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LncRNA FOXD3-AS1 promotes the progression of non-small cell lung cancer by regulating the miR-135a-5p/CDK6 axis

ABSTRACT: Long non-coding RNA (lncRNA) is essential to the development and progression of malignant human cancer. Growing evidence suggests that the lncRNA forkhead box D3 antisense 1 (FOXD3-AS1) is a crucial regulatory effector for multiple cancer types and is closely associated with poor prognosis. However, in most cases, the molecular mechanism underlying the role of FOXD3-AS1 in cancer development has not yet been fully elucidated. The present study focused on non-small cell lung cancer (NSCLC) in order to gain insight into how FOXD3-AS1 drives cancer progression. First, FOXD3-AS1 expression in NSCLC tissue samples was detected using reverse transcription-quantitative (RT-qPCR). Moreover, cell proliferation and apoptosis were determined using Cell Counting Kit-8 assays and flow cytometry, respectively. A luciferase reporter assay was then performed to determine whether there was a direct binding association between FOXD3-AS1 and microRNA (miR)-135a-5p. Lastly, a tumor subcutaneous xenograft model was established to examine the role of FOXD3-AS1 in tumor growth. FOXD3-AS1 was significantly overexpressed in NSCLC tissue samples and cell lines compared with normal tissue samples and cells. FOXD3-AS1 silencing expression significantly inhibited A549 and H1229 cell proliferation while inducing apoptosis compared with sh-NC group. The luciferase reporter assay demonstrated the direct binding interaction between FOXD3-AS1 and miR-135a-5p. Moreover, FOXD3-AS1 silencing led to the upregulation of miR-135a-5p in A549 and H1229 cells compared with sh-NC group. It was also demonstrated that miR-135a-5p could bind to the 3′ untranslated region of cyclin-dependent kinase 6 (CDK6) and negatively modulate its transcription. miR-135a-5p knockdown or CDK6 overexpression reversed the inhibition on cell proliferation and apoptosis following FOXD3-AS1 knockdown. Altogether, the present study suggests that FOXD3-AS1 sponges miR-135a-5p to promote cell proliferation and concomitantly inhibit apoptosis by regulating CDK6 expression in NSCLC cells.

SUBMITTER: Guo H

PROVIDER: S-EPMC8561623 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Long non‑coding RNA forkhead box D3 antisense RNA 1 (FOXD3‑AS1) functions as an oncogenic regulator in several types of cancer, including breast cancer, glioma and cervical cancer. However, the effects and mechanisms underlying FOXD3‑AS1 in cervical cancer (CC) are not completely understood. The present study aimed to investigate the biological functions and potential molecular mechanisms underlying FOXD3‑AS1 in CC progression. Reverse transcription‑quantitative PCR was performed to detect FOXD3‑AS1, microRNA (miR)‑128‑3p and LIM domain kinase 1 (LIMK1) expression levels in CC tissues and cells. Immunohistochemical staining and western blotting were conducted to assess LIMK1 protein expression levels in CC tissues and cells, respectively. Cell Counting Kit‑8 and BrdU assays were used to determine the role of FOXD3‑AS1 in regulating cell proliferation. CC cell migration and invasion were assessed by performing Transwell assays. Dual‑luciferase reporter assays were conducted to verify the binding between miR‑128‑3p and FOXD3‑AS1. FOXD3‑AS1 expression was significantly increased in CC tissues and cell lines compared with adjacent healthy tissues and normal cervical epithelial cells, respectively. High FOXD3‑AS1 expression was significantly associated with poor differentiation of tumor tissues, increased tumor size and positive lymph node metastasis. FOXD3‑AS1 overexpression significantly increased CC cell proliferation, migration and invasion compared with the negative control (NC) group, whereas FOXD3‑AS1 knockdown resulted in the opposite effects compared with the small interfering RNA‑NC group. Moreover, the results demonstrated that FOXD3‑AS1 targeted and negatively regulated miR‑128‑3p, which indirectly upregulated LIMK1 expression. Therefore, the present study demonstrated that FOXD3‑AS1 upregulated LIMK1 expression via competitively sponging miR‑128‑3p in CC cells, promoting CC progression.

Project description:IntroductionColon adenocarcinoma (COAD) is the third most common tumor of the digestive tract. Recent studies reported that lncRNA's abnormal expression might play a vital role in the occurrence and development of COAD.MethodsIn the present study, we investigated the expression of ILF3-AS1 in COAD cell lines, human normal colon epithelial cell line, patient tumor tissues and adjacent normal tissues by real-time quantitative PCR (RT-qPCR). Small interfering RNAs (siRNAs) were transfected into COAD cells to inhibit the expression of ILF3-AS1. The effects of ILF3-AS1 on cell proliferation, migration, invasion and apoptosis were measured by CCK-8 assay, transwell migration and invasion assay, and flow cytometry apoptosis assay, respectively. The direct binding of ILF3-AS1 and miR-619-5p/CAMK1D was validated by the luciferase reporter assay. The expression of CAMK1D and epithelial-mesenchymal transformation (EMT)-related proteins was detected by Western Blot analysis. Besides, in vivo experiments were conducted to demonstrate the oncogenic role of ILF3-AS1 in COAD.ResultsThe results showed that the expression of ILF3-AS1 was significantly higher in COAD tissue than in normal adjacent samples, and this conclusion was confirmed in the available public datasets. After ILF3-AS1 knockdown, the proliferation of COAD cell lines SW480 and HT29 was significantly inhibited. At the same time, the apoptosis was increased, and the invasion and migration abilities were decreased. After further exploring the mechanisms, we found that ILF3-AS1 serves as a competitive endogenous RNA of mir-619-5p. It can bind to mir-619-5p and reduce its expression, thus regulating the target gene CAMK1D. In addition, we found that high expression of ILF3-AS1 was significantly associated with tumor grade, tumor size, and distant metastasis in COAD samples. In vivo experiments confirmed that ILF3-AS1 promotes tumor growth in COAD models.ConclusionThe present study demonstrated that ILF3-AS1 plays an oncogenic role in COAD through regulating the miR-619-5p/CAMK1D axis, and inhibition of ILF3-AS1 may pave the way for COAD treatment.

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LncRNA FOXD3-AS1 promotes the progression of non-small cell lung cancer by regulating the miR-135a-5p/CDK6 axis

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