Starbase website (http://starbase.sysu.edu.cn/index.php) and RNA22 website (https://cm.jefferson.edu/rna22/) were used to predict the targeting sites for the interaction between MALAT1 and miR-26b.
Collection of clinical samples
From October 2015 to October 2017, 68 cases of primary BC were operated in the First Hospital of Lanzhou University, and the BC tissue samples and the corresponding adjacent tissue samples were collected. The BC patients ranged in age from 27 to 69 years old, with a median age of 51 years. All participants received routine treatment but without chemotherapy before operation, and all patients were confirmed by pathological diagnosis after the operation. This study was approved by Ethics Committee of the First Hospital of Lanzhou University, and carried out following the Declaration of Helsinki. The pathological diagnosis of patients is shown in Additional file 1: Table S1.
Cell culture and grouping
The BC cancer cells, MCF-7(ZQ0071), MDA-MB-231(ZQ0118), MDA-MB-468(ZQ0373) and normal mammary epithelial cells MCF-10 A were provided by the Shanghai Zhongqiao Xinzhou Co., Ltd. (Shanghai, China) and ATCC company, respectively. Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) with 10 % FBS, in a constant temperature incubator at 37 ℃ and 5 % CO2. After adhering to the culture dishes, the dells were digested with 0.25 % trypsin (Hyclone Company, Logan, Utah, USA) and passaged, and the logarithmic growth period cells were used for subsequent experiments. All cell lines have been tested for mycoplasma after their purchase and STR analysis was performed every month.
According to the needs of the experiment, the cells were divided into the following groups: sh-NC (transfected with gene interference negative control plasmids), sh-MALAT1 (transfected with gene interference MALAT1 interference plasmids), sh-NC + oe-NC (transfected with gene interference negative control plasmids and overexpression negative control plasmids), sh-METTL3 + oe-NC (transfected with interference METTL3 plasmid and over expression negative control plasmid), sh-NC + oe-MALAT1 (transfected with negative control plasmids and overexpression MALAT1 plasmids), sh-METTL3 + oe-MALAT1 (transfected with interference METTL3 plasmid and overexpression MALAT1 plasmid), NC-mimic (transfected with miR-26b overexpression negative control plasmid), miR-26b mimic (transfected with miR-26b overexpression plasmid), NC-inhibitor (transfected with miR-26b interference negative control plasmid), miR-26b inhibitor (transfected with miR-26b interference plasmid), sh-NC + NC-inhibitor (transfected with negative control plasmids of transfected gene interference and miR-26b interference plasmid), sh-MALAT1 + NC-inhibitor (transfected with interference MALAT1 plasmid and miR-26b interference negative control plasmid), sh-MALAT1 + miR-26b inhibitor (transfected with interference MALAT1 plasmid and miR-26b interference plasmid), NC-mimic + oe-NC (transfected with miR-26b negative overexpression control plasmid and negative overexpression control plasmid), miR-26b mimic + oe-NC (transfected with miR-26b overexpression plasmid and negative overexpression control plasmid), miR-26b mimic + oe-HMGA2 (transfected with miR-26b overexpression plasmid and HMGA2 overexpression plasmid), and sh-METTL3 + oe-HMGA2 (transfected with interference METTL3 plasmid and overexpression HMGA2 plasmid). All interference and overexpression plasmids were designed and provided by Invitrogen (Calsbad, CA, USA). The lipofectamine 2000 kit (product No. 11,668,019, Thermo Fisher Scientific Inc., CA, USA) was used for cell transfection. 1 × 105 cells were inoculated into each well in a six-well plate and cultured until cell confluence reached 60–70 %. Then, 250 µL of serum-free Opti-MEM medium (Gibco, Grand Island, New York, USA) was used to dilute 4 µg of target plasmids together with 10 µL of Lipofectamine 2000, and then mixed with light agitation. Next, the mixture was added to the culture system. After 6 h in culture, the medium was replaced with complete culture medium. After a further48 hours, cells were collected to detect the transfection efficiency for subsequent experiments [20, 21].
