Lung Adenocarcinoma Cell Line Research Articles

A novel anticancer effect of Licuala longecalyculata Furtado extracts on lung cancer cell line

Purpose: This study was designed to investigate anticancer effect of ethanol and aqueous extracts of Lygodium microphyllum (Cav.) R.Br., Lygodium salicifolium Presl, and Licuala longecalyculata Furtado on human lung adenocarcinoma A549 cell line, and to determine total phenolic and flavonoid contents of those medicinal plants.
 Methods: The three medicinal plants were extracted with ethanol and water using a rotary evaporator and freeze dryer, respectively. Cytotoxicity of the crude extracts at concentrations ranging from 3.125 to 200 μg/ml on A549 cells was determined using MTT assay at various times. Aluminium chloride colorimetric assay and the Folin-Ciocalteu colorimetric method were used to analyze total flavonoid and phenolic content, respectively.
 Results: L. longecalyculata Furtado extracted with ethanol strongly inhibited A549 cell proliferation in a dose- and time-dependent manner with an IC50 value of 12.00±0.41 μg/ml, whereas L. microphyllum (Cav.) R.Br. and L. salicifolium Presl did not. In addition to the morphological changes, an anticancer effect was observed in L. longecalyculata Furtado-treated A549 cells. L. longecalyculata Furtado showed the presence of phenolic and flavonoid compounds.
 Conclusion: These results indicate that in accordance with local wisdom, L. longecalyculata Furtado crude extract can empirically inhibit lung cancer cell line, but L. microphyllum (Cav.) R.Br. and L. salicifolium Presl cannot. However, further study of L. longecalyculata Furtado is needed to identify phytochemical compounds and their mechanism of anticancer action, in order to develop them as a new therapeutic agent for lung cancer management.

MIR99AHG inhibits EMT in pulmonary fibrosis via the miR-136-5p/USP4/ACE2 axis

BackgroundLong non-coding RNAs (lncRNAs) are closely related to the occurrence and development of cancer. Abnormally expressed lncRNA can be used as a diagnostic marker for cancer. In this study, we aim to investigate the clinical significance of MIR99AHG expression in lung adenocarcinoma (LUAD), and its biological roles in LUAD progression.MethodsThe relative expression of MIR99AHG in LUAD tissues and cell lines was analyzed using public databases and RT-qPCR. The biological functions of MIR99AHG were investigated using a loss-of-function approach. The effect of MIR99AHG on lung fibrosis was assessed by scratch assay, invasion assay and lung fibrosis rat model. FISH, luciferase reporter assay and immunofluorescence were performed to elucidate the underlying molecular mechanisms.ResultsLncRNA MIR99AHG expression level was downregulated in LUAD tissues and cell lines. Low MIR99AHG levels were associated with poorer patient overall survival. Functional analysis showed that MIR99AHG is associated with the LUAD malignant phenotype in vitro and in vivo. Further mechanistic studies showed that, MIR99AHG functions as a competitive endogenous RNA (ceRNA) to antagonize miR-136-5p-mediated ubiquitin specific protease 4 (USP4) degradation, thereby unregulated the expression of angiotensin-converting enzyme 2 (ACE2), a downstream target gene of USP4, which in turn affected alveolar type II epithelial cell fibrosis and epithelial–mesenchymal transition (EMT). In summary, the MIR99AHG/miR-136-5p/USP4/ACE2 signalling axis regulates lung fibrosis and EMT, thus inhibiting LUAD progression.ConclusionThis study showed that downregulated MIR99AHG leads to the development of pulmonary fibrosis. Therefore, overexpression of MIR99AHG may provide a new approach to preventing LUAD progression.

Establishing a Novel Gene Signature Related to Histone Modifications for Predicting Prognosis in Lung Adenocarcinoma.

