Lipofection of Single Guide RNA Targeting MMP8 Decreases Proliferation and Migration in Lung Adenocarcinoma Cells.
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ABSTRACT:
SUBMITTER: Maradiaga ODH
PROVIDER: S-EPMC8306211 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Objectives Evidence reveals that microRNAs (miRNAs) are abnormally expressed in lung adenocarcinoma (LUAD) tissue and are crucial in LUAD occurrence. Therefore, this study aims to find the miRNA which could regulate LUAD and to further explore its regulatory mechanism, thus providing a potential molecular target for LUAD. Methods miR-9-5p and ID4 expression in LUAD cells were measured by real-time quantitative PCR and western blot. Cell functional assays were conducted to detect the biological functions of LUAD cells. A dual-luciferase reporter assay was utilized to validate the binding relationship between miR-9-5p and ID4. Results miR-9-5p was highly expressed whereas ID4 was lowly expressed in LUAD. miR-9-5p facilitated LUAD cell progression by targeting ID4. Conclusion miR-9-5p promotes LUAD cell progression by modulating ID4 and may become a potential target for LUAD.
| S-EPMC8564129 | biostudies-literature
Project description:BackgroundIt is increasingly evidenced that long non-coding RNAs (lncRNAs) play an important role in various diseases. LncRNA LINC01194 acts as an oncogene in several cancer types. Nevertheless, the role of LINC01194 in lung adenocarcinoma (LUAD) has not yet been revealed.MethodsqRT-PCR was used to detect the expression of LINC01194, miR-641 and SETD7 mRNA, while western blot was exploited to examine SETD7 protein level. Cell proliferation was detected by colony formation and EdU assays. Transwell assays detected cell migration and invasion. TUNEL assay and flow cytometry analysis were used to detect cell apoptosis. RIP, RNA pull down and luciferase reporter assays detected the binding among LINC01194, miR-641 and SETD7.ResultsLINC01194 was significantly upregulated in LUAD tissues and cell lines. Knockdown of LINC01194 resulted in decreased cell proliferation, migration and invasion, and increased apoptosis. Mechanistic experiments unveiled that LINC01194 augmented SETD7 expression in LUAD cells by competitively interacting with miR-641. Rescue experiments showed that miR-641 inhibition and SETD7 overexpression rescued the repressing impacts on LUAD cell proliferation, migration and invasion caused by LINC01194 knockdown.ConclusionLINC01194 promotes the progression of LUAD by enhancing miR-641-targeted SETD7. The LINC01194/miR-641/SETD7 axis might provide new molecular targets for treating LUAD.
| S-EPMC7722326 | biostudies-literature
Project description:BackgroundMatrix metalloproteases (MMPs) constitute a family of enzymes capable of degrading different components of the extracellular matrix and are implicated in the invasion of tumor cells through the basement membrane. Polymorphisms in MMP genes may result in changes in the expression of MMPs being associated with the development and progression of cancer. We have investigated the association between three polymorphisms (-1607 1G/2G, +17 C/G and -77 A/G) in the human collagenases MMP1, MMP8 and MMP13 and the risk of development or progression of lung cancer.MethodsA hospital-based case-control study was designed including 501 lung cancer patients and 510 controls matched. Genotypes were determined by PCR-RFLP. Results were analyzed using unconditional logistic regression, Cox's proportional hazard regression, and the Kaplan-Meier method.ResultsThe MMP1 and MMP13 promoter polymorphisms were not associated with lung cancer risk, while the C/G polymorphism in MMP8 was associated with a statistically significant decreased risk of developing lung cancer (ORadj = 0.65; 95%CI = 0.45-0.93). The Kaplan-Meier analysis showed that the polymorphisms in MMP1, MMP8 and MMP13 not seem to modify the overall survival. Multivariate analysis revealed that MMP1, MMP8 and MMP13 polymorphisms are not independent prognostic factors for overall survival.ConclusionThis study suggests that the polymorphism in MMP8 is associated with a decreased lung cancer risk, which can be used as a prognostic marker in lung cancer.
