Isoflurane Preconditioning Alleviates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Inhibiting miR-195-3p Expression.

To investigate the role of microRNA-195-3p (miR-195-3p) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury. AC16 human cardiomyocyte cells were cultured and pretreated with different concentrations of isoflurane (ISO) (1%, 2%, and 3%), followed by 6 h each of hypoxia and reoxygenation to construct H/R cell models. The optimum ISO concentration was assessed based on the cell viability. miR-195-3p expression was regulated by in vitro cell transfection. Cell viability was determined by MTT assay, and apoptosis was evaluated by flow cytometry. The levels of myocardial injury and inflammation were determined by enzyme-linked immunosorbent assay. Compared with the control group, the cell viability of the H/R group had significantly decreased and that of ISO pretreatment had increased in a dose-dependent manner. Therefore, we selected a 2% ISO concentration for pretreatment. MiR-195-3p expression had significantly increased in the H/R group and decreased after 2% ISO pretreatment. Additionally, the number of apoptotic cells and the levels of lactate dehydrogenase, creatine kinase-myoglobin binding, cardiac troponin I, interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α had increased significantly. ISO preconditioning inhibited H/R-induced AC16 cell damage, whereas miR-195-3p overexpression reversed the protective effects of ISO on cardiomyocytes. The expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was reduced in the H/R-induced AC16 cells, and PTEN is a downstream target gene of miR-195-3p. Preconditioning with 2% ISO plays a protective role in H/R-induced AC16 cell damage by inhibiting miR-195-3p expression.

Inhibition of miR-195-3p protects against cardiac dysfunction and fibrosis after myocardial infarction.

Cardiac fibrosis following myocardial infarction is a major risk factor for heart failure. Recent evidence suggests that miR-195-3p is up-regulated in fibrotic diseases, including kidney and liver fibrosis. However, its function and underlying mechanisms in cardiac fibrosis after MI remain unknown. To investigate the role of miR-195-3p in MI-induced cardiac fibrosis, we established acute MI models by ligating adult C57B/L6 mice LAD coronary artery while sham-operated mice were used as controls. In vivo inhibition of miR-195-3p was conducted by intramyocardial injection of AAV9-anti-miR-195-3p. In vitro overexpression and inhibition of miR-195-3p were performed by transfecting cultured Cardiac Fibroblasts (CFs) with synthetic miRNA mimic and inhibitor. Our results showed that MI induced the expression of miR-195-3p and that inhibition of miR-195-3p reduced myofibroblast differentiation and collagen deposition and protected cardiac function. In vitro stimulation of CFs with TGF-ß1 resulted in a significant increase in miR-195-3p expression. Inhibition of miR-195-3p attenuated the TGF-ß1-induced expression of ECM proteins, migration, and proliferation. PTEN expression was significantly reduced in the hearts of MI mice, in activated CFs, and in CFs transfected with miR-195-3p mimic. Inhibition of miR-195-3p markedly restored PTEN expression in MI mice and TGF-ß1-treated CFs. In conclusion, this study highlights the crucial role of miR-195-3p in promoting cardiac fibrosis and dysfunction after MI. Inhibiting miR-195-3p could be a promising therapeutic strategy for preventing cardiac fibrosis and preserving cardiac function after MI. Additionally, the study sheds light on the mechanisms underlying the effects of miR-195-3p on fibrosis, including its regulation of PTEN/AKT pathway.

MiR-195-3p is a Novel Prognostic Biomarker Associated with Immune Infiltrates of Lung Adenocarcinoma.

BACKGROUND: MicroRNA-195-3p (miR-195-3p) plays an important role in some tumors, but its role in LUAD is unclear. This study explored the expression of miR-195-3p in LUAD and the relationship between the expression of miR-195-3p and the clinical and prognostic characteristics of LUAD patients. METHODS: MiR-195-3p expression and clinical information of LUAD patients were obtained from The Cancer Genome Atlas (TCGA). Kruskal-Wallis test, Wilcoxon signed rank test, logistic regression, and Cox regression were used to assess the relationship between the expression level of miR-195-3p and clinical features in LUAD tissues. Kaplan-Meier survival curves were used to analyze the effect of miR-195-3p expression levels on the prognosis of LUAD patients. Target genes of miR-195-3p were predicted by several software. GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and immune infiltration analysis were used to analyze the possible regulatory network of miR-195-3p. RESULTS: Compared with normal lung tissue, miR-195-3p is down expressed in LUAD tissue (P < 0.001). The low miR-195-3p expression in LUAD was significantly associated with N stage (P = 0.046), pathologic stage (P = 0.011), and gender (P = 0.010). Low miR-195-3p expression predicted a poorer overall survival (HR: 0.60; 95% CI: 0.45-0.81; P = 0.001) and disease-specific survival (HR: 0.55; 95% CI: 0.37-0.80; P = 0.002). The expression of miR-195-3p (HR: 0.488; 95% CI: 0.304-0.784; P = 0.003) was independently correlated with OS in LUAD patients. High expression of miR-195-3p genes, including ABCC2, AGMAT, ARNTL2, ATP6V0A4, CDC25A, CDK1, FAM111B, GJB2, GRIP1, HMGA2, HOXA9, KIF14, SYT2, and TFAP2A, were associated with poor OS in LUAD. GO and KEGG analysis suggested that miR-195-3p was related to the phagosome pathway. MiR-195-3p may promote the function of B cells, dendritic cells, eosinophils, immature dendritic cells, macrophages, Mast cells, NK cells, plasmacytoid dendritic cells, and follicular helper T cells. CONCLUSION: Low miR-195-3p expression is significantly associated with poor survival in LUAD, which may be a promising prognostic biomarker for LUAD.

