2.1. Cell culture, antibodies and reagents
LoVo colorectal cancer cell (CRC) line is derived from Bioresource Collection and Research Center (Hsinchu, Taiwan). CRC cells were mantained in Dulbecco’s Modified Eagle’s Medium-low glucose (D5523-1L, Sigma Aldrich, Missouri, USA) supplemented with 10% fetal bovine serum (Gibco Life Technologies Co., Grand Island, NY, USA), 1% penicillin/streptomycin, sodium bicarbonate at 37 0C, with 5% CO2 with 80-90% humidified condition.The cell medium was changed after 48h subculture. Oxaliplatin powder and 1-(4, 5-Dimethylthiazol-2-yl)-3, 5-diphenylformazan (MTT) was purchased from Sigma-Aldrich (Sigma Chemical Co., Merck KGaA, Darmstadt, Germany). followed the antibodies that we used are CTDSPL (Novus Biologicals Co., Centennial CO, USA); E2F1, phosphorylated RB and RB (Cell Signaling Technology, Inc. Beverly, MA, USA); cyclin D1, cyclin E1, cyclin B1, cyclin A1, PCNA, ERK1/2, phosphorylated-ERK1/2, AKT, phosphorylated-AKT, p53, phosphorylated-p53, p19, p27, p15/16, Ki67 and GAPDH (Santa Cruz Biotechnology, Inc. Santa Cruz, California, USA). Secondary antibodies are HRP conjugated-Goat anti-mouse and anti-Rabbit IgG antibodies was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
2.2. Establishment of Oxaliplatin-resistant LoVo cancer cells
Generating stable Oxaliplatin resistant colorectal cancer cells, first, we exposed Oxaliplatin drug with dose dependent manner (0.0 to 30.0 µg/ml) in parental LoVo cancer cell for 24 h; resulted in greater than 50% cells death (IC50 of Oxaliplatin, 15.0 μg/ml). Further, added 15.0 μg/ml Oxaliplatin in the parental LoVo colon cancer cells (24 h) untill reached 80% confluence, and passaged twice in this same concentration (15.0 μg/ml) of Oxaliplatin. The repetition procedure was used, and increased doses of Oxaliplatin (20.0 and 25.0 μg/ml). Next, selected the cell population that demonstrated at least a 3-fold greater IC50 (45 μg/ml) to Oxaliplatin than the parental LoVo cell lines.
2.3 Microarray analysis
RNA was isolated from the LoVoWT and LoVoOXR cell lines using an RNA MiniPrep kit (Zymo Research Corporation, Irvine, CA, USA) followed by the manufacturer’s instructions. The microRNA expression profile was analyzed through miRNA Microarray Services (Service Code: 2h213102401; Human miRNA OneArray®). The fold change was calculated by comparing the expression level of different miRNAs in the LoVoOXR cells as compared that LoVoWT using a log2 format.
2.4 RNA isolation and quantitative reverse transcription PCR (RT-qPCR) assay
For Real Time-quantitative polymerase chain reaction (RT-qPCR) assay, the RNA was collected from the parental and Oxaliplatin-resistant LoVo cells using GeneJET RNA purification (Thermo Fisher Scientific, Lithuania) and then reverse transcribed to cDNA using an GScript First-Strand Synthesis Kit, cDNA synthesis kit, Oligo-dT primers 50uM and 10mM dNTP mix according to manufacturer’s protocol. For examining miRNA expression level, we used a Applied Biosystem Real-Time PCR and ORATM SEE qPCR Green ROX L Mix, 2X (HighQu professionally simple, USA) with a total reaction volume of 20 µL containing 10 µL ORATM SEE qPCR Mix, 300 nM (each) forward and reverse primers, and 500 ng cDNA. The primers for mir-100-5p: AACCCGUAGAUCCGAACUUGUG. mRQ 3’ Primer (10 µM), U6 Forward Primer (10 µM), and U6 Reverse Primer (10 µM) were from the Mir-X miRNA First-Strand Synthesis Kit and SYBR qRT-PCR user manual. U6 was used as an endogenous control. The gene expression was determined by cyclic threshold equation 2∆Ct, in which ∆Ct = (Ct microRNA – Ct U6 rRNA). All reactions were performed in triplicate.The primer for upstream target are GATA-1; forward: CAC TGA GCT TGC CAC ATC C, Reverse: ATG GAG CCT CTG GGG ATT A, CEBP-b; forward: CGC TTA CCT CGG CTA CCA, Reverse: ACG AGG AGG ACG TGG AGA G, FOXP3; forward: CTT CCT TGA ACC CCA TGC, Reverese: GGA GGA GTG CCT GTA AGT GG and the downstream target CTDSPL; Forward: AAC CCC AAG GAG GAC GAG, Reverse: CAG AAG AAG GAG CTA AGG ATG C. GAPDH; forward: GCA CCG TCA AGG CTG AGA AC, Reverse: ATG GTG GTG AAG ACG CCA GT was used as endegoneus control.
2.5 mRNA prediction and analysis
Three common miRNAs predictor target and binding sites algorithms that are publically available: http://www.microrna.org for miRanda algorithm, http://www.targetscan.org for TargetScan algorithm and http://mirtar.mbc.nctu.edu.tw/human/ for miRTar base algorithm were used to predict the putative targets of miR-100-5p. Integrated function and pathway analysis were performed using DAVID bioinformatics resources (http://david.abcc.ncifcrf.gov/) and significant features of predicted miRNA targets were clustered.
miR-100-5p Overexpression (mimic), Knockdown (inhibitor), scrambled miRNA (mimic NC or inhibitor NC) and sh-RNA were used to transfected into cells with confluent about 60-80%. jetPRIME®(Polyplus Transfection Inc, Illkirch, France) Kit transfection was used, the transfected cell were incubated in 5% CO2 at 37 0C humidified condition for 24hr, then used in further experimental.
