CFTR activation suppresses glioblastoma cell proliferation,migration and invasion

The aim of this study was to investigate the function of Cystic fibrosis transmembrane conductance regulator (CFTR) in human glioblastoma (GBM) cells. Data dining results of the Human Protein Atlas showed that low CFTR expression was associated with poor prognosis for GBM patients. We found that CFTR protein expression was lower in U87 and U251 GBM cells than that in normal humane astrocyte cells. CFTR activation significantly reduced GBM cell proliferation. In addition, CFTR activation significantly abrogated migration and invasion of GBM cells. Besides, CFTR activator Forskolin treatment markedly reduced MMP-2 protein expression. These effects of CFTR activation were significantly inhibited by CFTR inhibitor CFTRinh-172 pretreatment. Our findings suggested that JAK2/STAT3 signaling was involved in the anti-glioblastoma effects of CFTR activation. Moreover, CFTR overexpression in combination with Forskolin induced a synergistic anti-proliferative response in U87?cells. Overall, our findings demonstrated that CFTR activation suppressed GBM cell proliferation, migration and invasion likely through the inhibition of JAK2/STAT3 signaling.     

MiR-1266 promotes cell proliferation,migration and invasion in cervical cancer by targeting DAB2IP

Cervical cancer (CC) is one of the most prevalent cancers in women in the world. However, the pathogenesis is still very unclear, and the current screening methods are too expensive. Emerging evidence shows that miR-1266 has great influence on tumor cell migration and invasion. In order to clarify the role of miR-1266 in CC, we collected serum from CC, high-grade squamous intraepithelial lesion (HSIL), low-grade squamous intraepithelial lesion (LSIL) and normal control (NC), collected tissues from CC and control group (CG), and followed up 50 CC patients. We used HeLa and SiHa cells to clarify the roles of miR-1266 on cell proliferation, migration and invasion. The CC mouse model was conducted to prove the role of miR-1266 on tumorigenesis. qRT-PCR was used to measure the expressions of miR-1266 and DAB2IP mRNA. Western blot was used to determine the expression of DAB2IP protein. Cell counting kit-8 proliferation assay (CCK-8), Colony formation assay, Wound-healing assay and Transwell invasion assay were used to determine the cell survival, proliferative, migrative and invasive abilities. Our study found that miR-1266 had a rising trend in serum from NC to LSIL to HSIL to CC, and increased in CC tissues. High expression serum miR-1266 had lower overall survival rates than patients with miR-1266 low expression. MiR-1266 promoted cell viability, proliferation, migration and invasion by targeting DAB2IP. And miR-1266 could promote tumorigenesis in vivo. In conclusion, miR-1266 could be used as a new biomarker for diagnosis, prediction and treatment of CC in the future.     

MicroRNA-320a inhibits cell proliferation,migration and invasion by targeting BMI-1 in nasopharyngeal carcinoma

In the present study, we investigated the roles and molecular mechanisms of miR-320a in human nasopharyngeal carcinoma (NPC). miR-320a expression was strongly reduced in NPC tissues and cell lines. Overexpression of miR-320a significantly suppressed NPC cell growth, migration, invasion and tumor growth in a xenograft mouse model. A luciferase reporter assay revealed that miR-320a could directly bind to the 3′ UTR of BMI-1. Overexpression of BMI-1 rescued miR-320a-mediated biological function. BMI-1 expression was found to be up-regulated and inversely correlated with miR-320a expression in NPC. Collectively, our data indicate that miR-320a plays a tumor suppressor role in the development and progression of NPC and may be a novel therapeutic target against NPC.     

MiR-152 reduces human umbilical vein endothelial cell proliferation and migration by targeting ADAM17

As a cleavage enzyme of precursor TNF-α, the high expression level of ADAM17 in endothelial cells is an important factor in atherosclerosis. In this study, we demonstrate that ADAM17 is the target of miR-152. We found that miR-152 could reduce TNF precursor cleavage and inhibit cell proliferation and migration by targeting ADAM17 in human umbilical vein endothelial cells (HUVECs). Furthermore, the expression pattern of miR-152 and corresponding target ADAM17 was opposite in HUVECs under hypoxic conditions. The levels of circulating miR-152 in AS patient sera were lower than those detected in the sera of normal individuals. Our results indicate that miR-152 may be involved in the development of human atherosclerosis and could be used as diagnostic biomarker or therapeutic target in atherosclerosis.     

MiR-517a-3p accelerates lung cancer cell proliferation and invasion through inhibiting FOXJ3 expression

Aims

Aberrant expression of microRNAs (miRNAs) results in alterations of various biological processes (e.g., cell cycle, cell differentiation, and apoptosis) and cell transformation. Altered miRNAs expression was associated with lung carcinogenesis and tumor progression. This study aimed to investigate the function and underlying molecular events of miR-517a-3p on regulation of lung cancer cell proliferation and invasion.

