Cell lines and culture conditions
Human normal colon cell, CCD-112CoN, was acquired from American Type Culture Collection (ATCC), (Manassas, VA, USA) and human CRC HT-29-Red-Fluc cell was acquired from PerkinElmer, Inc. (Waltham, USA). In addition, three more human CRC cell lines, namely DLD-1, HCT-116 and SW480 were kindly provided by Professor Jun YU, Department of Medicine and Therapeutics, Institute of Digestive Diseases, The Chinese University of Hong Kong. The growth condition of CCD-112CoN cells were maintained with 10% fetal bovine serum (FBS), (Gibco, USA) in Eagle’s minimum essential medium (EMEM, ATCC, Manassas, VA). Whereas, HT-29, DLD-1, HCT-116 and SW480 were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco, USA) with 10% FBS. Cell culture was maintained at 37°C in 5% CO2 in 100% humidity.
Dicer-substrate mediated transfection
To knockdown RAMS11, Dicer-substrate mediated silencing was performed. HCT116 and SW480 cells were seeded and cultured in 6-well plate. Transfection experiment was performed when cell density reached 60-70% confluence. A lipid-based in vitro transfection was carried out by Lipofectamine 2000 (Invitrogen, USA), according to the manufacturer’s protocol. TriFECTa Kits were purchased from Integrated DNA Technologies (IDT, USA) which contained a Dicer-substrate negative control (DSi-NC), positive control (Dsi-HPRT-S1), transfection control (Dsi-TYE 563) and predesigned Dsi-RNAs (target genes) duplex. The duplex sequences for Dsi-RAMS11 were: 5’–GAAUAAACAGGAUGUCUCUCACUTT-3’ and 3’– GACUUAUUUGUCCUACAGAGAGUGAAA-5’. The Dsi-NC and Dsi-HPRT-S1 sequence were not provided by the manufacturer. The Dsi-NC and Dsi-HPRT-S1 sequence were not provided by the manufacturer. The transfection conditions were optimized in preliminary experiments.
Complementary DNA synthesis and quantitative RT-PCR (qRT-PCR)
The total RNA from the colon cells were extracted using RNeasy mini kit (Qiagen, Germany) according to their guidelines. The RNA concentration was measured by NanoDrop200 (Thermo Scientific, USA). Following the standard protocol, first-strand cDNA was synthesized using Superscript II and Random Hexamer (Invitrogen, USA). Master Mix LightCycler 480 SYBR Green I (Roche, Switzerland) was used to complete the quantitative reaction using LightCycler 480 Instrument II (Roche, Switzerland). In order to get consistent results, melting temperature (Tm) 60±2°C and 45 cycles of amplification were followed. Detection of PCR product was based on SYBR green fluorescence signals. The melting curve analysis was performed to ensure specific target detection. Here, GAPDH was considered as the housekeeping gene and relative expression was calculated by 2−△△Ct method.
Cell viability assay
After 24 hours of transfection, cells were trypsinized and counted by haemocytometer for seeding and performing cell proliferation assay using Cell Counting Kit-8 (CCK-8, Dojindo). 5 x 103 cells in 100µl of complete medium was seeded and cultured in a 96-well plate. According to CCK-8 cell proliferation assay protocol, 10 µL of CCK-8 solution was added to the well. After 3 hours incubation at 37°C + 5% CO2, the amount of formazan which represents the number of live cells were measured at absorbance 450 nM using SPECTROstar Nano Microplate Reader (BMG Labtech, Germany).
Colony formation assay
Colony formation assay was performed to measure the cell proliferation in vitro. After being transfected for 24 hours, 1 x 103 cells were seeded and cultured for around two weeks in 6-well plate in triplicates. After colony formation, the colonies were fixed with a mixture of methanol and acetic acid at a ratio of 3:1. A solution of 0.5% crystal violet in methanol was used to stain and visualize the colonies. The images were photographed and the number of colonies were counted by ImageJ software (NIH).
Migration assay
In migration assay, 5 x 104 cells in 70 µl DMEM with 10% FBS were carefully placed in both compartments of the Culture-Insert 2 Well (Ibidi LLC, Germany). After 24 hours of cells settling, the culture inserts were gently removed by using tweezers to create a space of ~500 µm for measuring the cell migration ability. Then, each well was filled with 1.5ml of complete medium. The photographs of the wound areas were taken using an inverted microscope (Nikon, Japan) at various time point of 0-hour, 24-hour and 48-hour respectively. The migration index indicating the size of the gap was measured using the MRI Wound Healing Tool in ImageJ (NIH).
Western blotting
Western blotting was performed using standard, established protocol as previously published (19). Briefly, protein isolation was performed using RIPA lysis and extraction buffer (Thermo Scientific, USA) with a supplement of cOmplete ULTRA Tablets, Mini EDTA-free, Easy pack Protease Inhibitor Cocktail (Roche, Switzerland). Protein concentration was quantified using BCA Protein Assay Kit (Thermo Fisher Scientific, USA), and similar amount of proteins were loaded and run on 8-12% SDS-PAGE at ambient temperature. Proteins were then transferred onto Immun-Blot PVDF Membrane (Bio-Rad Laboratories, Inc, USA), and followed by two hours blocking in 5% bovine serum albumin (BSA) (Hyclone BSA, GE Healthcare Life Science, USA) in Tris-buffer saline with a supplement of 0.1% tween 20 (TBST). Then the blocked-membrane were incubated overnight with primary antibodies: β-actin (#8457, Cell signalling technology, Inc., (CST, USA)), GAPDH (#2118, CST), AKT (#9272), Phophor-AKT (#9271, CST), AMPKα (#5832, CST), phosphor-AMPKα (#2535, CST), Bcl-2 (#2872, CST), Bcl-xL (#2764, CST), Beclin-1 (#3738, CST), Caspase-9 (#9502, CST), E-cadherin (#3195, CST), N-cadherin (#13116S, CST), LC3B (#2775, CST), p62 (#5114, CST), mTOR (#2972, CST), Phosphor-mTOR (#2535, CST), Snail (#3879, CST), Sox2 (#3579, CST), and Vimentin (#5741S, CST) at 4°C. The secondary anti-rabit IgG, Horseradish peroxide (HRP)-linked or anti-mouse IgG-HRP-linked (#7076, CST) antibody were added and incubated with the membrane for two hours. Afterwards, Western Lightning Plus-Electrochemiluminescence (ECL) (PerkinElmer, Inc., USA) was added to the membrane to visualize protein bands in a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc, USA). The relative protein expressions were quantified using ImageJ software (NIH) with β-actin or GAPDH as internal control.
Statistical Analysis
All data are presented as mean ± standard error of mean (SEM) of at least three or more independent experiments. The statistical differences of the experimental data were calculated by student’s t test or one way ANOVA using GraphPad Prism version 8.0 (GraphPad Software, Inc., San Diego, CA, USA). The value of P < 0.05 is considered statistically significant.