Background Epigenetic modifications have been revealed to play an important role in tumorigenesis and tumor development. This study aims to analyze the role of histone modifications and the prognostic values of histone modifications in lung adenocarcinoma (LUAD). The promoters and enhancers of protein encoding genes (PCGs) were the regions of enriched histone modifications. Methods Expression profiles and clinical information of LUAD samples were downloaded from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Histone modification data of LUAD cell lines were downloaded from Encyclopedia of DNA Elements (ENCODE) database. Limma R package was used to identify differentially expressed PCGs. To identify molecular subtypes, consensus clustering was conducted based on the expression of dysregulated PCGs with abnormal histone modifications. Univariate Cox regression analysis and stepwise Akaike information criterion (stepAIC) were utilized to establish a prognostic model. Results We identified a total of 699 epigenetic dysregulated genes with 122 of them significantly correlating with LUAD prognosis. We constructed three molecular subtypes (C1, C2, and C3) based on the 122 prognostic genes. C2 had the longest overall survival while C1 had the worst prognosis. In addition, three subtypes had differential immune infiltration and the response to immune checkpoint inhibitors. Moreover, we identified a risk model containing 5 epi-PCGs that had favorable performance to predict prognosis in different datasets. Conclusions This study further supported the critical histone modifications in LUAD development. Three subtypes may provide guidance for the immunotherapy of LUAD patients. Importantly, the prognostic model had great potential to predict LUAD prognosis.

Spray drying siRNA-lipid nanoparticles for dry powder pulmonary delivery.

While all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections.

Effects of arsenic and its main metabolites on A549 cell apoptosis and the expression of pro-apoptotic genes Bad and Bik

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.

Deregulation of PRDM5 promotes cell proliferation by regulating JAK/STAT signaling pathway through SOCS1 in human lung adenocarcinoma.

PRDM5 is considered a tumor suppressor in several types of solid tumors and is involved in multiple cellular processes. However, target genes regulated by PRDM5 in lung cancer and its potential mechanism are poorly defined. Survival analysis was conducted using Kaplan-Meier estimates based on the online databases. RNA-sequencing and bioinformatics analysis were performed to identify the differentially expressed genes in PRDM5-overexpressed A549 cells. We observed deregulated PRDM5 in several lung adenocarcinoma cell lines and its association with a poor prognosis. PRDM5 overexpression inhibited the proliferation of lung adenocarcinoma cells in vitro and suppressed tumor growth in a xenograft model. PRDM5 upregulated the promoter activity of SOCS1, which then inhibited the phosphorylation of JAK2 and STAT3. Our study suggests that the low expression of PRDM5 promotes the proliferation of lung adenocarcinoma cells by downregulating SOCS1 and then upregulating the JAK2/STAT3 signaling pathway.

Oral inhalation of cannabidiol delivered from a metered dose inhaler to alleviate cytokine production induced by SARS-CoV-2 and pollutants

Cannabidiol (CBD) was formulated as a metered dose inhaler (CBD-MDI) and evaluated in vitro for its efficacy as an inhaled dosage form against inflammation caused by the SARS-CoV-2 virus, lipopolysaccharide (LPS) from Escherichia coli, silica particles, nicotine, and coal tar. A CBD-MDI formulation was prepared with 50 mg of CBD in 10 mL for a CBD dose of 250 μg/puff. The formulation ingredients included CBD, absolute ethanol as a cosolvent, and HFA-134a as the propellant. High aerosol performance of CBD-MDI was obtained with mass median aerodynamic diameter of 1.25 ± 0.01 μm, geometric standard deviation of 1.75 ± 0.00, emitted dose of 244.7 ± 2.1 μg, and fine particle dose of 122.0 ± 1.6 μg. The cytotoxicity and anti-inflammatory effectiveness of CBD-MDI were performed in alveolar macrophage (NR8383) and co-culture of alveolar macrophage (NR8383) and human lung adenocarcinoma (A549) cell line. CBD delivered from an MDI was safe on respiratory cells and did not trigger an immune response in alveolar macrophages. CBD-MDI effectively reduced the generation of cytokines in immune cells treated with viral antigen S-RBD, bacterial antigen LPS, silica particles, and coal tar. The efficacy of CBD-MDI was comparable to budesonide. Furthermore, the findings demonstrated that the use of CBD-MDI was more effective in treatment rather than prevention when inflammation was induced by either a viral or bacterial stimulant.

