Licochalcone A reverses NNK-induced ectopic miRNA expression to elicit in vitro and in vivo chemopreventive effects

BackgroundChemoprevention is the best cost-effective way regarding cancers. MicroRNAs (miRNAs) have been reported to be differentially expressed during the development of lung cancer. However, if lung cancer prevention can be achieved through modulating miRNAs expression so far remains unknown.PurposeTo discover ectopically expressed miRNAs in NNK-induced lung cancer and clarify whether Licochalcone A (lico A) can prevent NNK-induced lung cancer by modulating miRNA expression.Study design and methodsA/J mice were used to construct a lung cancer model by intraperitoneal injection with physiological saline NNK (100 mg/kg). Chemopreventive effects of lico A against lung cancer at 2 mg/kg and 20 mg/kg doses were evaluated in vivo. MicroRNA array and RT-qPCR were used to assess the expression levels of miRNAs. MLE-12 cells were treated with 0.1 mg/ml NNK, stimulating the ectopic expression pattern of miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p. miR-144-3p mimics and inhibitors were used to manipulate miR-144-3p levels. The effects of lico A (10 μM) on cell cycle distribution, apoptosis, and the expression of CK19, RASA1, miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p in NNK-treated MLE-12 cells were studied.ResultsThe expression levels of miR-144-3p, miR-20a-5p, and miR-29c-3p increased, while those of let-7d-3p and miR-328-3p decreased in both NNK-induced A/J mice and MLE-12 cells. Lico A could reverse the NNK-induced ectopic miRNA (miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p) expression both in vivo and in vitro and elicit in vivo lung cancer chemopreventive effect against NNK. In MLE-12 cells, the overexpression of miR-144-3p elicited the same effect as NNK regarding the expression of lung cancer biomarker CK19; the silencing of miR-144-3p reversed the effect of NNK on cell cycle distribution and apoptosis. Lico A could reverse the effect of NNK on the expression of miR-144-3p, CK19, and RASA1 (predicted target of miR-144-3p).ConclusionThe present study suggests that miR-144-3p, miR-20a-5p, miR-29c-3p, let-7d-3p, and miR-328-3p were involved in the in vivo pathogenesis of NNK-induced lung cancer, and lico A could reverse the effect of NNK both in vivo and in vitro to elicit lung cancer chemopreventive effects through, at least partially, these five ectopically expressed miRNAs, especially miR-144-3p.     

miR-30 family promotes migratory and invasive abilities in CD133<Superscript>+</Superscript> pancreatic cancer stem-like cells

Pancreatic cancer is a deadly disease with a poor prognosis. Recently, miRNAs have been reported to be abnormally expressed in several cancers and play a role in cancer development and progression. However, the role of miRNA in cancer stem cells remains unclear. Therefore, our aim was to investigate the role of miRNA in the CD133⁺ pancreatic cancer cell line Capan-1M9 because CD133 is a putative marker of pancreatic cancer stem cells. Using miRNA microarray, we found that the expression level of the miR-30 family decreased in CD133 genetic knockdown shCD133 Capan-1M9 cells. We focused on miR-30a, -30b, and -30c in the miR-30 family and created pancreatic cancer cell sublines, each transfected with these miRNAs. High expression of miR-30a, -30b, or -30c had no effect on cell proliferation and sphere forming. In contrast, these sublines were resistant to gemcitabine, which is a standard anticancer drug for pancreatic cancer, and in addition, promoted migration and invasion. Moreover, mesenchymal markers were up-regulated by these miRNAs, suggesting that mesenchymal phenotype is associated with an increase in migration and invasion. Thus, our study demonstrated that high expression of the miR-30 family modulated by CD133 promotes migratory and invasive abilities in CD133⁺ pancreatic cancer cells. These findings suggest that targeted therapies to the miR-30 family contribute to the development of novel therapies for CD133⁺ pancreatic cancer stem cells.     