| S-EPMC2628929 | biostudies-literature
Project description:The proposed competing endogenous RNA (ceRNA) mechanism suggested that diverse RNA species, including protein-coding messenger RNAs and non-coding RNAs such as long non-coding RNAs, pseudogenes and circular RNAs could communicate with each other by competing for binding to shared microRNAs. The ceRNA network (ceRNET) is involved in tumor progression and has become a hot research topic in recent years. To date, more attention has been paid to the role of non-coding RNAs in ceRNA crosstalk. However, coding transcripts are more abundant and powerful than non-coding RNAs and make up the majority of miRNA targets. In this study, we constructed a mRNA-mRNA related ceRNET of lung adenocarcinoma (LUAD) and identified the highlighted TWIST1-centered ceRNET, which recruits SLC12A5 and ZFHX4 as its ceRNAs. We found that TWIST1/SLC12A5/ZFHX4 are all upregulated in LUAD and are associated with poorer prognosis. SLC12A5 and ZFHX4 facilitated proliferation, migration, and invasion in vivo and in vitro, and their effects were reversed by miR-194-3p and miR-514a-3p, respectively. We further verified that SLC12A5 and ZFHX4 affected the function of TWIST1 by acting as ceRNAs. In summary, we constructed a mRNA-mRNA related ceRNET for LUAD and highlighted the well-known oncogene TWIST1. Then we verified that SLC12A5 and ZFHX4 exert their oncogenic function by regulating TWIST1 expression through a ceRNA mechanism.
| S-EPMC6811597 | biostudies-literature
Project description:BackgroundExtensive studies revealed that long non-coding RNAs (lncRNAs) could act as a regulator in tumors, including lung adenocarcinoma (LUAD). LncRNA FTX transcript, XIST regulator (FTX) has been reported to regulate the biological behaviors of some cancers. Nevertheless, its functional role and molecular mechanism remain obscure in LUAD. Our current study concentrates on exploring the biological function of FTX in LUAD.MethodsRT-qPCR was used to test the expression of FTX, miR-335-5p or NUCB2 in LUAD cells. The effect of FTX on LUAD progression was investigated by colony formation, EdU, flow cytometry, TUNEL, transwell and western blot assays. The interaction between microRNA-335-5p (miR-335-5p) and FTX or nucleobindin 2 (NUCB2) was confirmed by luciferase reporter assay.ResultsRT-qPCR showed that FTX expression was up-regulated in LUAD cell lines. Loss-of-function assay indicated that FTX accelerated cell proliferation, migration and invasion, while inhibited cell apoptosis in LUAD. Besides, miR-335-5p, lowly expressed in LUAD cells, was discovered to be sponged by FTX. Subsequently, NUCB2 was identified as a target gene of miR-335-5p. Additionally, it was confirmed that NUCB2 functioned as an oncogene in LUAD. Rescue assays indicated that LUAD progression inhibited by FTX knockdown could be restored by NUCB2 up-regulation.ConclusionFTX played an oncogenic role in LUAD and contributed to cancer development via targeting miR-335-5p/NUCB2 axis.