Dual inhibition of BCL2L1 and MCL1 is highly effective against RET fusion-positive or MET exon 14 skipping mutation-positive lung adenocarcinoma cells

Non-small cell lung carcinomas (NSCLCs), especially lung adenocarcinomas (LUADs), harbor several driver mutations against which highly effective tyrosine kinase inhibitors (TKIs) are available. Although TKIs are generally effective against certain NSCLCs, primary or acquired resistance almost always develops. Driver mutations include RET fusion (∼1–2% of NSCLC cases) and MET exon 14 skipping mutation (METΔex14; ∼3–4%). Surprisingly, the LUAD cell line LC-2/ad with CCDC6-RET fusion thrived independently of RET signaling, and Hs-746T cells harboring METΔex14 plus amplification survived MET silencing. However, these two cell lines were highly sensitive to dual silencing of the representative anti-apoptotic BCL2 family members BCL2L1 and MCL1, undergoing extensive apoptosis in monolayer or 3D on-top culture systems. Moreover, we found that most LUAD cell lines and tissues expressed high levels of BCL2L1 and MCL1 mRNA but extremely low levels of BCL2. Together, these findings suggest that inhibiting BCL2L1 plus MCL1 may represent a new approach to treating LUAD cells irrespective of their driver mutations.

Circular RNA Circ_0001777 Suppresses Lung Adenocarcinoma Progression In Vitro and In Vivo.

Circular RNA_0001777 (circ_0001777) is reported to be down-regulated in lung cancer. Nevertheless, the function of circ_0001777 in lung adenocarcinoma is largely unclear. We explored the role of circ_0001777 in lung adenocarcinoma progression and the underlying molecular mechanism. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot assay were conducted to determine the expression of RNAs and proteins. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, 5-ethynyl-20-deoxyuridine, and colony formation assays were conducted to analyze cell proliferation ability. Flow cytometry was carried out to assess cell apoptosis rate. Cell migration and invasion abilities were analyzed by wound healing assay and transwell assays. Cell glycolytic metabolism was measured using a fluorescence-based glucose assay kit and a lactate oxidase-based colorimetric assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation assay were implemented to verify the intermolecular interactions. Circ_0001777 expression was reduced in lung adenocarcinoma tissues and cell lines. Circ_0001777 overexpression restrained the proliferation, migration, invasion, and glycolysis and promoted the apoptosis of lung adenocarcinoma cells. Circ_0001777 could directly bind to microRNA-942-5p (miR-942-5p). The anti-tumor effects of circ_0001777 overexpression in lung adenocarcinoma cells were reversed after miR-942-5p accumulation. miR-942-5p directly bound to the 3' untranslated region (3'UTR) of prickle planar cell polarity protein 2 (PRICKLE2), and PRICKLE2 silencing reversed the anti-tumor effects of miR-942-5p knockdown in lung adenocarcinoma cells. Circ_0001777 could regulate PRICKLE2 expression by absorbing miR-942-5p. Circ_0001777 overexpression markedly hampered tumor progression in vivo. Circ_0001777 suppressed the progression of lung adenocarcinoma by binding to miR-942-5p to induce PRICKLE2 expression.

Knockdown of RhoQ, a member of Rho GTPase, accelerates TGF-β-induced EMT in human lung adenocarcinoma

Lung cancer is the leading cause of cancer-related deaths worldwide, and the most common subtype of lung cancer is adenocarcinoma. RhoQ is a Rho family GTPase with primary sequence and structural similarities to Cdc42 and RhoJ. RhoQ is involved in neurite outgrowth via membrane trafficking and is essential for insulin-stimulated glucose uptake in mature adipocytes. However, the function of RhoQ in lung adenocarcinoma (LUAD) remains unclear. In this study, RhoQ siRNAs were introduced into A549 and PC-9 cells. Expression level of EMT-related genes and invasion ability were investigated using Western blot and transwell assay. To examine the relationship between RhoQ expression and prognosis of LUAD, Kaplan–Meier plotter was used. We discovered that suppressing RhoQ expression promoted TGF-β-mediated EMT and invasion in LUAD cell lines. Furthermore, RhoQ knockdown increased Smad3 phosphorylation and Snail expression, indicating that RhoQ was involved in TGF/Smad signaling during the EMT process. Moreover, Kaplan–Meier plotter analysis revealed that low RhoQ levels were associated with poor overall survival in patients with LUAD. In conclusion, these findings shed light on RhoQ's role as a negative regulator of TGF-β-mediated EMT in LUAD.