Circulating plasma microRNAs in colorectal neoplasia: A pilot study in assessing response to therapy

IntroductionCurrent serological surveillance markers to monitor colorectal cancer (CRC) or colorectal advanced adenomas (CAA) are hampered by poor sensitivity and specificity. The aim of this study is to identify and validate a panel of plasma microRNAs which change in expression after resection of such lesions.MethodsA prospectively maintained colorectal surgery database was queried for patients in whom both pre- and post-procedural serum samples had been obtained. An initial screening analysis of CRC and CAA patients (5 each) was conducted using screening cards for 380 miRNAs. Four identified miRNAs were combined with a previously described panel of 7 miRNAs that were diagnostically predictive of CRC and CAA. Differential miRNA expression was assessed using quantitative real-time polymerase chain reaction(qRT-PCR).ResultsFifty patients were included (n = 27 CRC, n = 23 CAA). There was no difference in age, gender, or race profile of CRC patients compared to CAA patients. Six miRNA were significantly increased after CRC resection (miR-324, let7b, miR-454, miR-374a, miR-122, miR-19b, all p<0.05), while three miRNAs were significantly increased following CAA resection (miR-454, miR-374a, miR-122, all p<0.05). Three miRNA were increased in common for both (miR-454, miR-374a, miR-122).DiscussionThe expression of miRNAs associated with neoplasia (either CRC or CAA) was significantly increased following surgical resection or endoscopic removal of CRC or CAA. Future studies should focus on the evaluation of these miRNAs in CRC and CAA prognosis.     

Integrated mRNA-MicroRNA Profiling of Human NK Cell Differentiation Identifies MiR-583 as a Negative Regulator of IL2Rγ Expression

Natural killer (NK) cells are innate immune effector cells that protect against cancer and some viral infections. Until recently, most studies have investigated the molecular signatures of human or mouse NK cells to identify genes that are specifically expressed during NK cell development. However, the mechanism regulating NK cell development remains unclear. Here, we report a regulatory network of potential interactions during in vitro differentiation of human NK cells, identified using genome-wide mRNA and miRNA databases through hierarchical clustering analysis, gene ontology analysis and a miRNA target prediction program. The microRNA (miR)-583, which demonstrated the largest ratio change in mature NK cells, was highly correlated with IL2 receptor gamma (IL2Rγ) expression. The overexpression of miR-583 had an inhibitory effect on NK cell differentiation. In a reporter assay, the suppressive effect of miR-583 was ablated by mutating the putative miR-583 binding site of the IL2Rγ 3′ UTR. Therefore, we show that miR-583 acts as a negative regulator of NK cell differentiation by silencing IL2Rγ. Additionally, we provide a comprehensive database of genome-wide mRNA and miRNA expression during human NK cell differentiation, offering a better understanding of basic human NK cell biology for the application of human NK cells in immunotherapy.     

Role of Deregulated microRNAs in Breast Cancer Progression Using FFPE Tissue

MicroRNAs (miRNAs) contribute to cancer initiation and progression by silencing the expression of their target genes, causing either mRNA molecule degradation or translational inhibition. Intraductal epithelial proliferations of the breast are histologically and clinically classified into normal, atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). To better understand the progression of ductal breast cancer development, we attempt to identify deregulated miRNAs in this process using Formalin-Fixed, Paraffin-Embedded (FFPE) tissues from breast cancer patients. Following tissue microdissection, we obtained 8 normal, 4 ADH, 6 DCIS and 7 IDC samples, which were subject to RNA isolation and miRNA expression profiling analysis. We found that miR-21, miR-200b/c, miR-141, and miR-183 were consistently up-regulated in ADH, DCIS and IDC compared to normal, while miR-557 was uniquely down-regulated in DCIS. Interestingly, the most significant miRNA deregulations occurred during the transition from normal to ADH. However, the data did not reveal a step-wise miRNA alteration among discrete steps along tumor progression, which is in accordance with previous reports of mRNA profiling of different stages of breast cancer. Furthermore, the expression of MSH2 and SMAD7, two important molecules involving TGF-β pathway, was restored following miR-21 knockdown in both MCF-7 and Hs578T breast cancer cells. In this study, we have not only identified a number of potential candidate miRNAs for breast cancer, but also found that deregulation of miRNA expression during breast tumorigenesis might be an early event since it occurred significantly during normal to ADH transition. Consequently, we have demonstrated the feasibility of miRNA expression profiling analysis using archived FFPE tissues, typically with rich clinical information, as a means of miRNA biomarker discovery.     

