Primer sequences used for Real- time PCR.

<p>Primer sequences used for Real- time PCR.</p

miR-17-5p overexpression and subsequent paclitaxel treatment caused change in the expression of pro-apoptotic and anti- apoptotic proteins and also stimulated ROS generation in A549-T24 cells.

<p>(A) Western Blot analysis of apoptotic marker proteins. Western Blot analysis of change in expression of pro- and anti-apoptotic proteins (Bax, Bcl-2, P53, cleaved caspase3, cleaved PARP) in A549-T24 cells following miR-17-5p overexpression and paclitaxel treatment. Briefly, T24-miR-NC or T24-miR-17-5p cells were treated with 24 nM or 50 nM paclitaxel for 24 h and cell lysates were prepared for Western blotting using antibodies against Bax, Bcl-2, P53, cleaved caspase3, cleaved PARP. GAPDH served as the loading control. (B) Measurement of relative caspase3 mRNA level by qRT- PCR. Relative caspase3 mRNA was estimated by qRT-PCR in T24-miR-17-5p cells compared to T24-miR-NC cells following 24 nM or 50 nM paclitaxel treatment for 24 h. (C–D) miR-17-5p overexpression and subsequent paclitaxel treatment stimulated ROS generation in A549-T24 cells. T24-miR-NC or T24-miR-17-5p cells were treated with 24 nM or 50 nM paclitaxel for 24 h. ROS generation were estimated by staining the H2-DCFDA staining and flow cytometry. NC1, T1, NC2 and T2 represent the same as mentioned earlier. (E) Amelioration of paclitaxel induced cytotoxicity following miR-17-5p overexpression in A549-T24 cells by NAC. T24-miR-NC or T24-miR-17-5p cells were pre- incubated with 1 mM NAC for 4 h and then treated with 24 nM or 50 nM paclitaxel for 24 h. Cell viability was measured by MTT assay. Data are represented as the mean ± S.E. (*p<0.05 vs. control, where n = 3).</p

BECN1 is a direct target of miR-17-5p.

<p>(A) Schematic representation of the 3′- UTR of human BECN1 transcript. Predicted miR-17-5p binding site was depicted. The numbers (+135–141) represented the nucleotides that were predicted to base pair with the miR-17-5p seed sequence. (B) miR-17-5p directly binds to the 3′ UTR of BECN1 gene in A549-T24 cells. A549-T24 cells were co- transfected with renilla luciferase reporter plasmids (<i>pCI-neo-RL-Bec-3</i>′<i>UTR-wt</i> or <i>pCI-neo-RL-Bec-3</i>′<i>UTR-mut</i>), firefly luciferase plasmids (pGL3-FF) and pre-miR-17-5p precursor or anti-miR-17-5p or pre-miR-negative control precursor RNA using lipofectamine2000 transfection reagent. After 48 h, cells were harvested and lysed with passive lysis buffer. Luciferase activity was measured by using Promega dual luciferase reporter assay kit. The results were represented as relative fold repression (Renilla luc/Firefly luc activity) compared to control cells. (*p<0.05, **p<0.03 vs control, n = 4).</p

miR-17-5p Downregulation Contributes to Paclitaxel Resistance of Lung Cancer Cells through Altering Beclin1 Expression

<div><p>Non- small- cell lung cancer (NSCLC) is one of the most leading causes of cancer-related deaths worldwide. Paclitaxel based combination therapies have long been used as a standard treatment in aggressive NSCLCs. But paclitaxel resistance has emerged as a major clinical problem in combating non-small-cell lung cancer and autophagy is one of the important mechanisms involved in this phenomenon. In this study, we used microRNA (miRNA) arrays to screen differentially expressed miRNAs between paclitaxel sensitive lung cancer cells A549 and its paclitaxel-resistant cell variant (A549-T24). We identified miR-17-5p was one of most significantly downregulated miRNAs in paclitaxel-resistant lung cancer cells compared to paclitaxel sensitive parental cells. We found that overexpression of miR-17-5p sensitized paclitaxel resistant lung cancer cells to paclitaxel induced apoptotic cell death. Moreover, in this report we demonstrated that miR-17-5p directly binds to the 3′-UTR of beclin 1 gene, one of the most important autophagy modulator. Overexpression of miR-17-5p into paclitaxel resistant lung cancer cells reduced beclin1 expression and a concordant decease in cellular autophagy. We also observed similar results in another paclitaxel resistant lung adenosquamous carcinoma cells (H596-TxR). Our results indicated that paclitaxel resistance of lung cancer is associated with downregulation of miR-17-5p expression which might cause upregulation of BECN1 expression.</p></div

A549-T24 cells exhibit heightened level of autophagy with downregulated miR-17-5p expression compared to paclitaxel sensitive A549 cells.

<p>(A) Expression status of certain autophagic marker proteins BECN1, MAP-LC3, p62 and GAPDH (loading control) were measured by Western blotting. (B–C) Relative BECN1 and LC3-II mRNA expression levels were quantified by qRT-PCR analysis in A549 and A549-T24 cells, <i>bars</i> represent mean ± S.E. (<i>*</i>p<0.03 vs control, where n = 3) (D) Downregulation of miR-17-5p expression in paclitaxel resistant lung cancer cells (A549-T24) compared to A549 cells. Taqman qRT-PCR was performed to detect the relative levels of miR-17-5p in A549 and A549-T24 cells. Results were normalized to snU6 expression level and represented as mean ± S.E. from three independent replicates. <i>(**p<0.001</i> vs control, n = 3).</p

Micro-RNA profiles of paclitaxel resistant and paclitaxel sensitive A549 cells.