STK11 mutation affects the killing effect of NK cells to promote the progression of lung adenocarcinoma.

STK11 is a frequently mutated tumor suppressor in lung adenocarcinoma (LUAD). STK11 mutations also lead to dramatic changes in the tumor microenvironment. Studies have shown that strengthening the killing effect of NK cells is vital for effective cancer treatment. Nonetheless, the mechanism of STK11 mutation in modulating the killing effect of NK cells on LUAD cells remains unclear. The expression of STK11, Ki67, and IFN-γ in tumor tissues was evaluated by immunohistochemistry. The contents of T cells, NK cells, and macrophages were analyzed by immunofluorescence assay. The expression of IL2, IL6, and IFN-γ was detected by ELISA. STK11 expression in LUAD cell line was evaluated by qRT-PCR. CCK-8 and colony formation assay were used to measure proliferative ability of LUAD cells and the viability of NK cells. Flow cytometry was utilized to analyze cell apoptosis and NK cell content. Transwell assay was utilized to measure the chemotactic capability of NK cells. In vivo experiments validated the effect of abnormal expression of STK11 on tumor growth. LUAD patients with STK11 mutation had a high tumor proliferative ability. Forced expression of STK11 could substantially constrain the proliferation of LUAD cells and induce cell apoptosis. In addition, STK11 deletion significantly reduced the infiltration level of NK cells and inhibited the viability and chemotactic ability of NK cells as well as their killing effect on LUAD cells. In vivo animal experiment results demonstrated that STK11 deletion significantly reduced NK cell infiltration and promoted LUAD tumor growth. This study revealed the mechanism of STK11 mutation regulating NK cytotoxicity and promoting tumor development, providing scientific basis for the exploration of STK11-related LUAD therapeutic targets and theoretical reference for developing new NK cell-based immunotherapy.

Linc00996 is a favorable prognostic factor in LUAD: Results from bioinformatics analysis and experimental validation.

Background: Linc00996 has been reported in a variety of malignant tumors, but its potential role and significance in lung adenocarcinoma (LUAD) are not fully understood. The authors investigated the expression and biological behavior of Linc00996 in LUAD and elucidated the function of its potential target genes. Materials and methods: The data of Linc00996 expression in cancers were derived from GEPIA. GEO and TCGA datasets were used to identify the differential expression of Linc00996 in LUAD and analyze the respective correlation between different expression levels and LUAD stage and survival prognosis. We further elucidated the potential biological processes and pathways involved with Linc00996 in LAUD by GSEA. ssGSEA was applied to explore the relationship between Linc00996 and immune activity. Finally, the clinical impact of Linc00996 was assessed in 61 patients with LUAD, and the biological functions of Linc00996 were determined by a series of experiments in vitro, such as CCK8, colony formation, migration, and invasion assays. Results: Compared with adjacent normal lung tissues, Linc00996 was significantly downregulated in LUAD, and its expression was negatively correlated with T stage, N stage, and pathological stage. An in vitro study suggested that enhanced Linc00996 expression could inhibit cell proliferation, clonal formation, migration, and invasion in LUAD cell lines. Via GSEA and ssGSEA, we observed that Linc00996 might be connected with immune infiltration in LUAD, and Linc00996 might inhibit tumorigenesis and metastasis by regulating antigen processing and presentation, JAK-STAT3, and cell adhesion molecular signaling pathways. Conclusion: Linc00996 is a novel tumor suppressor in LUAD and may suppress the tumorigenesis and metastasis of LUAD via the tumor-related signaling pathway, such as antigen processing and presentation, JAK-STAT3, and cell adhesion molecular signaling pathways.