MicroRNAs expression profiles as diagnostic biomarkers of gastric cancer: a systematic literature review

Objective: The early identification of gastric cancer (GC) represents a major clinical challenge. We conducted a systematic review of studies evaluating the miRNA expression profiling as a diagnostic tool in GC.

Methods: We performed a search of PubMed, ISI Web of Science and SCOPUS databases for studies on diagnostic miRNAs and GC, published in English up to October 2017. Eligibility criteria included case-control studies evaluating blood or tissue-based miRNA expression profiles, and incorporating at least two detection phases (screening and validation).

Results: We included 27 eligible studies, that reported on 97 deregulated miRNAs either in blood or tissue, out of which 30 were reported in at least two studies. Among 22 studies on tissue-diagnostic miRNAs, 13 consistently upregulated miRNAs (miR-214, miR-21, miR-103, miR-107, miR-196a, miR-196b, miR-7, miR-135b, miR-222, miR-23b, miR-25, miR-92 and miR-93), and six consistently downregulated miRNAs (miR-148a, miR-375, miR-133b, miR-30a, miR-193a and miR-204) were reported. Ten miRNAs with inconsistent direction of expression in tissues were identified. Among the five studies performed on blood samples, only one miRNA was consistently upregulated (miR-20a).

Conclusions: This review shows that some tissue or blood miRNAs may be considered as potential biomarkers for GC diagnosis, that urgently needs to be confirmed from large prospective studies.     

Novel miRNA genes hypermethylated in breast cancer

MicroRNAs play an important role in the regulation of expression of many genes involved in cancer pathogenesis. One of the causes of miRNA level deregulation in tumors is the methylation of CpG islands in the promoter regions of the genes that encode them. Hypermethylation may lead to the suppression of miRNA gene expression and, as a consequence, to a decrease in their inhibitory effect on target gene mRNAs. A search for new miRNA genes hypermethylated in breast cancer has been carried out in the present study. The methylation of five miRNA genes associated with breast cancer (miR-132, miR-1258, miR-107, miR-130b, miR-137) has been as studied using a representative set of 41 breast cancer samples by methylation-specific PCR. Three new genes, MIR-132, MIR-137 and MIR-1258, with a high frequency of hypermethylation (41, 37 and 34%, respectively) have been identified in breast cancer. The methylation of these genes in the breast tissues of ten donors without cancer pathology in anamnesis was only found in single cases. These results enable the involvement of three miRNAs (miR-132, miR-137, miR-1258) and the methylation of the genes that encode them in the pathogenesis of breast cancer to be suggested.     

Decreased Expression of miR-21, miR-26a,miR-29a,and miR-142-3p in CD4+ T Cells and Peripheral Blood from Tuberculosis Patients