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from tissues or cells with TRIzol (Invitrogen) method following the manufacturer’s protocol. RNA purity was examined by Nanodrop2000 micro-UV spectrophotometer (1011U, Nanodrop, USA). RNA was reversely transcribed into cDNA following protocols of the TaqMan MicroRNA Assays Reverse Transcription primer (4,427,975, Applied Biosystems, USA) and PrimeScript RT reagent kit (RR047A, Takara, Japan). All primers were designed and synthesized by TaKaRa (Additional file 2: Table S2). According to a reaction system prepared by the fast SYBR Green PCR kit (Applied Biosystems), quantitative PCR was detected by an ABI7500 quantitative PCR instrument (7500, ABI, USA). GAPDH and U6 were used as internal reference genes of MALAT1 and miR-26b, respectively. The expression levels of target genes were calculated by the relative quantitative method (2−ΔΔCT method). ΔΔCt = ΔCt experimental group − ΔCt control group, where ΔCt = Ct (target gene)−Ct (internal reference), and the relative transcription level of target gene mRNA = 2−ΔΔCt. Each sample was set with three replicates and each test was repeated three times.
Western blot (WB)
Total protein in tissue or cell was extracted by RIPA lysate solution containing PMSF (P0013C, Beyotime, Shanghai, China). The prepared cells were incubated on ice for 30 min, and then the supernatant was centrifuged (4 ℃, 8000 g) for 10 min. A BCA test kit was used to detect the total protein concentration (P0012, Beyotime). Fifty µg protein samples were dissolved in 2 × SDS sample buffer and boiled for 10 min, and SDS-PAGE gel electrophoresis was performed followed by transfer of proteins to a PVDF membrane. After blocking in 5 % non-fat milk for 1 h, PVDF membranes were incubated overnight with primary antibodies: METTL3 (1:1000, ab19552, Abcam, Cambridge, UK), HMGA2 (1:1000, ab207301, Abcam), E-cadherin (1:500, ab15148, Abcam), Snail (1:500, ab82846, Abcam), Vimentin (1:2000, ab137321, Abcam), N-cadherin (1:1000, ab18203, Abcam), and GAPDH (ab19485, 1:2500, Abcam). Then, PVDF membranes were incubated for 1 h with corresponding HRP labeled goat-anti-rabbit IgG H&L (HRP) (ab97051, 1:2000, Abcam). According to the instructions of the ECL detection kit (product No. BB-3501, Amersham, UK), equal amounts of liquid A and B were mixed in the dark and then dripped onto the PVDF membranes. The film was photographed using a specific image analysis system (Bio-Rad company, USA), and analyzed using Quantity-One (v4.6.2) software. The relative protein content was expressed by the gray value of corresponding protein bands/the gray value of internal reference bands. The mean of triplicate experiment results was calculated [20, 21].
The tumor tissue samples were fixed with 10 % neutral formalin solution, dehydrated, embedded in paraffin, and sectioned by an ultrathin sectioning machine. The sections were then dewaxed with xylene, rehydrated with graded alcohol, and incubated with 3 % hydrogen peroxide to block the activity of endogenous peroxidase. The sections were boiled in 10 mM sodium citrate (pH 6.0) for 30 min, sealed in 10 % normal goat serum for 15 min, and then incubated overnight with antibodies against METTL3 (1:500, ab195352, Abcam), HMGA2 (1:500, ab207301, Abcam), Snail (1:2000, ab224731, Abcam), N-cadherin (1:1000, ab18203, Abcam), and Vimentin (1:500, ab137321, Abcam) in a wet chamber at 4 ℃. The next day, the sections were washed with PBS and incubated with secondary antibody for 1 h at room temperature prior to visualization as above.
