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Objective:To explore the value of microRNA(miR)-124-3p and its target gene aryl hydrocarbon receptor (AHR) in the diagnosis and prognostic evaluation of gastric cancer, and the related molecular mechanisms in regulating proliferation and invasion of gastric cancer cell.Methods:The clinical and prognostic characteristics of patients with gastric adenocarcinoma expressing miR-124-3p were obtained from The Cancer Genome Atlas database and Genotype-Tissue Expression database. The correlation between miR-124-3p expression level and pathological stage, TNM stage, overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI) in patients with gastric adenocarcinoma were studied by bioinformatics analysis. The interaction sites between miR-124-3p and AHR mRNA were predicted by Target Scan 7.1 online tool. The target binding sites of miR-124-3p in AHR mRNA were verified by subcutaneous tumorigenesis experiment in mice, immunohistochemistry, dual luciferase assay, quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Nine male Balb/c nude mice, aged 4 to 6 weeks with weight of (18.43±0.29) g were injected with miR-124-3p simulant (miR-124-3p group), negative control simulant (negative control group) and 0.9% sodium chloride solution (sodium chloride control group) through the tail vein. Gastric cancer cell lines (MKN-45, AGS) were transfected with RNA simulants (including miR-124-3p simulant, negative control simulant and 0.9% sodium chloride solution). The expression of AHR and Catenin β 1 gene ( CTNNB1) at mRNA level, the expression of AHR and β-catenin at protein level in 3 mice groups and the effects of miR-124-3p transfection on the proliferation and invasion of transfected gastric cancer cells were analyzed. Pearson correlation analysis and Holm-Sidak corrected multiple t test were used for statistical analysis. Results:Low expression of miR-124-3p was positively correlated with severe pathological stages and TNM stages in patients with gastric adenocarcinoma ( R2=0.83 and 0.86, P=0.031 and 0.023). High expression of miR-124-3p was positively correlated with OS, DSS and PFI ( R2=1.00, 0.99 and 0.99, P=0.029, 0.044 and 0.049). The results of subcutaneous tumorigenesis experiment in mice demonstrated that the number of apoptotic cells in the tumor of miR-124-3p group was more than that of negative control group and sodium chloride control group ((43.33±1.86)/high power field (HPF) vs. (20.00±1.73)/HPF and (18.67±1.76)/HPF), and the differences were statistically significant ( t=8.55 and 8.33, P=0.013 and 0.014). The results of immunohistochemistry showed that the optical density of AHR protein in mice tumor tissue of miR-124-3p group was lower than that of negative control group and sodium chloride control group (0.081±0.008 vs. 0.276±0.019 and 0.273±0.018), and the differences were statistically significant ( t=9.06 and 7.51, P=0.012 and 0.017). The results of dual luciferase assay indicated that the fluorescence intensity in wild-type AHR MKN-45 cells transfected with miR-124-3p simulant was lower than that of negative control group (0.293±0.020 vs. 1.000±0.032), and the difference was statistically significant ( t=18.56, P<0.001). The results of RT-qPCR demonstrated that the mRNA levels of AHR and CTNNB1 in MKN-45 cells transfected with miR-124-3p simulant were both lower than those in untreated MKN-45 cells (0.51±0.09 vs. 1.02±0.02, 0.46±0.03 vs. 1.03±0.01), and the differences were statistically significant ( t=4.51 and 16.60, P=0.046 and 0.004). The results of Western blotting experiments showed that the relative protein expression levels of AHR and β-catenin of MKN-45 cells transfected with miR-124-3p simulant were lower than those of transfected with 0.9% sodium chloride solution and negative control simulant (3 332.94±81.25 vs. 9 041.60±439.79 and 8 276.54±562.52, 2 725.79±167.57 vs. 9 701.94±410.02 and 8 081.66±275.84), and the differences were statistically significant ( t=15.49, 7.91, 17.35 and 19.42, P=0.004, 0.016, 0.003 and 0.003). Conclusions:MiR-124-3p is correlated with diagnosis and prognosis of gastric cancer. MiR-124-3p induces apoptosis of gastric cancer cells in vitro and vivo by negatively regulating AHR expression at mRNA and protein level, thereby down-regulating the expression of CTNNB1 mRNA and β-catenin pathway-related protein. Therefore, miR-124-3p may become a potential diagnostic and prognostic marker of gastric cancer.
