Cd44 Epithelial Isoform Inversely Associates with Invasive Characteristics of Colorectal Cancer

et al. 2020

J Exp Clin Cancer Res

Background CD8+ tumor-infiltrating T lymphocytes (T-TILs) in the tumor microenvironment (TME) play an important role in tumor development, and miRNAs regulate tumor cell interactions with the microenvironment. T-TIL-based tumor immunotherapy provides a promising treatment strategy in diffuse large B-cell lymphoma (DLBCL). MiRNAs tend to be attractive targets for novel antitumor interventions. Methods Weighted gene coexpression network analysis (WGCNA), CIBERSORT analysis and Cox regression analysis were used to identify CD8+ T-TIL-related miRNAs. RT-PCR, western blotting, immunohistochemistry (IHC), in situ hybridization (ISH), luciferase reporter assay, coimmunoprecipitation and ubiquitination analyses were used to detect miRNA, mRNA and protein expression and their combination. The viability and function of CD8+ T cells after stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), cytotoxicity assay, functional avidity assessment, flow cytometry and Cell Counting Kit-8 (CCK-8) assay. DLBCL cell lines, primary cells and a murine xenograft model established with A20 cell injection were used as in vitro and in vivo experimental models. Results MiR-340-5p was positively correlated with CD8+ T-TILs in DLBCL patients, and KMT5A was a direct target gene of miR-340-5p. CD8+ T-cell function was significantly enhanced by miR-340-5p mimics both in vitro and in vivo, which was reversed by KMT5A overexpression. We demonstrated that COP1/CD73 was involved in the downstream mechanism of the miR-340-5p/KMT5A axis involving ubiquitination. In vivo, we validated an improved CD8+ T-TIL infiltration rate and tumor suppression with miR-340-5p treatment. Furthermore, miR-340-5p directly regulated the biological activity of DLBCL cells without CD8+ T-cell participation. Conclusions MiR-340-5p promoted CD8+ T-TIL infiltration and antitumor function by regulating KMT5A and COP1 and further activating CD73 ubiquitination. MiR-340-5p is potentially a novel target for DLBCL immunotherapy.

Section: Methodsmentioning

confidence: 99%

“…The histochemical score (H-score) was achieved by multiplying the staining intensity and the percentage of positive cells and ranged from 0 to 300. The mean of H-score was considered the cut-off point [ 18 ]. Any cell with CD8-positive staining was counted as CD8 + T-TIL.…”

Section: Methodsmentioning

confidence: 99%

MiRNA-340-5p mediates the functional and infiltrative promotion of tumor-infiltrating CD8+ T lymphocytes in human diffuse large B cell lymphoma

et al. 2020

J Exp Clin Cancer Res

“…Slides were rst scanned at low magni cation (10x magni cation) and 10 high magni cation elds (400x magni cation) were assessed. For scoring gene expression, intensity of staining was classi ed into 0 (no expression), 1 (weak expression), 2 (moderate expression) and 3 (high expression); and the percentage of positive cells was categorized as 1 (positive cells ≤25%), 2 (25%< positive cells ≤50%), 3 (50% < positive cells ≤75%) and 4 (positive tumor cells >75%) (13). The histochemical score (H-score) was achieved by multiplying the staining intensity and the percentage of positive cells and ranged from 0 to 300.…”

Section: Western Blottingmentioning

confidence: 99%

“…The histochemical score (H-score) was achieved by multiplying the staining intensity and the percentage of positive cells and ranged from 0 to 300. The mean of H-score was considered as the cut-off point (13). Any cell with CD8-positive staining was counted as CD8 + T-TIL.…”

Section: Western Blottingmentioning

confidence: 99%

MiRNA-340-5p Mediates the Functional and Infiltrative Promotion of Tumor-Infiltrating CD8+ T Lymphocytes in Human Diffuse Large B Cell Lymphoma

