Vitamin D-mediated tsRNA-07804 triggers mitochondrial dysfunction and suppresses non-small cell lung cancer progression by targeting CRKL.

OBJECTIVE: tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs with various functions in multiple cancers. Nevertheless, whether vitamin D executes its function in mitochondrial dysfunction and non-small cell lung cancer (NSCLC) progression through tsRNAs remains obscure. METHODS: Differentially expressed tsRNAs between control and vitamin D-treated H1299 cells were acquired by small RNA sequencing. Cell and animal experiments were implemented to elucidate the impacts of vitamin D and tsRNA on mitochondrial dysfunction and NSCLC progression. Dual-luciferase reporter assay, quantitative real-time PCR, western blot and recovery experiments were applied to determine the mechanism of tsRNA in NSCLC. RESULTS: We discovered that vitamin D receptor resulted in decreased mitochondrial-related functions and vitamin D caused mitochondrial dysfunction of NSCLC cells. tsRNA-07804 was remarkably upregulated in vitamin D-treated H1299 cells. Functional experiments indicated that vitamin D led to mitochondrial dysfunction, repressed the proliferation, migration, invasion, and promoted apoptosis of H1299 cells via regulating tsRNA-07804. Mechanistically, tsRNA-07804 induced mitochondrial dysfunction and inhibited the malignancy of H1299 cells by suppressing CRKL expression. In vivo experiments showed that vitamin D inhibited the tumor growth in NSCLC by increasing tsRNA-07804 expression. Moreover, clinical sample analysis unveiled that tsRNA-07804 had a negative correlation with CRKL. CONCLUSIONS: In conclusion, our study proved that vitamin D induced mitochondrial dysfunction and suppressed the progression of NSCLC through the tsRNA-07804/CRKL axis. Overall, these results unveiled that tsRNA-07804 might act as a potential therapeutic target for NSCLC.

Budding uninhibited by benzimidazoles 1 overexpression is associated with poor prognosis and malignant phenotype: A promising therapeutic target for lung adenocarcinoma.

BACKGROUND: The budding uninhibited by benzimidazoles (BUB) family is involved in the cell cycle process as mitotic checkpoint components. Abnormal proliferation is a vital process in the development of lung adenocarcinoma (LUAD). Nevertheless, the roles of BUB1 in LUAD remain unclear. In this study, we evaluated the prognostic value and biological functions of BUB1 in LUAD using data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), clinical LUAD samples, and in vitro experiments. METHODS: The expression, prognostic significance, functions, immune infiltration, and methylation of BUB1 in LUAD were comprehensively analyzed using TCGA, GEO, Gene Expression Profiling Interactive Analysis, Metascape, cBioPortal, MethSurv, and cancerSEA databases. Furthermore, we performed a battery of in vitro experiments and immunohistochemistry (IHC) to verify the bioinformatics results. RESULTS: Multivariate analysis revealed that BUB1 overexpression was an independent prognostic factor (hazard ratio = 1.499, p = 0.013). Functional enrichment analysis showed that BUB1 was correlated with cell cycle, proliferation, DNA repair, DNA damage, and invasion (p < 0.05). Finally, in vitro experiments showed that downregulation of BUB1 inhibited the proliferation, migration, and invasion of LUAD cells and promoted LUAD cell apoptosis. IHC also showed that BUB1 was overexpressed in LUAD (p < 0.001) and was significantly associated with poor prognosis (p < 0.001). CONCLUSIONS: Our bioinformatics and IHC analyses revealed that BUB1 overexpression was an adverse prognostic factor in LUAD. In vitro experiments demonstrated that BUB1 promoted tumor cell proliferation, migration, and invasion in LUAD. These results indicated that BUB1 was a promising biomarker and potential therapeutic target in LUAD.

CircRNA hsa_circ_0087862 Acts as an Oncogene in Non-Small Cell Lung Cancer by Targeting miR-1253/RAB3D Axis.