Naphthoquinones and triterpenoids from Arnebia euchroma (Royle) Johnst and their hypoglycemic and lipid-lowering effects

A new pentacyclic triterpenoid, 2-hydroxy-1-ene-hydroxyhopanone (19), and a new benzoxepin-5-one, 3-(4-methyl-3-penten-1-yl)-6-hydroxy-9-methoxy-2H-1-benzoxepin-5-one (25), along with 26 known compounds (1–18, 20–24, 26–28), were isolated from the roots of Arnebia euchroma (Royle) Johnst. The structures of the new compounds were elucidated by extensive spectroscopic analyses. The absolute configurations of shikonofurans 9–13 were determined by quantum chemical ECD calculations and CD spectra comparison for the first time. Pharmacological study revealed that naphthoquinones 1–5, 7, and 8 had obvious cytotoxicity toward human lung adenocarcinoma A549 cell line. Meanwhile, the hypoglycemic and lipid-lowering effects of isolated compounds were assessed by checking their inhibitory effects on key enzymes regulating glucose and lipid metabolism. Results showed that compounds 1, 3, 5, 6, 8, 18, and 19 could inhibit the activity of ATP-citrate lyase (ACL); compound 7 could inhibit the activity of acetyl-CoA carboxylase (ACC1); while compounds 8 and 19 showed inhibitory effects on protein tyrosine phosphatase 1B (PTP1B). Among them, the naphthoquinone 6, steroid 18, and triterpenoid 19 showed moderate inhibitory effects on ACL and PTP1B, but didn't exhibit obvious cytotoxicity. This study demonstrated that compounds 6, 18, and 19 show great promising for the development of new agents for the treatment of metabolic diseases.

The role of LINC01419 in regulating the cell stemness in lung adenocarcinoma through recruiting EZH2 and regulating FBP1 expression

BackgroundRecent years have witnessed a growing academic interest in the effects of lncRNAs on tumors. LINC01419 is found to facilitate proliferation and metastasis of lung adenocarcinoma (LUAD) cells, but there is a great deal of uncertainty about how LINC01419 works on LUAD cell stemness. For this reason, the focus of this research is centered on the regulatory impact of LINC01419 on LUAD cell stemness.MethodsFor the detection of the expression level of LINC01419 in LUAD, qRT-PCR was performed. And how oe-LINC01419 and sh-LINC01419 affected LUAD cell proliferation as well as stem cell sphere-formation were examined by CCK-8 and cell sphere-forming assays. In addition, whether LINC01419 could recruit EZH2 and regulate FBP1 expression were determined by bioinformatics analysis, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP). Western blot was utilized to detect the protein expression levels of FBP1, CD44, CD133, and ALDH-1 as well.ResultsOn the basis of the findings from those assays, an up-regulation of LINC01419 level was demonstrated in LUAD cell lines, and a remarkable upregulation of it in CD44 + LUAD cells. In LUAD cells, proliferation and stem cell sphere-formation that were attenuated by LINC01419 knockdown were discovered to be facilitated by LINC01419 overexpression. And a binding relationship between LINC01419 and EZH2 was determined by RIP assay. Besides, EZH2 was capable of binding to FBP1 promoter region, as found by ChIP-PCR assay. Finally, it was demonstrated by in vitro experiments that LINC01419 could inhibit FBP1 expression by recruiting EZH2, resulting in promotion of LUAD cell proliferation and stemness.SignificanceTo summarize, our findings demonstrate a cancer-promoting role of LINC01419 in LUAD. LINC01419, by recruiting EZH2 and regulating FBP1 expression, contributes to LUAD cell stemness. According to these findings, the potential of LINC01419 to be the target for LUAD treatment is hence determined, which also adds more possibility to the enrichment of therapeutic strategies for lung cancer stem cells.

EP08.02-055 Targeting Lung Adenocarcinoma Cells with a Novel Disulfide Reductase Inhibitor

High-throughput phenotypic screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular effect, but additional strategies are required to identify and characterize their targets and mechanisms of action. Here we characterize the novel small molecule LCS3, identified in a phenotypic screen of ∼190,000 compounds for its ability to selectively impair the proliferation of human lung adenocarcinoma (LUAD) cell lines, and investigate its mechanism of action.

MiR-486-5p specifically suppresses SAPCD2 expression, which attenuates the aggressive phenotypes of lung adenocarcinoma cells.

