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ETS Proto-Oncogene 1-activated muskelin 1 antisense RNA drives the malignant progression of hepatocellular carcinoma by targeting miR-22-3p to upregulate ETS Proto-Oncogene 1.

ABSTRACT: Long noncoding RNA muskelin 1 antisense RNA (MKLN1-AS) acted as an oncogenic regulator in hepatocellular carcinoma (HCC). This study was performed to investigate the functional mechanism of MKLN1-AS. MKLN1-AS, microRNA-22-3p (miR-22-3p) and ETS Proto-Oncogene 1 (ETS1) levels were examined using reverse transcription-quantitative polymerase-chain reaction. Protein expression was detected by Western blot. The target relation was analyzed by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Cell proliferation ability was determined through cell counting kit-8 assay, colony formation assay and ethylenediurea assay. Angiogenesis was examined by tube formation assay. Cell migration and invasion were assessed via transwell assay. In vivo research was conducted by xenograft tumor model in nude mice. MKLN1-AS was upregulated in HCC tissues and cells. ETS1 promoted the ETS1 expression by binding to the 582-596 sites. Silence of MKLN1-AS suppressed cell growth, angiogenesis, migration, and invasion. MKLN1-AS interacted with miR-22-3p in HCC cells. The function of MKLN1-AS downregulation was relieved by miR-22-3p inhibition in HCC cells. ETS1 was validated as a target of miR-22-3p, and MKLN1-AS upregulated the ETS1 expression by sponging miR-22-3p. Overexpression of miR-22-3p retarded HCC progression by downregulating the level of ETS1. Tumor growth in vivo was also enhanced by MKLN1-AS through the regulation of miR-22-3p/ETS1 axis. These data demonstrated that ETS1-mediated MKLN1-AS contributed to the malignant phenotypes of HCC cells via depending on the miR-22-3p/ETS1 regulatory axis.

SUBMITTER: Pan G

PROVIDER: S-EPMC8805956 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice.Methods344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated.ResultsRemarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes.ConclusionsThe MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.

Project description:Accumulating evidences revealed that long noncoding RNAs (lncRNAs) have been participated in cancer malignant progression, including glioblastoma multiforme (GBM). Despite much studies have found the precise biological role in the regulatory mechanisms of GBM, however the molecular mechanisms, particularly upstream mechanisms still need further elucidated. RT-QPCR, cell transfection, western blotting and bioinformatic analysis were executed to detect the expression of EGR1, HNF1A-AS1, miR-22-3p and ENO1 in GBM. Cell proliferation assay, colony formation assay, wound healing, migration and invasion assays were performed to detect the malignant characters of GBM cells. The molecular regulation mechanism was confirmed by luciferase reporter assay, ChIP and RIP. Finally, orthotopic mouse models were established to examine the effect of HNF1A-AS1 in vivo. In the current study, we analyzed clinical samples to show that the HNF1A-AS1 expression is upregulated and associated with poor patient survival in GBM. Functional studies revealed that HNF1A-AS1 knockdown markedly inhibits malignant phenotypes of GBM cells, whereas overexpression of HNF1A-AS1 exerts opposite effect. Mechanistically, the transcription factor EGR1 forced the HNF1A-AS1 expression by directly binding the promoter region of HNF1A-AS1. Furthermore, combined bioinformatics analysis with our mechanistic work, using luciferase reporter assays and RIP, we first demonstrated that HNF1A-AS1 functions as a competing endogenous RNA (ceRNA) with miR-22-3p to regulate ENO1 expression in GBM cells. HNF1A-AS1 directly binds to miR-22-3p and significantly inhibits miR-22-3p expression, while ENO1 expression was increased. miR-22-3p inhibitor offsets the HNF1A-AS1 silencing induced suppression in malignant behaviors of GBM cells. ENO1 was verified as a direct target of miR-22-3p and its expression levels was negatively with the prognosis in GBM patients. Taken together, our study illuminated the definite mechanism of HNF1A-AS1 in promoting GBM malignancy, and provided a novel therapeutic target for further clinical application.

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ETS Proto-Oncogene 1-activated muskelin 1 antisense RNA drives the malignant progression of hepatocellular carcinoma by targeting miR-22-3p to upregulate ETS Proto-Oncogene 1.

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