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SLC34A2 axis
Biomedicine & Pharmacotherapy 120 (2019) 109457
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Long non-coding RNA MEG3 regulates migration and invasion of lung cancer stem cells via miR-650/SLC34A2 axis Yongjuan Zhaoa, Zhenxing Zhub, Shaomin Shia, Jing Wanga, Ning Lia, a b
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Department of Respiratory, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China Department of Hematology and Oncology, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lung cancer stem cells MEG3 miR-650 SLC34A2 NSCLC
Long non-coding RNA maternally expressed gene 3 (MEG3) is related to the occurrence and development of nonsmall cell lung cancer (NSCLC). However, the function and underlying molecular mechanisms of MEG3 in lung cancer stem cells (LCSCs) are still unclear. LCSCs were determined in lung cancer cells using fluorescenceactivated cell sorting (FACS). qRT-PCR and western blot were performed to examine the expressions of MEG3, miR-650, solute carrier family 34 member 2 (SLC34A2), octamer-binding transcription factor 4 (Oct4), and CD133. Sphere assay was employed to evaluate sphere-forming ability. Cell migration and invasion were analyzed by Transwell assay. The relationships among MEG3, miR-650, and SLC34A2 were validated by luciferase reporter, RIP, and RNA pulldown assays. We found MEG3 was downregulated in LCSCs. MEG3 depletion strengthened stem cell-like characteristics and sphere-forming ability in LCCs. Upregulation of MEG3 suppressed migration and invasion in LCCs and LCSCs. miR-650 was bound to MEG3 and upregulated in LCSCs. miR-650 inhibitor alleviated si-MEG3-induced promotion of stem cell-like characteristics in lung cancer cells (LCCs) H1299. Furthermore, miR-650 mimic attenuated the MEG3 upregulation-mediated inhibition of migration and invasion. In addition, SLC34A2 was a target of miR-650 and downregulated in LCSCs. miR-650 mimic induced stem cell-like characteristics in LCCs, which was weakened by overexpression of SLC34A2. In contrast, the repression of SLC34A2 mitigated the miR-650 silencing-induced inhibition of migration and invasion in LCCs and LCSCs. Besides, MEG3 regulated SLC34A2 expression by sponging miR-650. Importantly, SLC34A2 weakened MEG3-mediated stem cell-like state and cell metastasis. Our data suggested MEG3 was involved in stem cell-like state of LCCs and curbed migration and invasion through miR-650/SLC34A2 axis in NSCLC.
1. Introduction Lung cancer causes the most cancer-related deaths and non-small cell lung cancer (NSCLC) accounts for the majority of cases [1,2]. Although early diagnosis and more effective treatment strategies have diminished the mortality rates in recent years [3,4], the development of recurrence, metastasis and chemoresistance is still inevitable [5,6]. A correlation has been found between resistance to radiation, chemotherapeutic treatments, or tumor recurrence and lung cancer stem cells (LCSCs) [7–9]. It has been revealed that CD44 and CD133 are widely used to sort cancer stem cells (CSCs) from normal cancer cells [5,10,11]. Moreover, LCSCs possess the property to form spheres, which are thought to be enriched stem cells [11,12]. Therefore, eliminating LCSCs may provide a therapeutic avenue for overcoming chemoresistance, metastasis and relapse in NSCLC patients. Long non-coding RNAs (lncRNAs) have been reported to play key
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roles in variety of biological processes, including stem cell biology [13,14]. LncRNA maternally expressed gene 3 (MEG3) has been reported to be suppressed in various cancers [15,16]. Notably, MEG3 is significantly downregulated in NSCLC tissues and regulates NSCLC cell proliferation and apoptosis through the activation of p53 [17]. Moreover, MEG3 is markedly reduced in chemotherapy-resistant lung cancer tissues, and upregulation of MEG3 attenuates the autophagy and increases the sensitivity of vincristine in chemotherapy of NSCLC [18]. Those data suggested that MEG3 may be required for the development of NSCLC. In addition, a study has indicated that loss of MEG3 enhances cell proliferation, migration, invasion, sphere-forming ability, and CSC properties, whereas decreases the chemosensitivity to gemcitabine in human pancreatic cancer [19]. However, the function and underlying molecular mechanisms of MEG3 in LCSCs remain largely unknown. As we all known, lncRNAs that may act as microRNA (miRNA) sponges. Accumulating evidence has indicated that microRNA-650
Corresponding Author at: Department of Respiratory, China-Japan Union Hospital of Jilin University, No.126, Xiantai Street, Changchun, Jilin, China. E-mail address: [email protected] (N. Li).
https://doi.org/10.1016/j.biopha.2019.109457 Received 22 May 2019; Received in revised form 9 September 2019; Accepted 12 September 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.5. Migration and invasion assays
(miR-650) could act as an oncogene in the multiple cancers, including human lung adenocarcinoma [20–22]. Recently, miR-650 is reported to be correlated with neoplastic transformation of liver cancer stem cells [23]. Nevertheless, whether MEG3 participates in cancer stem cell maintenance of NSCLC by interacting with miR-650 has not been reported. It is known that miRNA can regulate a large fraction of proteincoding genes, which form a regulatory network during cell differentiation and development. Yang et al. have reported that solute carrier family 34 member 2 (SLC34A2) is downregulated in A549 and H1299 cells and overexpression of SLC34A2 inhibits the viability and invasion in NSCLC [24]. Furthermore, SLC34A2 has been reported to maintain the stem cell-like characteristics in CD147+ breast cancer stem cells (BCSCs) [25]. However, whether SLC34A2 is responsible for the MEG3/ miR-650 axis of stem cell state of NSCLC is not fully understood. In present study, we sought to investigate the effect of MEG3 on stem cell-like state and metastasis in NSCLC cells and explore the role of MEG3/miR-650/ SLC34A2 axis in this process.
1 × 105 of transfected LCCs or LCSCs suspended in 150 μL serumdeprived culture medium were seeded into the upper compartment of Transwell plates with 8.0 μm pore (Corning; cat number: 3422). For cell invasion assay, the membranes of the upper compartments were precoated with Matrigel (Solarbio, Beijing, China; cat number: 356234). Dilute the Matrigel (10 u L) with serum-free cold cell culture medium DMEM and added 100 ul of dilute gel to the 24-well transwell upper chamber (4 °C). Then incubate the transwell at 37 °C for at least 4–5 h. And un-coated ones were used for cell migration assay. The cells were cultured for 24 h, and cells migrated to the underside of the upper compartment membrane in response to culture medium supplemented with 10% FBS in the lower compartment were fixed with methanol and stained with 0.1% crystal violet (Sigma). The number of cells on the lower side of the filter were counted in 5 randomly picked view under microscope (Nikon Corp, Tokyo, Japan). Each migration or invasion group was tested with three replicates.
2. Materials and methods 2.1. Cell culture
2.6. RNA extraction and qRT-PCR assays
NSCLC cell line H1299 and human kidney cell line 293 T were obtained from the American Type Culture Collection (Manassas, VA, USA). H1299 cells and 293 T cells were cultured in complete Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) or RPMI 1640 medium (Thermo Fisher Scientific), respectively, supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37 °C in a humidified incubator containing 5% CO2.
