RNA methylation-mediated LINC01559 suppresses colorectal cancer progression by regulating the miR-106b-5p/PTEN axis

Long noncoding RNAs (lncRNAs) regulate multiple biological effects in cancers. Recently, RNA methylation has been found to modify not only coding RNAs but also some noncoding RNAs. How RNA methylation affects lncRNAs to affect colorectal cancer (CRC) progression remains elusive. The expression of LINC01559 was explored through RNA sequencing, quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). The preliminary exploration of its function was performed using Western blotting (WB) and immunohistochemistry (IHC). Functional experiments in vitro and in vivo were conducted to explore the biological functions of LINC01559 in CRC. The LINC01559/miR-106-5p/PTEN axis was verified through fluorescence in situ hybridization (FISH), luciferase assays, and rescue experiments. RIP-sequencing, m6A RNA immunoprecipitation (MeRIP) assays and bioinformatic analysis were conducted to determine the upstream mechanism of LINC01559. The results showed that LINC01559 was downregulated in CRC compared with normal controls. Lower expression of LINC01559 in CRC patients predicted a poor prognosis. In addition, PTEN was found to be positively correlated with LINC01559, and miR-106b-5p could be the link between LINC01559 and PTEN. Then, silencing LINC01559 restored the malignant phenotype of CRC cells, while cotransfection of miR-106b-5p inhibitor neutralized this effect. Mechanistically, we found abundant m6A modification sites on LINC01559. Then, we uncovered these sites as potential targets of METTL3 through experiments in vivo. The results revealed a negative functional regulation of the LINC01559/miR-106b-5p/PTEN axis in CRC progression and explored a new mechanism of METTL3-mediated m6A modification on LINC01559. These results elucidate a novel potential therapeutic target for CRC treatment.

(Invitrogen, ThermoFisher Scientific, Carlsbad, CA, USA) was used to transient siRNA and overexpression vector transfection.

Western blot analysis (WB)
Total proteins were prepared from CRC using RIPA buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate) with proteinase inhibitor cocktail (Solarbio, Beijing, China). The lysates were centrifuged at 12,000 rpm for 15 min at 4°C and protein concentration was measured by BCA kit (Beyotime Biotechnology, Beijing, China). Equal quantities of protein were electrophoresed through a 10% sodium dodecyl sulfate/polyacrylamide gel and transferred to PVDF membranes (Millipore, Massachusetts, MA). The PVDF membranes were blocked with skim milk powder dissolved by TBST (5%) at room temperature for 1 h, and then incubated with primary antibodies at 4°C overnight.
The primary antibodies included anti-N-cadherin (No. 66219-1-Ig), anti-E-cadherin from Cell Signaling Technology (MA, USA). Secondary antibodies hybridized the membrane at room temperature for 1 h. Then the membranes were visualized by the chemiluminescence kit (Absin, Shanghai, China). WB strips were detected through Image-Pro Plus 6.0 and analyzed through Student's t test.

Wound healing assay
Cells were cultured in standard conditions until 80-90% confluence and treated with mitomycin C (10 µg/ml) during the wound healing assay. The cell migration was assessed by measuring the movement of cells into the acellular area created by a sterile insert. The wound closure was observed after 48 h.

Transwell assays
To assess the migration and invasiveness of HCT116 and SW480 cells, we used Transwell chambers (Corning, NY, USA). Briefly, 3×10 5 cells in serum-free medium were placed in the upper chamber. Dulbecco's modified Eagle's medium (500 ml) supplemented with 10% fetal bovine serum was added to the lower chamber. After incubation in a humidified atmosphere containing 5% CO2 at 37°C for 72 h, Giemsa staining of A375 cells that had migrated or invaded into the lower chamber was performed. Stained cells were photographed under an IX53 inverted microscope (NIKON, Tokyo, Japan), and the Image-Pro Plus software program (Media Cybernetics, Rockville, MD) was used to count the cells.

Cell proliferation assay
Cells were seeded in 96-well plates at 0.8-1×10 3 per well. Cell proliferation was evaluated using Cell Counting Kit-8 (Dojin Laboratories, Tokyo, Japan) according to manufacturer's instructions. We collected cell samples at 24h, 48h, 72h and 96h, respectively. Then 10 µl of CCK-8 solution was added to culture medium, and incubated for 2h. The absorbance at 450 nm wavelength was determined with a reference wavelength of 570 nm.
Following staining with Hoechst 33342 at room temperature for 30 min in darkness and one or two washes with PBS, the cells were observed using a Micro system (ImageXpress, Downingtown, PA, U.S.A.). Five fields were randomly selected and photographed, and the number of EdU-positive cells was calculated.

Tube formation assay
Twenty-four-well plates were coated with 60 ml Matrigel (BD Biosciences, USA) at 37 °C for 1 h for gel formation. A total of 1×10 5 stably transfected cells in medium containing 10% FBS were plated into the pre-solidified Matrigel and started the process to form capillary tubes and networks once seeded on Matrigel. Six hours after incubation, plates were observed under microscope and photographed (Nikon, Japan).
The numbers of branching points generating at least three tubules were counted.

The statistical analysis of survival data
The data of DFS and follow-up data (current status, survival) was obtain through telephone follow-up survey, periodic re-examination and readmission. CRC recurrence or death were regarded as terminal point event. The days of DFS were calculated from date of diagnosis to date of terminal point event occurrence. The expression of LINC01559 was detected through q-PCR assays in 41 pairs of CRC tissues and adjacent normal tissues. The high level of LINC01559 was designed as the expression above the median, and the low level was designed as the expression below the median. The outcome variable 1 represented CRC recurrence or death due to CRC and 0 represented censoring (loss of follow-up, endpoint inoccurrence or others).
Survival data were obtained by the Kaplan-Meier method, with significance assessed by the log-rank test in SPSS.

Legends to Supplemental Material Supplemental Methods and Materials
Additional file 1: Figure S1. Key signaling pathways in CRC from KEGG.
Additional file 16: Figure S11. qRT-PCR assay was utilized to estimate the over-expressed efficiency of OV-METTL3 in HCT116 and SW480 cells and si-METTL3 efficiency of SW480.