The vast majority of Mycobacterium tuberculosis (M. tuberculosis) infected individuals are protected from developing tuberculosis and T cells are centrally involved in this process. MicroRNAs (miRNA) regulate T-cell functions and are biomarker candidates of disease susceptibility and treatment efficacy in M. tuberculosis infection. We determined the expression profile of 29 selected miRNAs in CD4⁺ T cells from tuberculosis patients and contacts with latent M. tuberculosis infection (LTBI). These analyses showed lower expression of miR-21, miR-26a, miR-29a, and miR-142-3p in CD4⁺ T cells from tuberculosis patients. Whole blood miRNA candidate analyses verified decreased expression of miR-26a, miR-29a, and miR-142-3p in children with tuberculosis as compared to healthy children with LTBI. Despite marked variances between individual donor samples, trends of increased miRNA candidate expression during treatment and recovery were observed. Functional in vitro analysis identified increased miR-21 and decreased miR-26a expression after re-stimulation of T cells. In vitro polarized Interleukin-17 positive T-cell clones showed activation-dependent miR-29a up-regulation. In order to characterize the role of miR-29a (a described suppressor of Interferon-γ in tuberculosis), we analyzed M. tuberculosis specific Interferon-γ expressing T cells in children with tuberculosis and healthy contacts but detected no correlation between miR-29a and Interferon-γ expression. Suppression of miR-29a in primary human T cells by antagomirs indicated no effect on Interferon-γ expression after in vitro activation. Finally, classification of miRNA targets revealed only a moderate overlap between the candidates. This may reflect differential roles of miR-21, miR-26a, miR-29a, and miR-142-3p in T-cell immunity against M. tuberculosis infection and disease.     

Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach

Background

While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia.

Methodology/Principal Findings

A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue.

Conclusions/Significance

This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.     

Epigallocatechin gallate up-regulation of miR-16 and induction of apoptosis in human cancer cells

Epigallocatechin gallate (EGCG) is a major type of green tea polyphenols and is known to have cancer prevention effect. MicroRNAs (miRNAs) are 19 to 25 nucleotides and are believed to be important in gene regulation. In the present study, the influence of EGCG on the expressions of miRNAs in human cancer cells was investigated as this has not yet been reported. By miRNA microarray analysis, EGCG treatment was found to modify the expressions of some of the miRNAs in human hepatocellular carcinoma HepG2 cells, 13 were up-regulated and 48 were down-regulated. miR-16 is one of the miRNAs up-regulated by EGCG and one of its target genes is confirmed to be the anti-apoptotic protein Bcl-2. EGCG treatment induced apoptosis and down-regulated Bcl-2 in HepG2 cells. Transfection with anti-miR-16 inhibitor suppressed miR-16 expression and counteracted the EGCG effects on Bcl-2 down-regulation and also induction of apoptosis in cells. Results from the present study confirm the role of miR-16 in mediating the apoptotic effect of EGCG and also support the importance of miRNAs in the regulation of the biological activity of EGCG.     

Targeting of Integrin ��1 and Kinesin 2�� by MicroRNA 183

MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H₂O₂. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells.     

The synergistic protection of EGCG and quercetin against streptozotocin (STZ)-induced NIT-1 pancreatic β cell damage via upregulation of BCL-2 expression by miR-16-5p

EGCG and quercetin are flavonoids which usually co-exist in edible plants and they exhibit anti-diabetes effects. This study aimed to explore the mechanisms by which quercetin and EGCG synergistically protected pancreatic β-cells from streptozotocin-induced apoptosis. EGCG, quercetin, and their combinations (both 15 μM) all reversed STZ-induced cells damage and enhanced glucose-stimulated insulin secretion, with the combination being more effective than a single compound. At the molecular level, the EGCG-quercetin combination upregulated BCL-2 expression and caused a greater reduction in miR-16-5p level than EGCG alone or quercetin alone. Overexpression of miR-16-5p could offset the down-regulated apoptotic genes caused by the synergistic action of the combination. These findings suggest that EGCG and quercetin exert synergistic anti-diabetes effect, possibly via decreasing the expression of miR-16-5p that targets directly BCL-2. This is the first report on a miRNA-based mechanism underlying the synergistic protective effect of EGCG and quercetin against pancreatic cell damage.     

Recurrence prediction using microRNA expression in hormone receptor positive breast cancer during tamoxifen treatment

Abstract

Purpose: To identify miRNAs associated with distant recurrence during tamoxifen treatment and build a recurrence prediction model.

Materials and methods: We measured the expression of five miRNAs (miR-134, miR-125b-5P, miRNA-30a, miR-10a-5p and miR-222). A total of 176 tumour tissues from 176 patients who had hormone receptor positive breast cancer with tamoxifen treatment were used to measure miRNA expression using quantitative real-time PCR (qRT-PCR).