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Objective:To explore the expression levels and the clinical significance of serum secreted frizzled-related protein 5 (SFRP5) and miR-124-3p in patients with hypertension during pregnancy.Methods:Ninety-eight patients with hypertension during pregnancy diagnosed from Jan. 2019 to Feb. 2022 were selected as the observation group. According to the degree of the condition of patients, they were divided into 41 cases of pregnancy hypertension, 32 cases of mild preeclampsia, and 25 cases of severe preeclampsia, and 80 healthy subjects during the same period were selected as the control group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression level of serum SFRP5 in patients, real-time fluorescence quantitative method (qRT-PCR) was used to detect the expression level of miR-124-3p. The relationship between SFRP5, miR-124-3p levels and clinicopathological indicators in patients with hypertension in pregnancy was analyzed, Pearson correlation analysis was used to analyze the correlation between SFRP5 and miR-124-3p. Multivariate Logistic regression was used to analyze the risk factors of hypertension in pregnancy.Results:Compared with the control group, the serum SFRP5 expression level of the observation group [ (33.78±5.21) ng/L vs (43.34±8.56) ng/L] was down-regulated, while the miR-124-3p level [ (2.16±0.41) vs (1.01±0.17) ] was up-regulated, and the serum SFRP5 level of the observation group decreased with the severity of the disease[ (38.43±6.37) ng/L (33.18±5.14) ng/L (26.94±3.38) ng/L], while the level of miR-124-3p increased with the severity of the disease[ (1.62±0.24) (2.19±0.43) (3.01±0.69) ], the difference was statistically significant ( P<0.05) . The expression levels of SFRP5 and miR-124-3p in the serum of patients with hypertension in pregnancy were related to the age, pregnancy, pre-pregnancy BMI, and fasting blood glucose level of patients ( P<0.05) , but not related to the gestational age of patients ( P>0.05) . Bioinformatics TargetScan website showed that SFRP5 and miR-124-3p had binding sites. Pearson correlation analysis showed that SFRP5 and miR-124-3p were negatively correlated ( r=-0.610, P<0.05) . Multivariate Logistic regression analysis showed that SFRP5 was a protective factor for pregnancy-induced hypertension in pregnant women, and miR-124-3p was a risk factor ( P<0.05) . Conclusion:The serum levels of SFRP5 and miR-124-3p are abnormally expressed in patients with hypertension during pregnancy, and there is a certain relationship with the degree of disease. Both are involved in the occurrence and development of hypertension during pregnancy.
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AIM: To investigate the effect of lncRNA MALAT1 on the proliferation, migration and angiogenesis of retinal vascular endothelial cells and its molecular mechanism.METHODS: The expression levels of lncRNA MALAT1 in plasma of normal control group, diabetic without retinopathy group and diabetic retinopathy group were detected by qPCR and the effect of glucose culture on the expression levels of lncRNA MALAT1 were detected by qPCR too. The expression level of miR-124-3p was detected by qRT-PCR; Western blotting was used to detect the expression level of SOX7; The targeting relationship between lncRNA MALAT1 and miR-124-3p, miR-124-3p and SOX7 were detected by the dual-luciferase reporter system; CCK-8 assay was used to detect cell proliferation activity; Transwell assay was used to detect the migration ability of cells; Angiogenesis of hRMECs cells was measured by in vitro tube formation assay.RESULTS:The expression level of lncRNA MALAT1 in plasma of diabetic retinopathy patients was significantly higher than that of diabetic without retinopathy group and normal control group(P<0.001). In vitro glucose culture significantly promoted the expression of lncRNA MALAT1 in hRMECs cells, as well as the proliferation, migration and angiogenesis of hRMECs cells(all P<0.05). Knockdown of lncRNA MALAT1 significantly inhibited the proliferation, migration and tubule formation of hRMECs cells(all P<0.05). Dual-luciferase reporter gene assay showed that lncRNA MALAT1 targeted with miR-124-3p, and miR-124-3p targeted with SOX7. Overexpression of miR-124-3p significantly inhibited the proliferation, migration and tubule formation of hRMECs cells(all P<0.05). Overexpression of lncRNA MALAT1+miR-124-3p, miR-124-3p+SOX7, and knockdown of lncRNA MALAT1+overexpression of SOX7 all significantly eliminated the inhibitory effect of hRMECs cells(all P<0.05).CONCLUSION: lncRNA MALAT1 promote the proliferation, migration and angiogenesis of retinal endothelial cells in diabetic retinopathy by down-regulating the negative regulation of miR-124-3p on SOX7. Therefore, abnormal upregulation of lncRNA MALAT1 in patients with diabetic retinopathy is a potential biomarker.
