<p>Figure S1 No significant difference in the serum levels of IL-4, IL-6, TNF-α and TGF-β between NSCLC patients and healthy controls.</p><p>Comparison of pro-inflammatory cytokines levels in serum from healthy donors and NSCLC patients. The concentration of IL-4 (A), IL-6 (B), TNF-α (C) and TGF-β (D) in serum from 37 healthy donors and 37 NSCLC patients was measured by ELISA. The significance of the differences between the healthy donors and patients was assessed using the Student’s t test.</p><p>1 Figure S2 Repression of mir-101 is involved in the tumorigenic activity of IL-1 in NSCLC cells.</p><p>(A) IL-1 exerts a tumor-promoting effect in NSCLC cells H460 (top) or H1299 cells (bottom). Left, MTT assays; right, transwell migration assays. The assays were performed 24 h post-treatment of IL-1.</p><p>(B) The effect of IL-1β treatment on expression of miR-101 in H1299 cells. The level of miR-101 was determined by quantitative real-time PCR (qRT-PCR) assays 48 h after IL-1β treatment, with U6 serving as an internal normalized reference.</p><p>(C) Restoring mir-101 expression in IL-1-treated H460 cells. IL-1 treated-H460 cells were respectively transfected with 0.5 nmol/L, 1 nmol/L or 2.5 nmol/L of miR-101 mimics, and qRT-PCR analyses of miR- 101 levels were performed 24 h post-transfection.</p><p>(D) Ectopic miR-101 overrode the tumorigenic effect of IL-1 in H1299 cells. IL-1 treated H1299 cells were respectively transfected with Ctrl RNA or miR-101 mimics, and the MTT (left) and transwell migration assays (right) were performed 24 h post-transfection.</p><p>The average values ± s.d. of three separate experiments were plotted. *P<0.05, **P<0.01. Results shown are representative of three independent experiments.</p><p>2 Figure S3 IL-1β down-regulates mir-101 via the COX-2/HIF-1α pathway in NSCLC cells.</p><p>(A) qRT-PCR assays of the effect of IL-1β on primary and mature mir-101 expression in H460 cells. Cells were treated with IL-1β, and the assays were performed 24 h post treatment. -actin mRNA and U6 respectively served as internal normalized references for pri-miR-101 and miR-101.</p><p>3 (B) Immunostaining of HIF-1α protein (green) in H460 cells. Cells were treated with CoCl2 (middle) or IL- 1β (bottom). Representative staining images are shown on the left, and quantification of nuclear HIF-1 staining fluorescent densities is shown at the right (mean ± SD). Nuclei were stained with DAPI (blue); scale bar: 20 m.</p><p>(C) qRT-PCR assays of the effect of CoCl2 on primary and mature mir-101 expression in H460 cells. Cells were treated with CoCl2, and the assays were performed 5 h post-treatment. -actin mRNA and U6 respectively served as internal normalized references for pri-miR-101 and miR-101.</p><p>(D) Western blot assays of the effect of IL-1 on HIF-1 protein expression (lane 2), with -actin serving as a loading reference.</p><p>(E) Knockdown of HIF-1 completely abolished the repression of mir-101 by IL-1 in H460 cells. IL-1- treated cells were transfected with scrambled siRNA (Scr-siR) or HIF-1 siRNA (HIF-1-siR), and the assays were performed 24 h post-transfection. Top, qRT-PCR assays of the effect of HIF-1-siR on IL-1- mediated repression of mir-101; bottom, western blot assays of HIF-1 protein, with -actin serving as a loading reference.</p><p>(F) Overexpression of COX-2 strongly repressed mir-101 expression. Top, qRT-PCR analysis of miR-101 level, with U6 serving as an internal normalized reference; bottom, western blotting assays of COX-2 protein, with -actin serving as a loading reference.</p><p>(G) Knockdown of COX-2 significantly increased mir-101 expression. Top, qRT-PCR analysis of miR-101 level, with U6 serving as an internal normalized reference; bottom, Western blotting assays of COX-2 protein, with -actin serving as a loading reference. </p><p>(H) Knockdown of Cox-2 significantly attenuated the repression of mir-101 by IL-1 in H460 cells. IL-1- treated cells were transfected with Scr-siR or COX-2 siRNA (COX-2-siR), and the assays were performed 24 h post-transfection. Left, qRT-PCR assays of the effect of COX-2 siR on IL-1-mediated repression of mir-101; right, western blot assays of HIF-1 and COX-2 proteins, with -actin serving as a loading reference.