The Matrigel (YB356234, Shanghai Yu Bo Biotech Co., Ltd., Shanghai, China) preserved at − 80 ℃ was melted at 4℃ overnight. Then, 200 µL serum-free medium and 200 µL Matrigel were fully mixed at 4 ℃ to dilute the Matrix gel. Next, 50 µL diluted matrix gel was added to each Transwell plate upper chamber, placed in the incubator, and incubated for 2–3 h for solidification. Next, 2 × 104 MCF-7 cells were added to the upper chamber of each well, and 800 µL medium containing 20 % FBS to the lower chamber, and incubated at 37 ℃ for 24 h. Next, the Transwell plate was immersed in 10 % formaldehyde for 10 min and then rinsed three times with clear water. The fixed cells were stained with 0.1 % crystal violet (Solarbio Co., Beijing, China) for 30 min, and the cells on the surface were then wiped off with cotton ball. Finally, the samples were observed, photographed, and counted under a microscope. In the Transwell migration experiment, there was no need to deposit the matrix glue, and the incubation time was 16 h. Cells were counted in four randomly selected fields of view .
Methylated-RNA immunoprecipitation (Me-RIP)
Total RNA was isolated from BC cells by the Trizol method. The mRNA in total RNA was separated and purified by PolyATtract® mRNA Isolation Systems (product No. A-Z5300, Eide Technology Co., Ltd., Beijing, China). An IP buffer (20 mm Tris, pH 7.5,140 mM NaCl, 1 % NP-40, 2 mM EDTA), along with anti-m6A antibody (1:500, ab151230, Abcam) or anti-IgG antibody IgG (ab109489, 1:100, Abcam) were added and incubated with protein A/G magnetic beads for 1 h for binding. The purified mRNA and bead-antibody complex was added to the IP buffer along with ribonuclease inhibitor and protease inhibitor and incubated overnight at 4 ℃. The RNA was eluted with elution buffer and purified by phenol chloroform extraction. MALAT1 expression level was assessed by qRT-PCR, and each experiment was repeated three times [23, 24].
Photoactivatable ribonucleoside enhanced crosslinking immunoprecipitation (PAR-CLIP)
BC cells were incubated with 200 mM 4-thiopyridine (4SU) (Sigma-Aldrich, St. Louis, MO, USA) for 14 h, and then cross-linked with 0.4 J/cm2 at 365 nm. The lysates were immunoprecipitated with METTL 3 antibody and incubated overnight at 4 ℃. RNA was labeled with [g-32P]-ATP and observed by autoradiography. The precipitate was digested by protease K to remove the protein, and the relative quantitative expression of MALAT1 was examined using qRT-PCR assay.
Dual luciferase reporter assay
To construct luciferase reporter vector, HMGA2-3′UTR and MALAT1 cDNA fragments containing miR-26b binding site were inserted into pGL3 plasmids. We constructed the binding site mutations of the HMGA2-3′UTR-MUT and MALAT1-MUT fragments by the point mutation method and inserted them into pGL3 plasmid. All the vector sequences were confirmed by gene sequencing. Then, pGL3-MALAT1, pGL3-MALAT1-MUT, pGL3-HMGA2-3′UTR, and pGL3-HMGA2-3′UTR-MUT recombinant vectors and Renilla internal reference plasmids were transfected into HEK-293 cells by liposome transfection with miR-26b mimic or NC-mimic, respectively. Cells were collected and lysed after 48 h of transfection. A luciferase detection kit (K801-200, Biovision, Milpitas, CA, USA) was used for detection of reporter genes by a Dual-Luciferase gene analysis system (Promega, Madison, WI, USA). Renilla luciferase was regarded as the reference gene, and the relative luciferase (RLU) activity of target reporter genes was calculated as the ratio of RLU activity of firefly luciferase to that of renilla luciferase .