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@#［摘要］ 目的：探讨盐酸石蒜碱（lycorine hydrochloride，LH）对肝癌HCCLM3 细胞恶性生物学行为的影响及其对circASH2L/ miR-124-3p轴的调控作用。方法：将HCCLM3细胞分为不同浓度LH处理组（LH-L、LH-M、LH-H组）和Con、si-NC、si-circASH2L、 LH+pcDNA、LH+pcDNA-circASH2L 组；以CCK-8 法、平板克隆形成实验、流式细胞术、细胞划痕实验和Transwell 小室实验分别 检测HCCLM3 细胞的增殖、克隆形成、凋亡、迁移和侵袭；qPCR法检测HCCLM3 细胞中circASH2L和miR-124-3p 的表达量；双荧 光素酶报告基因实验检测circASH2L 和miR-124-3p 的靶向关系；WB 法检测cleaved-caspase3、cleaved-caspase9、E-cadherin、 N-cadherin 蛋白表达量。结果：与Con 组比较，LH不同浓度组细胞增殖抑制率升高（P<0.05），凋亡率与cleaved-caspase3、cleavedcaspase9 、E-cadherin 蛋白水平升高（均P<0.05），miR-124-3p 的表达水平升高（P<0.05），克隆形成数与侵袭细胞数减少（均 P<0.05），划痕愈合率与N-cadherin 蛋白水平降低（P<0.05），circASH2L 的表达水平降低（P<0.05），且不同浓度组间比较差异有统 计学意义（P<0.05）。circASH2L 可负向调控miR-124-3p。与si-NC 组比较，si-circASH2L 组细胞增殖抑制率升高（P<0.05），凋亡 率与cleaved-caspase3、cleaved-caspase9、E-cadherin 蛋白水平升高（均P<0.05），划痕愈合率与N-cadherin 蛋白水平降低（均 P<0.05），克隆形成数与侵袭细胞数减少（均P<0.05）。与LH+pcDNA组比较，LH+pcDNA-circASH2L 组miR-124-3p 的表达水平 降低（P<0.05），细胞增殖抑制率降低（P<0.05），凋亡率与cleaved-caspase3、cleaved-caspase9、E-cadherin 蛋白水平降低（均 P<0.05），克隆形成数与侵袭细胞数增多（均P<0.05），划痕愈合率与N-cadherin 蛋白水平升高（均P<0.05）。结论：LH可通过调控 circASH2L/miR-124-3p 轴来抑制肝癌细胞HCCLM3 的增殖、迁移、侵袭并诱导其凋亡。
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With the understanding of microRNA (miRNA or miR) functions in tumor initiation, progression, and metastasis, efforts are underway to develop new miRNA-based therapies. Very recently, we demonstrated effectiveness of a novel humanized bioengineered miR-124-3p prodrug in controlling spontaneous lung metastasis in mouse models. This study was to investigate the molecular and cellular mechanisms by which miR-124-3p controls tumor metastasis. Proteomics study identified a set of proteins selectively and significantly downregulated by bioengineered miR-124-3p in A549 cells, which were assembled into multiple cellular components critical for metastatic potential. Among them, plectin (PLEC) was verified as a new direct target for miR-124-3p that links cytoskeleton components and junctions. In miR-124-3p-treated lung cancer and osteosarcoma cells, protein levels of vimentin, talin 1 (TLN1), integrin beta-1 (ITGB1), IQ motif containing GTPase activating protein 1 (IQGAP1), cadherin 2 or N-cadherin (CDH2), and junctional adhesion molecule A (F11R or JAMA or JAM1) decreased, causing remodeling of cytoskeletons and disruption of cell-cell junctions. Furthermore, miR-124-3p sharply suppressed the formation of focal adhesion plaques, leading to reduced cell adhesion capacity. Additionally, efficacy and safety of biologic miR-124-3p therapy was established in an aggressive experimental metastasis mouse model
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Objective To investigate different expression levels between young and old bone marrow mesenchymal stem cells in microRNAs (miRNAs) that are significantly conserved between humans and mice.Additional studies have been conducted to discover changes in miRNA expression in old mice relative to that in young adults and discussed the roles of miRNAs in primary osteoporosis.Methods MiRNAs that are highly conserved between human and mice,and are expressed at significantly different levels in the bone marrow mesenchymal stem cells of young and old people were identified by searching the Gene Expression Omnibus (GEO) database.Human bone mesenchymal stem cells (hBMSCs) were transfected with miRNA mimics,and their relative alkaline phosphatase (ALP) activity levels were then determined.Micro-CT scanning