Single-cell transcriptomic sequencing data reveal aberrant DNA methylation in SMAD3 promoter region in tumor-associated fibroblasts affecting molecular mechanism of radiosensitivity in non-small cell lung cancer.

OBJECTIVE: Non-small cell lung cancer (NSCLC) often exhibits resistance to radiotherapy, posing significant treatment challenges. This study investigates the role of SMAD3 in NSCLC, focusing on its potential in influencing radiosensitivity via the ITGA6/PI3K/Akt pathway. METHODS: The study utilized gene expression data from the GEO database to identify differentially expressed genes related to radiotherapy resistance in NSCLC. Using the GSE37745 dataset, prognostic genes were identified through Cox regression and survival analysis. Functional roles of target genes were explored using Gene Set Enrichment Analysis (GSEA) and co-expression analyses. Gene promoter methylation levels were assessed using databases like UALCAN, DNMIVD, and UCSC Xena, while the TISCH database provided insights into the correlation between target genes and CAFs. Experiments included RT-qPCR, Western blot, and immunohistochemistry on NSCLC patient samples, in vitro studies on isolated CAFs cells, and in vivo nude mouse tumor models. RESULTS: Fifteen key genes associated with radiotherapy resistance in NSCLC cells were identified. SMAD3 was recognized as an independent prognostic factor for NSCLC, linked to poor patient outcomes. High expression of SMAD3 was correlated with low DNA methylation in its promoter region and was enriched in CAFs. In vitro and in vivo experiments confirmed that SMAD3 promotes radiotherapy resistance by activating the ITGA6/PI3K/Akt signaling pathway. CONCLUSION: High expression of SMAD3 in NSCLC tissues, cells, and CAFs is closely associated with poor prognosis and increased radiotherapy resistance. SMAD3 is likely to enhance radiotherapy resistance in NSCLC cells by activating the ITGA6/PI3K/Akt signaling pathway.

Effects of Hyperthyroidism on Venous Thromboembolism: A Mendelian Randomization Study.

Objective: Observational studies show the correlation between thyroid dysfunction and risk of venous thromboembolism. However, the causal effects remain uncertain. Our study was conducted to evaluate whether thyroid function and dysfunction were causally linked to the risk of venous thromboembolism. Methods: Publicly available summary data of thyrotropin (TSH) and free thyroxine (FT4), hypothyroidism, and hyperthyroidism were obtained from the ThyroidOmics Consortium and the UK Biobank. With single nucleotide polymorphisms (SNPs) as instrumental variables, the casual effects of genetically predicted TSH and FT4 and hypo- and hyperthyroidism on venous thromboembolism outcome were estimated through Mendelian randomization analysis methods (inverse variance weighted (IVW), MR-Egger, weighted median, simple mode, and weighted mode). Cochran's Q test was performed to evaluate the heterogeneity and horizontal pleiotropy. Results: Our study selected 15 FT4-, 36 TSH-, 3 hyperthyroidism-, and 79 hypothyroidism-associated SNPs as instrumental variables. The IVW analysis results showed that the odds ratio of venous thromboembolism for hyperthyroidism was 1.124 (95% confidence interval: 1.019-1.240; p = 0.019), demonstrating the casual effect of hyperthyroidism not FT4, TSH, and hypothyroidism on venous thromboembolism. No heterogeneity or horizontal pleiotropy was observed according to Cochran's Q test. Conclusion: Our Mendelian randomization analysis supports the causal effect of hypothyroidism on risk of venous thromboembolism. There is no evidence that genetically predicted TSH, FT4, and hypothyroidism have casual effects on venous thromboembolism. Future studies should be conducted to elucidate the underlying pathophysiological mechanisms.

Exosomal microRNA-506 inhibits biological activity of lung adenocarcinoma cells and increases sensitivity to cisplatin-based hyperthermia.