Results: The five miRNAs were all up-regulated in distant recurrence cases within 5?years after surgery and during tamoxifen treatment. Kaplan-Meier survival analyses based on expression cut-offs determined by receiver characteristics curves (ROC) showed that high expression of miR-134, miR-125b-5P, miRNA-30a, miR-10a-5p and miR-222 were significantly (log-rank p-value =0.006, p-value <0.0001, p-value <0.0001, p-value <0.0001 and p-value <0.0001, respectively) associated with short relapse-free time. Our results were used to build a combined 3 miRNAs expression model. It could be used to categorize high-risk subset of patients with short relapse-free survival (AUC =0.891, p-value <0.0001).

Conclusions: Distant recurrence during tamoxifen treatment of hormone positive breast cancer might be affected by tamoxifen resistance related miRNAs. Such distant recurrence can be predicted using miRNA measurement.     

Influence of miRNA-106b and miRNA-135a on butyrate-regulated expression of p21 and Cyclin D2 in human colon adenoma cells

Epigenetic and posttranslational modifications of the expression of cell cycle-relevant genes or proteins like p21, e.g., by miRNAs are crucial mechanisms in the development or prevention of colon cancer. The present study investigated the influence of butyrate and trichostatin A (TSA) as histone deacetylase inhibitors on the expression of colon cancer-relevant miRNA (miR-135a, miR-135b, miR-24, miR-106b, miR-let-7a) in LT97 colon adenoma cells as a model of an early stage of colon carcinogenesis. The impact of distinct miRNAs (miR-106b, miR-135a) on butyrate-mediated regulation of p21 and Cyclin D2 gene and protein expression as well as the effect on LT97 cell proliferation (non-transfected, miR-106b and miR-135a mimic transfected) was analyzed. Butyrate and partial TSA reduced the expression of miR-135a, miR-135b, miR-24 and miR-let-7a (~0.5-fold, 24 h) and miR-24, miR-106b and miR-let-7a (~0.5–0.7-fold, 48 h) in LT97 cells. Levels of p21 mRNA and protein were significantly increased by butyrate and TSA (~threefold and 4.5-fold, respectively, 24 h) in non-transfected but not in miR-106b transfected LT97 cells. Levels of Cyclin D2 mRNA were significantly reduced by butyrate and TSA (~0.3-fold, 24 h) in non-transfected and miR-135a-transfected LT97 cells, whereas protein levels were predominantly not influenced. MiR-106b and miR-135a significantly reduced butyrate-/TSA-mediated inhibition of LT97 cell proliferation (72 h). These results indicate that butyrate is able to modify colon cancer-relevant miRNAs like miR-106b and miR-135a which are involved in the regulation of cell cycle-relevant genes like p21 and might influence inhibition of adenoma cell proliferation.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0500-4) contains supplementary material, which is available to authorized users.     

Estradiol downregulates miR-21 expression and increases miR-21 target gene expression in MCF-7 breast cancer cells

Select changes in microRNA (miRNA) expression correlate with estrogen receptor α (ERα) expression in breast tumors. miR-21 is higher in ERα positive than negative tumors, but no one has examined how estradiol (E₂) regulates miR-21 in breast cancer cells. Here we report that E₂ inhibits miR-21 expression in MCF-7 human breast cancer cells. The E₂-induced reduction in miR-21 was inhibited by 4-hydroxytamoxifen (4-OHT), ICI 182 780 (Faslodex), and siRNA ERα indicating that the suppression is ERα-mediated. ERα and ERβ agonists PPT and DPN inhibited and 4-OHT increased miR-21 expression. E₂ increased luciferase activity from reporters containing the miR-21 recognition elements from the 3′-UTRs of miR-21 target genes, corroborating that E₂ represses miR-21 expression resulting in a loss of target gene suppression. The E₂-mediated decrease in miR-21 correlated with increased protein expression of endogenous miR-21-targets Pdcd4, PTEN and Bcl-2. siRNA knockdown of ERα blocked the E₂-induced increase in Pdcd4, PTEN and Bcl-2. Transfection of MCF-7 cells with antisense (AS) to miR-21 mimicked the E₂-induced increase in Pdcd4, PTEN and Bcl-2. These results are the first to demonstrate that E₂ represses the expression of an oncogenic miRNA, miR-21, by activating estrogen receptor in MCF-7 cells.     

