Due to the lack of early clinical symptoms, the majority of ESCC patients are diagnosed at the advanced stages. The prognosis for esophageal carcinoma is poor, and the 5-year relative survival rate of patients with distant metastasis is only 5% [
6]. Therefore, identifying novel biomarkers and molecular targets is urgently needed for improving the outcomes of ESCC patients.
Circular RNAs (circRNAs), a class of non-coding transcripts generated by pre-mRNA back splicing, is characterized by a covalently closed loop without 5′ caps and 3′ tails [
7]. CircRNAs have drawn increasing attentions for their important participation in the genesis and development of human cancers at transcriptional, post-transcriptional, and translational levels [
8]. CircRNAs could act as miRNA sponges to affect the biological activity and function of their target mRNAs [
9]. For example, circARHGAP10 accelerated cell proliferation and migration in non-small-cell lung cancer by targeting the miR-150-5p/GLUT1 axis [
10]. Hsa_circ_0068871 facilitated bladder cancer progression via up-regulating FGFR3 expression and activating STAT3 signaling by serving as a sponge of miR-181a-5p [
11]. In recent years, several circRNAs such as ciRS-7 [
12], circPRKCI [
13] and circGSK3β [
14], have been found to be aberrantly expressed in ESCC and play important roles in cancer process. CircNTRK2 (hsa_circ_0087378), located at chr9:87356806–87,367,000 with a length of 237 bp, is formed by the circularization of 12–14 exons of Pre-NTRK2. A previous report by Yuan et al. demonstrated that hsa_circ_0087378 was down-regulated in ER-positive breast cancer, and hsa_circ_0087378/miR1260b/SFRP1 axis was proposed as a vital regulatory pathway [
15]. According to the GEO database (GSE131969), circNTRK2 is identified as the most up-regulated circRNAs among all candidates. Thus, circNTRK2 was selected as a research object for further function and mechanism analysis in ESCC.
In the current study, we verified that circNTRK2 was up-regulated in ESCC tissues and cells. High circNTRIK2 was associated with TNM stage, lymph node metastasis and poor prognosis. Functionally, knockdown of circNTRK2 repressed ESCC cell proliferation, invasion and EMT in vitro, and slowed tumor growth in vivo. Mechanistically, circNTRK2 facilitated NRIP1 expression by sponging endogenous miR-140-3p. Our findings reveal a novel regulatory mechanism of circNTRK2 in ESCC and proposed a promising therapeutic target for ESCC patients.
Materials and methods
Patient tissue specimens
The study was permitted by the Ethics Committee of The First Affiliated Hospital of Henan University of Chinese Medicine and performed according to the Declaration of Helsinki Principles. Informed written consents were signed by each patient for using their tissues. Tumor tissue samples and adjacent normal tissues (at least 3 cm from the edge of cancer tissues) were obtained from 56 patients with a definite pathological diagnosis of ESCC. None of the participants received preoperative radiotherapy or chemotherapy. All specimens were immediately snap-frozen in liquid nitrogen and stored at − 80 °C until further used.
Cell culture
Human ESCC cell lines (Eca-109, EC-9706, KYSE-30, KYSE-150, TE-1) were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human normal esophageal epithelial cell line Het-1A was obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). ESCC cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin. Het-1A cells were maintained in bronchial epithelial cell growth medium (BEGM, BulletKit, Lonza, MD). All cells were kept in a humidified incubator under an atmosphere of 5% CO2 at 37 °C.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA in ESCC tissues and cells was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). For circRNA and mRNA, the cDNA was synthesized by using SuperScript VILO cDNA Synthesis kit (Invitrogen; Carlsbad, CA, USA). For miRNA, reverse transcription was performed by using miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen, Beijing, China). The quantitative PCR was conducted using SYBR Premix Ex Taq II (TaKaRa, Dalian, China) on a LightCycler 480 system (Roche, Basel, Switzerland). The relative gene expression was calculated by using the 2-ΔΔCt method with U6 as the reference gene for miRNA and GAPDH as the internal control for circRNA and mRNA. All primers were obtained from Songon (Shanghai, China) and listed as follows:
circNTRK2: F, 5′-TCTCGGTCTATGCTGTGGTG-3′, R, 5′-CATTCGCTGCAGTTCCATAA-3′;
miR-140-3p: F, 5′-CAGTGCTGTACCACAGGGTAGA-3′, R, 5′-TATCCTTGTTCACGACTCCTTCAC-3′;
NRIP1: F, 5′-GAGCACTCCACCTTTACTTACAT-3′, R, 5′-CAATCATACCTATCGGTTTATCTG-3′;
GAPDH: F, 5′-TGTTCGTCATGGGTGTGAAC-3′, R, 5′-ATGGCATGGACTGTGGTCAT-3′;
U6: F, 5′-ATTGGAACGATACAGAGAAGATT-3′, R, 5′-GGAACGCTTCACGAATTTG-3′.