Total RNAs were isolated from cells by using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and reversely transcribed into complementary DNA (cDNA) using the TaqMan MicroRNA Reverse Transcription Kit (Biosystems, Foster City, CA, USA) or the Primer Script RT reagent kit (TaKaRa, Dalian, China). qRT-PCR was performed using a SYBR Green PCR Master Mix (Biosystems) in line with the procedure of instructions. Relative expressions of MEG3, miR-650, octamer-binding transcription factor 4 (Oct4), CD133 and SLC34A2 were calculated on the basis of the 2−ΔΔCt method, normalizing to U6 small nuclear RNA (snRNA) or β-actin, respectively. The primers were listed as follows：MEG3 forward, 5′-CTGCCCATCTACACCTCACG-3′, and reverse, 5′-CTCTCCGCCGTCTGCGCTAGGGGCT-3′; miR-650 forward, 5′AGAGGAGGCAGCGCTCT-3′, and reverse, 5′- CAGTGCGTGTCGTGG AGT -3′; SLC34A2 forward, 5′-GAGAACATCGCCAAATGC-3′, and reverse, 5′GCAACCACAGAGGACCAG-3′; Oct4 forward, 5′-GTGCCGTGAAGCTGG AGAA-3′, and reverse, 5′-TGGTCGTTTGGCTGAATACCTT-3′; CD133 forward: 5′-ACAGCGATCAAGGAGACCAA-3′, and reverse: 5′-GTCAAG TTCTGCATCCACGG-3′; β-actin forward, 5′-GGACCTGACTGACTAC CTC-3′, and reverse, 5′-TACTCCTGCTTGCTGAT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′, and reverse, 5′-AACGCTTCACGAATTT GCGT-3′.
2.2. Isolation of LCSCs by fluorescence-activated cell sorting (FACS) LCSCs were isolated by sorting CD44+CD133+ populations using anti-CD133-PE and anti-CD44-FITC antibodies (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, H1299 cells were incubated with anti-CD133-PE and anti-CD44-FITC antibodies on ice for 30 min in the dark. After washed with cold PBS, CD44+CD133+ cells were purified by flow cytometry (BD Biosciences). All CD44-/CD133- cells were called LCCs. 2.3. Sphere formation assay Cells were plated as single-cell suspensions in low-attachment plates 24-well plates (Corning, Corning, NY, USA; cat number: 3524) at a density of 100 cells/well. Cells were grown in serum-free Mammo Cult Human Medium (Stem Cell Technologies, Vancouver, BC, Canada) for 1 or 7 days. All spheres in each well were assessed via a Zeiss Axiovert microscope (Carl Zeiss, Thornwood, NY, USA). Three replicates were involved in each group.
2.7. Western blot assay Proteins were extracted using radioimmunoprecipitation assay buffer. The protein concentration was measured using a BCA assay kit (Beyotime, Shanghai, China). Proteins (20 μg of each sample) were separated by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% fat-free milk powder in Tris-buffered saline with 0.1% Tween 20 buffer and then incubated at 4 °C overnight with following primary antibodies against Oct4 (Abcam, Cambridge, MA, USA; cat number: ab27985), CD133 (Abcam; cat number: ab19898), SLC34A2 (Abcam; cat number: ab228474), β-actin (Abcam; cat number: ab8226). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA). The protein signals were visualized using enhanced chemiluminescence (Beyotime). Densitometry values were normalized to levels of β-actin and analyzed by ImageJ software (Media Cybernetics, Rockville, MD, USA).
2.4. Cell transfection Cells (70% confluence in 6-well plates) were transfected with small interfering RNA (siRNA) for MEG3 (si-MEG3), siRNA for SLC34A2 (siSLC34A2), siRNA negative control (si-NC), MEG3 overexpression plasmid (MEG3), SLC34A2 overexpression plasmid (SLC34A2), pcDNA 3.0 vector (pcDNA), miR-650 mimic (miR-650), negative control mimic (miR-NC), miR-650 inhibitor (anti-miR-650) or negative control inhibitor (anti-miR-NC) using Lipofectamine 3000 reagent (Thermo Fisher Scientific) according to the manufacturer's protocols. miR-650 mimic (Sigma, St. Louis, MO, USA; cat number: HMI0881); miR-650 inhibitor (Sigma; cat number: HSTUD0881). siRNA for MEG3, SLC34A2, MEG3 or SLC34A2 overexpression plasmid were purchased from Vigene Bioscicences. The transfected cells after 48 h were used for following experiments. 2
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We found that cell metastasis ability was higher in LCSCs than that of LCCs. Then, LCCs or LCSCs were transfected with pcDNA 3.0 vector or MEG3 overexpression plasmid. Also, following transfection efficiency assay revealed that the transfection of MEG3 overexpression plasmid was efficient to induce the higher expressions of MEG3 in LCCs and LCSCs (Fig. 2A). The results further revealed that upregulation of MEG3 decreased the number of migration and invasion cells in LCCs and LCSCs (Fig. 2B and C).
2.8. Luciferase reporter assay Bioinformatics analysis was introduced to analyze the binding sites between MEG3 and miR-650 using DIANA Tools and the binding sites between miR-650 and SLC34A2 using miRDB database. MEG3 fragments containing the wild-type (MEG3-WT) or mutated (MEG3-MT) binding sites of miR-650 were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA). The same method was used for the construction of SLC34A2-WT or SLC34A2-MT. For the association between miR-650 and MEG3, 293 T cells were co-transfected with miR-650 or miR-NC and MEG3-WT or MEG3-MT using Lipofectamine 3000. For the association between miR650 and SLC34A2, 293 T cells were co-transfected with miR-650 or miR-NC and SLC34A2-WT or SLC34A2-MT. For the association among miR-650, MEG3 and SLC34A2, 293 T cells were transfected with SLC34A2-WT, SLC34A2-WT + miR-650, SLC34A2-WT + miR650+pcDNA3.0 vector, or SLC34A2-WT + miR-650+MEG3. Then, the luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.
3.3. miR-650 is bound to MEG3 LncRNAs that act as sponges to bind miRNAs and prevent them from performing normal regulatory activities. To address whether MEG3 acts in this way, the potential binding sites of miR-650 and MEG3 were predicted by DIANA Tools (Fig. 3A). Then, a luciferase reporter assay was carried out to validate whether MEG3 binds miR-650. The results indicated that luciferase activity was decreased in 293 T cells transfected with miR-650 mimic in MEG3-WT group, while it showed little impact on the luciferase activity of MEG3-MT group (Fig. 3B). Subsequently, RIP assay was conducted with antibody against Ago2 or IgG. As shown in Fig. 3C, MEG3 was enriched in LCSCs transfected with miR-650 mimic in RIP-Ago2 group compared with that in RIP-IgG group. Moreover, MEG3 was pulled down by Bio-miR-650-WT, while it failed to show any efficacy in Bio-miR-650-MT group (Fig. 3D). Meanwhile, a significant abundance of miR-650 was observed in Bioprobe-MEG3-WT group compared with that in Bio-probe-MEG3-MT group (Fig. 3D), indicating that MEG3 was interacted with miR-650.
2.9. RNA immunoprecipitation (RIP) and RNA pulldown assays RIP assay was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore) according to the manufacturer’s instructions. Briefly, the cells were harvested and incubated with magnetic beads and anti-Ago2 antibody (Abcam), and IgG was used as a negative control. The beads were washed and incubated with proteinase K to digest the protein at 24 h after incubation. For RNA pulldown assay, probes for MEG3 or miR-650 were biotinylated (GENEWIZ, Suzhou, China) and transfected into LCCs. After 48 h, the cells were harvested and lysed. The samples were incubated with Dynabeads M280 Streptavidin (Solarbio). The bound RNAs were analyzed by qRTPCR.
3.4. miR-650 is implicated in the function of MEG3 in LCCs and LCSCs
3. Results
Given the above results, the effect of miR-650 on the MEG3-mediated function of LCCs and LCSCs was further explored. Firstly, we evaluated the level of miR-650 in LCCs and LCSCs. Higher expression of miR-650 was displayed in LCSCs compared with that of LCCs (Fig. 4A). Then, downregulation of MEG3 increased the expression of miR-650, which was weakened by miR-650 inhibitor introduction (Fig. 4B). Additionally, miR-650 depletion attenuated the si-MEG3-induced increase of Oct4 and CD133 levels in LCCs (Fig. 4C and D). Interestingly, enforced expression of MEG3 led to the inhibition of miR-650 expression, while the introduction of miR-650 mimic abated the repression effect (Fig. 4E and F). Subsequently, the analysis of cell migration and invasion ability revealed that miR-650 mimic receded the MEG3 upregulation-induced suppression of migration and invasion in LCCs and LCSCs (Fig. 4G and H).
