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SUPPLEMENTARY DATA
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METHODS
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RNA Extraction
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Total RNA extraction was descripted in our previous study1. For plasma, total RNAs
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were extracted from 400µl of plasma using the mirVana PARIS Kit (Ambion)
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according to the manufacturer’s protocol, and eluted with 105 µl of pre-heated (95℃)
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Elution solution. To allow for the normalization of sample-to-sample variation in the
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RNA isolation step, 10µl of 0.05µM synthetic C. elegans miR-39 (synthetic RNA
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oligo-nucleotides synthesized by GenePharma) was added to each denatured sample
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after combining the plasma sample with Denaturing Solution. For the frozen tissues,
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total RNA was extracted using TRIzol reagent (Invitrogen) according to the
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manufacturer’s protocol, and finally resuspended in 60µl of pre-heated (95 ℃ )
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nuclease-free water.
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Quantitative Reverse-transcriptase Polymerase Chain Reaction (qRT-PCR)
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qRT-PCR assays were descripted in our previous study1. The reverse-transcriptase
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reaction was carried out using a Taqman MicroRNA Reverse Transcription Kit
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(Applied Biosystems). cDNA was synthesized in 5 µl volumes containing 1.67 µl of
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RNA extract, 0.5µl of 10×reverse transcription buffer, 0.05 µl of 100 mM dNTPs,
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0.063 µl of RNase Inhibitor (20 U µl-1), 0.33 µl of Mutiscribe Reverse Transcriptase
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(50 U µl-1), 0.5 µl of gene-specific primer and 1.887 µl of nuclease-free water. The
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reactions were incubated at 16℃ for 30 min, followed by 42℃ for 30 min, then 85
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℃ for 5 min before being held at 4℃. The synthesized cDNA was diluted 2 fold by
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nuclear-free water. Quantitative PCR reactions were carried out using 2µl of cDNA
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solution, 5 µl of TaqMan 2×Perfect Master Mix (Takara), 0.25 µl of gene-specific
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primers/probe (TaqMan® MicroRNA Assays, Applied Biosystems. Table S1) and
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2.75 µl of nuclease-free water in a final volume of 10 µl, and run on a Bio-Rad IQ5
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(Bio-Rad Laboratories, Inc) thermocycler. The reaction mixtures were incubated at 95
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℃ for 2 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 30 s. The cycle
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threshold (Ct) values were calculated with the Bio-Rad iQ5 2.1 Standard Edition
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Optical System Software 2.1.94.0617.
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The plasma miRNA concentrations were calculated using a standard curve
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generated using synthetic miR-25. Briefly, the standards of miR-25 and C. elegans
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miR-39 were purchased from GenePharma, and used to perform the standard curve of
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miR-25. C. elegans miR-39, which lacks sequence homology to human miRNAs, was
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selected to normalize the experimental qRT-PCR data. Known quantities of synthetic
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C. elegans miR-39 were diluted to produce Ct values within the Ct value ranges of the
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miRNA standard curves. We empirically added 10 μl of 0.05 mM synthetic C.
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elegans miR-39 to 400 μl of plasma after combining the plasma sample with
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Denaturing Solution. The C. elegans miR-39 was amplified as well as miR-25. The
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following formula was used for adjusting the Ct values of miR-25 in all plasma
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samples: Normalized_ Ct value for the miRNA in the sample = Raw_ Ct value -
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[(SpikeIn_ Average_ Ct value of the given sample) - (Median_SpikeIn_Ct)]2. The
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normalized_ Ct value was then used to calculate the concentration of miR-25 upon the
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miR-25 standard curve. The standard reference miRNAs were amplified for each
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reaction. The expression of miR-25, TOB1 and p57 from tissue or cell samples was
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normalized using the 2- ⊿ ⊿ Ct method from the Ct values of interest relative to
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RNU6BB or β-actin. All of the primer sequences are shown in Table S1.
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SUPPLEMENTARY FIGURE LEGEND
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Figure S1. The migration and invasion of AGS, BGC-823 and MKN-45 cells
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after transfection with agomir-25. After AGS, BGC-823 and MKN-45 cells were
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transfected with Mock, agomir-NC (600nM) or agomir-25 (600nM) for 24 h,
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respectively, 5 × 105 cells were used to perform the migration (A) and invasion (B)
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assays. Histogram reveals the values of absorbance at 570nm for migration, or at
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560nm for invasion. Agomir-25 significantly increased the migration and invasion of
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AGS, BGC-823 and MKN-45 cells when compared with agomir-NC (P < 0.05, or P <
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0.01). All assays were repeated in duplicates.
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Figure S2. Correlation between miR-25 expression and migration and invasion
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abilities of GC cells. (A) Correlation between the miR-25 expression and migration
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abilities in AGS, BGC-823, MKN-45, HGC-27 and SGC-7901 cells (P < 0.05, R2 =
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0.9262). (B) Correlation between the miR-25 expression and invasion abilities in
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AGS, BGC-823, MKN-45, HGC-27 and SGC-7901 cells (P < 0.05, R2 = 0.8473).
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Figure S3. MiR-25 regulates proliferation of GC cells. (A) After HGC-27 and
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SGC-7901 cells were transfected with Mock, antagomir-NC (800nM) or antagomir-25
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(800nM) for 24 h, respectively, and (B) AGS, BGC-823 and MKN-45 cells were
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transfected with Mock, gomir-NC (600nM) or agomir-25 (600nM) for 24 h,
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respectively, 1×104 cells were used to perform the proliferation assays. Histogram
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reveals the values of absorbance at 450nm for proliferation. Notes: “ns”, no
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significance, and “**”, P < 0.01.
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Figure S4. TOB1 expression in gastric mucosa tissues and GC cell lines. The
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expression of TOB1 was detected via qRT-PCR assays. Scatter shows TOB1
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expression in the gastric mucosa tissues of 10 healthy subjects. Bar shows TOB1
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expression in the GC cell lines of AGS, BGC-823, MKN-45, HGC-27 and SGC-7901.
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β-actin serves as an internal reference. All assays were repeated in duplicates.
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Figure S5.MiR-25-induced the loss of TOB1 regulates proliferation of GC cells.
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(A) After HGC-27 and SGC-7901 cells were transfected with negative control (NC)
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(100nM), si-TOB1 (100nM), Empty vector (200ng), or TOB1 vector (200ng) for 24 h,
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respectively, and (B) AGS, BGC-823, MKN-45, HGC-27 and SGC-7901 cells were
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transfected with agomir-NC (600nM) or agomir-25 (600nM), or cotransfected with
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agomir-25 (600nM) and TOB1 vector (200ng) for 24 h, respectively, 1×104 cells were
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used to perform the proliferation assays. Histogram reveals the values of absorbance
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at 450nm for proliferation. Notes: “*”, P < 0.05, and “**”, P < 0.01. All assays were
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performed in triplicates.
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Reference
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Li BS, Zhao YL, Guo G, Li W, Zhu ED, Luo X et al (2012). Plasma
microRNAs, miR-223, miR-21 and miR-218, as novel potential biomarkers
for gastric cancer detection. PloS one 7: e41629.
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Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK,
Pogosova-Agadjanyan EL et al (2008). Circulating microRNAs as stable
blood-based markers for cancer detection. Proceedings of the National
Academy of Sciences of the United States of America 105: 10513-10518.