50 nM biotin labeled bio-miR-26b-probe and bio-NC probe (Jinkairui Bioengineering Co., Ltd., Wuhan) were used to transfect the cells, which were collected after 48 in culture. The cells were placed on ice in Pierce IP lysis buffer (Thermo Fisher Scientific Inc.) for 30 min, and then centrifuged to collect the supernatant cell lysate. Each lysate was added with an equal amount of beads (Thermo Fisher Scientific Inc.) and incubated overnight at 4 ℃. The next day, the precipitates were collected by centrifugation, washed twice with precooled cracking buffer, and then eluted with high salt buffer. Finally, the supernatant was collected by centrifugation. The RNA combined with miR-26b was purified by the Trizol method and the enrichment of MALAT1 was detected by qRT-PCR .
RNA Immunoprecipitation (RIP)
Cells were lysed in an equal volume RIPA lysate (p0013B, Beyotime) for 30 min. Lysates were centrifuged at 4 ℃, and 14,000 rpm for 10 min to obtain the supernatant. The binding of MALAT1 to Ago2 was detected by a RIP kit (Millipore, USA). In brief, the coprecipitation system was added with 50 µL magnetic beads, and then resuspended in 100 µL RIP wash buffer, followed by addition of 5 µg rabbit anti-Ago2 antibody (ab186733, 1:50, Abcam). The reaction system was incubated at 4 ℃ for at least 6 h. After washing in buffer, the bead-antibody complex was resuspended with 900 µL RIP wash buffer and 100 µL cell supernatant. The samples were placed on the magnetic base to collect the bead-protein complex, digested by protease K, and then the RNA was extracted for subsequent PCR detection. Rabbit anti-IgG (ab172730, 1:100, Abcam) was used as negative control, and the experiment was repeated three times.
Tumorigenesis assay in nude mice
Eighteen BALB/C female nude mice (SLAC Laboratory Animal Co., Ltd., Shanghai, China) aged 4–5 weeks and weighing 15–18 g were randomly assigned into three groups of six mice. The MCF-7 cell lines stably transfected with sh-NC + oe-NC, sh-METTL3 + oe-NC and sh-METTL3 + oe-HMGA2 were selected for subcutaneous establishment of the BC cell line MCF-7 as xenografts in the nude mice. For this purpose, MCF-7 cell lines in the logarithmic growth stage were prepared into a suspension with a concentration of about 1 × 107 cells/ml. The prepared cell suspension was injected into the left armpit of the mice, and the subsequent tumor growth was recorded. After four weeks, mice were killed by cervical dislocation. Tumor tissues were resected and weighed with a balance. The expression of proteins in tumor tissues was examined using Western blot.
For construction of the model of lung metastasis of BC, 3 × 106 MCF-7 cells infected with sh-NC + oe-NC, sh-METTL3 + oe-NC or sh-METTL3 + oe-HMGA2 were injected into nude mice via a tail vein. After 5 weeks, the pathological changes of lung tissue were observed by hematoxylin and eosin staining (HE staining) and the metastasis of tumor nodules were observed.
All the above animal experiments had been approved by the hospital animal protection and use committee, and conformed to management and use principles.
Lung tissue of nude mice was collected, fixed in 10 % neutral formalin, then embedded in paraffin and dewaxed with xylene. Then, the tumor sections were stained with hematoxylin, washed with distilled water, immersed in 95 % ethanol, stained with eosin, hydrated with gradient ethanol, dehydrated with xylene, dried and fixed with neutral resin, and then observed under optical microscope.
All data were represented as the mean ± standard deviation. All analyses were completed with SPSS 24.0 software (IBM, Chicago, IL, USA). The comparisons between paired or unpaired groups were analyzed with paired or unpaired t-test, respectively. The data of multiple groups were compared with Tukey’s test-corrected one-way analysis of variance (ANOVA). Data among groups at different times were compared by Bonferroni test-corrected two-way ANOVA or repeated measurement ANOVA. P < 0.05 was statistically significant.