Abstract Bone metastasis is one of the main complications of lung cancer and most important factors that lead to poor life quality and low survival rate in lung cancer patients. However, the regulatory mechanisms underlying lung cancer bone metastasis are still poor understood. Here, we report that microRNA-182 (miR-182) plays a critical role in regulating osteoclastic metastasis of lung cancer cells. We found that miR-182 was significantly upregulated in both bone-metastatic human non–small cell lung cancer (NSCLC) cell line and tumor specimens. We further demonstrated that miR-182 markedly enhanced the ability of NSCLC cells for osteolytic bone metastasis in nude mice. Mechanistically, miR-182 promotes NSCLC cells to secrete Interleukin-8 (IL-8) and in turn facilitates osteoclastogenesis via activating STAT3 signaling in osteoclast progenitor cells. Importantly, systemically delivered IL-8 neutralizing antibody inhibits NSCLC bone metastasis in nude mice. Collectively, our findings identify the miR-182/IL-8/STAT3 axis as a key regulatory pathway in controlling lung cancer cell-induced osteolytic bone metastasis and suggest a promising therapeutic strategy that targets this regulatory axis to interrupt lung cancer bone metastasis. Subject terms: Lung cancer, Metastasis Introduction Lung cancer is the leading cause of cancer-related death worldwide, causing ∼18% of cancer-related deaths [[56]1]. More than 65% of lung cancer patients have local or distant metastasis at diagnosis, with bone metastasis as the most prevalent malignant clinical symptom [[57]2]. In particular, approximately 30–40% of non-small cell lung cancer (NSCLC) patients develop bone metastasis, with an average survival rate of about 6 months [[58]3, [59]4]. However, effective intervention strategies for lung cancer bone metastasis are still lacking due to a poor understanding of the regulatory mechanisms. Recent studies have advanced our understanding of the process of breast and prostate cancer bone metastasis. Bone-metastatic breast and prostate cancer cells are shown to secrete various inflammatory factors or growth factors, including IL-11, IL-6, IL-8, TGF-β, TNF, and EGF, and directly activate osteoclast progenitor cells [[60]5–[61]8]. Moreover, recent studies on breast cancer cells report that tumor-derived Jagged1 promotes osteoclast formation and bone absorption by activating the Notch signaling pathway in osteoclasts [[62]9], while cancer cell secreted-IL-11 functions as a pro-osteolytic factor by activating the JAK1/STAT3 pathway in osteoclast progenitor cells [[63]10, [64]11]. In addition, prostate cancer cell-secreted PTHrP may stimulate the differentiation and maturation of osteoclast cells by altering the homeostasis between the osteoclastogenesis inducer RANKL and its decoy receptor Osteoprotegerin (OPG) in metastatic niches [[65]12–[66]15]. These processes together promote osteoclast differentiation in metastatic niches, thereby increasing osteolytic bone lesions and facilitating the bone metastasis of breast and prostate cancer cells. Similarly, more than 70% of lung cancer bone metastasis display osteolytic bone destruction [[67]16]. However, unlike recent advances in our understanding of breast and prostate cancer bone metastasis, how lung cancer bone metastasis is regulated remains largely unexplored. MicroRNAs (miRNAs) are known as an emerging class of post-transcriptional gene regulators in eukaryotic cells, which have been shown to play critical roles in all stages of tumor progression [[68]17, [69]18]. In particular, miRNAs play a critical role in modulating cellular pathways implicated in osteolytic bone destruction and expression of key cytokines and/or chemokines in bone microenvironment [[70]19–[71]21]. For instance, overexpression of miR-155 in RAW264.7 cells blocks osteoclastogenesis by repressing MITF and PU.1, two crucial transcription factors for osteoclast differentiation [[72]22], while breast cancer cell-induced miR-141 and miR-219 downregulation in osteoclasts promote osteoclast