Main methods

Transfected miR-517a-3p mimics or inhibitors into 95D and 95C cells respectively, the effects of miR-517a-3p on lung cancer cell proliferation, migration, and invasion were detected. Bioinformatics software forecasted potential target genes of miR-517a-3p and dual luciferase reporter gene system and western blot verified whether miR-517a-3p regulates FOXJ3 expression directly.

Key findings

MiR-517a-3p was differentially expressed in lung cancer 95D and 95C cell lines that have different metastatic potential. Manipulation of miR-517a-3p expression changed lung cancer cell proliferation, migration and invasion capacity. MiR-517a-3p directly regulated FOXJ3 expression by binding to FOXJ3 promoter.

Significance

This study demonstrated that miR-517a-3p promoted lung cancer cell proliferation and invasion by targeting of FOXJ3 expression.     

Cell quiescence correlates with enhanced glioblastoma cell invasion and cytotoxic resistance

Glioblastoma (GBM) tumor cells exhibit drug resistance and are highly infiltrative. GBM stem cells (GSCs), which have low proliferative capacity are thought to be one of the sources of resistant cells which result in relapse/recurrence. However, the molecular mechanisms regulating quiescent-specific tumor cell biology are not well understood. Using human GBM cell lines and patient-derived GBM cells, Oregon Green dye retention was used to identify and isolate the slow-cycling, quiescent-like cell subpopulation from the more proliferative cells in culture. Sensitivity of cell subpopulations to temozolomide and radiation, as well as the migration and invasive potential were measured. Differential expression analysis following RNAseq identified genes enriched in the quiescent cell subpopulation. Orthotopic transplantation of cells into mice was used to compare the in vivo malignancy and invasive capacity of the cells. Proliferative quiescence correlated with better TMZ resistance and enhanced cell invasion, in vitro and in vivo. RNAseq expression analysis identified genes involved in the regulation cell invasion/migration and a three-gene signature, TGFBI, IGFBP3, CHI3L1, overexpressed in quiescent cells which correlates with poor GBM patient survival.     

miR-363-3p靶向 TWIST1调控上皮间质转化抑制非小细胞肺癌A549细胞迁移和侵袭

目的探讨miR-363-3p靶向TWIST1调控非小细胞肺癌A549细胞迁移和侵袭的分子机制。 方法将体外培养的A549细胞分为空白对照组（细胞未做任何处理）;模拟物对照组（转染miR-363-3p模拟物阴性对照）;模拟物组（转染miR-363-3p模拟物）;后期实验另设：模拟物+ pcDNA组（转染miR-363-3p模拟物和pcDNA3.1空载体质粒）;模拟物+ TWIST1组（转染miR-363-3p模拟物和pcDNA3.1-TWIST1过表达质粒）。采用RT-PCR检测细胞中miR-363-3p的表达,Western blot检测细胞中TWIST1蛋白和上皮间质转化（EMT）相关蛋白Vimentin、E-cadherin的表达,Transwell小室实验检测细胞的迁移和侵袭。双荧光素酶报告基因实验检测miR-363-3p和TWIST1的靶向关系。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果与空白对照组和模拟物对照组比较,模拟物组A549细胞中miR-363-3p表达水平（1.00±0.08、0.97±0.05比3.82±0.45）升高,TWIST1 mRNA （1.00±0.06、0.98±0.06比0.39±0.02）和蛋白的表达水平（0.81±0.05、0.78±0.06比0.42±0.02）降低,差异具有统计学意义（P均< 0.05）。与空白对照组和模拟物对照组比较,模拟物组A549细胞Vimentin蛋白表达水平（0.58±0.04、0.55±0.05比0.36±0.03）降低,E-cadherin蛋白的表达水平（0.22±0.02、0.25±0.03比0.47±0.03）增高,迁移细胞数（85.75±5.45、83.52±6.85比53.05±4.50）和侵袭细胞数（128.26±6.15、125.95±8.05比71.64±5.75）降低,差异有统计学意义（P均< 0.05）。与模拟物对照组比较,模拟物组细胞中Vimentin蛋白的表达水平（0.55±0.05比0.36±0.03）降低,而E-cadherin蛋白表达水平（0.25±0.03比0.47±0.03）升高,迁移细胞数（83.52±6.85比53.05±4.50）和侵袭细胞数（125.95±8.05比71.64±5.75）均减少（P均< 0.05）。TWIST1是miR-363-3p潜在的靶基因。与模拟物+ pcDNA3.1组比较,模拟物+ TWIST1组A549细胞中Vimentin蛋白的表达水平（0.32±0.02比0.42±0.03）升高,而E-cadherin蛋白表达水平（0.56±0.04比0.38±0.03）降低,A549细胞的迁移数（49.45±4.22比67.52±5.05）和侵袭数（72.45±5.73比108.35±6.56）增加（P均< 0.05）。 结论miR-363-3p可通过靶向TWIST1调控EMT抑制A549细胞迁移和侵袭。     