PURPOSE: Circular RNAs (circRNAs) have been found to regulate several human tumors. The present study was to explore the mechanism of hsa_circ_0087862 in regulating non-small cell lung cancer (NSCLC). METHODS: Totally 102 NSCLC cases were enrolled. NCI-H1359 and A549 cells were transfected. Cells viability, apoptosis, migration and invasion were determined by CCK-8 assay, flow cytometry, scratch test and transwell experiment, respectively. Luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay were performed. Xenograft tumor experiments were performed using nude mice. hsa_circ_0087862, miR-1253 and RAB3D expression in tissues/cells were detected by qRT-PCR. RAB3D and Ki67 protein expressions in cells/tissues were researched by Western blot and immunohistochemistry. Apoptosis of xenograft tumor tissue cells was detected using Tunel assay. RESULTS: hsa_circ_0087862 was significantly up-regulated in NSCLC patients, which was associated with poor prognosis (P < 0.05). hsa_circ_0087862 down-regulation prominently weakened NSCLC cells viability, migration, invasion and enhanced apoptosis (P < 0.01). hsa_circ_0087862 overexpression exhibited the opposite results in NSCLC cells. miR-1253 was sponged by hsa_circ_0087862. miR-1253 expression in NSCLC tissues was negatively correlated with hsa_circ_0087862 (P < 0.001). RAB3D expression in NSCLC was directly inhibited by miR-1253. miR-1253 down-regulation or RAB3D overexpression dramatically reversed NSCLC cells phenotype induced by hsa_circ_0087862 down-regulation. hsa_circ_0087862 down-regulation markedly inhibited tumor growth in vivo (P < 0.01). In xenograft tumor tissues, hsa_circ_0087862 down-regulation obviously decreased expression of RAB3D, Ki67 and increased apoptosis. CONCLUSION: hsa_circ_0087862 acted as an oncogene in NSCLC by targeting miR-1253/RAB3D.

MiR-101-3p inhibits the growth and metastasis of non-small cell lung cancer through blocking PI3K/AKT signal pathway by targeting MALAT-1.

BACKGROUND: MiR-101-3p is an important tumor suppressor miRNA in many human cancers. This study was to investigate the role of miR-101-3p in the progression of non-small cell lung cancer (NSCLC) and its potential underlying mechanism. METHODS: In this study, the endogenous expression of miR-101-3p or MALAT-1 in cells was modulated by cell transfection assays. The regulatory interaction of miR-101-3p and MALAT-1 was examined by Luciferase reporter gene and RNA pull-down assays. The effect of miR-101-3p or MALAT-1 on NSCLC cells was evaluated by cell proliferation assays, wound-healing assays and transwell invasion assays. The mice tumor model was established to test the role of miR-101-3p and MALAT-1 in the growth and metastasis of NSCLC in vivo. RESULTS: The relative expression of miR-101-3p in NSCLC cells was significantly decreased; while MALAT-1 was significantly increased. Moreover, overexpression of miR-101-3p could significantly inhibit the proliferation, migration and invasion of NSCLC cells in vitro, as well as MALAT-1 expression. Further studies confirmed that miR-101-3p could specifically repress MALAT-1 expression through direct binding; MALAT-1 overexpression completely reversed the miR-101-3p-induced suppression on the viability, migration and invasion of NSCLC cells and MALAT-1 expression. Finally, we confirmed that miR-101-3p could block the MALAT-1-induced activation of PI3K/AKT signal pathway and resulted in the inhibition on the growth and metastasis of NSCLC cells in vivo. CONCLUSION: MiR-101-3p inhibited the growth and metastasis of NSCLC through blocking PI3K/AKT signal pathway by targeting MALAT-1.

Prognostic value of matrix metalloproteinase-7 expression in patients with non-small cell lung cancer.

The prognostic value of matrix metalloproteinase-7 (MMP-7) for survival of patients with non-small cell lung cancer (NSCLC) remains controversial. We performed a meta-analysis of the literatures to clarify its impact. Trials were selected for meta-analysis if they provided an independent assessment of MMP-7 in NSCLC and reported the analysis of survival data based on MMP-7 status. Pooled hazard ratio (HR) with 95% confidence interval (95% CI) was used to evaluate the associations between MMP-7 expression and survival of NSCLC patients. Heterogeneity and publication bias were also assessed. Seven studies involving 1,446 patients were identified. The combined HR for all studies was 1.28 (95% CI 0.86-1.91; P = 0.22). Subgroup analysis revealed that MMP-7 overexpression had a favorable impact on survival in Caucasians (HR = 0.74; 95% CI 0.55-0.99; P = 0.043) but showed a poor survival prognosis in Asians (HR = 1.74; 95% CI 1.05-2.88, P = 0.031). Its effect also appeared significant when the analysis was restricted to Asian patients with squamous cell cancer (HR =3.42; 95% CI 1.92-6.11, P = 0.000) and adenocarcinoma (HR = 2.1; 95% CI 1.34-3.29, P = 0.001). Our meta-analysis suggests that there are ethnic differences in the clinical significance of MMP-7 expression for patients with NSCLC.

