Ll models of C33AE6, C33AE7, C33AE6/ E7, HT-Ll models of C33AE6, C33AE7, C33AE6/ E7, HT-3E6,

May 21, 2018

Ll models of C33AE6, C33AE7, C33AE6/ E7, HT-
Ll models of C33AE6, C33AE7, C33AE6/ E7, HT-3E6, HT-3E7, and HT-3E6/E7 by transfecting HPV16 E6, -E7, and -E6/E7 oncogenes with lentivirus vectors into the HPV-negative C33A and HT-3 cells in our lab. The C33A-vector (C33A-V) and HT-3 vector (HT-3 V) cells were established by transfecting C33A and HT-3 cells with lentivirus vectors that did not code for the HPV16 E6, -E7, or -E6/E7 proteins as controls. Stable transfections were treated with 10 g/ml puromycin for 3 weeks. The transfection efficiency was tested with Western blotting, which revealed that the transfected cells successfully expressed E6, -E7, or -E6/E7 proteins. All cells were maintained in a humidified incubator set at 37 and 5 CO2. The miRNA expression profiles were identified in HT-3E6/E7, HT3 V and HT-3 cell lines using an LC Sciences microRNA microarray(Hangzhou, China) containing 2578 human mature microRNAs based on Sanger miRBase Release 20.0. Differentially expressed miRNAs wereSYBR Green-based real-time quantification of miRNAs was used to determine miR-3156-3p expression as previously described. Total RNA was extracted using the Trizol reagent (Invitrogen). The PD173074MedChemExpress PD173074 quality of total RNA is assessed by ultraviolet spectrophotometer, the total RNA ration of A260/A280 between 1.8 and 2.0 was considered as high quality. Then, 1 g of total RNA was subsequently reverse-transcribed to cDNA with a miR-3156-3p-specific stem-loop-like RT primer(RIBOBIO,Guangzhou, China) following the manufacturer’s protocol. Then, qRT-PCR was performed using SYBR Green mix with primers specific to miR-3156-3p(RIBOBIO,Guangzhou, China). Small nuclear RNA RNU6 was used as an endogenous control. Relative quantification of the miRNA expression was calculated with the 2-CT method.qRT-PCR for mRNAcDNAs were synthesized using a transcriptor first strand cDNA synthesis kit (Roche). Then, qRT-PCR for mRNA was performed using FastStart Universal SYBR Green Master (Roche). The primers used for qRT-PCR include, for SLC6A6, forward 5′- GCT TCC CGT ACC TCT GCT AC-3′ and antisense 5′-TGG CCT ATG ATG ATC TCC AA-3′. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. RelativeXia et al. Virology Journal (2017) 14:Page 11 ofFig. 7 Sequence alignments of miR-3156-3p mimics and inhibitor were assessed using PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27107493 NCBI blastquantification of the mRNA expression was calculated with the 2-CT method.Cell proliferation assay and apoptosis analysiswere fixed and stained with crystal violet. Then stained cells were visualized and counted under a light microscope. The assays were performed in triplicate.Tube formation assayCell proliferation was assessed with a Cell Counting Kit8 (CCK-8) assay kit (Dojindo, Japan). Hela, Siha and Caski cells were separately cultured in 96-well plates overnight at a density of 5000 cells/well then transfected with miR-3156-3p mimics or an inhibitor as described above. At 1, 2, 3, 4 and 5 days after transfection, 10 l of CCK8 solution was added to each well for 1 h and absorbance readings at 450 nm were obtained in triplicate using a spectrophotometric plate reader. The data were obtained from the measurement of 4 replicate wells for each data point. For the apoptosis analysis, cells were harvested after transfection for 48 h by trypsinization, washed twice using cold PBS and were subsequently stained with Annexin V-FITC and propidium iodide using Annexin V apoptosis detection kit FITC (ebioscience,San Diego, CA 92121 USA) at room temperature, and apoptosis analys.