| S-EPMC7092578 | biostudies-literature
Project description:It has previously been reported that lung cancer has the highest morbidity and mortality rate worldwide; however, the pathogenesis underlying lung cancer has not been fully elucidated. The aim of the present was primarily to assess the influence of microRNA (miR)-106a-5p on the biological behaviors of lung cancer cells. In the present study, bioinformatics analysis was used to analyze the expression characteristics of miR-106a-5p and its relationship with the prognosis of patients with lung adenocarcinoma (LUAD) in The Cancer Genome Atlas. A dual luciferase reporter assay was performed to verify the binding of miR-106a-5p and liver kinase B1 (LKB1). The Cell Counting Kit-8, colony formation and Transwell assays were utilized to detect cell viability, proliferation and migration, respectively. Protein and RNA expression levels were examined by western blotting and reverse transcription-quantitative PCR analysis, respectively. It was observed that miR-106a-5p was highly expressed in LUAD and associated with poor prognosis. miR-106a-5p promoted the proliferation and migration of LUAD cells, and inhibited autophagy. By contrast, LKB1 inhibited cell proliferation and migration, promoted autophagy and blocked the cancer-promoting effects of miR-106a-5p. Overexpression of miR-106a-5p inhibited the phosphorylation of AMP-activated protein kinase (AMPK) and tuberin (TSC2), and promoted the phosphorylation of mTOR. By contrast, overexpression of LKB1 blocked the promotion of mTOR phosphorylation, and the inhibition of AMPK and TSC2 phosphorylation caused by miR-106a-5p. In summary, the results of the present study indicated that miR-106a-5p regulated the phosphorylation of the AMPK pathway by targeting LKB1, and was involved in the proliferation, migration and autophagy of LUAD cells.
| S-EPMC8543179 | biostudies-literature
Project description:Semaphorin 5A (SEMA5A), a member of the semaphorin family, plays an important role in axonal guidance. Previously, the authors identified another possible role of SEMA5A as a prognostic biomarker for non?smoking women with lung adenocarcinoma in Taiwan, and this phenomenon has been validated in other ethnic groups. However, the functional significance of SEMA5A in lung adenocarcinoma remains unclear. Therefore, we assessed the function of SEMA5A in three lung adenocarcinoma cell lines in this study. Kaplan?Meier Plotter for lung cancer was conducted for survival analyses. Reverse transcription?quantitative PCR (RT?qPCR) and western blot analysis were performed to investigate the expression and post?translational regulation of SEMA5A in lung adenocarcinoma cell lines. A pre?designed PyroMark CpG assay and 5?aza?2'?deoxycytidine treatment were used to measure the methylation levels of SEMA5A. The biological functions of lung adenocarcinoma cells overexpressing SEMA5A were investigated by microarrays, and validated both in vitro (proliferation, colony formation and migration assays) and in vivo (tumor xenografts) experiments. The results revealed that the hypermethylation of SEMA5A and the cleavage of the extracellular domain of SEMA5A were responsible for the downregulation of the SEMA5A levels in lung adenocarcinoma cells (A549 and H1299) as compared to the normal controls. Functional analysis of SEMA5A?regulated genes revealed that they were involved in cellular growth and proliferation. The overexpression of SEMA5A in A549 and H1299 cells significantly decreased the proliferation (P<0.01), colony formation (P<0.001) and migratory ability (P<0.01) of the cells. The suppressive effects of SEMA5A on the proliferative and migratory ability of the cells were also observed in both in vitro and in vivo experiments using brain metastatic Bm7 lung adenocarcinoma cells. On the whole, the findings of this study suggest a suppressive role for SEMA5A in lung adenocarcinoma involving the inhibition of the proliferation and migration of lung transformed cells.
| S-EPMC6910195 | biostudies-literature
Project description:Lung cancer is the most common cancer in males and females and ~40% of lung cancer cases are adenocarcinomas. Previous studies have demonstrated that myristoylated alanine rich protein kinase C substrate (MARCKS) is upregulated in several types of cancer and is associated with poor prognosis in patients with breast cancer. However, its expression level and role in lung adenocarcinoma remain unknown. Therefore, the aim of the present study was to investigate the expression level and biological functions of MARCKS like 1 (MARCKSL1), a member of the MARCKS family, in lung adenocarcinoma. The expression level of MARCKSL1 was examined in human lung adenocarcinoma tissues and cell lines. MARCKSL1-specific small interfering RNAs effectively suppressed its expression level and significantly inhibited the proliferation, migration and invasion of lung adenocarcinoma cells. Additionally, the role of MARCKSLI in the regulation of metastasis was examined. Silencing MARCKSL1 decreased the expression of the epithelial-mesenchymal transition (EMT)-associated proteins E-cadherin, N-cadherin, vimentin and snail family transcriptional repressor 2, and decreased the phosphorylation level of AKT. The results obtained in the current study suggested that MARCKSL1 promoted the progression of lung adenocarcinoma by regulating EMT. MARCKSLI may have prognostic value and serve as a novel therapeutic target in lung adenocarcinoma.
