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CircRNA hsa_circ_0070934 functions as a competitive endogenous RNA to regulate HOXB7 expression by sponging miR-1236-3p in cutaneous squamous cell carcinoma.

ABSTRACT: Circular ribonucleic acids (circRNAs) serve a vital role in the pathological processes of a number of diseases. Previous microarray results of circRNA expression revealed that hsa_circ_0070933 and hsa_circ_0070934, two circRNAs associated with the La ribonucleoprotein 1B gene, were highly expressed in cutaneous squamous cell carcinoma (CSCC). The present study aimed to explore the specific role of these circRNAs in CSCC. Through reverse transcription-quantitative PCR, hsa_circ_0070933 and hsa_circ_0070934 expression levels in CSCC cell lines and a human keratino-cyte cell line were detected. Additionally, direct interactions between miR-1236-3p and HOXB7 or circ-0070934 were identified using RNA binding protein immunoprecipitation assays and dual-luciferase reporter assays. Cell Counting Kit-8, 5-ethynyl-2?-deoxyuridine, Transwell invasion and flow cytometry assays were used to assess the roles of miR-1236-3p or circ-0070934 in cell invasion, proliferation and apoptosis. Subsequently, in vivo tumor formation assays were used to verify the role of circ-0070934 in CSCC. The results demonstrated that the expression of circ-0070934 was stably upregulated in a number of CSCC cell lines compared with that in normal human keratinocytes. Knockdown of circ-0070934 inhibited the invasive and proliferative potential of CSCC cells and promoted apoptosis both in vivo and in vitro. In addition, circ-0070934 modulated HOXB7 expression through competitive binding with miR-1236-3p. In conclusion, the results of the present study demonstrated the effects of the circ-0070934/miR-1236-3p/HOXB7 regulatory axis on CSCC and provided a novel insight for the pathogenesis of CSCC.

SUBMITTER: Zhang D

PROVIDER: S-EPMC7307596 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Project description:BackgroundAccumulating evidence indicates that circRNAs may serve as essential regulators in the progression of several human cancers, but the function and mechanism of circRNAs in intrahepatic cholangiocarcinoma (ICC) are largely unknown.MethodsRNA-seq was used to assess differentially expressed circRNAs between 4 ICC and peritumor tissues. Quantitative RT-PCR and in situ hybridization were used to determine the circHMGCS1-016 expression in ICC tissues. The function and mechanism of circHMGCS1-016 were further identified via in vivo experiments. The clinical characteristics and prognostic significance of circHMGCS1-016 were analyzed by a retrospective study. The functions of circHMGCS1-016 were assessed via modifying circRNA expression in ICC cells. Moreover, the molecular mechanisms of circHMGCS1-016 in ICC cells were explored by circRNA precipitation, miRNA immunoprecipitation, SILAC and luciferase reporter assays.ResultsWe identified that compared with peritumor tissues, ICC tissues expressed hsa_circ_0008621 (circHMGCS1-016) high by RNA-seq, which was further identified by qRT-PCR and in situ hybridization. Moreover, the expression of circHMGCS1-016 was revealed to be associated with survival and recurrence of ICC patients. By regulating circHMGCS1-016 expression, we found that elevated circHMGCS1-016 promoted ICC development both in vitro and in vivo. By SILAC and circRNA-pull down, we demonstrated that circHMGCS1-016 induced ICC cell invasion and reshaped the tumor immune microenvironment via the miR-1236-3p/CD73 and GAL-8 axis. In ICC tissues, we uncovered that a high level of circHMGCS1-016 was positively associated with CD73 and GAL-8 expression and negatively related to the CD8+ T cells infiltration, which was further validated by establishing a humanized mouse tumor model. Importantly, we displayed that ICC patients with high levels of circHMGCS1-016 in tumor tissues benefited less from anti-PD1 treatment compared to those with low levels of circHMGCS1-016.ConclusionsCircHMGCS1-016 is a forceful contributor in ICC development and immune tolerance via miR-1236-3p/CD73 and GAL-8 axis. CircHMGCS1-016 can be explored as a new potential biomarker and therapeutic target for PD1-resistant ICC.

Project description:Long non‑coding RNAs (lncRNAs) and microRNAs (miRNAs) are essential for the progression of tumors, including cutaneous squamous cell carcinoma (CSCC). The present study aimed to examine the competing endogenous RNA (ceRNA) network in CSCC. Differentially expressed genes in CSCC were analyzed using the GSE66359 microarray data set, and the upstream miRNAs and lncRNAs were predicted using online database analysis (TargetScan 7.1, mirDIP 4.1, miRSearch V3.0, miRDB and RNA22 2.0) and were verified in clinical tissues. RNA pull‑down and dual luciferase reporter gene assays were used to verify the targeting relationships among lncRNA human histocompatibility leukocyte antigen complex P5 (HCP5), miR‑138‑5p and enhancer of zeste homolog 2 (EZH2). Cell lines with a high and low HCP5 expression were screened, and a pcDNA‑3.1‑HCP5 overexpression vector, small interfering RNA against HCP5, miR‑138‑5p mimics and miR‑138‑5p inhibitors were transfected into the CSCC cells. Cell viability, invasion, migration, apoptotic rate and autophagy were evaluated. The effects of HCP5 on autophagy and apoptosis of CSCC cells were verified in vivo using Ki67 and TUNEL staining. EZH2 was demonstrated to be upregulated in CSCC cells. miR‑138‑5p target sequences were identified in HCP5 and EZH2. HCP5 was revealed to function as a putative ceRNA of miR‑138‑5p to positively regulate EZH2, and EZH2 was shown to regulate autophagy and apoptosis of CSCC cells through the STAT3/VEGFR2 pathway. HCP5 overexpression decreased miR‑138‑5p levels, increased EZH2 levels and promoted cell malignant behaviors and autophagy but decreased the apoptosis rate. These trends were opposite when HCP5 was silenced. In conclusion, HCP5 may competitively bind to miR‑138‑5p to regulate EZH2 in CSCC cells, promoting autophagy and reducing apoptosis through the STAT3/VEGFR2 pathway. This study may provide a new perspective for understanding the molecular mechanism and treatment of CSCC.

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CircRNA hsa_circ_0070934 functions as a competitive endogenous RNA to regulate HOXB7 expression by sponging miR-1236-3p in cutaneous squamous cell carcinoma.

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