2.7 Protein calculation
Protein derived from cell extract which was using the Bradford reagent (BIO-RAD, Hercules, CA, USA) to calculate protein estimation according to the manufacturer’s instruction using bovine serum albumin (BSA) as a standard (2.0mg/ml). In brief, the calculation standard curve prepare with a range of protein concentrations (0.0, 0.1, 0.2, 0.3, 0.4 and 0.5mg/ml) from diluted BSA (0.5mg/ml final concentration). 198µl Bradford reagent was mixed with 2.0µl of protein samples in a 96-wells plate. Next, measuring the optical density (OD) of sample was at 595nm using a multiwall ELISA plate reader (Molecular Devices, Palo Alto, CA, USA) and calculated the protein amounts with previously performed BSA standard curve.
2.8 Western blotting
Followed the method of Huang, 2014  with minor modifications, we examined the protein expression. In brief, at 14,000 rpm for 15-20 min the protein was centrifuged and 45.0µg protein with equal amount was boiled (95 ⁰C, 10 min) together with appropriate 5x sample buffer [10% SDS, 50% Glycerol, 0.1% Bromophenol blue, 250mM Tris-Hcl (pH-6.8) and 5% β-mercaptoethanol], and then loaded on 10%-12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) at 80-100V in certain time. Next, transferred proteins on polyvinylene difluoride membrane (PVDF) (Millipore Corporation limited, Bedford, MA, USA) in transfer buffer (25mM Tris base, 190 mM glycine and 20% methanol) using a BIO-RAD transfer system (Bio-Rad Laboratories, Munich, Germany). Incubated protein in membrane with blocking buffer [5% (w/v) BSA, 1xTransfer Buffer Saline (TBS) and 0.1% Tween-20] for 1 h at RT. Hiring primary antibodies (1/1000- 1/2000 dilution in 1xTBST) at 4 ⁰C for overnight. Thereafter, washed membrane three times with 1xTBST and incubated with secondary antibodies (1:4500) anti-rabbit and anti-goat HRP-secondary antibodies. Next, detection the protein signals was using custom made Immobilon®Western chemiluminescent reagent (Millipore Corporation limited, Billerica, MA, USA) on an Image Bright i500 imaging systems (InvitrogenTM iBright TM Imaging system, Thermo fisher Scientific, USA). Next, analysis the densitometry was used the ImageJ program 1.50 (National Institutes of Health, Bethesda, MD, USA). GAPDH and HDAC1 were used as endogenous control for normalization protein.
2.9 Cell Viability Assay
Trypan blue staining method was used to measure the cell viability and counting cell. In brief, at 15,00xg for 3-5 min trypsinized LoVoWT and LoVoOXR cells were centrifuged then resuspended with 1xPBS. After that, 10.0μl of cells suspension was mixed with 30.0µl of 0.3% trypan blue stained solution for 5 min. Then, the number of colouring cells was counted using a haemocytometer under microscope. Living cell displayed colourless, while dead cells were blue colour.
2.10 MTT [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium-bromide] Assay
Followed the procedure of Hsu (2019) with minor modification MTT method was used to measure anti-cells viability effect of cytotoxic drugs through alteration colour of MTT from light orange into a soluble purple formazon crystals using mitochondrial succinate dehydrogenase. In brief, LoVoWT and LoVoOXR cells (5x104) were cultivated triplicate in DMEM-Low Glucose with 10% FBS in 24 well plates for 24h. After that, cells were treated with increasing concentration of oxaliplatin ( 0, 5, 10, 15, 20, 25 and 30µg/ml). After 24h exposure, remove treated medium followed by adding medium with 100µl MTT solution (15mg/50ml) then incubated at 37 ºC for 2-5 hr until purple precipitate was formed. Thereafter, the supernatant was discard and then add 150µl of DMSO per well for dissolving blue MTT formazon crystals. The absorbance was measured at 570nm using a multi-wall ELISA plate reader (Molecular Devices, Palo Alto, CA, USA). IC50 wastaken as the concentration of 50 percent death of cells population.
Cell survival (percentage) = [1-(ODexperiment sample/ ODcontrol sample)] x 100%. (n=3)
2.11 Immunocytochemistry Assay
The cell proliferation rate of LoVoWT and LoVoOXR cancer cells was measured by immunocytochemistry assay using Ki67 staining. Briefly, LoVoWT and LoVoOXR cells were cultivated with density 5x103 cell per well of 8 well chamber slides, incubation for 24 h. Then, fixed cells with 4% formaldehyde for 30-45 min at room temperature. Thereafter, washed the fixed cells twice with PBS and permeabilization solution in 0.1% PBST (0.1% TRITON X-100 in 1XPBS solution) for 15 min at room temperature. Cells were blocked in blocking solution [4% (w/v) bovine serum albumin + 0.1% Tween-20 in PBS] for 1-2 h at RT and incubated overnight in primary antibodies diluted with blocking solution at 4°C, washed 3 times 5 min with 0.1% PBST, and then incubated with secondary antibodies were diluted 1:500 for fluorophores Alexa 488 at room temperature for 1-2 hr, and washed 3 times 5 min with 0.1% PBST. Coverslips were washed in distilled water prior mounting on slide with DAPI.
2.12 Statistical Analyses
The analyses datas were done in three independent biological triplicates and values were represented as mean ± standard deviation (SD). By SigmaPlot software version 11.0 (Systat Software Inc., San Jose, CA, USA) for windows with GraphPad Prism software 7.0 the mean value data were carried out. The distribution data was used one-way analysis of variance was applied followed by Student t-test. Moreover, all values were concluded as representative statistically significant with the probability was less than 0.05% (P<0.05).