Exosomal microRNAs (miRNAs) play a vital role in the occurrence and development of lung adenocarcinoma (LUAD). Based on the bioinformatics analyses, the current study sought to explore the effects of exosomal miR-506 on LUAD cell biology and the efficacy of cisplatin (CDDP)-based hyperthermia (HT). After sample preparation, we identified decreased miR-506 and elevated ATAD2. LUAD cells were subsequently transfected with miR-506 mimic, oe-ATAD2 and PI3K/AKT signaling pathway inhibitor LY294002 to analyze effects of the miR-506/ATAD2/PI3K/AKT axis on cell biological processes and chemoresistance. Effects of exosomal miR-506 on sensitivity of LUAD cells to CDDP-based HT were further assessed in a co-culture system of BMSC-derived exosomes and LUAD cells, which was also validated in tumor-bearing nude mice. miR-506 down-regulated ATAD2 to inhibit the PI3K/AKT signaling pathway, thereby inhibiting the malignant phenotypes of LUAD cells and augmenting LUAD cell sensitivity to CDDP-based HT. Further, BMSCs-derived exosomes harboring miR-506 sensitized LUAD cells to DDP/HT both in vitro and in vivo. Collectively, our findings revealed that exosomal miR-506 sensitized LUAD cells to CDDP-based HT by inhibiting ATAD2/PI3K/AKT signaling pathway, offering a potential therapeutic target for LUAD treatment.

Retraction Notice to: Silencing of lncRNA LINC00857 Enhances BIRC5-Dependent Radio-Sensitivity of Lung Adenocarcinoma Cells by Recruiting NF-κB1.

[This retracts the article DOI: 10.1016/j.omtn.2020.09.020.].

miR-126-5p enhances radiosensitivity of lung adenocarcinoma cells by inhibiting EZH2 via the KLF2/BIRC axis.

Radiotherapy is a common method for the treatment of lung adenocarcinoma, but it often fails due to the relative non-susceptibility of lung adenocarcinoma cells to radiation. We aimed to discuss the related mechanisms by which miR-126-5p might mediate radiosensitivity of lung adenocarcinoma cells. The binding affinity between miR-126-5p and EZH2 and between KLF2 and BIRC5 was identified using multiple assays. A549 and H1650 cells treated with X-ray were transfected with miR-126-5p mimic/inhibitor, oe-EZH2, or si-KLF2 to detect cell biological functions and radiosensitivity. Finally, lung adenocarcinoma nude mouse models were established. miR-126-5p and KLF2 were poorly expressed, while EZH2 and BIRC5 were upregulated in lung adenocarcinoma tissues and cells. miR-126-5p targeted EZH2 to promote the KLF2 expression so as to inhibit BIRC5 activation. Both in vitro and in vivo experiments verified that elevated miR-126-5p inhibited cell migration and promoted apoptosis to enhance the sensitivity of lung adenocarcinoma cells to radiotherapy via the EZH2/KLF2/BIRC5 axis. Collectively, miR-126-5p downregulated EZH2 to facilitate the sensitivity of lung adenocarcinoma cells to radiotherapy via KLF2/BIRC5.

Fe3O4 magnetic nanoparticle-enhanced radiotherapy for lung adenocarcinoma via delivery of siBIRC5 and AS-ODN.