Confirmation of circular structure
To confirm the circular structure of circNTRK2, the circular and linear transcripts of NTRK2 were amplified by divergent and convergent primers in both complementary DNA (cDNA) and genomic DNA (gDNA) from ESCC cells. Then, agarose gel was used to separate the PCR products. In theory, circNTRK2 is amplified by divergent primers in cDNA but not gDNA. Additionally, Sanger sequencing was performed to verify the sequence of circNTRK2. Meanwhile, total RNA (5 μg) extracted from ESCC cells was incubated with RNase R (3 U/μg, Epicenter, Madison, WI, USA) for 20 min at 37 °C, followed by qRT-PCR to measure the expression levels of circular and linear NTRK2.
Cell transfection
To down-regulate circNTRK2, small interfering RNAs (siRNAs) targeting back splice junction of circNTRK2 (si-circ #1, si-circ #2, si-circ #3) were synthesized by GenePharma (Shanghai, China). NRIP1-specific siRNA (si-NRIP1) was used to silence NRIP1. Non-targeting control siRNA (si-NC) was used as a control. The sequences of siRNAs were listed as follows: si-circ #1, 5′- GCATGAAAGGTGCAAACCCAA − 3′; si-circ #2, 5′- GGCATGAAAGGTGCAAACCCA − 3′; si-circ #3, GTTTGGCATGAAAGGTGCAAA; si-NRIP1, 5′- GAGGAUCAGAACUUUAACATT-3′; si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′. MiR-140-3p mimics (miR-140-3p) and its matched control (miR-NC), miR-140-3p inhibitor (anti-miR-140-3p) and its matched control (anti-miR-NC) were obtained from GenePharma (Shanghai, China). To construct circNTRK2-overexpression plasmid (circNTRK2), the full length circNTRK2 cDNA was synthesized and then inserted into pLCDH-ciR vector (Geneseed, Guangzhou, China), while mock vector with no circNTRK2 sequence was used as a control. To establish NRIP1-overexpression plasmid (NRIP1), the coding region of NRIP1 cDNA was subcloned into pcDNA3.1 expression vector (Geneseed, Guangzhou, China). Cells were screened with puromycin (2 μg/ml) for 4 weeks to establish overexpression and control cell lines. ESCC cells were transfected with these oligonucleotides or plasmids at appropriate doses using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual.
Cell counting kit-8 (CCK-8) assay
CCK-8 (Bimake, Shanghai, China) was used to evaluate ESCC cell viability. Cells (2 × 103/well) were inoculated into 96-well plates. After incubation of 24 h, 48 h, 72 h, or 96 h at suitable condition, 10 μl CCK8 solution and 90 μl medium were added to each well. Two hours later, the absorbance at 450 nm was measured using a Varioskan Flash Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA).
5-Ethynyl-2′- deoxyuridine (EdU) assay
The proliferative ability of ESCC cells was detected by using a Cell-Light™ EdU DNA Cell Proliferation Kit (RiboBio, Guangzhou, China) following the manufacture’s guideline. Images of randomly selected fields were obtained under a fluorescence microscope (Leica, Wetzlar, Germany).
ESCC cells were added into 6-well plates at a density of 1000 cells/well and incubated at 37 °C for half a month. After fixing with 75% ethanol and staining with 0.1% crystal violet, the colonies with more than 50 cells were counted.
Transwell invasion assay
Transwell assay was implemented to determine the invasive capability of ESCC cells in 24-well Boyden chambers (Corning Incorporated, Corning, NY, USA) with pre-coated Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, 600 μl RPMI 1640 medium containing 10% FBS was added to the lower chamber, while 1 × 105 cells in 200 μl serum-free medium was plated into the upper chamber. After incubation at 37 °C with 5% CO2 for 24 h, cells remaining on the upper surfaces of the transwell chambers were removed, and cells traversed to the bottom surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The invasive cells were captured by a light microscope (Olympus, Japan) and counted from five randomly chosen fields.
Flow cytometry analysis
ESCC cells (5 × 105/well) were seeded into 6-well plates and cultured for 48 h at 37 °C. Then, cells were stained with an Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) and subjected to a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) for apoptosis detection.
Western blot assay
Total protein from ESCC cells was extracted using RIPA lysis and extraction buffer (Thermo Fisher Scientific, Rockford, IL, USA) and quantified by BCA Protein Assay Kit (Beyotime, Shanghai, China). Subsequently, 30 μg protein samples were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Piscataway, NJ, USA). After blocking in nonfat milk overnight at 4 °C, the membranes were incubated with primary antibody against E-cadherin (1:500, Abcam, Cambridge, MA, USA), vimentin (1:1000, Abcam), Cleaved PARP (1:1000; Abcam), Cleaved caspase-3 (1:500, Abcam), NRIP1 (1:500, Abcam), and GAPDH (1:10,000, Abcam) overnight at 4 °C, and then were probed with HRP-conjugated secondary antibody for 2 h at room temprature. ECL Western Blotting Detection Kit (Solarbio, Beijing, China) was used to detect the protein bands.