3.1. MEG3 depletion strengthens stem cell-like characteristics in LCCs
3.5. SLC34A2 is a target of miR-650
Firstly, to investigate the role of MEG3 in LCSCs, CD44+CD133+ cells were sorted from H1299 cells, namely LCSCs (Fig. 1A). Next, the basal expression of MEG3 was measured in LCSCs and LCCs which had been isolated from H1299 cells using FACS. The results showed a significant decrease in MEG3 expression in LCSCs compared with that in LCCs (Fig. 1B). After that, small interfering RNA for MEG3 was transfected in LCCs. As expected, the introduction of si-MEG3 strikingly reduced MEG3 level (Fig. 1C). Then, LCSC markers were detected in LCCs transfected with si-NC or si-MEG3. Compared with si-NC group, MEG3 knockdown cells demonstrated elevated expressions of Oct4 and CD133 (Fig. 1D and E), suggesting that MEG3 depletion promoted stem cell-like state of LCCs. Moreover, sphere-forming ability was enhanced in si-MEG3 groups compared with that of si-NC group at day 7 (Fig. 1F).
It is accepted that miRNAs exert their functions by regulating the expression of downstream target genes. Therefore, we searched for targets of miR-650 using miRDB Tools, which presented the potential binding sites of miR-650 and SLC34A2 (Fig. 5A). Next, luciferase reporter assay and RNA pulldown assay were used to further validate the interaction of SLC34A2 and miR-650. After over-expressing of miR-650, the luciferase activity of the SLC34A2-WT reporter containing miR-650 binding sites was found to be attenuated in 293 T cells, while it was not observed in SLC34A2-MT group (Fig. 5B). Moreover, RIP assay disclosed more enrichment of SLC34A2 in miR-650 group than that in miR-NC group (Fig. 5C). According to the results above, miR-650 was interacted with SLC34A2 by putative complementary sites.
2.10. Statistical analysis Data are presented as the mean ± standard deviation (SD). The experiments were performed three times (n = 3). All statistical analyses were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). For comparing the differences between them, paired Student’s ttest and one-way analysis of variance (ANOVA) were used. A value of P less than 0.05 was considered to be statistically significant.
3.6. SLC34A2 participates in miR-650-induced stem cell-like state and metastasis in NSCLC
3.2. Upregulation of MEG3 inhibits cell migration and invasion in LCCs and LCSCs
The mRNA level of SLC34A2 was greatly decreased in LCSCs compared with that in LCCs (Fig. 6A). Moreover, an obvious repression of SLC34A2 was observed in LCCs transfected with miR-650 mimic, which
In the following experiments, we tested the hypothesis that overexpression of MEG3 influences the migration and invasion of NSCLC. 3
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Fig. 1. MEG3 depletion strengthens stem cell-like characteristics in LCCs. (A) LCSCs were determined in H1299 cells using FACS. (B) The level of MEG3 was examined in LCCs and LCSCs. (C) The level of MEG3 was measured in LCCs transfected with si-NC or si-MEG3. (D and E) The mRNA and protein levels of Oct4 and CD133 in LCCs transfected with si-NC or si-MEG3. (F) Sphere assay was performed in LCCs transfected with si-NC or si-MEG3. **P < 0.01, ***P < 0.001.
Fig. 2. Upregulation of MEG3 inhibits cell migration and invasion in LCCs and LCSCs. LCCs and LCSCs were transfected with pcDNA3.0 vector or MEG3 overexpression plasmid, respectively. (A) The level of MEG3 was examined in LCCs and LCSCs. (B and C) Cell migration and invasion abilities were evaluated by Transwell assay. **P < 0.01.
3.7. MEG3 regulates SLC34A2 expression by sponging miR-650
was undermined by SLC34A2 overexpression (Fig. 6B). Furthermore, the results of western blot and qRT‐PCR both indicated that overexpression of miR-650 markedly increased the expressions of stemness markers Oct4 and CD133 at protein and mRNA levels (Fig. 6C and D). In addition, the effects of SLC34A2 silencing on miR-650 inhibitor-induced impediment of migration and invasion were further studied. After the transfection, SLC34A2 protein expression was effectively increased in LCCs and LCSCs transfected with miR-650 inhibitor, while it was undermined by si- SLC34A2 (Fig. 6E and F). Furthermore, we found that SLC34A2 depletion drastically attenuated the miR-650 inhibitorinduced repression of cell migration and invasion in LCCs and LCSCs (Fig. 6G and H).
Considering the effects of MEG3, miR-650, and SLC34A2 on cell function in LCCs and LCSCs, we further explored the associations among the MEG3, miR-650, and SLC34A2. As shown in Fig. 7A, luciferase activity was significantly decreased in 293 T cells in SLC34A2WT + miR-650 mimic group, while it was abated by inhibition of MEG3. Moreover, the effects of miR-650 and MEG3 on SLC34A2 protein expression were investigated in LCSCs. Upregulation of MEG3 increased the expression of SLC34A2, which was attenuated by introduction of miR-650 (Fig. 7B). In contrast, miR-650 inhibition abrogated the MEG3 depletion-induced loss of the SLC34A2 expression (Fig. 7C). To address 4
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Fig. 3. miR-650 is bound to MEG3. (A) The potential binding sites of miR-650 and MEG3 were predicted by DIANA Tools. (B) The luciferase activity was measured in 293 T cells co-transfected with MEG3-WT or MEG3-MT and miR-650 mimic or miR-NC. (C) The expression of MEG3 was detected in LCCs after Ago2 or IgG RIP assay. (D) The level of MEG3 in the sample pulled down by biotinylated miR-650 and the expression of miR-650 in the sample pulled down by biotinylated MEG3 probe. **P < 0.01, ***P < 0.001.
