Research ArticleAPPLIED SCIENCES AND ENGINEERING

Unveiling massive numbers of cancer-related urinary-microRNA candidates via nanowires

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Science Advances  15 Dec 2017:
Vol. 3, no. 12, e1701133
DOI: 10.1126/sciadv.1701133
  • Fig. 1 Nanowire-induced electrostatic collection of urine EVs followed by in situ extraction of EV-encapsulated miRNAs.

    (A) Schematic illustrations for urine EV collection and in situ extraction of urine EV–encapsulated miRNAs using a nanowire-anchored microfluidic device. (B) A schematic illustration (gray rods, nanowires; transparent cyan areas, PDMS) and an inset illustration on the lower left showing a cross-sectional image (yellow and blue represent nanowires and PDMS, respectively) for buried nanowires after poring, curing, and peeling off PDMS, and a vertical cross-sectional FESEM image of buried nanowires; nanowires and PDMS are highlighted as yellow and blue, respectively, and the white dotted line indicates a PDMS edge. Scale bar, 1 μm. (C) A schematic illustration and an inset illustration on the lower left showing a cross-sectional image for growing nanowires from the buried nanowires (nanowire-embedded PDMS), and a vertical cross-sectional image of the nanowire-embedded PDMS. Scale bar, 1 μm. (D) A schematic illustration and an inset illustration on the lower left showing a cross-sectional image for bonding the nanowire-embedded PDMS substrate to the microfluidic herringbone-structured PDMS substrate, an image of the nanowire-anchored microfluidic device (bonding the nanowire-embedded PDMS and the microfluidic herringbone-structured PDMS substrates) with PEEK tubes for an inlet and an outlet (scale bar, 1 cm), and a laser micrograph of the microfluidic herringbone structure on PDMS (scale bar, 1 mm). (E) A schematic illustration of the nanowire-embedded PDMS (gray rods, nanowires; transparent cyan areas, PDMS), and an overview of FESEM image for the nanowire-embedded PDMS (scale bar, 1 μm) after being exposed to lysis buffer. (F) A schematic illustration of nanowires on the Si substrate (gray rods, nanowires; dark cyan areas, Si substrate; faded cyan areas, Cr layer), and an overview of FESEM image for the nanowires on the Si substrate after being exposed to lysis buffer. Scale bar, 1 μm.

  • Fig. 2 In situ extraction of miRNAs using the nanowire-anchored microfluidic device.

    (A) Scatterplot of normalized intensities of miRNAs extracted from the collected EVs on nanowires in the device versus the ultracentrifuged EVs. Each point corresponds to a different miRNA type (that is, species). The boundary between pink and cyan represents the same level of miRNA expression for the two approaches. (B) Histogram of miRNA species for nanowires (pink) and ultracentrifugation extraction (cyan). Error bars show the SD for a series of measurements (n = 3). (C) Scatterplot of normalized intensities of miRNAs extracted from the collected EVs using the nanowire-anchored microfluidic device versus miRNA expression extracted from the collected EVs when using a commercially available kit. Each point corresponds to a different miRNA type (species). The boundary between pink and gray represents the same level of miRNA expression for the two approaches. (D) Histogram of miRNA species for nanowires (pink) and the commercially available kit (gray). Error bars show the SD for a series of measurements (n = 3). a.u., arbitrary units.

  • Fig. 3 EV collection onto the nanowires.

    (A) A schematic illustration for the experimental process and calculation of collection efficiency. (B) Size distribution of the urinary free-floating objects in the untreated urine. Error bars show the SD for a series of measurements (n = 3). (C) Size distribution of the urinary free-floating objects in the flow-through fraction of the urine being processed by the device (pink) and in the ultracentrifuged urine (cyan). Error bars show the SD for a series of measurements (n = 3). (D) Fluorescently (PKH26) labeled EVs collected on nanowires. Red denotes PKH26-labeled EVs on nanowires. Scale bar, 500 μm. (E) An FESEM image of nanowires after introduction of PKH26-labeled EVs. White arrows indicate collected EVs. Scale bar, 200 nm. (F) Detection of EVs in urine on nanowires (pink) and a 96-well plate (cyan) using an antibody of CD63 or CD81. The measured concentration of the urinary free-floating objects was 1.4 × 108 ml−1. N.D. indicates fluorescence intensity was not detected. The black dotted line shows the signal level at 3 SD above the background. Error bars showing the SD for a series of measurements of nanowires and a 96-well plate (n = 24 and 3, respectively).

