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Abstract Background: Circular RNA (circRNA), a group of non-coding RNA, is pivotal in the progression of various cancers, including Non-Small Cell Lung Cancer (NSCLC). Some circRNAs have been reported to be implicated in the progression of NSCLC, however, the function and molecular mechanism of hsa_circ_0000317 (circ_0000317) in NSCLC have not been fully understood. Methods: The significantly differentially expressed circRNA in NSCLC tissues, circ_0000317, was screened out by microarray. Circ_0000317, microRNA(miR)-494-3p and Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) expressions in NSCLC tissues were respectively probed by quantitative real-time polymerase chain reaction and western blot assay. MTT and Transwell assays were adopted to examine the growth, migration, and invasion of NSCLC cells. Bioinformatics, luciferase reporter gene assay, RNA immunoprecipitation, and RNA pull-down assay were conducted to probe the relationships among circ_0000317, miR-494-3p, and PTEN. Results: Circ_0000317 expression level was reduced in NSCLC tissues and cell lines. Circ_0000317 expression in NSCLC patients was associated with TNM stage and lymphatic metastasis. Circ_0000317 overexpression restrained the proliferation, migration, and invasion of NSCLC cells, but co-transfection of miR-494-3p mimics partially reversed this effect. In addition, circ_0000317, was identified as a competitive endogenous RNA, which could sponge miR-494-3p to increase PTEN expression and activate PI3K/AKT pathway. Conclusion: Circ_0000317, inhibits NSCLC progression via modulating miR-494-3p/PTEN/PI3K/AKT pathway.
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Objective:To explore the mechanism of miR-494 negatively regulating ROCK1 and PTEN in inhibiting apoptosis of pancreatic cells and participating in the occurrence and development of acute pancreatitis.Methods:Pancreatic acinar cells AR42J from rats were treated by caerulein, and then the levels of amylase, tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1) and IL-6 in the supernatant of cell culture were detected by ELISA to verify the cell model of acute pancreatitis. RT-PCR was used to detect the expression of miR-494 in normal AR42J cells (control group) and acute pancreatitis cell model (model group). Flow cytometry was used to detect the apoptosis of the control group, negative control miRNA transfected acute pancreatitis cell model (negative control group) and miR-494 transfected acute pancreatitis cell model (miR-494 transfection group). Western blot was used to detect the expression of ROCK1 and PTEN in the control group, negative control group and miR-494 transfection group.Results:The levels of amylase, TNF-α, IL-1 and IL-6 in the supernatant of AR42J cells treated with caerulein for 8 h and 12 h were significantly higher than those at 0 h and the control group ( P<0.05), indicating that the model was successfully constructed. The expression levels of miR-494 at 8 h, 12 h and 24 h after the establishment of acute pancreatitis cell model were significantly higher than those at 4 h and the control group ( P < 0.05). The apoptosis rate of the model group was significantly higher than that of the control group ( P<0.05), and the apoptosis rate of the miR-494 transfection group was significantly lower than that of the model group ( P<0.05). The expression levels of ROCK1 and PTEN in the miR-494 transfection group were significantly lower than those in the model group and negative control group ( P<0.05). Conclusions:When acute pancreatitis occurs, overexpression of miR-494 can inhibit the expression of pro-apoptotic protein, thus inhibiting the apoptosis of pancreatic acinar cells and promoting the development of acute pancreatitis.
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Objective To investigate the effect and related mechanism of microRNA (miR)-494 on the hepatic ischemia-reperfusion injury (HIRI). Methods Twenty-four male SD rats were randomly divided into four groups (n=6 in each group). In the sham operation group, abdominal surgery without hepatic ischemia-reperfusion was performed. In the HIRI group, partial liver ischemia was performed for 60 min, followed by 6 h perfusion. In the HIRI+agomir-miR-494 group, intraperitoneal injection of agomir-miR-494 (20 μL) was daily given within preoperative 7 d. In HIRI+agomir-NC group, an equivalent quantity of agomir-NC was daily injected intraperitoneally within preoperative 7 d. The expression level of miR-494 messenger RNA(mRNA) in the liver tissues in each group was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression levels of liver injury and oxidative stress related indexes were measured by relevant kits. The histopathological changes of the liver in each group were observed. The quantity of apoptotic cells and cytoplasmic histone-related DNA fragments in the liver tissues of rats was detected by relevant kits. The expression levels of the proteins related to the phosphatidylinositol-3-kinase(PI3K)/protein kinase(AKT) signaling pathway were measured by Western blot. Results The expression level of miR-494 mRNA in the rat liver tissues in the HIRI+agomir-miR-494 group was significantly higher than that in the HIRI+agomir-NC group (P < 0.01). The levels of the serum liver injury and oxidative stress related indexes in the HIRI+agomir-miR-494 group were significantly lower than those in the HIRI+agomir-NC group (all P < 0.01). Compared with those in the HIRI+agomir-NC group, the quantity of cellular necrosis was significantly reduced, the cell integrity was considerably increased and the quantity of TUNELpositive cells was evidently decreased in the HIRI+agomir-miR-494 group (all P < 0.05). The expression levels of poly ADP-ribose polymerase(PARP), cysteinyl aspartate specific proteinase-3(Caspase-3) and Bax in the HIRI+agomirmiR-494 group were significantly lower than those in the HIRI+agomir-NC group (all P < 0.05). The quantity of DNA fragments in the HIRI+agomir-miR-494 group was significantly less than that in the HIRI+agomir-NC group (P < 0.01). The expression levels of p-AKT, p-mammalian target of rapamycin(mTOR) and p-p70S6K in the HIRI+agomir-miR-494 group were significantly higher than those in the HIRI+agomir-NC group (all P < 0.05). Conclusions miR-494 can alleviate the severity of HIRI in rats by activating the PI3K/AKT signaling pathway.