Subcellular fractionation
The RNA from nuclear and cytoplasm fractions was isolated by using a Cytoplasmic and Nuclear RNA Purification Kit (Norgen, Thorold, ON, Canada). In brief, ESCC cells were incubated with lysis solution on ice for 10 min and then centrifuged for 3 min at 12,000 g. The supernatant was collected for cytoplasmic RNA extraction and the nuclear pellet was used for nuclear RNA. qRT-PCR was used to measure the relative expression of circNTRK2 in different fractions. GAPDH was used as the cytoplasmic control, while U6 was used as the nuclear control.
Dual-luciferase reporter assay
To evaluate the direct binding between miR-140-3p and circNTRK2, the wild type or mutant sequences containing the binding sites of miR-140-3p in circNTRK2 were inserted into a pmirGLO vector (Promega Corporation, Madison, WI, USA), named as circNTRK2-wt and circNTRK2-mut, respectively. Subsequently, the luciferase reporter (100 ng) and miR-140-3p mimic or miR-NC (40 nM) were co-transfected into ESCC cells. After 48 h, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) with renilla luciferase activity as an internal reference. Similar procedures were conducted to examine the binding between miR-140-3p and NRIP1. Luciferase reporters including wild type NRIP1-wt and mutated types (NRIP1-mut1: nt 893–900, NRIP1-mut2: nt 3086–3092, and double mutation: NRIP1-mut1 + 2) were constructed.
RNA immunoprecipitation (RIP) assay
Magna RNA immunoprecipitation kit (Millipore, Billerica, MA, USA) was used to validate the binding between circNTRK2 and miR-140-3p. Briefly, ESCC cells were washed with PBS and lysed in RIP lysis buffer. Subsequently, cell lysates were incubated with RIP buffer containing magnetic beads coupled with human anti-Argonaute 2 antibody (Ago2; Millipore) or non-specific anti-IgG (Millipore). After elution, qRT-PCR was used to determine the level of circNTRK2 in immunoprecipitated RNA.
RNA pull-down assay
ESCC cells were transfected with the wild type biotin-labelled miR-140-3p (Bio-miR-140-3p-wt), mutant biotin-labeled miR-140-3p (Bio-miR-140-3p-mut), and non-specific negative control (Bio-miR-NC). After 48 h, cell lysates were incubated with streptavidin magnetic beads, followed by qRT-PCR to measure the level of circNTRK2 in RNA complexes.
Mice xenograft models
All animal experiments were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Henan University of Chinese Medicine and performed following the guidelines of National Institutes of Health. Male BALB/c nude mice aged 4–5 weeks were obtained from Shanghai Laboratory Animal Company (SLAC, Shanghai, China). KYSE-150 cells (5× 106) stably infected with lentivirus vectors encoding shRNA against circNTRK2 (sh-circNTRK2) or a non-silencing negative control (sh-NC) were subcutaneously injected into the right armpit of nude mice (n = 5 per group). The length and width of xenograft tumors were measured at indicated time points, and tumor volumes were calculated according with the formula: volume (mm3) = width2 × length/2. At 25 days after inoculation, all mice were sacrificed and xenografts were dissected for further analysis.
Statistical analysis
Data analysis was performed by using GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA, USA). All continuous data were presented as mean ± standard deviation (SD). Differences in two groups were assessed by Student’s t-test, while one-way analysis of variance (ANOVA) was utilized in multiple groups. Chi-square test was applied to evaluate the relationship between circNTRK2 expression and clinicopathological parameters. Kaplan-Meier method was employed to determine the survival rate and log-rank test was used to compare the difference. Pearson’s correlation coefficients were used to detect the correlation. P < 0.05 was set as statistically significant.
Discussion
Although there is a slight decline in the global incidence of ESCC in recent years, it is still a primary cause of cancer-related mortality worldwide [
17]. CircRNAs have drawn increasing attentions for their important roles in the initiation and progression of human cancers [
18]. However, much is still undiscovered about the precise roles of circRNAs in ESCC. A deeper understanding of the mechanisms of circRNAs is vital to discover the promising biomarkers and targets for ESCC patients. Based on the information from GEO database (GSE131969), we selected circNTRK2 to elucidate its biological significance and underlying mechanisms in ESCC. Our results demonstrated that circNTRK2 served as a sponge for miR-140-3p to relieve its inhibition on NRIP1, thus contributing to cell proliferation and invasion in ESCC.