  • Fig. 4 In situ extraction of cancer-related miRNAs using the nanowire-anchored microfluidic device; heat maps of the miRNA expression array for noncancer lung, pancreatic, liver, bladder, and prostate cancer donor urine samples (n = 3).

    For intuitive understanding of the expression level of each miRNA and easy comparison between each group, we used color gradations showing signal intensity variation. Black, logarithmic signal intensity of 5; blue, logarithmic signal intensity less than or equal to 2; and yellow, logarithmic signal intensity greater than or equal to 8. Each column in the heat maps represents the logarithmic signal intensities of each miRNA corresponding to the color gradation.

  • Fig. 5 Down-regulated and overexpressed miRNAs extracted from Fig. 4 between noncancer donors and each cancer donor.

    Extracted miRNAs were the second smallest logarithmic signal intensities in one group larger than the three pulsing second largest logarithmic signal intensities in the other group. The symbols − and + show noncancer and cancer donors, respectively. Pink lines highlight minimums in one group that were larger than the three pulsing maximums in the other group, giving highlighted miRNAs an edge over other miRNAs. Green and orange lines highlight cancer-specific down-regulated miRNAs and overexpressed miRNAs, respectively.

  • Table 1 Comparison among three methodologies.
    Fabricated deviceExoQuickUltracentrifugation
    Collected objectsExosomes
    Microvesicles
    EV-free miRNAs
    Exosomes
    Microvesicles
    Exosomes
    Collection mechanismElectrostatic interaction
    between nanowire surface
    charge and collected objects
    Polymer-based capture of objects ranging
    from 60 to 180 nm in diameter, according to
    the kit manufacturer’s instruction manual
    Balance between applied
    forces and density of the
    collected objects
    Sample volume1 ml1 ml1 ml for small RNA quantification
    20 ml for urinary miRNA profiling
    Processing time40 min870 min300 min
    Collection efficiency (small RNA yield)0.194 ± 0.028 ng/μl0.120 ± 0.015 ng/μl0.159 ± 0.077 ng/μl
    Extraction species of urinary
    miRNAs being identified
    749, 822, 1111 (n = 3)337, 355, 491 (n = 3)171, 261, 352 (n = 3)
    200 to 300*

    *From Cheng et al. (31).

    • Table 2 Potential cancer-related urinary miRNAs as indicated in Fig. 5.

      Blank spaces in the “Biological functions” column indicate that biological functions of the miRNAs have not been reported.