MicroRNA-195-3p promotes hepatic stellate cell activation and liver fibrosis by suppressing PTEN expression.

Liver fibrosis is a reversible wound healing reaction characterized by abnormal accumulation of extracellular matrix (ECM) in response to liver injury. Recent studies have shown that it can be epigenetically regulated, especially by microRNAs (miRNAs). It has been acknowledged that activation of hepatic stellate cells (HSCs) is a pivotal step in the initiation and progression of liver fibrosis. Notably, our results showed that miR-195-3p was increased in HSCs isolated from CCl4-treated mice and that the increase was more pronounced as the degree of liver fibrosis increased. Moreover, treatment of LX-2 cells, a human immortalized hepatic stellate cell line, with TGF-ß1 resulted remarkable upregulation of miR-195-3p. Gain-of-function and loss-of-function experiments have suggested that the increased levels of miR-195-3p inhibit the expression of phosphatase and tension homolog deleted on chromosome 10 (PTEN), a negative regulator of the PI3K/Akt/mTOR signaling pathway in liver fibrosis, thereby contributing to HSC activation and proliferation and promoting the expression of profibrotic genes, such as α-SMA and collagen I, in LX-2 cells, which accelerates the accumulation of fibrous extracellular matrix deposition in the liver, while knockdown of miR-195-3p induced the opposite effect. Taken together, these results provide evidence for the harmful role of miR-195-3p in CCl4-treated mouse liver fibrosis.

Long noncoding RNA TINCR facilitates hepatocellular carcinoma progression and dampens chemosensitivity to oxaliplatin by regulating the miR-195-3p/ST6GAL1/NF-κB pathway.

BACKGROUND: Long non-coding RNAs (lncRNA) have an essential role in progression and chemoresistance of hepatocellular carcinoma (HCC). In-depth study of specific regulatory mechanisms is of great value in providing potential therapeutic targets. The present study aimed to explore the regulatory functions and mechanisms of lncRNA TINCR in HCC progression and oxaliplatin response. METHODS: The expression of TINCR in HCC tissues and cell lines was detected by quantitative reverse transcription PCR (qRT-PCR). Cell proliferation, migration, invasion, and chemosensitivity were evaluated by cell counting kit 8 (CCK8), colony formation, transwell, and apoptosis assays. Luciferase reporter assays and RNA pulldown were used to identify the interaction between TINCR and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) via miR-195-3p. The corresponding functions were verified in the complementation test and in vivo animal experiment. RESULTS: TINCR was upregulated in HCC and associated with poor patient prognosis. Silencing TINCR inhibited HCC proliferation, migration, invasion, and oxaliplatin resistance while overexpressing TINCR showed opposite above-mentioned functions. Mechanistically, TINCR acted as a competing endogenous (ceRNA) to sponge miR-195-3p, relieving its repression on ST6GAL1, and activated nuclear factor kappa B (NF-κB) signaling. The mouse xenograft experiment further verified that knockdown TINCR attenuated tumor progression and oxaliplatin resistance in vivo. CONCLUSIONS: Our finding indicated that there existed a TINCR/miR-195-3p/ST6GAL1/NF-κB signaling regulatory axis that regulated tumor progression and oxaliplatin resistance, which might be exploited for anticancer therapy in HCC.

MicroRNA-195-3p inhibits cyclin dependent kinase 1 to induce radiosensitivity in nasopharyngeal carcinoma.

MicroRNAs (miRNAs) are revealed to participate in the progression of multiple malignancies, including nasopharyngeal carcinoma (NPC). This work is intended to decipher the function of microRNA-195-3p (miR-195-3p) in regulating the radiosensitivity of NPC cells and its mechanism. MiR-195-3p and cyclin-dependent kinase 1 (CDK1) expressions were detected in NPC tissues and cells using qRT-PCR and Western blot, respectively. Moreover, radiation-resistant cell lines were induced by continuous irradiation with different doses. Furthermore, the CCK-8 experiment, colony formation assay and flow cytometry were utilized to examine the growth, apoptosis and cell cycle of radioresistant cells. Bioinformatics prediction and dual-luciferase reporter gene assay were applied to prove the targeting relationship between miR-195-3p and CDK1 mRNA 3'UTR. The data showed that miR-195-3p was remarkably down-modulated in NPC tissues and was associated with increased tumor grade, lymph node metastasis and clinical stage of the patients. MiR-195-3p expression was significantly down-modulated in radiation-resistant NPC tissues and NPC cell lines relative to radiation-sensitive NPC tissues and human nasopharyngeal epithelial cells, while CDK1 expression was notably up-modulated. MiR-195-3p overexpression inhibited the growth of NPC cells, decreased radioresistance, promoted apoptosis, and impeded the cell cycle progression. CDK1 was a target gene of miR-195-3p, and CDK1 overexpression counteracted the effects of miR-195-3p on NPC cell growth, apoptosis, cell cycle progression and radiosensitivity. In summary, miR-195-3p improves the radiosensitivity of NPC cells by targeting and regulating CDK1.