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Objective To investigate the different expression levels of death-associated protein kinase ( DAPK) related miR-191 and phosphatase and tensin homolog deleted on chromosome ten ( PTEN) related miR-494 from fine-needle aspiration biopsy ( FNAB) samples and blood of both benign and malignant thyroid nodules, and to find new clinical molecular diagnostic markers. Methods FNAB specimens and peripheral venous blood were collected from 113 patients with thyroid nodules (48 cases of malignant and suspected malignant thyroid nodules, 38 cases of nodular goiter, and 27 cases of thyroid adenomas). The expression levels of miR-191 and miR-494 were detected by realtime fluorescence quantitative reverse transcription PCR ( qRT-PCR ) . qRT-PCR were applied to detect miR-191 and miR-494 expression level in 98 patients with thyroid nodules and peripheral circulation. Receiver operating characteristic curves ( ROC curves) were used to evaluate the sensitivity and specificity of miR-191 and miR-494 to diagnose malignant thyroid nodules. Results (1) The sensitivity of FNAB in diagnosing thyroid cancer was 91. 7％ (44/48) and the specificity was 90. 9％ (30/33),the diagnostic accuracy was 91. 35％. (2) In FNAB samples, as well as in peripheral circulation, the relative expression of miR-191 in thyroid cancer group is significantly lower than that of the benign group, while the relative expression of miR-494 in thyroid cancer group is significantly higher than that of the benign group (P<0. 05). (3) The sensitivity and specificity of miR-191 and miR-494 were acceptable (area under the ROC curve>0. 7). Sensitivity and specificity of miR-191 in FNAB and peripheral circulation were 76. 9％, 73. 5％ and 61. 5％, 64. 1％; miR-494 were 63. 6％,76. 5％ and 72. 7％, 84. 6％respectively. (4) In thyroid cancer FNAB samples and peripheral circulation, the differences between the relative expression level of miR-191 and miR-494, and the clinical characteristics of age, gender, nodule size, and calcification, with or without cervical lymph node enlargement, thyroid function and thyroid antibodies with or without abnormalities were not statistically significant(P>0. 05). Conclusion MiR-191 and miR-494 can be used as molecular diagnostic markers for early diagnosis of thyroid carcinoma with adjunctive FNAB.
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Objective To investigate the expression levels of miR-494 in breast cancer tissues and cell lines,and its correlations with the clinical stages,metastasis and prognosis of breast cancer.Methods The expression levels of miR--494 in 5 breast cancer cell lines,including MDA-MB-231,MDA-MB-453,HCC-1937,MDA-MB-468 and MCF-7,and normal breast cell line HBL-100,were detected by quantitative real-time polymerase chain reaction (qRT-PCR).The breast cancer tissues and normal adjacent tissues from 54 patients with breast cancer were collected,and their expression levels of miR-494 were determined by qRT-PCR.Then,the correlations of miR-494 levels with clinical stages,metastasis and prognosis of breast cancer were analyzed.Results The expression levels of miR-494 in MDA-MB-231,MDA-MB-453,HCC-1937,MDA-MB-468 and MCF-7 cells were significantly lower than that in HBL-100 cells (t =212.9,37.73,27.53,10.61 and 19.46,respectively,and all P ＜0.05).The expression levels of miR-494 in breast cancer tissues were also significantly lower than that in normal adjacent tissues (t =5.80,P ＜ 0.01).Moreover,the expression of miR-494 was significantly related to the clinical stage (x2 =17.41,P ＜0.05),tumor grade (x2 =5.33,P ＜0.05),C-erbB-2 expression (x2 =9.83,P ＜ 0.05) and the percentage of Ki-67 positive cells ((2 =6.13,P ＜ 0.05) of breast cancer.Conclusion The expression levels of miR-494 are significantly dowrregulated in breast cancer tissues and cell lines,and related to the clinical stage,metastasis and prognosis of breast cancer,indicating that miR-494 may serve as a new biomarker for the diagnosis and prognosis of breast cancer.