MiR-486-5p expression is restrained in lung adenocarcinoma (LUAD). However, much less has been understood on its role in LUAD. We aimed to explore the biofunctions of miR-486-5p in LUAD. A differential expression analysis based on The Cancer Genome Atlas-LUAD dataset was done to screen the differently expressed miRNAs and mRNAs. MiR-486-5p and SAPCD2 mRNA expression was analyzed by qRT-PCR, and protein level of SAPCD2 was assayed by western blot. Upregulation and downregulation of miR-486-5p or SAPCD2 were achieved by cell transfection. For cell function assays, the proliferation of cancer cells was examined by MTT assay. Cell apoptosis was assessed by flow cytometry and microscopy. Transwell assay was applied to evaluate cell migration and invasion. A dual-luciferase detection was employed to determine the miRNA-mRNA targeting relationship. MiR-486-5p expression was notably reduced in LUAD tissue and cell lines. Upregulating miR-486-5p restrained the anti-apoptotic and proliferative abilities, as well as cell migratory and invasive phenotypes in LUAD cells. SAPCD2 was determined as one target of miR-486-5p. Also, SAPCD2 forced expression was able to attenuate the inhibitory impacts of miR-486-5p on the malignant phenotypes of LUAD cells. MiR-486-5p suppressed cell malignant progression in LUAD by targeting SAPCD2, suggesting that the two may be targets for LUAD treatment.

The Impact of Endemic Iris taochia Ethanolic Extracts on Human Lung Adenocarcinoma Cells

Iris taochia is an elegant endemic plant in Türkiye and it has limited distribution. In this study, cytotoxic effects of ethanolic extracts from different parts and concentrations extracted from I. taochia collected from the surroundings of Tortum (Erzurum), on A549 human lung adenocarcinoma cell line were investigated. Cytotoxicity of extracts were evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) method. Apopototic activity of IC50 values of extracts were evaluated with Annexin V and Caspase 3/7 assays. Ultrastructural changes of IC50 doses treated cells were investigated by transmission electron microscopy. As a result, it was determined that ethanol extract of I. taochia showed significant cytotoxic activity on A549 cells after 24 hours the extract a dose-dependent reduction in cell viability. IC50 values of above and below ground parts ethanolic extracts were determined as 7 µg/ml and 20 µg/ml respectively. Specifically, apoptosis inducing effect was increased at 7 and 20 µg/ml concentrations by 24 hours. We found that I. taochia ethanol extracts had antiproliferative and apoptotic effects on the human lung adenocarcinoma cells A549. However, further studies at molecular level are required to support our findings and to elucidate chemoproteventive and chemotherapeutic effects of I. taochia on lung cancer.

Protective roles of cytoplasmic p21Cip1/Waf1 in senolysis and ferroptosis of lung cancer cells

Therapy-induced senescent cancer cells increase the expression of the cyclin-dependent kinase inhibitors p16Ink4a and p21Cip1/Waf1 . Given that p21 regulates not only the cell cycle but also cell death, we investigated the roles of p21 in cell death using a p16-negative A549 human lung adenocarcinoma cell line. Senescence was induced by doxorubicin (DXR) or pemetrexed (PEM). The protein expression of p21 was examined by immunoblot. Cell death, reactive oxygen species (ROS) and lipid peroxidation were determined by flow cytometry. ABT-263 and ABT-737 were used as senolytic drugs. In vivo growth of A549 cells with different levels of p21 and their sensitivity to PEM were examined in xenograft models. DXR-induced senescent A549 cells increased the expression of cytoplasmic p21, and the sensitivity to ABT-263 was augmented in p21-knockout A549 (A549-KOp21) cells. A similar senolytic effect was observed when PEM was combined with ABT-737. PEM alone induced a higher level of non-apoptotic cell death, ferroptosis, in A549-KOp21 cells than in A549 cells. Although there was no difference in the level of lipid peroxidation, ROS levels were higher in PEM-treated A549-KOp21 cells than in PEM-treated A549 cells. A loss of p21 increased the sensitivity of A549 cells to PEM both in vitro and in vivo. A clinical database analysis showed that CDKN1Ahigh lung adenocarcinoma patients had a poorer prognosis compared to CDKN1Alow patients. Cytoplasmic p21, which was increased in therapy-induced senescent lung cancer cells, plays protective roles in senolysis and ferroptosis.