      Cancer types (down-regulation/overexpression)miRNA namesBiological functions
      Lung (down-regulation)miR-3127-3p*
      miR-3130-5p*
      miR-3131*
      miR-3141*
      miR-3150b-5p*
      miR-3151-3p*
      miR-3151-5p*
      miR-3154*
      miR-3160-3p*
      miR-3160-5p*
      miR-378a-5pSuppressing cell proliferation and inducing
      apoptosis (52)
      miR-520c-3pTumor suppressor (5357)
      miR-526b-3pTumor suppressor (58)
      miR-3150a-3p
      miR-3162-5p
      miR-4254
      Lung (overexpression)miR-3117-5p*
      miR-3118*
      miR-3121-3p*
      miR-3121-5p*
      miR-3126-5p*
      miR-3128*
      miR-3133*
      miR-3134*
      miR-3136-3p*
      miR-3136-5p*
      miR-3139*
      miR-3142*
      miR-3143*
      miR-3145-3p*
      miR-3163*Inhibiting non–small cell lung cancer cell
      growth (59)
      miR-3166*
      miR-3167*
      miR-16-1-3pRepressing gastric cancer cell invasion and
      metastasis (60)
      miR-424-3pPotential metastasis-related miRNAs (61)
      miR-519c-5p
      miR-525-5p
      miR-551b-5p
      miR-558Promoting tumorigenesis (62)
      miR-921
      miR-942-3p
      miR-3126-3p
      miR-3127-5pReducing non–small cell lung cancer cell proliferation (63)
      miR-3129-5p
      miR-3144-5p
      miR-3150a-5p
      miR-3152-5p
      miR-3155a
      miR-3157-3p
      miR-3159
      miR-3165
      miR-3678-3p
      miR-4321
      miR-4521Significantly up-regulated miRNA in cancer
      stem cells (64)
      miR-4800-3p
      miR-4999-5p
      miR-5096
      miR-5187-5p
      miR-6874-5p
      Pancreatic (down-regulation)miR-372-3p*Tumor suppressor (65)
      miR-378b*
      miR-520b*Inhibiting cellular migration and invasion (66)
      miR-1266-3p*
      miR-3605-5p*
      miR-3612*
      miR-4645-3p*
      miR-4694-3p*
      miR-4752*
      miR-6816-3p*
      miR-8087*
      let-7f-2-3p
      miR-15a-3pInducing apoptosis in human cancer cell lines (67)
      miR-20a-3p
      miR-33b-3p
      miR-34c-5pTumor suppressor (68)
      miR-93-5p
      miR-130a-5p
      miR-135a-5pInhibiting tumor metastasis (69)
      miR-135b-5p
      miR-185-5pTumor suppressor (70)
      miR-203a-3p
      miR-302d-5p
      miR-337-3pTumor suppressor (71)
      miR-378c
      miR-422aDown-regulated in colon cancer (72)
      miR-449c-5p
      miR-483-5p
      miR-506-3pInducing differentiation (73)
      miR-511-5p
      miR-520c-3pTumor suppressor (5357)
      miR-654-3p
      miR-668-5p
      miR-670-5p
      miR-671-3p
      miR-744-3p
      miR-1178-3p
      miR-1254
      miR-1284Down-regulated in lymph node metastatic sites (74)
      miR-1323Modulating radioresistance (75)
      miR-2116-5p
      miR-2355-3p
      miR-3132
      miR-3138
      miR-3164
      miR-3186-3p
      miR-3189-3p
      miR-3198
      miR-3200-5p
      miR-3657
      miR-3667-5p
      miR-3680-5p
      miR-3692-5p
      miR-3713
      miR-3921
      miR-3936
      miR-4273Increasing colorectal cancer risk (76)
      miR-4299
      miR-4306
      miR-4316
      miR-4319
      miR-4421
      miR-4429
      miR-4435
      miR-4441
      miR-4473
      miR-4506
      miR-4633-5p
      miR-4658
      miR-4733-5p
      miR-4733-3p
      miR-5004-3p
      miR-5194
      miR-5197-5p
      miR-5571-5p
      miR-6083
      miR-6717-5p
      miR-6720-5p
      miR-6767-3p
      miR-6781-3p
      miR-6811-3p
      miR-6821-3p
      miR-6828-5p
      miR-6832-5p
      miR-6837-3p
      miR-6841-5p
      miR-6853-5p
      miR-6871-3p
      miR-6875-5p
      miR-6878-5p
      miR-7112-3p
      miR-7703
      miR-7848-3p
      miR-7856-5p
      Pancreatic (overexpression)let-7i-3p
      miR-183-5pPromoting cancer proliferation, invasion, and
      metastasis (77)
      miR-202-5pIncreasing TGFBR1 and TGFBR2 protein expressions (78)
      miR-409-5pPromoting tumorigenesis (79)
      miR-4661-5p
      miR-4800-3p
      miR-5587-5p
      Liver (down-regulation)let-7i-2-3p
      miR-520c-3pTumor suppressor (5357)
      Liver (overexpression)miR-4521*
      let-7c-3p
      let-7i-5p
      miR-16-1-3pRepressing gastric cancer cell invasion and metastasis (60)
      miR-26a-1-3p
      miR-28-5pSuppressing insulin-like growth factor 1 expression (80)
      miR-105-5p
      miR-195-3p
      miR-200b-5p
      miR-219a-2-3p
      miR-297Promoting cell proliferation and invasion (81)
      miR-300Inhibiting pituitary tumor transforming gene
      expression (82)
      miR-330-3pPromoting invasion and metastasis (83)
      miR-374b-5p
      miR-431-5p
      miR-454-5pPromoting tumorigenesis (84)
      miR-513c-5p
      miR-548ax
      miR-593-5p
      miR-623
      miR-664a-5p
      miR-942-3p
      miR-1205
      miR-1276
      miR-1288-3p
      miR-1297Promoting cell proliferation (85)
      miR-3678-3p
      miR-4283
      miR-4295Promoting cell proliferation and invasion (86)
      miR-4439
      miR-4524b-5p
      miR-4703-3p
      miR-4768-5p
      miR-4800-3p
      miR-5187-5p
      miR-5696
      miR-7161-5p
      Bladder (down-regulation)let-7f-2-3p
      miR-520c-3pTumor suppressor (5357)
      miR-4783-5p
      Bladder (overexpression)miR-16-1-3pRepressing gastric cancer cell invasion and metastasis (60)
      miR-23b-3pRegulating chemoresistance of gastric cancer cell (87)
      miR-28-5pSuppressing insulin-like growth factor 1 expression (80)
      miR-92a-2-5p
      miR-142-3pPromoting malignant phenotypes (88)
      miR-195-3pPromoting tumorigenesis and inhibiting apoptosis (89)
      miR-196b-5p
      miR-299-3pReducing Oct4 gene expression (90)
      miR-492
      miR-513b-5p
      miR-601
      miR-619-5p
      miR-1285-3p
      miR-3155a
      miR-3162-5p
      miR-3678-3p
      miR-4283
      miR-4295Promoting bladder cancer cell proliferation (91)
      miR-4311
      miR-4531
      miR-5096
      miR-5187-5p
      Prostate (down-regulation)miR-15a-3pInducing apoptosis in human cancer cell lines (67)
      miR-135b-5p
      miR-520c-3pTumor suppressor (5357)
      miR-4783-5p
      miR-7849-3p
      Prostate (overexpression)miR-4531*
      miR-28-5pSuppressing insulin-like growth factor 1 expression (80)
      miR-103a-2-5p
      miR-105-5pInhibiting the expression of tumor-suppressive genes (92)
      miR-124-3pRegulating cell proliferation, invasion, and apoptosis (93)
      miR-151a-5pTumor cell migration and invasion (94)
      miR-151b
      miR-200a-5p
      miR-300Promoting cell proliferation and invasion (95)
      miR-424-3p
      miR-519c-5p
      miR-551b-5p
      miR-617
      miR-873-3p
      miR-921
      miR-1288-3p
      miR-3124-5p
      miR-3155a
      miR-3917
      miR-4283
      miR-4727-3p
      miR-5096
      miR-5187-5p
      miR-6074
      miR-6874-5p
      miR-6892-5p