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Project description:Purpose:Recent studies have shown that STIP1 is associated with proliferation and migration in numerous types of tumors; however, the role of STIP1 in lung adenocarcinoma is still poorly understood. Therefore, the aim of this study was to evaluate the role of STIP1 in lung adenocarcinoma, in vitro and in vivo. Methods:The expression of STIP1 in lung adenocarcinoma was assessed by immunohistochemistry, RT-qPCR, and Western blot. The effects of STIP1 on the proliferation of lung adenocarcinoma cells were detected by the cell counting kit-8 assay; the effect of STIP1 on adhesion of lung adenocarcinoma cells was detected by Giemsa staining, while the cell scratch and Transwell assays were employed to examine the effect of STIP1 on the migratory ability of lung adenocarcinoma cells. Finally, apoptosis was evaluated by Hoechst staining and flow cytometry. Results:The expression level of STIP1 in lung adenocarcinoma tissue was significantly higher than that in adjacent normal tissue (P<0.05). Compared with that in nontransfected controls, cell proliferation, adhesion, and migration, as well as vimentin protein expression and levels of phosphorylated JAK2/STAT3, were significantly decreased (P<0.05) in A549 lung adenocarcinoma cells transfected with STIP1 shRNA, whereas E-cadherin protein expression and rates of apoptosis were significantly increased in these cells (P< 0.05). Conclusion:Elevated expression of STIP1 in lung adenocarcinoma may enhance the proliferative, adhesive, and migratory ability, and reduce the apoptosis of lung adenocarcinoma cells through the JAK2/STAT3 signaling pathway and epithelial-mesenchymal transition (EMT), thereby promoting the recurrence and metastatic potential of this cancer. The results indicate that STIP1 may be an effective therapeutic target for the treatment of lung adenocarcinoma.
| S-EPMC6890180 | biostudies-literature
Project description:Piwi-like RNA-mediated gene silencing 1 (PIWIL1) has been identified as a novel extremely highly expressed cancer-testis (CT) gene in lung adenocarcinoma. However, the exact function and mechanism of PIWIL1 in lung adenocarcinoma remains unclear. Herein, we sought to investigate the role of PIWIL1 in the occurrence and development of lung adenocarcinoma. We examined the expression pattern of PIWIL1 in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples, and validated it by Real-Time PCR (RT-PCR) in additional 21 paired lung adenocarcinoma tissues and 16 normal tissues. Subsequently, we explored the biological function of PIWIL1 in A549 and H1299 cell lines by gain and loss-of-function analyses. Using TCGA lung adenocarcinoma data, we further performed coexpression and Gene Ontology (GO) analyses, and analyzed the association of DNA methylation levels in PIWIL1 promoter region with its expression. Finally, we evaluated its expression in different mutation status of significantly mutated genes (SMGs) in TCGA lung adenocarcinoma data. We observed that PIWIL1 was expressed in testis and lung adenocarcinoma but not in other normal tissues, and its high expression was associated with shortened survival of lung cancer patients. Overexpression of PIWIL1 could facilitate the proliferation, invasion and migration of lung adenocarcinoma cells and vice versa. GO analysis revealed that PIWIL1 upregulated genes were enriched in embryonic development, cell proliferation and regulation of transcription. Moreover, promoter DNA hypomethylation of PIWIL1 could contribute to its aberrant expression in tumors. Interestingly, PIWIL1 expression was significantly higher in patients without hepatocyte growth factor (HGF) or serine/threonine kinase 11 (STK11) mutation (P = 0.006 and 0.005, respectively). PIWIL1 is an epidriver gene in lung adenocarcinoma, indicating a potential target for further therapy.
| S-EPMC5774002 | biostudies-literature