miR-106b-5p, miR-17-5p to predict recurrence and progression in breast DCIS model based on TGFβ paradox.

e23019 Background: Ductal carcinoma in situ (DCIS) is a well-known precursor of invasive ductal carcinoma (IDC). Part of patients show disease recurrence as DCIS or IDC after local treatment, but there are no established markers for prediction of recurrence. Methods: Authors analyzed 30 patients diagnosed as pure DCIS, recurrent DCIS, and IDC progressed in DCIS background. miRNA was extracted from archival tissue, and hierarchical clustering of miRNA microarray was performed. We selected highly expressed miR-17-5p and miR-106b-5p as marker for recurrence of DCIS. Two miRNAs were transfected to MCF-12 and MCF-7 cell line. Cell proliferation assay and Western blot analysis was performed for analyzing the interaction between cell proliferation and TGFβ downstream pathway. Results: miR-106b-5p single and combined miR-106b-5p and miR-17-5p transfected MCF-12 cell line showed increased proliferation index compared to un-transfected cell line. In MCF-7, miR-106b and miR-17-5p transfected cell line showed inferior proliferation index compared to un-transfected cell line. Western blot analysis showed minimal increased expression of SMAD4, phosphorylated SMAD2 (pSMAD2) in miR-106b-5p and miR-17-5p transfected MCF-12 cell line. However, decreased expression of TGFBR2 and no interval change of SMAD4 and pSMAD2 was detected in miR-106b-5p and miR-17-5p transfected MCF-7 cell line. Conclusions: miR-106-5p, miR-17-5p showed increased expression in recurrent DCIS or IDC based on miRNA hierarchical microarray. miRNA transfected MCF-12 cell line showed increased proliferation index and activated TGFβ downstream pathway. miRNA transfection might have made normal cell line to pre-cancerous cell line and TGFβ pathway might have influenced to promote tumor proliferation, based on TGFβ paradox hypothesis.

Flavoprotein miniSOG Cytotoxisity Can Be Induced By Bioluminescence Resonance Energy Transfer

In this study, we investigated the possibility of phototoxic flavoprotein miniSOG (photosensitizer) excitation in cancer cells by bioluminescence occurring when luciferase NanoLuc oxidizes its substrate, furimazine. We have shown that the phototoxic flavoprotein miniSOG expressed in eukaryotic cells in fusion with NanoLuc luciferase is activated in the presence of its substrate, furimazine. Upon such condition, miniSOG possesses photoinduced cytotoxicity and causes a 48% cell death level in a stably transfected cell line.

Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R)

This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.