BACKGROUND: Radiotherapy is the mainstay treatment for lung adenocarcinoma, yet remains highly susceptible to resistance. Fe3O4 magnetic nanoparticles (MNPs) possess the ability to induce biological therapeutic effects. Herein, the current study set out to explore the effects of Fe3O4 MNPs on radiosensitivity of lung adenocarcinoma cells. METHODS: Fe3O4 MNPs loaded with both negatively-charged small interfering RNA against baculoviral IAP repeat containing 5 (siBIRC5) and oligodeoxynucleotide antisense (AS-ODN) to generate co-delivery NPs, followed by evaluation. Gel retardation assay was further performed to determine the binding ability of Fe3O4 MNPs to AS-ODN/siBIRC5. The radiosensitizing effect of NPs on lung adenocarcinoma cells was determined in the absence or the presence of NPs or radiotherapy. A549 and H460 tumor-bearing mice were established, where tumor tissues were subjected to immunohistochemistry. RESULTS: NPs were successfully prepared and characterized. BIRC5 expression levels were augmented in tissues of lung cancer patients. Fe3O4 MNPs enhanced the uptake of siBIRC5 and AS-ODN by lung adenocarcinoma cells. The presence of NPs under magnetic field reduced the BIRC5 expression and elevated the DR5 expression in lung adenocarcinoma cells. Lung adenocarcinoma cells treated with NPs exhibited inhibited tumor cell migration and increased DNA damage. After magnetic field treatment, tumors were better suppressed in the tumor-bearing mice treated with NPs, followed by radiotherapy. CONCLUSION: Findings obtained in our study indicated that Fe3O4 MNPs-targeted delivery of siBIRC5 and AS-ODN enhances radiosensitivity, providing an innovative solution for the current clinically existing lung adenocarcinoma patients with radiotherapy resistance with a low risk of toxicity.

Silencing of lncRNA LINC00857 Enhances BIRC5-Dependent Radio-Sensitivity of Lung Adenocarcinoma Cells by Recruiting NF-κB1.

Lung adenocarcinoma (LUAD) is a predominant type of lung cancer in never-smoker patients. In this study, we identified a long noncoding RNA (lncRNA) LINC00857 that might regulate radio-sensitivity of LUAD cells. Expression of LINC00857 and baculoviral IAP repeat containing 5 (BIRC5) was determined to be upregulated in LUAD cells and tissues using qRT-PCR and western blot analysis. The correlation between LINC00857 and nuclear factor kappa B subunit 1 (NF-κB1) was verified using RNA immunoprecipitation and chromatin immunoprecipitation assays, while the binding relationship between NF-κB1 and BIRC5 was determined by dual-luciferase reporter assay. It was suggested that LINC00857 could recruit NF-κB1 in BIRC5 promoter region. BIRC5 promoter activity was repressed in response to small interfering-LINC00857 (si-LINC00857) in LUAD cells. Silencing LINC00857 or BIRC5 reduced proliferation and colony formation but enhanced apoptosis and radio-sensitivity of LUAD cells. The experiment in vivo verified the function of silencing LINC00857 on enhancing radio-sensitivity of LUAD cells. Our results reveal a functional regulatory LINC00857-NF-κB1-BIRC5 triplet in LUAD cells, suggesting LINC00857 as a potential target for LUAD treatment.

Exosomal microRNA-26b-5p down-regulates ATF2 to enhance radiosensitivity of lung adenocarcinoma cells.

Lung adenocarcinoma (LUAD), as the most common subtype of non-small cell lung cancer, is responsible for more than 500 000 deaths worldwide annually. In this study, we identify a novel microRNA-26b-5p (miR-26b-5p) and elucidated its function on LUAD. The survival rate of parent LUAD cells and radiation-resistant LUAD cells were determined using clonogenic survival assay. We overexpressed or inhibited miR-26b-5p in LUAD, and the correlation between activating transcription factor 2 (ATF2) and miR-26b-5p was determined using integrated bioinformatics analysis and dual-luciferase reporter gene assay. Exosomes derived from A549 cell lines were then detected using Western blot assay, followed by co-transfection with radiation-resistant A549R cells. LUAD tissues and serum were collected, followed by miR-26b-5p relative expression quantification using RT-qPCR. miR-26b-5p was identified as the most differentially expressed miRNA and was down-regulated in LUAD. Radiation-resistant cells were more resistant to X-radiation compared with parent cells. miR-26b-5p overexpression and X-irradiation led to enhanced radiosensitivity of LUAD cells. ATF2 was negatively targeted by miR-26b-5p. Exosomal miR-26b-5p derived from A549 cells could be transported to irradiation-resistant LUAD cells and inhibit ATF2 expression to promote DNA damage, apoptosis and radiosensitivity of LUAD cells, which was verified using serum-based miR-26b-5p. Our results show a regulatory network of miR-26b-5p on radiosensitivity of LUAD cells, which may serve as a non-invasive biomarker for LUAD.