MiR-34a inhibits the proliferation,migration, and invasion of oral squamous cell carcinoma by directly targeting SATB2

In various kinds of carcinomas, the special AT-rich sequence-binding protein 2 (SATB2) with its atypical expression promotes the metastasis and progression of the tumor, though in the oral squamous cell carcinoma (OSCC) its inherent mechanism and the status of SATB2 remain unclear. The role played by the SATB2 expression in the OSCC cell lines and tissue samples in the target of miR-34a downstream is the intended endeavor of this study. In te OSCCs the miR-34a expression was determined by quantitative real-time polymerase chain reaction (q-PCR), while the SATB2 expression in the cell lines and tissue samples in OSCC was analyzed with the q-PCR and the western blot. Studies in both in vitro and in vivo of the effects of miR-34a on the initiation of OSCC were conducted. As a direct target of the miR-34a the SATB2 was verified with the luciferase reporter assay. In cases where the miR-34a levels were low, the SATB2 in OSCCs seemed to be overexpressed. Besides, both in the in vitro and in vivo a suppression of migration, invasion, and cell growth was caused by miR-34a by down regulating the SATB2 expression. The SATB2 being a direct target of miR-34a was confirmed by the cotransfection of miR-34a mimics specifically the decrease in the expression of luciferase of SATB2–3′UTR-wt reporter. As a whole, our study confirmed the inhibition of miR-34a in the invasion, proliferation, and migration of the OSCCs, playing a potential tumor suppressor role with SATB2 as its downstream target.     

MicroRNA-497 inhibition of ovarian cancer cell migration and invasion through targeting of SMAD specific E3 ubiquitin protein ligase 1

Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. Understanding the molecular mechanism underlying ovarian cancer progression facilitates the development of promising strategy for ovarian cancer therapy. Previously, we observed frequent down-regulation of miR-497 expression in ovarian cancer tissues. In this study, we investigated the role of miR-497 in ovarian cancer metastasis. We found that endogenous miR-497 expression was down-regulated in the more aggressive ovarian cancer cell lines compared with the less aggressive cells. Exogenous expression of miR-497 suppressed ovarian cancer cell migration and invasion, whereas reduction of endogenous miR-497 expression induced tumor cell migration and invasion. Mechanistic investigations confirmed pro-metastatic factor SMURF1 as a direct target of miR-497 through which miR-497 ablated tumor cell migration and invasion. Further studies revealed that lower levels of miR-497 expression were associated with shorter overall survival as well as increased SMURF1 expression in ovarian cancer patients. Our results indicate that down-regulation of miR-497 in ovarian cancer may facilitate tumor metastasis. Restoration of miR-497 expression may be a promising strategy for ovarian cancer therapy.     