<p>miRNA array profiles of paclitaxel sensitive (C1 and C2) and resistant (Tx1 and Tx2) lung cancer cell lines was shown. Supervised hierarchical clustering of cell lines based on their differential miRNAs expression with ΔLMR≥2 between the two groups was exhibited. Each column represents a cell line and each row a probe set. The heat map indicates high (red) or low (blue) level of expression relative to the mean as per the scale shown in the figure.</p

miR-17-5p modulates BECN1 expression in A549-T24 cells.

<p>(A) A549-T24 cells were transfected either with 100 nM pre-miR-negative control (T24-miR-NC) or pre-miR-17-5p (T24-miR-17-5p) precursor RNA. After 24 h, cell lysates were prepared for Western blotting with antibody against BECN1, MAP-LC3, p62 and GAPDH was used as loading control. (B–C) Relative BECN1 and LC3-II mRNA expression levels were quantified by qRT-PCR analysis in T24-miR-NC and T24-miR-17-5p cells, <i>bars</i> represent mean ± S.E. from three independent experiments (**p<0.01 vs control, where n = 3).</p

miR-17-5p is functionally involved in the paclitaxel response to A549 cells.

<p>(A) A549-T24 cells were either transfected with 100 nM pre-miR-negative control (T24-miR-NC) or pre-miR-17-5p (T24-miR-17-5p) precursor RNA and were seeded into 96 well plates at a density of 1×10⁴ cells per well. After 24 h, cells were treated with 0, 12, 24, 50, 100, 200 nM paclitaxel for another 24 h. The cell viability was assessed by MTT assay. Data are presented as % of cell viability measured in cells treated with paclitaxel. <i>Columns</i>, mean of three independent experiments; <i>bars</i>, mean ±S.E. (*p<0.05 vs negative control, p<0.03 vs A549 control, where n = 4). (B–C) Cells (T24-miR-NC and T24-miR-17-5p) were subsequently treated with 24 nM and 50 nM paclitaxel for 24 h and are subjected to FACS analysis after being stained by Trypan blue. Histogram represents the Red fluorescence intensity (FL3-H) vs. counts plot where dose dependent increase of cell death occurs. The results represent the best of data collected from three experiments with similar results.</p

miR-17-5p overexpression and subsequent paclitaxel treatment induced mitochondrial pathway mediated apoptosis in A549-T24 cells.

<p>(A–B) Overexpression of miR-17-5p induced apoptosis in paclitaxel resistant lung cancer cells. (A) T24-miR-NC or T24-miR-17-5p cells were treated either with 24 nM or 50 nM paclitaxel for another 24 h. Cells were then harvested for apoptosis analysis by annexinV- FITC/PI staining and flowcytometry. The % of early apoptotic cells (annexinV-FITC positive/PI negative cells) and late apoptotic cells (annexinV-FITC positive/PI positive cells) were determined. The results represented are the best of data collected from three independent experiments with similar results. (C–D) miR-17-5p overexpression and subsequent treatment with paclitaxel induced collapse of mitochondrial membrane potential in A549-T24. T24-miR-NC and T24-miR-17-5p cells were treated with 24 nM and 50 nM paclitaxel for 24 h. Cells were harvested and stained with JC-1 fluorescent dye. Histogram represents drop in red fluorescence (FL2-H) intensity vs. cell counts plot where mitochondrial membrane potential decreases following miR-17-5p overexpression and subsequent paclitaxel treatment. NC1, T1, NC2 and T2 represents T24-miR-NC cells treated with 24 nM paclitaxel, T24-miR-17-5p cells treated with 24 nM paclitaxel, T24-miR-NC cells treated with 50 nM paclitaxel and T24-miR-17-5p cells treated with 50 nM paclitaxel respectively. The results represent the best of data collected from three independent experiments with similar results. (E) Western blot analysis to detect the release of the cytochrome-<i>c</i> in the cytosol from mitochondria in A549 T24 cells following miR-17-5p overexpression and subsequent paclitaxel treatment.</p

Inhibitor-Induced Conformational Stabilization and Structural Alteration of a Mip-Like Peptidyl Prolyl <i>cis-trans</i> Isomerase and Its C-Terminal Domain

<div><p>FKBP22, an <i>Escherichia coli</i>-encoded PPIase (peptidyl-prolyl <i>cis</i>-<i>trans</i> isomerase) enzyme, shares substantial identity with the Mip-like pathogenic factors, caries two domains, exists as a dimer in solution and binds some immunosuppressive drugs (such as FK506 and rapamycin) using its C-terminal domain (CTD). To understand the effects of these drugs on the structure and stability of the Mip-like proteins, rFKBP22 (a chimeric FKBP22) and CTD⁺ (a CTD variant) have been studied in the presence and absence of rapamycin using different probes. We demonstrated that rapamycin binding causes minor structural alterations of rFKBP22 and CTD⁺. Both the proteins (equilibrated with rapamycin) were unfolded via the formation of intermediates in the presence of urea. Further study revealed that thermal unfolding of both rFKBP22 and rapamycin-saturated rFKBP22 occurred by a three-state mechanism with the synthesis of intermediates. Intermediate from the rapamycin-equilibrated rFKBP22 was formed at a comparatively higher temperature. All intermediates carried substantial extents of secondary and tertiary structures. Intermediate resulted from the thermal unfolding of rFKBP22 existed as the dimers in solution, carried an increased extent of hydrophobic surface and possessed relatively higher rapamycin binding activity. Despite the formation of intermediates, both the thermal and urea-induced unfolding reactions were reversible in nature. Unfolding studies also indicated the considerable stabilization of both proteins by rapamycin binding. The data suggest that rFKBP22 or CTD⁺ could be exploited to screen the rapamycin-like inhibitors in the future.</p></div