Ku70作为RNA解旋酶调节miR-124的加工成熟及神经细胞的分化

目的 Ku70蛋白主要通过其DNA结合特性参与双链DNA断裂（DSB）的非同源端连接（NHEJ）修复,有报道称其具有RNA结合功能,本文探索Ku70是否具有RNA解旋酶活性并影响miRNA加工成熟。方法 利用RNA免疫共沉淀（RIP）测序结合生物信息学分析Ku蛋白结合的RNA;蛋白质印迹法（Western blot,WB）结合定量反转录PCR（qRT-PCR）检测Ku蛋白与miRNAs的表达关系;生物膜干涉技术（BLI）实验分析Ku蛋白与RNA的结合能力;电泳迁移率变动分析（EMSA）实验确定Ku70及Ku80的RNA解旋酶活性;形态学检测结合WB分析Ku70调节miR-124引起的神经细胞功能变化;免疫荧光结合形态学分析寨卡病毒（ZIKV）感染后Ku70及miR-124的变化与神经元分化关联。结果 研究发现,Ku70蛋白具有RNA解旋酶活性,并通过其RNA解旋酶活性影响miRNA加工成熟。Ku70缺失引起许多miRNAs上调,其中包括神经细胞特异的miR-124。在人神经前体细胞（hNPCs）和人神经母细胞瘤细胞（SH-SY5Y）中敲低Ku70可促进 miR-124的成熟,从而导致上述细胞向神经元分化。本文进一步发现,ZIKV感染影响了Ku70及miR-124的表达,导致细胞形态的分化。结论 本研究揭示了Ku70的一种新功能,即Ku70有可能参与miRNA的成熟调控和神经细胞的分化,并且可能是ZIKV病毒致小头症的原因之一。     

肺鳞状细胞癌和腺癌PD-L1蛋白及相关miRNA表达差异的研究

摘要 目的：探讨肺鳞状细胞癌（鳞癌）和腺癌PD-L1蛋白及相关miRNA表达的差异。方法：2019年5月至2020年11月来我院就诊的非小细胞肺癌初治患者纳入本项研究；按照病理类型，将患者分为腺癌组和鳞癌组；H&E染色检测免疫细胞数量；免疫组化检测PD-L1、ki-67、PD-1、CTLA-4和LAG-3的表达；miRNA测序筛选鳞癌和腺癌间差异表达的miRNA。结果：H&E染色结果显示鳞癌组微环境中免疫细胞的数量为86.86±8.96个/高倍视野（HPF），腺癌组的数量为26.29±3.99个/HPF（t=6.173，P＜0.001）；肺鳞癌组微环境免疫细胞PD-1、CTLA-4和LAG-3阳性表达的比例分别为53.71±6.88%、35.29±3.25%和34.43±3.29%，腺癌组阳性表达的比例分别为22.29±3.80%、13.43±2.32%和24.00±1.98%（t=3.997，P=0.002；t=5.476，P＜0.001；t=2.719，P=0.019）；肺鳞癌组患者PD-L1蛋白阳性表达的比例为76.67%，腺癌组的比例为36.67%（P=0.001）；肺鳞癌PD-L1（miR-135、miR-24和miR-30b等）和PD-1（miR-802、miR-155和miR-3127-5p等）相关miRNA的表达均显著高于腺癌。结论：肺鳞癌PD-L1蛋白及相关miRNA的表达、微环境免疫细胞PD-1、CTLA-4和LAG-3阳性比例均显著高于腺癌。