miR-650 in a negative-feedback mechanism. In consequence, MEG3 can also indirectly stimulate SLC34A2 expression through inhibiting the level of miR-650. It is well documented that CSCs exist in many cancers and are closely associated with cancer progression, metastasis, and chemoresistance [9,26]. To fully address their potentials as cancer markers or drug targets, a better understanding of the underlying molecular mechanism of CSC maintenance is required. Oct4 is detected in CSCs enriched from tumor tissues or cell lines, which is related to its function in tumorigenesis, metastasis and resistance to anticancer therapies [27]. Moreover, the loss of Oct4 expression is associated with stem cell differentiation [28]. To further explore the effect of MEG3 on stem cell-like state of LCCs, the expressions of Oct4 and CD133 selected as surface markers of LCSCs were subsequently analyzed. Si-MEG3-mediated silence of MEG3 increased the expressions of Oct4 and CD133, suggesting that MEG3 may be involved in maintaining cancer stem cell phenotypes. Consistent with the data, suppression of MEG3 strikingly increased sphere-forming ability, revealing that MEG3 depletion may contribute to induce the stem cell-like state in LCCs. In addition, upregulation of MEG3 inhibited the migration and invasion in LCCs and LCSCs, suggesting that MEG3 may act as a tumor-suppressor in the development of NSCLC, which is consistent with previous study [18]. Crosstalk between lncRNAs and miRNAs has been involved in progression of NSCLC. For instance, linc00673 regulates NSCLC proliferation, migration, invasion and epithelial mesenchymal transition by interacting with miR-150-5p [29]. Moreover, lncRNA colon cancerassociated transcript-1(CCAT1)/miR-130a-3p axis enhances cisplatin
whether MEG3 regulates phenotypic changes through miR-650/ SLC34A2 axis, LCCs were introduced si-MEG3 and SLC34A2 overexpression plasmid. The transfection of SLC34A2 overexpression plasmid rescued si-MEG3-mediated SLC34A2 downregulation (Fig. 8A). As expect, ectopic expression of SLC34A2 also weakened the si-MEG3induced upregulation of Oct4 and CD133 on mRNA and protein levels in LCCs (Fig. 8B and C). In LCCs and LCSCs, enhanced expression of MEG3 also promoted the protein level of SLC34A2, which was undermined by loss of SLC34A2 (Fig. 8D and E). Interestingly, enforced expression of MEG3 led to the inhibition of cell migration and invasion, while the introduction of si- SLC34A2 abated the repression effect (Fig. 8F and G). Therefore, we thought that MEG3 may play an important role through miR-650/ SLC34A2 axis. 4. Discussion NSCLC has a low 5-year overall survival rate, which is considered to be post-treatment enrichment of CSCs with abilities of cancer initiation and further maintenance of tumors [9]. In the present study, LCSCs were determined in H1299 cells using FACS. We demonstrated that MEG3/miR-650/SLC34A2 axis exhibited a double-negative feedback loop in regulation of LCSCs maintenance. MEG3 was significantly downregulated in LCSCs. Moreover, MEG3 had a critical role in inhibiting LCSCs properties and tumorigenesis in vitro. Here, we described a mechanism in which MEG3 functioned as a competing endogenous RNA (ceRNA) by sponging miR-650 to promote the expression of SLC34A2. Interestingly, SLC34A2 is also repressed by
Fig. 4. miR-650 is implicated in the function of MEG3 in LCCs and LCSCs. (A) The level of miR-650 was examined in LCCs and LCSCs. (B) The abundance of miR650 in LCCs transfected with si-NC, si-MEG3, si-MEG3+anti-miR-NC, or si-MEG3+anti-miR-650. (C and D) The mRNA and protein levels of Oct4 and CD133 in LCCs treated as described in Fig. 4B. (E and F) LCCs and LCSCs were transfected with pcDNA3.0 vector, MEG3 overexpression plasmid, MEG3 overexpression plasmid + miR-NC, or MEG3 overexpression plasmid + miR-650 mimic, respectively. qRT-PCR was performed to measure the expression of miR-650. (G and H) Cell migration and invasion abilities were evaluated by Transwell assay. **P < 0.01, ***P < 0.001. 5
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Fig. 5. SLC34A2 is a target of miR-650. (A) The putative binding site of miR-650 and SLC34A2. (B) The luciferase activity was measured in 293 T cells cotransfected with SLC34A2-WT or SLC34A2-MT and miR-650 mimic or miR-NC. (C) The expression of SLC34A2 was detected in LCCs after Ago2 or IgG RIP assay. *P < 0.05, ***P < 0.001.
Notably, restoration of SLC34A2 rescued the MEG3 silence on cell phenotypic changes. Meanwhile, knockdown of SLC34A2 also receded the effect of gain of MEG3 on cell migration and invasion. A novel regulatory mechanism MEG3/miR-650/SLC34A2 was first observed in LCSCs, establishing a comprehensive regulatory network that adapts to environmental changes in the maintenance of LCSCs. However, there are some limitations. Our study only investigated the partial roles of MEG3 in vitro. The role of MEG3 in vivo will be validated in future. Moreover, the functions of SLC34A2 and miR-650 alone should be explored. In conclusion, we disclosed MEG3 as a cancer suppressor, which exerts a crucial role in the stem cell-like state and metastasis in NSCLC. Besides, our findings shed light on the interaction between MEG3 and miR-650 for the first time, and revealed that MEG3 positively regulates posttranscriptional expression of SLC34A2 by sponging miR-650 in NSCLC. MEG3/miR-650/SLC34A2 axis may be implicated in stem celllike state and cell migration and invasion, thus probably providing a novel therapeutic avenue in NSCLC.
resistance of NSCLC cells by targeting sex-determining region Y-box 4 (SOX4) [30]. miR-650 has been reported to be upregulated in NSCLC [31]. In this report, we found that MEG3 negatively regulated the expression of miR-650. Besides, the ectopic expression of miR-650 was observed in LCSCs. Interestingly, the inhibition of miR-650 attenuated the MEG3 silencing-mediated promotion of stem cell-like state. It was also observed that miR-650 weakened the effect of upregulation of MEG3 on migration and invasion in LCCs and LCSCs. Those findings demonstrated that MEG3 was involved in stem cell-like state and metastasis in NSCLC by interacting with miR-650, forming a reciprocal negative regulatory loop. In general, feedback loops are widely observed between miRNAs and their targets. In a previous report, miR650 promotes the hepatocellular carcinoma metastasis via suppressing the expression of large tumor suppressor kinase 2 [32]. Here, we revealed that SLC34A2 was a target of miR-650. Thus, it is probably that there is a negative regulation between SLC34A2 and miR-650. However, the effect of SLC34A2 on the function of miR-650 in NSCLC needs further study. SLC34A2, a member of SLC34 family of sodium-driven phosphate cotransporters [33]. A previous study disclosed that SLC34A2 inhibits tumor growth and metastasis ability in H1299 subcutaneous tumor model and lung metastasis model [17]. Moreover, SLC34A2 could be a favorable prognostic marker in lung cancer patients [34]. In present study, a significant reduce of SLC34A2 was observed in LCSCs. Overexpression of SLC34A2 ameliorated miR-650-induced promotion of the cancer stem cell-like characteristics. Furthermore, the inhibition of SLC34A2 restored cell migration and invasion, which was blocked by downregulation of miR-650. More importantly, MEG3 and miR-650 competitively regulated the SLC34A2 expression, indicating that MEG3, functioning as a ceRNA, links the network of miR-650 and SLC34A2.
Declaration of Competing Interest All authors declare that they have no financial and non-financial conflicts of interest.
Acknowledgement Not applicable.
Fig. 6. SLC34A2 participates in miR-650-induced stem cell-like state and metastasis in NSCLC. (A) The level of SLC34A2 was examined in LCCs and LCSCs. (B) The expression of SLC34A2 in LCCs transfected with miR-NC, miR-650 mimic, miR-650 mimic + pcDNA3.0 vector, or miR-650 mimic + SLC34A2 overexpression plasmid. (C and D) The mRNA and protein levels of Oct4 and CD133 in LCCs treated as described in Fig. 6B. (E and F) LCCs and LCSCs were transfected with anti-miRNC, anti-miR-650, anti-miR-650+si-NC, or anti-miR-650+si-SLC34A2, respectively. Western blot was performed to measure the expression of SLC34A2. (G and H) Cell migration and invasion abilities were evaluated by Transwell assay in LCCs and LCSCs. **P < 0.01, ***P < 0.001. 6
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Fig. 7. MEG3 regulates SLC34A2 expression by sponging miR-650. (A) The luciferase activity was measured in 293 T cells transfected with SLC34A2-WT, SLC34A2-WT + miR-650 mimic, SLC34A2-WT + miR-650 mimic + pcDNA3.0 vector, or SLC34A2-WT + miR-650 mimic + MEG3 overexpression plasmid. (B) The expression of SLC34A2 in LCSCs transfected with pcDNA3.0 vector, MEG3 overexpression plasmid, MEG3 overexpression plasmid + miR-NC, or MEG3 overexpression plasmid + miR-650 mimic. (C) The expression of SLC34A2 in LCSCs transfected with si-NC, si-MEG3, si-MEG3+anti-miR-NC, or si-MEG3+anti-miR-650. **P < 0.01, ***P < 0.001.
Fig. 8. The effect of SLC34A2 on the MEG3-mediated function on LCCs and LCSCs. (A) The protein level of SLC34A2 was examined in LCCs transfected with siNC, si-MEG3, si-MEG3+pcDNA, or si-MEG3+SLC34A2 overexpression plasmid. (B and C) The mRNA and protein levels of Oct4 and CD133 in LCCs treated as described in Fig. 8A. (D and E) LCCs and LCSCs were transfected with pcDNA3.0 vector, MEG3 overexpression plasmid, MEG3 overexpression plasmid + si-NC, or MEG3 overexpression plasmid + si-SLC34A2, respectively. Western blot was performed to measure the expression of SLC34A2. (F and G) Cell migration and invasion abilities were evaluated by Transwell assay. *P < 0.05, **P < 0.01, ***P < 0.001.