Chitooligosaccharides promote radiosensitivity in colon cancer line SW480.

AIM: To investigate the anti-proliferation and radiosensitization effect of chitooligosaccharides (COS) on human colon cancer cell line SW480. METHODS: SW480 cells were treated with 0, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/mL of COS for 48 h. CCK-8 assay was employed to obtain the cell survival ratio of SW480 cells, and the anti-proliferation curve was observed with the inhibition ratio of COS on SW480 cells. The RAY + COS group was treated with 1.0 mg/mL of COS for 48 h, while both the RAY and RAY+COS groups were exposed to X-ray at 0, 1, 2, 4, 6 and 8 Gy, respectively. Clonogenic assay was used to analyze cell viability in the two groups at 10 d after treatment, and a cell survival curve was used to analyze the sensitization ratio of COS. The RAY group was exposed to X-ray at 6 Gy, while the RAY+COS group was treated with 1.0 mg/mL of COS for 48 h in advance and exposed to X-ray at 6 Gy. Flow cytometry was employed to detect cell cycle and apoptosis rate in the non-treatment group, as well as in the RAY and RAY + COS groups after 24 h of treatment. RESULTS: COS inhibited the proliferation of SW480 cells, and the inhibition rate positively correlated with the concentration of COS (P < 0.01). Cell viability decreased as radiation dose increased in the RAY and RAY+COS groups (P < 0.01). Cell viabilities in the RAY+COS group were lower than in the RAY group at all doses of X-ray exposure (P < 0.01), and the sensitization ratio of COS on SW480 cells was 1.39. Compared with the non-treatment group, there was a significant increase in apoptosis rate in both the RAY and RAY + COS groups; while the apoptosis rate in the RAY+COS group was significantly higher than in the RAY group (P < 0.01). In comparing these three groups, the percentage of G2/M phase in both the RAY and RAY + COS groups significantly increased, and the percentage of the S phase and G0/G1 phase was downregulated. Furthermore, the percentage in the G2/M phase was higher, and the percentage in the S phase and G0/G1 phase was lower in the RAY + COS group vs RAY group (P < 0.01). CONCLUSION: COS can inhibit the proliferation of SW480 cells and enhance the radiosensitization of SW480 cells, inducing apoptosis and G2/M phase arrest.

Anti-proliferation effect of zoledronic acid on human colon cancer line SW480.

OBJECTIVE: To investigate the anti-proliferation effect and mechanism of zoledronic acid (ZOL) on human colon cancer line SW480. METHODS: SW480 cells were treated with 0, 12.5, 25, 50, 100 and 200 µmoL/L of ZOL for 48 h, and CCK-8 assay was employed to obtain the survival rate of SW480 cells. SW480 cells were treated with 25 µmoL/L of ZOL for 0, 12, 24, 48 and 72 h, and then the survival rate was obtained. SW480 cells of the ZOL group were treated with 25 µmoL/L of ZOL for 48 h, while cells of the CsA + ZOL group were pretreated with 10 µmoL/L of CsA for 0.5 h and then treated with 25 µmoL/L of ZOL for 48 h. Then the survival rates of SW480 cells of the control group, ZOL group and CsA + ZOL group were determined. Flow cytometry was employed to detect the apoptosis rate and the mitochondrial transmembrane potential (△Ψm) of the three groups and Western blot was used to detect the expressions of cyt C in the cytosol of the three groups. RESULTS: ZOL inhibited the proliferation of SW480 cells, and the inhibition rate positively correlated with the concentration of ZOL and the action time (P < 0.01). The cell survival rate and the △Ψm of the ZOL group were greatly lower than those of the control group, while the apoptosis rate and the expression of cyt C in the cytosol were obviously higher than those of the control group. All the differences showed distinctly statistical significances (P < 0.01). The cell survival rate and the △Ψm of the CsA + ZOL group were all lower than those of the control group, but substantially higher than those of the ZOL group; while the apoptosis rate and the expression of cyt C in the cytosol were higher than those of the control group, but distinctly lower than those of the ZOL group. All the differences were statistically significant (P < 0.01). CONCLUSIONS: ZOL can induce the apoptosis in human colon cancer line SW480 and then inhibit the proliferation of SW480 cells directly by opening the mitochondrial permeability transition pore abnormally, decreasing △Ψm, and releasing the cyt C into the cytosol. And the effect enhances with the increases of the concentration of ZOL and the action time.