et al. 2020

Preprint

Background: CD8+ tumor-infiltrating T lymphocytes (T-TILs) in the tumor microenvironment (TME) play an important role in tumor development, and miRNAs regulate tumor cell interactions with the microenvironment. T-TIL-based tumor immunotherapy provides a promising treatment strategy in diffuse large B-cell lymphoma (DLBCL). MiRNAs tend to be attractive targets for novel antitumor interventions.Methods: Weighted gene coexpression network analysis (WGCNA), CIBERSORT analysis and Cox regression analysis were used to identify CD8+ T-TIL-related miRNAs. RT-PCR, western blotting, immunohistochemistry (IHC), luciferase reporter assay, coimmunoprecipitation and ubiquitination analyses were used to detect miRNA, mRNA and protein expression and their combination. The viability and function of CD8+ T cells after stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), cytotoxicity assay, functional avidity assessment, flow cytometry and Cell Counting Kit-8 (CCK-8) assay. DLBCL cell lines, primary cells and a murine xenograft model established with A20 cell injection were used as in vitro and in vivo experimental models.Results: MiR-340-5p was positively correlated with CD8+ T-TILs in DLBCL patients, and KMT5A was a direct target gene of miR-340-5p. CD8+ T-cell function was significantly enhanced by miR-340-5p mimics both in vitro and in vivo, which was reversed by KMT5A overexpression. We demonstrated that COP1/CD73 was involved in the downstream mechanism of the miR-340-5p/KMT5A axis involving ubiquitination. In vivo, we validated an improved CD8+ T-TIL infiltration rate and tumor suppression with miR-340-5p treatment. Furthermore, miR-340-5p directly regulated the biological activity of DLBCL cells without CD8+ T-cell participation.Conclusions: MiR-340-5p promoted CD8+ T-TIL infiltration and antitumor function by regulating KMT5A and COP1 and further activating CD73 ubiquitination. MiR-340-5p is potentially a novel target for DLBCL immunotherapy.

“…Slides were rst scanned at low magni cation (10x magni cation), and 10 high magni cation elds (400x magni cation) were assessed. For scoring gene and miRNA expression, the intensity of staining was classi ed into 0 (no expression), 1 (weak expression), 2 (moderate expression) and 3 (high expression), and the percentage of positive cells was categorized as 1 (positive cells ≤25%), 2 (25%< positive cells ≤50%), 3 (50% < positive cells ≤75%) and 4 (positive tumor cells >75%) (18,19). The histochemical score (H-score) was achieved by multiplying the staining intensity and the percentage of positive cells and ranged from 0 to 300.…”

Section: Ihc and Ishmentioning

confidence: 99%

MiRNA-340-5p mediates the functional and infiltrative promotion of tumor-infiltrating CD8+ T lymphocytes in human diffuse large B cell lymphoma

et al. 2020

Preprint

Background: CD8+ tumor-infiltrating T lymphocytes (T-TILs) in the tumor microenvironment (TME) play an important role in tumor development, and miRNAs regulate tumor cell interactions with the microenvironment. T-TIL-based tumor immunotherapy provides a promising treatment strategy in diffuse large B-cell lymphoma (DLBCL). MiRNAs tend to be attractive targets for novel antitumor interventions. Methods: Weighted gene coexpression network analysis (WGCNA), CIBERSORT analysis and Cox regression analysis were used to identify CD8+ T-TIL-related miRNAs. RT-PCR, western blotting, immunohistochemistry (IHC), in situ hybridization (ISH), luciferase reporter assay, coimmunoprecipitation and ubiquitination analyses were used to detect miRNA, mRNA and protein expression and their combination. The viability and function of CD8+ T cells after stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), cytotoxicity assay, functional avidity assessment, flow cytometry and Cell Counting Kit-8 (CCK-8) assay. DLBCL cell lines, primary cells and a murine xenograft model established with A20 cell injection were used as in vitro and in vivo experimental models. Results: MiR-340-5p was positively correlated with CD8+ T-TILs in DLBCL patients, and KMT5A was a direct target gene of miR-340-5p. CD8+ T-cell function was significantly enhanced by miR-340-5p mimics both in vitro and in vivo, which was reversed by KMT5A overexpression. We demonstrated that COP1/CD73 was involved in the downstream mechanism of the miR-340-5p/KMT5A axis involving ubiquitination. In vivo, we validated an improved CD8+ T-TIL infiltration rate and tumor suppression with miR-340-5p treatment. Furthermore, miR-340-5p directly regulated the biological activity of DLBCL cells without CD8+ T-cell participation. Conclusions: MiR-340-5p promoted CD8+ T-TIL infiltration and antitumor function by regulating KMT5A and COP1 and further activating CD73 ubiquitination. MiR-340-5p is potentially a novel target for DLBCL immunotherapy.