Polymorphisms in DNA repair genes of XRCC1, XPA, XPC, XPD and associations with lung cancer risk in Chinese people.

BACKGROUND: The carcinogenic chemicals and reactive oxygen species in tobacco can result in DNA damage. DNA repair genes play an important role in maintaining genome integrity. Genetic polymorphisms of DNA repair genes and smoking may contribute to susceptibility of lung cancer. METHODS: In this hospital-based case-control study, we investigated the relationship between 13 tagging single nucleotide polymorphisms (SNPs) in base excision repair pathway and nucleotide excision repair pathway genes, smoking, and lung cancer susceptibility. Thirteen tag SNPs were genotyped in 265 lung cancer patients and 301 healthy controls. Logistic regression and multifactor dimensionality reduction method were applied to explore the association and high-order gene-gene and gene-smoking interaction. RESULTS: In single tag SNP analysis, XPA rs2808668, XPC rs2733533, and XPD rs1799787 were significantly associated with lung cancer susceptibility. Joint effects analysis of XPA rs2808668, XPC rs2733533 and XPD rs1799787 showed that there was an increased risk of lung cancer with increasing numbers of risk alleles. Haplotype analysis showed that XRCC1 (rs25487, rs1799782, rs3213334) GCC had a positive association with lung cancer. Analysis of gene-gene and gene-smoking interaction by multifactor dimensionality reduction showed that a positive interaction existed between the four genes and smoking. The two-factor model, including XPC rs2755333 and smoking, had the best prediction ability for lung cancer. Compared with the C/C genotype of XPC rs2733533 and no smoking, the combination of genotype A carriers with XPC rs2733533 and heavy smokers (≥30 pack-year) had a 13.32-fold risk of lung cancer. CONCLUSION: Our results suggest multiple genetic variants in multiple DNA repair genes may jointly contribute to lung cancer risk through gene-gene and gene-smoking interactions.

Screening and identification of lung cancer metastasis-related genes by suppression subtractive hybridization.

BACKGROUND AND OBJECTIVE: Lung cancer metastasis is a complicated process in which multiple stages and multiple genes are involved. There is an urgent need to use new molecular biology techniques to get more systematic information and have a general idea of the molecular events that take place in lung cancer metastasis. The object of this study was to construct the subtracted cDNA libraries of different metastatic potential lung cancer cell lines, NL9980 and L9981, which were established and screened from human lung large cell carcinoma cell line, WCQH-9801. METHOD: The forward and reverse subtracted cDNA libraries were constructed in the large cell lung cancer cell lines NL9980 and L9981 with the same heredity background but different metastatic potential, by suppression subtractive hybridization (SSH). The positive clones were preliminarily screened by blue-white colony and precisely identified by PCR. The forward and reverse subtracted libraries were screened and identified by dot blot so as to obtain the clones corresponding to gene segments with differential expression. DNA sequencing was performed to analyze the sequences of differential expression segments, which were then searched and compared using the Basic Local Alignment Search Tool from The National Center for Biotechnology Information NCBI BLAST tools. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed to confirm the differential expressed genes both on RNA and protein levels. RESULTS: The forward and reverse subtracted cDNA libraries of the different large cell lung cancer cell lines with metastatic potential were successfully constructed. With blue-white colony and dot blot, 307 positive clones in the forward subtracted library and 78 positive clones in the reverse subtracted library were obtained. Fifty-five clones were successfully sequenced in the forward subtracted library while 31 clones were successfully sequenced in the reverse subtracted library. One new expressed sequence tag (EST) segment was identified from the reverse subtracted cDNA library and was successfully submitted to GenBank and embodied by GenBank. For the differentially expressed genes between L9981 and NL9980 screened by SSH, four genes, ANXA2, KRT18, ACTG1 was upregulated in L9981 cells compared to NL9980 cells. Annexin A2 (which was encoded by ANXA2), γ-actin (which was encoded by ACTG1), and aldose reductase (which was encoded by AKR1B1) proteins were upregulated in L9981 cells compared to NL9980 cells by western blotting. CONCLUSION: The forward and reverse subtracted cDNA libraries of different metastatic potential large cell lung cancer cell lines were successfully constructed by SSH. A series of genes have been screened out to have significantly different expression levels between lung cancer cell lines NL9980 and L9981. A new EST segment that may represent a new metastasis-related gene has been identified. Consistent with the result of SSH, both quantitative real-time RT-PCR and western Blotting confirmed the upregulation of ANXA2, ACTG1 and AKR1B1 in lung cancer cell line L9981 compared with NL9980. These three genes may play important roles in lung cancer metastasis.