</p><p>The average values ± s.d. of three separate experiments were plotted. **P<0.01, ***P<0.001. Results shown are representative of three independent experiments.</p><p>4 Figure S4 miR-101 up-regulates let-7 family miRNAs by targeting Lin28B in NSCLC cells.</p><p>(A) Transfection of miR-101 mimics into IL-1-treated H460 cells completely overrode the effect of IL-1 on the expression of let-7 family miRNAs. IL-1-treated cells were transfected with miR-101 mimics or Ctrl RNA. qRT-PCR assays of let-7 family miRNA levels 48 h post-transfection, with U6 serving as an internal normalized reference.</p><p>(B) Ectopic expression of Flag-Lin28B significantly rescued the effect of miR-101 overexpression on expression of let-7 family miRNAs. H1299 cells were transfected with miR-101 mimics or co-transfected with miR-101 mimics and p3xFlag-Lin28B or control p3xFlag vectors. qRT-PCR assays of let-7 family miRNA levels 48 h post-transfection, with U6 serving as an internal normalized reference.</p><p>The average values ± s.d. of three separate experiments were plotted. **P<0.01, ***P<0.001. Results shown are representative of three independent experiments.</p><p>5 Figure S5 Comparison of the expression of Lin28B and miR-101 in H460 and H1299 cells. (A) qRT-PCR analyses of endogenous miR-101 expression in H460 and H1299 cells, with U6 serving as an internal reference. (B) Western blot assays of endogenous Lin28B protein levels in H460 and H1299 cells, with -actin serving as a loading control. (C) qRT-PCR analyses miR-101 levels in anti-miR-101-transfected H460 cells (left) or miR-101 mimics- transfected H1299 cells (right). The cells were respectively transfected with 100 nmol/L of anti-miR-101 and 50 nmol/L of miR-101 mimics, and qRT-PCR analyses of miR-101 levels were performed 24 h post- transfection. The average values ± s.d. of three separate experiments were plotted. *P<0.05. Results shown are representative of three independent experiments.</p><p>6 Figure S6 miR-101 plays a tumor suppressive role in NSCLC cells. (A) Inhibition of mir-101 promoted the proliferation of H460 cells. Top, MTT assays; bottom, western blot analyses of PCNA and Lin28B proteins, with -actin serving as a loading control.</p><p>(B) Inhibition of mir-101 promoted soft-agar colony formation. Top, quantitative results of soft agar foci per field; bottom, representative images.</p><p>(C) Inhibition of mir-101 promoted transwell cell migration. Top, quantitative results of migratory cells per </p><p>7 field; bottom, representative images.</p><p>(D) Inhibition of mir-101 reduced xenograft tumor growth in nude mice. The curves show the time course of tumor growth, and the inset photographs are representative xenografted tumors 25 days after inoculation.</p><p>H460 cells were transfected with Ctrl RNA or anti-miR-101, and the assays were performed 24 h post transfection except where indicated otherwise. The average values ± s.d. of three separate experiments were plotted. *P<0.05, **P<0.01, ***P<0.001. Results shown are representative of three independent experiments.</p><p>8 Figure S7 Repression of miR-101 is critical to IL-1-mediated induction of Cox-2 and Lin28B in NSCLC cells.</p><p>(A) Both activation of NF-B and repression of mir-101 are required for IL-1 to enhance Lin28B expression in NSCLC cells. Left, western blot of Lin28B protein in cells with indicated treatment with - actin serving as a loading control; right, qRT-PCR of Lin28B mRNA levels in cells with indicated treatment.</p><p>(B) IL-1β up-regulated Lin28B in NSCLC cells via the IL-1/COX-2 axis. Western blot of Lin28B and COX-2 proteins in cells with indicated treatment, with -actin serving as a loading control. </p><p>(C) Knockdown of miR-101 expression restored the stimulatory effect of IL-1 on COX-2 levels in Aspirin- or Celecoxib-treated H460 cells. Western blot of COX-2 proteins in cells with indicated treatment, with - actin serving as a loading control. </p><p>9 Table S1 Sequences of chemically synthesized DNA and RNA oligonucleotides</p><p>10</p>