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Long non-coding RNA MEG3 regulates migration and invasion of lung cancer stem cells via miR-650/SLC34A2 axis Yongjuan Zhaoa, Zhenxing Zhub, Shaomin Shia, Jing Wanga, Ning Lia, a b
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Department of Respiratory, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China Department of Hematology and Oncology, China-Japan Union Hospital of Jilin University, Changchun, Jilin, China
A R T I C LE I N FO
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Keywords: Lung cancer stem cells MEG3 miR-650 SLC34A2 NSCLC
Long non-coding RNA maternally expressed gene 3 (MEG3) is related to the occurrence and development of nonsmall cell lung cancer (NSCLC). However, the function and underlying molecular mechanisms of MEG3 in lung cancer stem cells (LCSCs) are still unclear. LCSCs were determined in lung cancer cells using fluorescenceactivated cell sorting (FACS). qRT-PCR and western blot were performed to examine the expressions of MEG3, miR-650, solute carrier family 34 member 2 (SLC34A2), octamer-binding transcription factor 4 (Oct4), and CD133. Sphere assay was employed to evaluate sphere-forming ability. Cell migration and invasion were analyzed by Transwell assay. The relationships among MEG3, miR-650, and SLC34A2 were validated by luciferase reporter, RIP, and RNA pulldown assays. We found MEG3 was downregulated in LCSCs. MEG3 depletion strengthened stem cell-like characteristics and sphere-forming ability in LCCs. Upregulation of MEG3 suppressed migration and invasion in LCCs and LCSCs. miR-650 was bound to MEG3 and upregulated in LCSCs. miR-650 inhibitor alleviated si-MEG3-induced promotion of stem cell-like characteristics in lung cancer cells (LCCs) H1299. Furthermore, miR-650 mimic attenuated the MEG3 upregulation-mediated inhibition of migration and invasion. In addition, SLC34A2 was a target of miR-650 and downregulated in LCSCs. miR-650 mimic induced stem cell-like characteristics in LCCs, which was weakened by overexpression of SLC34A2. In contrast, the repression of SLC34A2 mitigated the miR-650 silencing-induced inhibition of migration and invasion in LCCs and LCSCs. Besides, MEG3 regulated SLC34A2 expression by sponging miR-650. Importantly, SLC34A2 weakened MEG3-mediated stem cell-like state and cell metastasis. Our data suggested MEG3 was involved in stem cell-like state of LCCs and curbed migration and invasion through miR-650/SLC34A2 axis in NSCLC.
1. Introduction Lung cancer causes the most cancer-related deaths and non-small cell lung cancer (NSCLC) accounts for the majority of cases [1,2]. Although early diagnosis and more effective treatment strategies have diminished the mortality rates in recent years [3,4], the development of recurrence, metastasis and chemoresistance is still inevitable [5,6]. A correlation has been found between resistance to radiation, chemotherapeutic treatments, or tumor recurrence and lung cancer stem cells (LCSCs) [7–9]. It has been revealed that CD44 and CD133 are widely used to sort cancer stem cells (CSCs) from normal cancer cells [5,10,11]. Moreover, LCSCs possess the property to form spheres, which are thought to be enriched stem cells [11,12]. Therefore, eliminating LCSCs may provide a therapeutic avenue for overcoming chemoresistance, metastasis and relapse in NSCLC patients. Long non-coding RNAs (lncRNAs) have been reported to play key
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roles in variety of biological processes, including stem cell biology [13,14]. LncRNA maternally expressed gene 3 (MEG3) has been reported to be suppressed in various cancers [15,16]. Notably, MEG3 is significantly downregulated in NSCLC tissues and regulates NSCLC cell proliferation and apoptosis through the activation of p53 [17]. Moreover, MEG3 is markedly reduced in chemotherapy-resistant lung cancer tissues, and upregulation of MEG3 attenuates the autophagy and increases the sensitivity of vincristine in chemotherapy of NSCLC [18]. Those data suggested that MEG3 may be required for the development of NSCLC. In addition, a study has indicated that loss of MEG3 enhances cell proliferation, migration, invasion, sphere-forming ability, and CSC properties, whereas decreases the chemosensitivity to gemcitabine in human pancreatic cancer [19]. However, the function and underlying molecular mechanisms of MEG3 in LCSCs remain largely unknown. As we all known, lncRNAs that may act as microRNA (miRNA) sponges. Accumulating evidence has indicated that microRNA-650
Corresponding Author at: Department of Respiratory, China-Japan Union Hospital of Jilin University, No.126, Xiantai Street, Changchun, Jilin, China. E-mail address: [email protected] (N. Li).
https://doi.org/10.1016/j.biopha.2019.109457 Received 22 May 2019; Received in revised form 9 September 2019; Accepted 12 September 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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2.5. Migration and invasion assays
(miR-650) could act as an oncogene in the multiple cancers, including human lung adenocarcinoma [20–22]. Recently, miR-650 is reported to be correlated with neoplastic transformation of liver cancer stem cells [23]. Nevertheless, whether MEG3 participates in cancer stem cell maintenance of NSCLC by interacting with miR-650 has not been reported. It is known that miRNA can regulate a large fraction of proteincoding genes, which form a regulatory network during cell differentiation and development. Yang et al. have reported that solute carrier family 34 member 2 (SLC34A2) is downregulated in A549 and H1299 cells and overexpression of SLC34A2 inhibits the viability and invasion in NSCLC [24]. Furthermore, SLC34A2 has been reported to maintain the stem cell-like characteristics in CD147+ breast cancer stem cells (BCSCs) [25]. However, whether SLC34A2 is responsible for the MEG3/ miR-650 axis of stem cell state of NSCLC is not fully understood. In present study, we sought to investigate the effect of MEG3 on stem cell-like state and metastasis in NSCLC cells and explore the role of MEG3/miR-650/ SLC34A2 axis in this process.
1 × 105 of transfected LCCs or LCSCs suspended in 150 μL serumdeprived culture medium were seeded into the upper compartment of Transwell plates with 8.0 μm pore (Corning; cat number: 3422). For cell invasion assay, the membranes of the upper compartments were precoated with Matrigel (Solarbio, Beijing, China; cat number: 356234). Dilute the Matrigel (10 u L) with serum-free cold cell culture medium DMEM and added 100 ul of dilute gel to the 24-well transwell upper chamber (4 °C). Then incubate the transwell at 37 °C for at least 4–5 h. And un-coated ones were used for cell migration assay. The cells were cultured for 24 h, and cells migrated to the underside of the upper compartment membrane in response to culture medium supplemented with 10% FBS in the lower compartment were fixed with methanol and stained with 0.1% crystal violet (Sigma). The number of cells on the lower side of the filter were counted in 5 randomly picked view under microscope (Nikon Corp, Tokyo, Japan). Each migration or invasion group was tested with three replicates.
2. Materials and methods 2.1. Cell culture
2.6. RNA extraction and qRT-PCR assays
NSCLC cell line H1299 and human kidney cell line 293 T were obtained from the American Type Culture Collection (Manassas, VA, USA). H1299 cells and 293 T cells were cultured in complete Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA, USA) or RPMI 1640 medium (Thermo Fisher Scientific), respectively, supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37 °C in a humidified incubator containing 5% CO2.