differentiation and osteolytic bone metastasis [[73]23]. Our recent study also showed that NSCLC cell-derived exosomal miR-17-5p promote osteoclast differentiation by targeting PTEN [[74]24], but it has been largely unknown whether and how miRNAs in lung cancer cells are directly involved in regulating the osteolytic bone metastasis. In the present study, we discovered that miR-182 acts as a critical regulator in controlling lung cancer bone metastasis. This miRNA was previously reported to regulate tumor occurrence, progression, and distant metastasis in various types of cancers, including melanoma [[75]25], breast cancer [[76]26], and lung cancer [[77]27, [78]28]. We found that miR-182 was upregulated in bone-metastatic NSCLC cells and tumors and further showed that this miRNA significantly promoted the osteolytic bone metastasis of NSCLC cells in nude mice. Our mechanistic studies revealed that miR-182 enhanced IL-8 expression in NSCLC cells by targeting the NF-κB signaling inhibitor gene KLHL21 [[79]29], and thus increased IL-8 secretion from NSCLC cells to facilitate osteoclastogenesis via activating STAT3 signaling in osteoclast progenitor cells. Collectively, our findings indicate the miR-182/IL-8/STAT3 cascade as a novel regulatory axis in controlling osteoclast differentiation and osteolytic lesion development in metastatic niches, providing new mechanistic insights into lung cancer bone metastasis and potential therapeutic targets for the treatment of lung cancer bone metastasis. Materials and methods RNA oligonucleotides and plasmids miR-182 mimics, anti-miR-182, and their cognate negative control RNAs were purchased from RiboBio (Guangzhou, China). Human KLHL21 coding sequences were cloned into the p3×Flag-CMV-14 expression vector (Sigma) to construct p3×Flag-KLHL21. For reporter pRL-TK-KLHL21 3′-untranslated region (3′-UTR), the human KLHL21 3′-UTR was cloned downstream of the Renilla luciferase gene in pRL-TK (Promega). Eight nucleotides in KLHL21 3′-UTR corresponding to 5′ part of miR-182 were deleted in the pRL-KLHL21 3′-UTR Mut construct. All constructs were confirmed by DNA sequencing. Cell lines, cell infection, and transfection Human NSCLC cell line A549 and murine pre-osteoclast cell line RAW264.7 were obtained from the American Type Culture Collection (ATCC) and cultured according to their guidelines. All cell lines have been authenticated using STR profiling. Mycoplasma contamination testing was performed and the cells were proved to be mycoplasma free. A549 subline stably expressing luciferase (referred to as A549-luc) was generated from the parental cell line, as previously reported [[80]30, [81]31]. Generation of the bone-metastatic A549 subline was carried out as previously reported [[82]10, [83]32]. In brief, A549-luc cells were inoculated into the left cardiac ventricle of BALB/c athymic nude mice (6–8 weeks old), and then the bone tissue-resided A549-luc cells were isolated from mouse bones indicated by Bioluminescence imaging (BLI). After in vitro cultivation, the isolated A549-luc cells were administered again into the left cardiac ventricle. After three cycles of selection, the obtained A549-luc subline was defined as bone-metastatic subline (referred to as A549-BM; Fig. [84]S1A, B). The pseudovirus of GV369-miR-182 and control viruses were purchased from GENECHEM (Shanghai, China). NSCLC cells were infected with indicated viruses and subjected to antibiotic selection for enrichment before the assays. Cell transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. For RNA oligonucleotide transfection, 50 nmol/L of miRNA mimics and 100 nmol/L of antisense oligonucleotides were used. RNA isolation and real-time quantitative PCR (qPCR) The assays were performed as we described previously [[85]17, [86]18]. Total RNAs were extracted from cells or tissues with TRIzol reagent (Invitrogen). miRNA and mRNA levels were quantified by quantitative reverse transcription PCR (qPCR) using SYBR Green (Takara), with U6 small nuclear RNA and β-actin as internal normalized references,