Additional file 1: Table S1. of The regulatory and predictive functions of miR-17 and miR-92 families on cisplatin resistance of non-small cell lung cancer

Differential miRNA expression in A549/DDP cells compared with A549 cells. (DOC 127 kb

Disturbance of the let-7/LIN28 double-negative feedback loop is associated with radio- and chemo-resistance in non-small cell lung cancer

<div><p>Radio- and chemo-resistance represent major obstacles in the therapy of non-small-cell lung cancer (NSCLC) and the underlying molecular mechanisms are not known. In the present study, during induction of radio- or chemo-resistance in NSCLC cells, dynamic analyses revealed that decreased expression of let-7 induced by irradiation or cisplatin resulted in increased expression of its target gene <i>LIN28</i>, and increased expression of LIN28 then contributed to further decreased expression of let-7 by inhibiting its maturation and biogenesis. Moreover, we showed that down-regulation of let-7 and up-regulation of LIN28 expression promoted resistance to irradiation or cisplatin by regulating the single-cell proliferative capability of NSCLC cells. Consequently, in NSCLC cells, let-7 and LIN28 can form a double-negative feedback loop through mutual inhibition, and disturbance of the let-7/LIN28 double-negative feedback loop induced by irradiation or chemotherapeutic drugs can result in radio- and chemo-resistance. In addition, low expression of let-7 and high expression of LIN28 in NSCLC patients was associated significantly with resistance to radiotherapy or chemotherapy. Therefore, our study demonstrated that disturbance of the let-7/LIN28 double-negative feedback loop is involved in the regulation of radio- and chemo-resistance, and that let-7 and LIN28 could be employed as predictive biomarkers of response to radiotherapy or chemotherapy in NSCLC patients.</p></div

Dynamic analyses of expression of let-7 and LIN28 during induction of radio- or chemo-resistance in A549 cells.

<p><b>(A)</b> To varying degrees, expression of let-7 miRNAs was down-regulated during induction of radio- or chemo-resistance. <b>(B)</b> To varying degrees, expression of LIN28A and LIN28B was up-regulated during induction of radio- or chemo-resistance.</p

Let-7 and LIN28 were putative regulators of single-cell proliferative capability.

<p><b>(A)</b> Colony-formation assay of A549/IR and A549/DDP cells transfected with let-7 mimics or si-LIN28A. <b>(B)</b> Colony-formation assay of A549 cells transfected with let-7 inhibitors or LIN28A plasmids (n = 3, *<i>P</i> < 0.05).</p

LIN28 blocked let-7 maturation specifically.

<p><b>(A)</b> si-LIN28 increased the maturation of let-7 in A549/IR and A549/DDP cells and LIN28 plasmid decreased the maturation of let-7 in A549 cells, respectively. <b>(B)</b> Inhibition of LIN28 decreased resistance to irradiation in A549/IR cells or to cisplatin in A549/DDP cells significantly. <b>(C)</b> Overexpression of LIN28 increased resistance to irradiation or cisplatin in A549 cells significantly (n = 3, *<i>P</i> < 0.05).</p

Let-7 family miRNAs regulated radio- and chemo-resistance by targeting <i>LIN28</i>.

<p><b>(A)</b> Luciferase assays of let-7 targeting effects on <i>LIN28</i>. Mutations were generated in the <i>LIN28A</i> 3′-UTR and <i>LIN28B</i> 3′-UTR sequence in the complementary site for the seed region of let-7 as indicated. <b>(B)</b> mRNA and protein levels of LIN28 were decreased in A549/IR and A549/DDP cells after transfection of let-7 mimics and increased in A549 cells after transfection of let-7 inhibitors, as detected by RT-PCR and western blotting. <b>(C)</b> Overexpression of let-7 decreased resistance to irradiation in A549/IR cells or to cisplatin in A549/DDP cells significantly. <b>(D)</b> Inhibition of let-7 increased resistance to irradiation or cisplatin in A549 cells significantly (n = 3, *<i>P</i> < 0.05).</p

Expression of let-7 family miRNAs was down-regulated in radio- or chemo-resistant cells.

<p><b>(A)</b> Members of the let-7 family (schematic). <b>(B)</b> Down-regulation of expression of let-7 family miRNAs in microarray experiments was validated by RT-PCR (n = 3, *<i>P</i> < 0.05).</p