Up to now, increasing circRNAs have been discovered to be associated with the pathophysiological events in ESCC. For example, hsa_circ_0006948 was up-regulated in ESCC, and induced HMGA2 expression to facilitate ESCC progression via miR-490-3p [
19]. Hsa-circ_0000654 expression was increased in ESCC tissues, and knockdown of circ_0000654 repressed cell growth and metastasis through miR-149-5p/STAT3 axis [
20]. Circular RNA ciRS-7 promoted ESCC growth and metastasis via serving as a miR-876-5p sponge to increase MAGE-A family expression [
21]. In the current study, circNTRK2 was confirmed as a circular RNA through Sanger sequencing, PCR and RNase R treatment. CircNTRK2 expression was elevated in ESCC tissues and cells. Moreover, high circNTRK2 expression was associated with advanced TNM stage, lymph node metastasis and poor prognosis. Knockdown of circNTRK2 inhibited ESCC cell proliferation, invasion and EMT, and enhanced apoptosis, while overexpression of circNTRK2 displayed the contrary effect. These data suggested the carcinogenicity of circNTRK2 in ESCC. However, another study showed that hsa_circ_0087378 (circNTRK2) was down-regulated in tumor tissues and cell lines in ER-positive BC, and hsa_circ_0087378/miR-1260b/SFRP1 was concluded as its possible regulatory mechanism [
15]. The controversy may be attributed to the cell-type specific features of circular RNA expression [
22].
In recent years, circRNAs are known as competing endogenous RNA (ceRNA) to influence miRNAs stability and expression, thereby alleviating their inhibition of target genes [
23]. By using subcellular fractionation assay, circNTRK2 was found to predominantly exist in the cytoplasm, implying that it may exert effect through post-transcriptional regulation. Hence, we speculated that circNTRK2 was involved in the regulation of ESCC through the similar ceRNA mechanism. On the basis of the prediction from bioinformatic tools and the data from luciferase reporter, RIP and RNA pull-down assays, miR-140-3p was confirmed as a direct target of circNTRK2. MiR-140-3p was previously demonstrated as a tumor-suppressor in some types of human malignancies, such as squamous cell lung cancer [
24], breast cancer [
25], hepatocellular carcinoma [
26] and cervical cancer [
27]. In the present study, miR-140-3p expression was down-regulated in ESCC tissues and cells, and was inversely correlated to circNTRK2 expression. Functionally, overexpression of miR-140-3p repressed cell proliferation and invasion, and promoted apoptosis, suggesting the anti-tumor effect of miR-140-3p in ESCC. However, miR-140-3p-induced suppression of cell proliferation and invasion was evidently reversed following the introduction of circNTRK2. From the above results, we concluded that circNTRK2 accelerated ESCC progression via sponging miR-140-3p.
Nuclear receptor-interacting protein 1 (NRIP1), also known as RIP140, was originally identified in breast cancer cells through its interaction with the estrogen receptor α [
28]. NRIP1 is associated with the regulation of various oncogenic signaling pathways and participates in the progression of solid tumors [
29]. For instance, down-regulation of NRIP1 by siRNA inhibited breast cancer cell growth in vitro and in vivo [
30]. NRIP1 was demonstrated as an independent predictor of poor survival for cervical cancer patients [
31]. However, NRIP1 was found as a tumor-suppressor in some other malignancies, such as nasopharyngeal carcinoma [
32], hepatocellular carcinoma [
33], and colon cancer [
34]. In our study, NRIP1 was verified as a target of miR-140-3p in ESCC cells, and NRIP1 was highly expressed in ESCC tissues. Moreover, circNTRK2 overexpression led to an increase of NRIP1, while this effect was attenuated by the restoration of miR-140-3p. Furthermore, NRIP1 expression was positively associated with circNTRK2, while was negatively correlated to miR-140-3p in ESCC tissues. Thus, a conclusion was reached that circNTRK2 functioned as a miR-140-3p sponge to abolish its inhibition of NRIP1. Loss-of-function experiments revealed that silencing of NRIP1 suppressed cell proliferation and invasion, but facilitated apoptosis in ESCC. Moreover, the anti-proliferation and anti-invasion effects induced by circNTRK2 knockdown were greatly abrogated by the overexpression of NRIP1. To sum up, circNTRK2 promoted ESCC progression by sponging miR-140-3p and stimulating NRIP1. Our study elucidated a circNTRK2-miR-140-3p-NRIP1 regulatory axis in ESCC (Fig.
6K). In addition to the “miRNA sponges” function, circRNAs can also interact with RNA binding proteins, regulate modulate mRNAs stability, modulate gene transcription, and act as translation templates of proteins [
18,
35]. The other possible action mechanisms of circNTRK2 in ESCC will be further investigated in our future research.
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