was employed to quantitatively characterize cortical and cancellous bones of young and old mice,and to confirm that these mice accurately modeled natural aging osteoporosis.Simultaneously,we investigated differences in expression levels of miRNAs that influence ALP activity in hBMSCs in the two groups of mice.Correlations between miRNA expression levels,and parameters of bone mass and bone strength were studied.Results 28 miRNAs were found to be more than 2 fold up-regulated (down-regulated) with statistical significance (P＜0.05) in the GEO database.We also found that ALP activity was lower in hBMSCs transfected with 4 miRNAs (mir-124-3p,mir-126-3p,mir-128-3p,mir-424-5p,P＜0.05 or P＜ 0.01).The micro-CT scans indicated that the mice are accurately modeled natural aging osteoporosis.Expression of mir-124-3p increased significantly in older mice.This upregulation correlated positively with trabecular separation,and negatively with trabecular pattern factor in trabecular bone.However,in cortical bone,its expression correlated positively with trabecular separation,and negatively with bone volume fraction,trabecular number,and bone mineral density (P＜ 0.05).Conclusion Hsa-mir-124-3p,which is expressed differently in young and old bone marrow stromal cells,inhibited the osteogenic differentiation of hBMSCs.Upregulation of this miRNA in the bone tissue of aged mice may be related to the development of osteoporosis.
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@# Objective: To investigate the mechanism of lncRNA MALAT1 modulating proliferation and metastasis of cervical cancer cells via regulating miR-124-3p/IGF2BP1 axis. Methods: A total of 45 cases of cervical cancer tissues and corresponding paracancerous tissues resected from patients, who were admitted to the Department of Obstetrics and Gynecology of Guiyang Maternal and Child Health Hospital during April 2014 and December 2017, were included in this study; in addition, cervical cancer cell lines SiHa, Caski, HeLa and C33awere also collected for this study. qPCR was applied to detect the expression of MALAT1 in cervical cancer tissues and cell lines. MALAT1-knockdown vectors, miR-124-3p inhibitors and IGF2BP1-overexpression vectors were constructed and used to transfect cervical cancer cells, respectively; the influence of MALAT1 or MALAT1 knockdown on cell proliferation, invasion and epithelial mesenchymal transition (EMT) via miR-124-3p/IGF2BP1 axis were determined by CCK-8 assay, Transwell assay, Wb and immunofluorescence, respectively. The interaction between MALAT1, miR-124-3p, and IGF2BP1 were verified by dual luciferase reporter gene assay. Results: MALAT1 was up-regulated in cervical cancer tissues and cell lines (P<0.05 or P<0.01). Meanwhile, MALAT1 knockdown remarkably inhibited proliferation, invasion and EMT of cervical cancer cells (P<0.05 or P<0.01). Moreover, dual-luciferase reporter gene assay showed that MALAT1 directly interacted with miR-124-3p and down-regulated its expression, while miR-1243p negatively regulated IGF2BP1 expression. Our experiment further validated that MALAT1 knockdown suppressed proliferative, invasion and EMT of cervical cancer cells via inducing the inhibitory effect of miR-124-3p on IGF2BP1 (P<0.05 or P<0.01). Conclusion: MALAT1 promotes the proliferation, invasion and EMT of cervical cancer cells by down-regulating miR-124-3p/IGF2BP1 axis, which provides potential molecular targets for early diagnosis or treatment of cervical cancer.