TRIM36 enhances lung adenocarcinoma radiosensitivity and inhibits tumorigenesis through promoting RAD51 ubiquitination and antagonizing hsa-miR-376a-5p

The tripartite motif (TRIM) family proteins exhibit oncogenic or anti-oncogenic roles in various cancers. As a TRIM family member, TRIM36 is expressed in lung adenocarcinoma (LUAD), but its roles remain unexplored. Here, we set to investigate the clinical relevance and the biological actions of TRIM36 in LUAD. mRNA levels of TRIM36 and the Kaplan-Meier survival analysis for patients with LUAD were analyzed from The Cancer Genome Atlas (TCGA) database. Real-time PCR and western blotting were utilized to measure TRIM36 levels both in vivo and in vitro. We demonstrated that TRIM36 levels were significantly decreased in LUAD patients. The low expression of TRIM36 was correlated with a poor prognosis. Overexpression and knock-down studies illustrated that TRIM36 inhibits cell proliferation and promotes apoptosis in LUAD cell lines. LUAD cells were irradiated with 60Co. In addition, TRIM36 enhanced radiosensitivity in LUAD cell lines. Moreover, we found that TRIM36 expression was negatively associated with RAD51. Co-overexpressing RAD51 blocked TRIM36's effects on proliferation and apoptosis. TRIM36 formed a complex with RAD51, promoting its ubiquitination. Inhibiting hsa-miR-376a-5p enhanced apoptosis and the effects were mediated by TRIM36 in response to radiation. In conclusion, our results indicated that TRIM36 is anti-oncogenic in LUAD by promoting RAD1 ubiquitination. Hsa-miR-376a-5p acts upstream of TRIM36. TRIM36 and RAD1 may serve as prognostic indicators for LUAD. The interactions between TRIM36, RAD1 and hsa-miR-376a-5p can be future therapeutic targets to increase radiation sensitivity in LUAD patients.

MiR-30b-3p Inhibits the Proliferation and Invasion of Lung Adenocarcinoma by Targeting COX6B1

背景与目的肺腺癌（lung adenocarcinoma, LUAD）是当前最常见的肺癌亚型，微小RNA（microRNAs, miRNAs）是一类非编码小RNA，在细胞活动中发挥核心作用。miR-30b-3p在许多类型的癌症中均起到了关键作用，但关于其在肺腺癌中如何发挥作用的研究仍然很少。本研究通过探索miR-30b-3p在肺腺癌增殖和侵袭中的作用和机制，以期为临床上抑制肺腺癌的增殖和侵袭拓展新的方向。方法利用NCBI生物数据库查找肺腺癌中差异表达明显的miRNA，并查询其差异表达及生存曲线；采用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction, qRT-PCR）检测miR-30b-3p在各肺腺癌细胞系中的表达；5-乙炔基-2’脱氧尿嘧啶核苷（5-ethynyl-2'-deoxyuridine, EdU）细胞增殖实验和Transwell实验检测各组A549细胞增殖和侵袭能力的变化；使用在线预测数据网站确定miR-30b-3p的靶蛋白；Western blot验证COX6B1在不同组肺腺癌细胞中的表达情况；双荧光素酶实验证实miR-30b-3p与COX6B1是否存在结合位点。结果miR-30b-3p在肺腺癌组织和细胞中表达下调（P<0.05），低表达水平的miR-30b-3p与肺腺癌患者的不良预后有关（P=0.005,8）；过表达miR-30b-3p能够抑制肺腺癌细胞的增殖与侵袭能力（P<0.05）；双荧光素酶实验证明miR-30b-3p和COX6B1存在结合位点（P<0.05）；Western blot实验表明在肺腺癌细胞A549中，过表达miR-30b-3p能够下调COX6B1的表达（P<0.05）；EdU细胞增殖实验和Transwell侵袭实验表明，miR-30b-3p的过表达能够逆转上调COX6B1对肺腺癌细胞增殖和侵袭能力的促进作用（P<0.05）。结论miR-30b-3p在肺腺癌中起到了抑癌基因的作用，并能够通过调控COX6B1的表达来抑制肺腺癌的增殖和侵袭。