Total RNAs were isolated from cells by using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and reversely transcribed into complementary DNA (cDNA) using the TaqMan MicroRNA Reverse Transcription Kit (Biosystems, Foster City, CA, USA) or the Primer Script RT reagent kit (TaKaRa, Dalian, China). qRT-PCR was performed using a SYBR Green PCR Master Mix (Biosystems) in line with the procedure of instructions. Relative expressions of MEG3, miR-650, octamer-binding transcription factor 4 (Oct4), CD133 and SLC34A2 were calculated on the basis of the 2−ΔΔCt method, normalizing to U6 small nuclear RNA (snRNA) or β-actin, respectively. The primers were listed as follows：MEG3 forward, 5′-CTGCCCATCTACACCTCACG-3′, and reverse, 5′-CTCTCCGCCGTCTGCGCTAGGGGCT-3′; miR-650 forward, 5′AGAGGAGGCAGCGCTCT-3′, and reverse, 5′- CAGTGCGTGTCGTGG AGT -3′; SLC34A2 forward, 5′-GAGAACATCGCCAAATGC-3′, and reverse, 5′GCAACCACAGAGGACCAG-3′; Oct4 forward, 5′-GTGCCGTGAAGCTGG AGAA-3′, and reverse, 5′-TGGTCGTTTGGCTGAATACCTT-3′; CD133 forward: 5′-ACAGCGATCAAGGAGACCAA-3′, and reverse: 5′-GTCAAG TTCTGCATCCACGG-3′; β-actin forward, 5′-GGACCTGACTGACTAC CTC-3′, and reverse, 5′-TACTCCTGCTTGCTGAT-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′, and reverse, 5′-AACGCTTCACGAATTT GCGT-3′.
2.2. Isolation of LCSCs by fluorescence-activated cell sorting (FACS) LCSCs were isolated by sorting CD44+CD133+ populations using anti-CD133-PE and anti-CD44-FITC antibodies (BD Biosciences, Franklin Lakes, NJ, USA). Briefly, H1299 cells were incubated with anti-CD133-PE and anti-CD44-FITC antibodies on ice for 30 min in the dark. After washed with cold PBS, CD44+CD133+ cells were purified by flow cytometry (BD Biosciences). All CD44-/CD133- cells were called LCCs. 2.3. Sphere formation assay Cells were plated as single-cell suspensions in low-attachment plates 24-well plates (Corning, Corning, NY, USA; cat number: 3524) at a density of 100 cells/well. Cells were grown in serum-free Mammo Cult Human Medium (Stem Cell Technologies, Vancouver, BC, Canada) for 1 or 7 days. All spheres in each well were assessed via a Zeiss Axiovert microscope (Carl Zeiss, Thornwood, NY, USA). Three replicates were involved in each group.
2.7. Western blot assay Proteins were extracted using radioimmunoprecipitation assay buffer. The protein concentration was measured using a BCA assay kit (Beyotime, Shanghai, China). Proteins (20 μg of each sample) were separated by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% fat-free milk powder in Tris-buffered saline with 0.1% Tween 20 buffer and then incubated at 4 °C overnight with following primary antibodies against Oct4 (Abcam, Cambridge, MA, USA; cat number: ab27985), CD133 (Abcam; cat number: ab19898), SLC34A2 (Abcam; cat number: ab228474), β-actin (Abcam; cat number: ab8226). Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA). The protein signals were visualized using enhanced chemiluminescence (Beyotime). Densitometry values were normalized to levels of β-actin and analyzed by ImageJ software (Media Cybernetics, Rockville, MD, USA).
2.4. Cell transfection Cells (70% confluence in 6-well plates) were transfected with small interfering RNA (siRNA) for MEG3 (si-MEG3), siRNA for SLC34A2 (siSLC34A2), siRNA negative control (si-NC), MEG3 overexpression plasmid (MEG3), SLC34A2 overexpression plasmid (SLC34A2), pcDNA 3.0 vector (pcDNA), miR-650 mimic (miR-650), negative control mimic (miR-NC), miR-650 inhibitor (anti-miR-650) or negative control inhibitor (anti-miR-NC) using Lipofectamine 3000 reagent (Thermo Fisher Scientific) according to the manufacturer's protocols. miR-650 mimic (Sigma, St. Louis, MO, USA; cat number: HMI0881); miR-650 inhibitor (Sigma; cat number: HSTUD0881). siRNA for MEG3, SLC34A2, MEG3 or SLC34A2 overexpression plasmid were purchased from Vigene Bioscicences. The transfected cells after 48 h were used for following experiments. 2
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We found that cell metastasis ability was higher in LCSCs than that of LCCs. Then, LCCs or LCSCs were transfected with pcDNA 3.0 vector or MEG3 overexpression plasmid. Also, following transfection efficiency assay revealed that the transfection of MEG3 overexpression plasmid was efficient to induce the higher expressions of MEG3 in LCCs and LCSCs (Fig. 2A). The results further revealed that upregulation of MEG3 decreased the number of migration and invasion cells in LCCs and LCSCs (Fig. 2B and C).
2.8. Luciferase reporter assay Bioinformatics analysis was introduced to analyze the binding sites between MEG3 and miR-650 using DIANA Tools and the binding sites between miR-650 and SLC34A2 using miRDB database. MEG3 fragments containing the wild-type (MEG3-WT) or mutated (MEG3-MT) binding sites of miR-650 were cloned into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA). The same method was used for the construction of SLC34A2-WT or SLC34A2-MT. For the association between miR-650 and MEG3, 293 T cells were co-transfected with miR-650 or miR-NC and MEG3-WT or MEG3-MT using Lipofectamine 3000. For the association between miR650 and SLC34A2, 293 T cells were co-transfected with miR-650 or miR-NC and SLC34A2-WT or SLC34A2-MT. For the association among miR-650, MEG3 and SLC34A2, 293 T cells were transfected with SLC34A2-WT, SLC34A2-WT + miR-650, SLC34A2-WT + miR650+pcDNA3.0 vector, or SLC34A2-WT + miR-650+MEG3. Then, the luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.
3.3. miR-650 is bound to MEG3 LncRNAs that act as sponges to bind miRNAs and prevent them from performing normal regulatory activities. To address whether MEG3 acts in this way, the potential binding sites of miR-650 and MEG3 were predicted by DIANA Tools (Fig. 3A). Then, a luciferase reporter assay was carried out to validate whether MEG3 binds miR-650. The results indicated that luciferase activity was decreased in 293 T cells transfected with miR-650 mimic in MEG3-WT group, while it showed little impact on the luciferase activity of MEG3-MT group (Fig. 3B). Subsequently, RIP assay was conducted with antibody against Ago2 or IgG. As shown in Fig. 3C, MEG3 was enriched in LCSCs transfected with miR-650 mimic in RIP-Ago2 group compared with that in RIP-IgG group. Moreover, MEG3 was pulled down by Bio-miR-650-WT, while it failed to show any efficacy in Bio-miR-650-MT group (Fig. 3D). Meanwhile, a significant abundance of miR-650 was observed in Bioprobe-MEG3-WT group compared with that in Bio-probe-MEG3-MT group (Fig. 3D), indicating that MEG3 was interacted with miR-650.
2.9. RNA immunoprecipitation (RIP) and RNA pulldown assays RIP assay was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (Millipore) according to the manufacturer’s instructions. Briefly, the cells were harvested and incubated with magnetic beads and anti-Ago2 antibody (Abcam), and IgG was used as a negative control. The beads were washed and incubated with proteinase K to digest the protein at 24 h after incubation. For RNA pulldown assay, probes for MEG3 or miR-650 were biotinylated (GENEWIZ, Suzhou, China) and transfected into LCCs. After 48 h, the cells were harvested and lysed. The samples were incubated with Dynabeads M280 Streptavidin (Solarbio). The bound RNAs were analyzed by qRTPCR.
3.4. miR-650 is implicated in the function of MEG3 in LCCs and LCSCs
3. Results
Given the above results, the effect of miR-650 on the MEG3-mediated function of LCCs and LCSCs was further explored. Firstly, we evaluated the level of miR-650 in LCCs and LCSCs. Higher expression of miR-650 was displayed in LCSCs compared with that of LCCs (Fig. 4A). Then, downregulation of MEG3 increased the expression of miR-650, which was weakened by miR-650 inhibitor introduction (Fig. 4B). Additionally, miR-650 depletion attenuated the si-MEG3-induced increase of Oct4 and CD133 levels in LCCs (Fig. 4C and D). Interestingly, enforced expression of MEG3 led to the inhibition of miR-650 expression, while the introduction of miR-650 mimic abated the repression effect (Fig. 4E and F). Subsequently, the analysis of cell migration and invasion ability revealed that miR-650 mimic receded the MEG3 upregulation-induced suppression of migration and invasion in LCCs and LCSCs (Fig. 4G and H).