Diagnosis of the invasiveness of lung adenocarcinoma manifesting as ground glass opacities on high-resolution computed tomography.

BACKGROUND: To explore the diagnostic method in assessing the malignancy of pulmonary adenocarcinoma characterized by ground glass opacities (GGO) on computed tomography (CT). METHODS: Preoperative CT data for preinvasive and invasive lung adenocarcinomas were analyzed retrospectively. GGO lesions that were detected on lung windows but absent using the mediastinal window were subject to adjustment of the window width, which was reduced with the fixed interval of 100 HU until the lesions were no longer evident, with a fixed mediastinal window level of 40 HU. The shape, smoking habits, size of the lesion on the lung window, and window width at which lesions disappeared were compared and receiver operating characteristic curves were used to determine the optimal cut-off of the lesion size and window width to differentiate between these invasive and preinvasive lesions. RESULTS: Of the 209 lung adenocarcinomas, 102 were preinvasive (25 atypical adenomatous hyperplasia and 77 adenocarcinoma in situ), while 107 were invasive (78 minimally invasive adenocarcinoma and 29 invasive adenocarcinoma). The shape, lesion size, and window width at which lesions were no longer evident differed significantly between the two groups (P < 0.05). The size of 8.9 mm and a window width of 1250 HU were the optimal cut-off to differentiate between preinvasive and invasive lesions. CONCLUSION: The shape, size of the lesion, and window width on high-resolution CT may be useful in assessing the invasiveness of lung adenocarcinoma that manifests as GGO. Irregular lesions that disappear at window width <1250 HU, with a diameter of > 8.9 mm are more likely to be invasive.

Anti-proliferation and radiosensitization effects of chitooligosaccharides on human lung cancer line HepG2.

OBJECTIVE: To observe the anti-proliferation and radiosensitization effect of chitooligosaccharides (COS) on human lung cancer cell line HepG2. METHODS: CCK-8 assay was employed to obtain the inhibition ratio of COS on HepG2 cells at 24 h after treatment. The clonogenic assay was used to analyze the cell viability of RAY group and RAY + COS group with X-ray of 0, 1, 2, 4, 6 and 8 Gy, and the cell survival curve was used to analyze the sensitization ratio of COS. Flow cytometry was employed to detect cell cycle and apoptosis rate in control group, RAY group and RAY + COS group after 24 h treatment. RESULTS: COS inhibited the proliferation of HepG2 cells, and the inhibition rate positively correlated with the concentration of COS. The cell viability decreased with increasing exposure dose in RAY group and RAY + COS group. The cell viabilities of RAY + COS group were lower than those of RAY group at the dose of 4, 6 and 8 Gy (P < 0.05), and the sensitization ratio of COS was 1.19. There were higher percentage at G2/M phase and apoptosis rate, and lower percentage at S phase in RAY + COS group versus the other two groups (P < 0.01). CONCLUSIONS: COS can inhibit the proliferation of HepG2 cells, and enhance the radiosensitization of HepG2 cells, induce apoptosis and G2/M phase arrest.