LCNEC of the lung is a rare but highly aggressive tumor. LCNECs presents with a high gene mutation rate, and genetic alterations have a predictive value on chemotherapy outcome . However, the pattern and function of these dysregulated mRNAs and miRNAs have not been fully recognized. In this study, we identified 343 DEGs (including 133 upregulated and 210 downregulated genes) and 60 DEMs (including 31 upregulated and 29 downregulated miRNAs) in the LCNEC tissues compared with normal tissues.
We found several critical genes and pathways in the transcriptome with biological significance in this study. First, Stathmin 1 (STMN1), a member of the Stathmin family, acts as a microtubule destabilizer to regulate the cell cycle. The activation of STMN1 pathway promotes lung cancer cell invasion, migration and resistance to tyrosine kinase inhibitors [25, 26]. STMN1 overexpression predicts poor survival in multiple solid tumors including lung cancer . High STMN1 expression is also associated with cancer progression and chemoresistance in lung squamous cell carcinoma . Similar to our results, Shimizu et al. reported that all 17 LCNEC samples expressed a high level of STMN1 in their study, indicating that STMN1 might be a potential diagnostic marker for high-grade lung neuroendocrine tumors . Second, Wnt signaling pathway. It plays a role in the development of lung cancer . Wnt inhibitory factor 1 (WIF1) directly binds to Wnt protein to inhibit Wnt signaling pathway, functioning as a tumor suppressor. WIF1 is downregulated in both the lung squamous cell carcinomas and adenocarcinomas, and negative WIF1 expression is significantly associated with high malignancy and metastasis of lung cancers . However, WIF1 expression is not associated with survival . Third, tissue inhibitor of metalloproteinases (TIMPs). TIMPs are the most important inhibitors of metalloproteinase function, and TIMP3 is commonly downregulated in most cancer types . TIMP3 expression is also low in lung cancer and has a negative correlation with cancer stage and prognosis . Yun et al. found that interleukin-32 gamma suppressed lung tumor development by upregulating TIMP3 . Fourth, caveolins (CAVs), including CAV1, CAV2 and CAV3, are a family of proteins known to regulate cholesterol distribution, signal transduction, cell migration and endocytic vesicular trafficking . For female lung cancer patients who never smoked, low expression of CAV1 was associated with a worse overall survival . And the overexpression of CAV1 inhibited lung adenocarcinoma cell proliferation . Finally, NUF2. NUF2 was found in most of the GO terms. Many studies have clarified the relationship between NUF2 and cancer initiation or development. In an association study, Xu et al. found that NUF2 as a prognostic marker in breast cancer . The relationship between NUF2 and cancer was also demonstrated by Wang et al. NUF2 is a valuable prognostic biomarker to predict early recurrence of hepatocellular carcinoma after surgical resection . However, the association between NUF2 and LCNEC metastasis remains unclear, and further studies are still needed.
Of the ten top upregulated miRNAs, several have been demonstrated to be onco-miRNAs in lung cancer. MiR-1290 and miR-1246 promote cell stemness and invasiveness of non-small cell lung cancer (NSCLC) . MiR-301b-3p contributes to cell proliferation, invasion, drug resistance of NSCLC through repressing transforming growth factor-beta receptor II (TGFBR2) . MiR-183-5p and miR-18a-5p induce NSCLC progression by targeting PTEN and interferon regulatory 2 (IRF2), respectively [42, 43]. The suppression of miR-96-5p inhibited epithelial-mesenchymal transitioning (EMT) and metastasis of NSCLC . The downregulated miRNAs identified in this study also function as tumor suppressors in lung cancer. MiR-144-5p increased the radiosensitivity of NSCLC cells by repressing activating transcription factor 2 (ATF2)  and miR-451a decreased doxorubicin resistance in lung cancer via suppressing c-Myc . MiR-144-3p and MiR-30a-3p inhibited the progression of lung cancer via targeting EZH2 and DNA methyltransferase 3a [47, 48]. Long noncoding RNA RMRP sponged miR-1-3p to promote NSCLC cell proliferation and invasion . Moreover, miR-126-5p, miR-486-5p, miR-338-3p, miR-126-3p and miR-145-5p have all be shown to block the progression of lung cancer [50–53].
The pathway enrichment analysis revealed that the altered signaling pathways in LCNEC mainly focused on focal adhesion, cytokine-receptor interaction, hematopoietic cell lineage, and TGF-β signaling pathway, while the biological process mainly included response to wounding, inflammatory response, cell adhesion, biological adhesion, and vasculature development. Focal adhesion is a process of cellular attachment by linking the actin cytoskeleton to components of the extracellular matrix via integrins, and Focal adhesion kinase (FAK) plays a pivotal role in focal adhesion regulation . FAK promotes lung cancer progression and drug resistance [55, 56], and is essential for the formation of an aggressive phenotype of NSCLC with mutant KRAS . TGF-β signaling pathway regulates cell apoptosis, motility, invasion, extracellular matrix production, angiogenesis and immune function. In lung adenocarcinoma cells, TGF-β signaling pathway mediates EMT process mostly via the Smad pathways . However, the activation of TGF-β signaling pathway is suppressed in SCLC cells . Recently, Li et al. analyzed eight studies involving 579 patients with lung cancer and concluded that high TGF-β expression as an indicator of poor survival .
We identified TF-miRNA-mRNA regulation loops, such as module ETS1-miR195‐CD36, TAOK1‐miR7-1-3P‐GRIA1, E2F3‐miR195‐CD36, and TEAD1‐miR30A‐CTHRC1, which might play important roles in LCNEC. And 12 hub mRNAs were selected out, including TAOK1, STMN1, MAPK7, DUSP6, FZD3, BIRC5, F3, PDE4D, RGL1, CD36, GRIA1 and IL6R. ETS1 overexpression in breast cancers is associated with invasive features and predicts poor prognosis . ETS1 also promotes NSCLC cell migration and invasion . MAPK7, also known as ERK5. Jiang et al. showed that ERK5 was activated during lung cancer development, and ectopic expression of ERK5 promoted cell proliferation and G2/M cell cycle transition. Besides, they found that ERK5 is a potential regulator of radiosensitivity . Moncho-Amor et al. showed that DUSP6 plays a major role in the regulation of cell migration, motility and tumor growth in NSCLC cells .
Our study has some limitations. First, the number of samples is relatively small, which may cause some bias. Second, the verification experiments are lack, and wet laboratory experiments are warranted to confirm these newly found targets.