3.1. MEG3 depletion strengthens stem cell-like characteristics in LCCs
3.5. SLC34A2 is a target of miR-650
Firstly, to investigate the role of MEG3 in LCSCs, CD44+CD133+ cells were sorted from H1299 cells, namely LCSCs (Fig. 1A). Next, the basal expression of MEG3 was measured in LCSCs and LCCs which had been isolated from H1299 cells using FACS. The results showed a significant decrease in MEG3 expression in LCSCs compared with that in LCCs (Fig. 1B). After that, small interfering RNA for MEG3 was transfected in LCCs. As expected, the introduction of si-MEG3 strikingly reduced MEG3 level (Fig. 1C). Then, LCSC markers were detected in LCCs transfected with si-NC or si-MEG3. Compared with si-NC group, MEG3 knockdown cells demonstrated elevated expressions of Oct4 and CD133 (Fig. 1D and E), suggesting that MEG3 depletion promoted stem cell-like state of LCCs. Moreover, sphere-forming ability was enhanced in si-MEG3 groups compared with that of si-NC group at day 7 (Fig. 1F).
It is accepted that miRNAs exert their functions by regulating the expression of downstream target genes. Therefore, we searched for targets of miR-650 using miRDB Tools, which presented the potential binding sites of miR-650 and SLC34A2 (Fig. 5A). Next, luciferase reporter assay and RNA pulldown assay were used to further validate the interaction of SLC34A2 and miR-650. After over-expressing of miR-650, the luciferase activity of the SLC34A2-WT reporter containing miR-650 binding sites was found to be attenuated in 293 T cells, while it was not observed in SLC34A2-MT group (Fig. 5B). Moreover, RIP assay disclosed more enrichment of SLC34A2 in miR-650 group than that in miR-NC group (Fig. 5C). According to the results above, miR-650 was interacted with SLC34A2 by putative complementary sites.
2.10. Statistical analysis Data are presented as the mean ± standard deviation (SD). The experiments were performed three times (n = 3). All statistical analyses were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). For comparing the differences between them, paired Student’s ttest and one-way analysis of variance (ANOVA) were used. A value of P less than 0.05 was considered to be statistically significant.
3.6. SLC34A2 participates in miR-650-induced stem cell-like state and metastasis in NSCLC
3.2. Upregulation of MEG3 inhibits cell migration and invasion in LCCs and LCSCs
The mRNA level of SLC34A2 was greatly decreased in LCSCs compared with that in LCCs (Fig. 6A). Moreover, an obvious repression of SLC34A2 was observed in LCCs transfected with miR-650 mimic, which
In the following experiments, we tested the hypothesis that overexpression of MEG3 influences the migration and invasion of NSCLC. 3
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Fig. 1. MEG3 depletion strengthens stem cell-like characteristics in LCCs. (A) LCSCs were determined in H1299 cells using FACS. (B) The level of MEG3 was examined in LCCs and LCSCs. (C) The level of MEG3 was measured in LCCs transfected with si-NC or si-MEG3. (D and E) The mRNA and protein levels of Oct4 and CD133 in LCCs transfected with si-NC or si-MEG3. (F) Sphere assay was performed in LCCs transfected with si-NC or si-MEG3. **P < 0.01, ***P < 0.001.
Fig. 2. Upregulation of MEG3 inhibits cell migration and invasion in LCCs and LCSCs. LCCs and LCSCs were transfected with pcDNA3.0 vector or MEG3 overexpression plasmid, respectively. (A) The level of MEG3 was examined in LCCs and LCSCs. (B and C) Cell migration and invasion abilities were evaluated by Transwell assay. **P < 0.01.
3.7. MEG3 regulates SLC34A2 expression by sponging miR-650
was undermined by SLC34A2 overexpression (Fig. 6B). Furthermore, the results of western blot and qRT‐PCR both indicated that overexpression of miR-650 markedly increased the expressions of stemness markers Oct4 and CD133 at protein and mRNA levels (Fig. 6C and D). In addition, the effects of SLC34A2 silencing on miR-650 inhibitor-induced impediment of migration and invasion were further studied. After the transfection, SLC34A2 protein expression was effectively increased in LCCs and LCSCs transfected with miR-650 inhibitor, while it was undermined by si- SLC34A2 (Fig. 6E and F). Furthermore, we found that SLC34A2 depletion drastically attenuated the miR-650 inhibitorinduced repression of cell migration and invasion in LCCs and LCSCs (Fig. 6G and H).
Considering the effects of MEG3, miR-650, and SLC34A2 on cell function in LCCs and LCSCs, we further explored the associations among the MEG3, miR-650, and SLC34A2. As shown in Fig. 7A, luciferase activity was significantly decreased in 293 T cells in SLC34A2WT + miR-650 mimic group, while it was abated by inhibition of MEG3. Moreover, the effects of miR-650 and MEG3 on SLC34A2 protein expression were investigated in LCSCs. Upregulation of MEG3 increased the expression of SLC34A2, which was attenuated by introduction of miR-650 (Fig. 7B). In contrast, miR-650 inhibition abrogated the MEG3 depletion-induced loss of the SLC34A2 expression (Fig. 7C). To address 4
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Fig. 3. miR-650 is bound to MEG3. (A) The potential binding sites of miR-650 and MEG3 were predicted by DIANA Tools. (B) The luciferase activity was measured in 293 T cells co-transfected with MEG3-WT or MEG3-MT and miR-650 mimic or miR-NC. (C) The expression of MEG3 was detected in LCCs after Ago2 or IgG RIP assay. (D) The level of MEG3 in the sample pulled down by biotinylated miR-650 and the expression of miR-650 in the sample pulled down by biotinylated MEG3 probe. **P < 0.01, ***P < 0.001.
