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MiR?146a?5p targets TCSF and influences cell growth and apoptosis to repress NSCLC progression.

ABSTRACT: Several studies have indicated that microRNAs (miRs) mediate multiple pathways associated with tumorigenesis and progression. Our preliminary study experimentally verified that miR?146a?5p has a role in the biological behavior of non?small cell lung cancer (NSCLC) cells. To perform further investigation of miR?146a?5p, the present study evaluated miR?146a?5p by targeting its downstream gene tumor collagenase stimulatory factor (TCSF) to influence cell viability, proliferation and apoptosis in NSCLC. Online sequence prediction, a thorough search of the open source database The Cancer Genome Atlas (TCGA), immunohistochemistry (IHC) of TCSF in clinical lung cancer tissues, and a dual?luciferase assay, as well as assays to test viability, proliferation and apoptosis in vitro, were conducted to explain the targeted regulation association between miR?146a?5p and TCSF in NSCLC. The miRanda and TargetScanHuman database revealed that TCSF and miR?146a?5p had target binding sites. A luciferase reporter assay demonstrated that miR?146a?5p and TCSF did have complementary sequences (P<0.05). From the TCGA database, TCSF was highly expressed in lung adenocarcinoma and lung squamous cell carcinoma tissues when compared with normal lung tissues (P<0.05). Furthermore, the protein level of TCSF in cancerous lung tissues was determined by IHC, and it was concluded that TCSF protein was also upregulated in NSCLC tissues (P<0.001). A significant difference was identified following in vitro experiments for the NSCLC cell line A549, which revealed that miR?146a?5p and TCSF regulated cell viability, proliferation and apoptosis. In conclusion, the present study verified the target action association between TCSF and miR?146a?5p with high throughput data analysis and experimental results in NSCLC.

SUBMITTER: Huang WT

PROVIDER: S-EPMC6412506 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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miR‑146a‑5p targets TCSF and influences cell growth and apoptosis to repress NSCLC progression.

Huang Wen-Ting WT He Rong-Quan RQ Li Xiao-Jiao XJ Ma Jie J Peng Zhi-Gang ZG Zhong Jin-Cai JC Hu Xiao-Hua XH Chen Gang G

Oncology reports 20190225 4

Several studies have indicated that microRNAs (miRs) mediate multiple pathways associated with tumorigenesis and progression. Our preliminary study experimentally verified that miR‑146a‑5p has a role in the biological behavior of non‑small cell lung cancer (NSCLC) cells. To perform further investigation of miR‑146a‑5p, the present study evaluated miR‑146a‑5p by targeting its downstream gene tumor collagenase stimulatory factor (TCSF) to influence cell viability, proliferation and apoptosis in NS ...[more]

PMID: 30816543

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Project description:Our previous study demonstrated that the expression of miR‑146a‑5p was downregulated in non‑small cell lung cancer (NSCLC) tissue, which affected the progression and prognosis of patients with NSCLC. Thus, the present study was conducted to investigate the functional mechanism of miR‑146a‑5p in tumorigenesis and angiogenesis in NSCLC. Following the construction of a H460 NSCLC cell line in which miR‑146a‑5p was overexpressed via lentivirus transduction, the NSCLC chick embryo chorioallantoic membrane (CAM) model was established by transplanting miR‑146a‑5p‑overexpressing NSCLC cells into the CAM. Then, the size of the neoplasms within the CAM was measured, the vessel ratio was calculated, and the cellular morphology, metastasis and inflammation of tumor cell was observed using hematoxylin and eosin staining. The target genes of miR‑146a‑5p were predicted by 12 online software programs; these genes were then subjected to Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway annotations using the Database for Annotation, Visualization and Integrated Discovery 6.7 as well as constructed into a protein interaction network using protein‑protein interaction from Search Tool for the Retrieval of Interacting Genes/Proteins. The xenograft tumor size and angiogenesis conditions of the miR‑146a‑5p‑overexpressing group (volume 6.340±0.066 mm3, vessel ratio 9.326±0.083) was obviously restricted (P<0.001) when compared with the low expression group (volume 30.13±0.06 mm3, vessel ratio 16.94±0.11). In addition, marked necrosis along with inflammatory cell infiltration was observed with the HE‑stained slices from the miR‑146a‑5p low expression group. Regarding the results of the target gene prediction, cancer and toll‑like receptor signaling were the two most significant pathways represented among the target genes, while JUN, EGFR and RAC1 were the most relevant proteins among the selected potential targets of miR‑146a‑5p. In a CAM xenograft tumor model, overexpression of miR‑146a‑5p inhibited the tumorigenesis and angiogenesis of an NSCLC cell line. miR‑146a‑5p may act as a tumor suppressor gene in NSCLC and have moderate prognostic value in lung cancer.

Project description:BACKGROUND:microRNAs (miRNAs) are a small, endogenous non-coding RNAs that are involved in post-transcriptional gene regulation of many biological processes, including embryo implantation and placental development. In our previous study, miR-146a-5p was found expressed higher in the serum exosomes of pregnant sows than non-pregnant. The research on miR-146a-5p has been mainly related to human diseases, but there are few studies on its effects on the reproduction of sows in early pregnancy. OBJECTIVE:In this article, our motivation is to study the role of miR-146a-5p in the early pregnancy of sows on the cell proliferetion and apoptosis by targeting SMAD3 and SMAD4. METHODS:Bioinformatics software was used to identify the target genes of miR-146a-5p. The wildtype and mutant-type recombinant plasmids of dual-luciferase reporter with 3'-UTR of Smad3 or 3'- UTR of Smad4 were constructed, and co-transfected in porcine kidney cell (PK-15 cell) with miR- 146a-5p mimic, mimic-NC(M-NC), inhibitor and inhibitor-NC(IN-NC), then dual-luciferase activity analysis, qRT-PCR and Western blot were performed to verify the target genes. After the transfection of BeWo choriocarcinoma cell (BeWo cell) with miR-146a-5p mimic, M-NC, inhibitor and IN-NC, the mRNA expression of Caspase-3, BAX and Bcl-2 was measured using qRT-PCR, and the cell proliferation was measured using CCK-8 kit. RESULTS:The luciferase, mRNA and protein expression of Smad3 in PK-15 cells treated by Smad3- 3'-UTR-W co-transfected with miR-146a-5p mimic were significantly lower than that with miR- 146a-5p M-NC, and the results of Smad4 were similar to Smad3, but the protein expression had a trend to lower in mimic group. The expression level of Bcl-2 in the miR-146a-5p mimic group was significantly lower than that in the miR-146a-5p M-NC group, but the expression pattern of Caspase-3 was just opposite. The mimic of miR-146a-5p reduced the proliferation of BeWo cells, however the inhibitor increased. CONCLUSION:Smad3 and Smad4 are the direct target genes of miR-146a-5p. The expression of Smad3 and Smad4 were affected by the mimic and inhibitor of miR-146a-5p. miR-146a-5p affects cell apoptosis and proliferation by regulating their target genes. This study provided new data to understand the regulation mechanism of early pregnancy in sows.

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MiR?146a?5p targets TCSF and influences cell growth and apoptosis to repress NSCLC progression.

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miR‑146a‑5p targets TCSF and influences cell growth and apoptosis to repress NSCLC progression.

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