      *Marks showing miRNAs highlighted by pink lines in Fig. 5.

      Supplementary Materials

      • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/12/e1701133/DC1

        fig. S1. Schematic of the fabrication procedure for nanowires anchored into PDMS.

        fig. S2. Nanowires anchored into PDMS.

        fig. S3. FESEM images and EDS elemental mappings corresponding to FESEM images for PDMS without nanowires, PDMS with buried nanowires, and nanowire-embedded PDMS; scale bars, 1 μm.

        fig. S4. Mechanical stability of anchored nanowires and nonanchored nanowires.

        fig. S5. Extraction process of miRNAs in urine using the nanowire-anchored microfluidic device, ultracentrifugation, and commercially available kits.

        fig. S6. FESEM images and EDS elemental mappings of an STEM image of a single nanowire.

        fig. S7. Detection of EVs on ZnO nanowires, ZnO/Al2O3 core-shell nanowires, and no nanowires using an antibody of CD9.

        fig. S8. Zeta potential of EVs in urine.

        fig. S9. Size distribution of EVs collected by ultracentrifugation and EV-free miRNAs collected onto nanowires.

        fig. S10. Size distribution of the urinary free-floating objects.

        movie S1. EV collection followed by miRNA extraction in urine using the nanowire-anchored microfluidic device.

        data S1. Logarithmic signal intensities in noncancer miRNAs with those in cancer miRNAs.

      • Supplementary Materials

        This PDF file includes:

        • fig. S1. Schematic of the fabrication procedure for nanowires anchored into PDMS.
        • fig. S2. Nanowires anchored into PDMS.
        • fig. S3. FESEM images and EDS elemental mappings corresponding to FESEM images for PDMS without nanowires, PDMS with buried nanowires, and nanowire-embedded PDMS; scale bars, 1 μm.
        • fig. S4. Mechanical stability of anchored nanowires and nonanchored nanowires.
        • fig. S5. Extraction process of miRNAs in urine using the nanowire-anchored microfluidic device, ultracentrifugation, and commercially available kits.
        • fig. S6. FESEM images and EDS elemental mappings of an STEM image of a single nanowire.
        • fig. S7. Detection of EVs on ZnO nanowires, ZnO/Al2O3 core-shell nanowires, and no nanowires using an antibody of CD9.
        • fig. S8. Zeta potential of EVs in urine.
        • fig. S9. Size distribution of EVs collected by ultracentrifugation and EV-free miRNAs collected onto nanowires.
        • fig. S10. Size distribution of the urinary free-floating objects.
        • Legend for movie S1
        • Legend for data S1

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        Other Supplementary Material for this manuscript includes the following:

        • movie S1 (.mov format). EV collection followed by miRNA extraction in urine using the nanowire-anchored microfluidic device.
        • data S1 (Microsoft Excel format). Logarithmic signal intensities in noncancer miRNAs with those in cancer miRNAs.

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