miR-650 in a negative-feedback mechanism. In consequence, MEG3 can also indirectly stimulate SLC34A2 expression through inhibiting the level of miR-650. It is well documented that CSCs exist in many cancers and are closely associated with cancer progression, metastasis, and chemoresistance [9,26]. To fully address their potentials as cancer markers or drug targets, a better understanding of the underlying molecular mechanism of CSC maintenance is required. Oct4 is detected in CSCs enriched from tumor tissues or cell lines, which is related to its function in tumorigenesis, metastasis and resistance to anticancer therapies [27]. Moreover, the loss of Oct4 expression is associated with stem cell differentiation [28]. To further explore the effect of MEG3 on stem cell-like state of LCCs, the expressions of Oct4 and CD133 selected as surface markers of LCSCs were subsequently analyzed. Si-MEG3-mediated silence of MEG3 increased the expressions of Oct4 and CD133, suggesting that MEG3 may be involved in maintaining cancer stem cell phenotypes. Consistent with the data, suppression of MEG3 strikingly increased sphere-forming ability, revealing that MEG3 depletion may contribute to induce the stem cell-like state in LCCs. In addition, upregulation of MEG3 inhibited the migration and invasion in LCCs and LCSCs, suggesting that MEG3 may act as a tumor-suppressor in the development of NSCLC, which is consistent with previous study [18]. Crosstalk between lncRNAs and miRNAs has been involved in progression of NSCLC. For instance, linc00673 regulates NSCLC proliferation, migration, invasion and epithelial mesenchymal transition by interacting with miR-150-5p [29]. Moreover, lncRNA colon cancerassociated transcript-1(CCAT1)/miR-130a-3p axis enhances cisplatin
whether MEG3 regulates phenotypic changes through miR-650/ SLC34A2 axis, LCCs were introduced si-MEG3 and SLC34A2 overexpression plasmid. The transfection of SLC34A2 overexpression plasmid rescued si-MEG3-mediated SLC34A2 downregulation (Fig. 8A). As expect, ectopic expression of SLC34A2 also weakened the si-MEG3induced upregulation of Oct4 and CD133 on mRNA and protein levels in LCCs (Fig. 8B and C). In LCCs and LCSCs, enhanced expression of MEG3 also promoted the protein level of SLC34A2, which was undermined by loss of SLC34A2 (Fig. 8D and E). Interestingly, enforced expression of MEG3 led to the inhibition of cell migration and invasion, while the introduction of si- SLC34A2 abated the repression effect (Fig. 8F and G). Therefore, we thought that MEG3 may play an important role through miR-650/ SLC34A2 axis. 4. Discussion NSCLC has a low 5-year overall survival rate, which is considered to be post-treatment enrichment of CSCs with abilities of cancer initiation and further maintenance of tumors [9]. In the present study, LCSCs were determined in H1299 cells using FACS. We demonstrated that MEG3/miR-650/SLC34A2 axis exhibited a double-negative feedback loop in regulation of LCSCs maintenance. MEG3 was significantly downregulated in LCSCs. Moreover, MEG3 had a critical role in inhibiting LCSCs properties and tumorigenesis in vitro. Here, we described a mechanism in which MEG3 functioned as a competing endogenous RNA (ceRNA) by sponging miR-650 to promote the expression of SLC34A2. Interestingly, SLC34A2 is also repressed by
Fig. 4. miR-650 is implicated in the function of MEG3 in LCCs and LCSCs. (A) The level of miR-650 was examined in LCCs and LCSCs. (B) The abundance of miR650 in LCCs transfected with si-NC, si-MEG3, si-MEG3+anti-miR-NC, or si-MEG3+anti-miR-650. (C and D) The mRNA and protein levels of Oct4 and CD133 in LCCs treated as described in Fig. 4B. (E and F) LCCs and LCSCs were transfected with pcDNA3.0 vector, MEG3 overexpression plasmid, MEG3 overexpression plasmid + miR-NC, or MEG3 overexpression plasmid + miR-650 mimic, respectively. qRT-PCR was performed to measure the expression of miR-650. (G and H) Cell migration and invasion abilities were evaluated by Transwell assay. **P < 0.01, ***P < 0.001. 5
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Fig. 5. SLC34A2 is a target of miR-650. (A) The putative binding site of miR-650 and SLC34A2. (B) The luciferase activity was measured in 293 T cells cotransfected with SLC34A2-WT or SLC34A2-MT and miR-650 mimic or miR-NC. (C) The expression of SLC34A2 was detected in LCCs after Ago2 or IgG RIP assay. *P < 0.05, ***P < 0.001.
Notably, restoration of SLC34A2 rescued the MEG3 silence on cell phenotypic changes. Meanwhile, knockdown of SLC34A2 also receded the effect of gain of MEG3 on cell migration and invasion. A novel regulatory mechanism MEG3/miR-650/SLC34A2 was first observed in LCSCs, establishing a comprehensive regulatory network that adapts to environmental changes in the maintenance of LCSCs. However, there are some limitations. Our study only investigated the partial roles of MEG3 in vitro. The role of MEG3 in vivo will be validated in future. Moreover, the functions of SLC34A2 and miR-650 alone should be explored. In conclusion, we disclosed MEG3 as a cancer suppressor, which exerts a crucial role in the stem cell-like state and metastasis in NSCLC. Besides, our findings shed light on the interaction between MEG3 and miR-650 for the first time, and revealed that MEG3 positively regulates posttranscriptional expression of SLC34A2 by sponging miR-650 in NSCLC. MEG3/miR-650/SLC34A2 axis may be implicated in stem celllike state and cell migration and invasion, thus probably providing a novel therapeutic avenue in NSCLC.
resistance of NSCLC cells by targeting sex-determining region Y-box 4 (SOX4) [30]. miR-650 has been reported to be upregulated in NSCLC [31]. In this report, we found that MEG3 negatively regulated the expression of miR-650. Besides, the ectopic expression of miR-650 was observed in LCSCs. Interestingly, the inhibition of miR-650 attenuated the MEG3 silencing-mediated promotion of stem cell-like state. It was also observed that miR-650 weakened the effect of upregulation of MEG3 on migration and invasion in LCCs and LCSCs. Those findings demonstrated that MEG3 was involved in stem cell-like state and metastasis in NSCLC by interacting with miR-650, forming a reciprocal negative regulatory loop. In general, feedback loops are widely observed between miRNAs and their targets. In a previous report, miR650 promotes the hepatocellular carcinoma metastasis via suppressing the expression of large tumor suppressor kinase 2 [32]. Here, we revealed that SLC34A2 was a target of miR-650. Thus, it is probably that there is a negative regulation between SLC34A2 and miR-650. However, the effect of SLC34A2 on the function of miR-650 in NSCLC needs further study. SLC34A2, a member of SLC34 family of sodium-driven phosphate cotransporters [33]. A previous study disclosed that SLC34A2 inhibits tumor growth and metastasis ability in H1299 subcutaneous tumor model and lung metastasis model [17]. Moreover, SLC34A2 could be a favorable prognostic marker in lung cancer patients [34]. In present study, a significant reduce of SLC34A2 was observed in LCSCs. Overexpression of SLC34A2 ameliorated miR-650-induced promotion of the cancer stem cell-like characteristics. Furthermore, the inhibition of SLC34A2 restored cell migration and invasion, which was blocked by downregulation of miR-650. More importantly, MEG3 and miR-650 competitively regulated the SLC34A2 expression, indicating that MEG3, functioning as a ceRNA, links the network of miR-650 and SLC34A2.
Declaration of Competing Interest All authors declare that they have no financial and non-financial conflicts of interest.
Acknowledgement Not applicable.
Fig. 6. SLC34A2 participates in miR-650-induced stem cell-like state and metastasis in NSCLC. (A) The level of SLC34A2 was examined in LCCs and LCSCs. (B) The expression of SLC34A2 in LCCs transfected with miR-NC, miR-650 mimic, miR-650 mimic + pcDNA3.0 vector, or miR-650 mimic + SLC34A2 overexpression plasmid. (C and D) The mRNA and protein levels of Oct4 and CD133 in LCCs treated as described in Fig. 6B. (E and F) LCCs and LCSCs were transfected with anti-miRNC, anti-miR-650, anti-miR-650+si-NC, or anti-miR-650+si-SLC34A2, respectively. Western blot was performed to measure the expression of SLC34A2. (G and H) Cell migration and invasion abilities were evaluated by Transwell assay in LCCs and LCSCs. **P < 0.01, ***P < 0.001. 6
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Fig. 7. MEG3 regulates SLC34A2 expression by sponging miR-650. (A) The luciferase activity was measured in 293 T cells transfected with SLC34A2-WT, SLC34A2-WT + miR-650 mimic, SLC34A2-WT + miR-650 mimic + pcDNA3.0 vector, or SLC34A2-WT + miR-650 mimic + MEG3 overexpression plasmid. (B) The expression of SLC34A2 in LCSCs transfected with pcDNA3.0 vector, MEG3 overexpression plasmid, MEG3 overexpression plasmid + miR-NC, or MEG3 overexpression plasmid + miR-650 mimic. (C) The expression of SLC34A2 in LCSCs transfected with si-NC, si-MEG3, si-MEG3+anti-miR-NC, or si-MEG3+anti-miR-650. **P < 0.01, ***P < 0.001.
Fig. 8. The effect of SLC34A2 on the MEG3-mediated function on LCCs and LCSCs. (A) The protein level of SLC34A2 was examined in LCCs transfected with siNC, si-MEG3, si-MEG3+pcDNA, or si-MEG3+SLC34A2 overexpression plasmid. (B and C) The mRNA and protein levels of Oct4 and CD133 in LCCs treated as described in Fig. 8A. (D and E) LCCs and LCSCs were transfected with pcDNA3.0 vector, MEG3 overexpression plasmid, MEG3 overexpression plasmid + si-NC, or MEG3 overexpression plasmid + si-SLC34A2, respectively. Western blot was performed to measure the expression of SLC34A2. (F and G) Cell migration and invasion abilities were evaluated by Transwell assay. *P < 0.05, **P < 0.01, ***P < 0.001.
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