MiR-195-3p inhibits cell proliferation in cervical cancer by targeting BCDIN3D.

BACKGROUND: Cervical cancer (CC) is one of the most prevailing cancers among females. Accumulated studies concentrated on the regulatory role of micro RNA in cancers. This research is to explore the potential role of mir-195-3p in cervical cancer progression. METHODS: Bioinformatics tools were used to investigate differential expression of mir-195-3p and BCDIN3D in cervical cancer. RNA expression patterns of both mir-195-3p and BCDIN3D were detected by RT-PCR in CC cell lines. The protein expression of BCDIN3D was measured by Western Blot. Hela and Siha cell lines were transfected with mir-195-3p inhibitors, mir-195-3p mimics and BCDIN3D si-RNA, si-NC. Luciferase reporter assays were adopted to confirm the binding. The interplays between mir-195-3p and BCDIN3D were explored in CC cell lines. CCK-8 assays checked how mir-195-3p regulated cell proliferation and Ki67 was examined by Western blot for its protein expressions as a biomarker for CC cell proliferation. RESULTS: MiR-195-3p was downregulated while BCDIN3D was upregulated in cervical cancer cell lines. The binding was confirmed via Luciferase Assay. RT-PCR suggested that upregulation of mir-195-3p inhibited BCDIN3D and downregulation of BCDIN3D in return induced mir-195-3p. CCK-8 pointed out that overexpression of mir-195-3p inhibited the cell viability. Ki67 protein expression was inhibited by miR-195-3p mimics or silence of BCDIN3D. CONCLUSION: The present research led us to a conclusion that mir-195-3p might inhibit cervical cancer cell proliferation and was reversely regulated by BCDIN3D. This suggests that miR-195-3p mimics/ BCDIN3D si-RNA might be used in the treatments of cervical cancer in the future after various animal assays and clinical trials.

miR-195-3p alleviates homocysteine-mediated atherosclerosis by targeting IL-31 through its epigenetics modifications.

Atherosclerosis is a serious age-related disease, which has a tremendous impact on health care globally. Macrophage inflammation is crucial for the initiation and progression of atherosclerosis, and microRNAs (miRNAs) recently have emerged as potent modulators of inflammation, while the underlying mechanisms of its involvement in homocysteine (Hcy)-mediated macrophage inflammation of atherosclerosis remain largely unknown. Here, we demonstrated that elevated Hcy inhibits the expression of miR-195-3p, which in turn enhances IL-31 expression and thereby causes the secretion of macrophages pro-inflammatory factors IL-1ß, IL-6 and TNF-α and accelerate atherosclerosis. Furthermore, we identified that Hcy can induce DNA hypermethylation and H3K9 deacetylation of miR-195-3p promoter due to the increased the binding of DNMT3a and HDAC11 at its promoter. More importantly, Sp1 interacts with DNMT3a suppressed the binding of HDAC11 at miR-195-3p promoter and promoted its transcription. In summary, our results revealed a novel mechanism that transcriptional and epigenetic regulation of miR-195-3p inhibits macrophage inflammation through targeting IL-31, which provides a candidate diagnostic marker and novel therapeutic target in cardiovascular diseases induced by Hcy.

Chemokine CCL4 Induces Vascular Endothelial Growth Factor C Expression and Lymphangiogenesis by miR-195-3p in Oral Squamous Cell Carcinoma.

The inflammatory chemokine (C-C motif) ligand 4 (CCL4) plays an important role in the pathogenesis and progression of cancer. In particular, higher serum CCL4 levels in patients with oral squamous cell carcinoma (OSCC) are associated with a more advanced stage of disease. OSCC accounts for approximately 95% of oral cancer in Taiwan and has a poor prognosis, due to aggressive local invasion and metastasis, leading to recurrence. OSCC spreads preferentially through lymphatic vessels and has the propensity to metastasize to the cervical lymph nodes even in the early stage of disease. Vascular endothelial growth factor C (VEGF-C) is an essential regulator of lymphangiogenesis. In particular, VEGF-C is specific to lymphatic vessel development, and VEGF-C expression levels have been found to directly correlate with lymph node metastasis in OSCC. However, it is unclear as to whether CCL4 correlates with VEGF-C expression and lymphangiogenesis in OSCC. We found that CCL4 increased VEGF-C expression and promoted lymphangiogenesis in oral cancer cells in vitro and in vivo. miR-195-3p mimic reversed CCL4-enhanced VEGF-C expression. CCL4 stimulation of oral cancer cells augmented JAK2 and STAT3 phosphorylation. Thus, CCL4 may be a new molecular therapeutic target for inhibition of lymphangiogenesis and metastasis in OSCC.