miR-98-5p靶向PYGO2基因对宫颈癌细胞增殖、迁移和侵袭的影响及其作用机制研究

目的探讨miR-98-5p对宫颈癌细胞增殖、迁移和侵袭的影响以及其作用机制。 方法选取2016年1月至2019年1月郴州市第一人民医院收治的宫颈癌患者的癌组织和癌旁组织;予以miR-98-5p、si-PYGO2及anti-miR-98-5p单独或共培养Siha细胞,记为：miR-NC组、miR-98-5p组、si-NC组、si-PYGO2组、anti-miR-NC组、anti-miR-98-5p组、miR-98-5p+pcDNA组、miR-98-5p+pcDNA-PYGO2组。运用qRT-PCR检测宫颈癌组织和细胞中miR-98-5p和PYGO2 mRNA的表达水平;Western blot检测蛋白表达;MTT法检测细胞增殖活性;Transwell检测细胞迁移和侵袭;将WT-PYGO2、MUT-PYGO2分别与miR-NC、miR-98-5p共转染至Siha细胞中,双荧光素酶报告基因检测实验检测荧光活性。采用方差分析和t检验进行统计学分析。 结果与癌旁组织相比,宫颈癌组织中miR-98-5p表达水平降低（0.98±0.08比0.47±0.05）,PYGO2 mRNA （1.00±0.07比2.43±0.24）和蛋白表达水平（0.27±0.03比0.62±0.05）均升高（P均< 0.001）。与正常宫颈细胞Ect1/E6E7相比,宫颈癌细胞Siha、Hela、Caski中PYGO2 mRNA （0.98±0.09比2.76±0.23、2.46±0.24、2.55±0.21）和蛋白表达水平（0.21±0.03比0.62±0.06、0.51±0.05、0.57±0.06）升高;miR-98-5p的表达水平降低（1.00±0.08比0.34±0.04、0.56±0.05、0.46±0.04） （P均< 0.05）。与miR-NC组相比,miR-98-5p组宫颈癌Siha细胞活性（48 h：0.61±0.05比0.42±0.04,72 h：1.02±0.09比0.59±0.06）、迁移数量[ （112.46±10.27）个比（48.35±4.96）个]及侵袭数量[ （92.47±9.56）个比（39.46±3.52）个]均降低（P均< 0.05）。与si-NC组相比,si-PYGO2组宫颈癌Siha细胞活性（48 h：0.64±0.06比0.46±0.05,72 h：1.05±0.08比0.67±0.06）、Siha迁移数量[ （106.48±9.75）个比（42.16±4.25）个]和侵袭数量[ （87.63±8.11）个比（35.42±6.20）个]均降低（P均< 0.05）;Cyclin D1、MMP-2、MMP-9、MMP-14表达水平降低,p21、p27表达水平升高,差异有统计学意义（P均< 0.05）。与miR-NC组比较,miR-98-5p组转染WT-PYGO2的Siha细胞荧光素酶活性（0.38±0.04比0.99±0.08）降低（P < 0.05）,转染MUT-PYGO2的Siha细胞荧光素酶活性（1.03±0.08比1.01±0.09）差异无统计学意义（P > 0.05）。PYGO2过表达逆转了miR-98-5p过表达对宫颈癌Siha细胞的增殖、迁移和侵袭的抑制作用。 结论miR-98-5p可抑制宫颈癌细胞增殖、迁移和侵袭,其机制可能与其靶向调控PYGO2的表达有关,将可为宫颈癌的预防和治疗提供新靶点。     

MiR-26a regulates the expression of serum IGF-1 in patients with osteoporosis and its effect on proliferation and apoptosis of mouse chondrocytes

Objectives:To examine the effects of the regulation on IGF-1 by miR-26a on the serum of patients with osteoporosis (OP) and apoptosis and proliferation of chondrocytes of mice with OP.Methods:Totally 47 patients with OP treated in our hospital between July 2018 and November 2019 were selected as the research group, and 42 healthy individuals in physical examination over this period were selected as the control group. Serum was sampled from each participant in both groups, and miR-26a in the sampled serum was quantified and compared. In addition, chondrocytes were sampled from mice with OP. The changes of proliferation and apoptosis of the chondrocytes were analyzed via MTT and flow cytometry, and the levels of Caspase3, Caspase9, Bax, and Bcl-2 were quantified by western blot (WB) assay.Results:MiR-26a was expressed highly in the serum of patients with OP and chondrocytes of mice with OP, while IGF-1 was lowly expressed in them. According to the dual-luciferase reporter assay, there was a targeting correlation between miR-26a and IGF-1, and suppressing miR-26a significantly up-regulated the expression and protein level of IGF-1.Conclusions:MiR-26a can serve as a biological marker for the diagnosis of OP, and it can suppress the proliferation of chondrocytes and promote their apoptosis by regulating IGF-1.     

MiR-92a regulates brown adipocytes differentiation,mitochondrial oxidative respiration,and heat generation by targeting SMAD7

Brown adipocytes are rich in mitochondria and linked to the body's blood fat levels and obesity. MiR-92a is negatively correlated with the activity of brown adipocytes. This study aimed to explore the mechanism of miR-92a on brown adipocytes. The expression of miR-92a in C2C12 cell was detected by a quantitative real-time-polymerase chain reaction (qRT-PCR). C2C12 cells were induced to brown adipocytes. The direct target gene of miR-92a was determined using the dual-luciferase reporter assay. Brown adipocytes were treated with isoprenaline (Iso) and transfected by miR-92a inhibitor and siSMAD7. The expression of heat-producing genes and adipose differentiation genes related to brown adipocytes were detected by qRT-PCR and Western blot analysis. The expression of SMAD7, p-SMAD2, and p-SMAD3 were detected using Western blot analysis. The mitochondrial content was measured by mitotracker fluorescent staining. MiR-92a inhibitor significantly decreased the expression of miR-92a in C2C12 cells. MiR-92a inhibitor could upregulate the expression of Ucp1, Cox7a1, Elovl3, Ppargc1α, PPARγ, and FABP4, and its effect on Ucp1 was increased after the treatment of isoprenaline. Moreover, miR-92a inhibitor increased mitochondrial content, oxygen consumption rate (OCR) and the expression of SMAD7 and suppressed the expressions of p-SMAD2 and p-SMAD3, whereas miR-92a directly targeted SMAD7 to exert its inhibitory effects. SiSMAD7 reversed the effects of the inhibitor on heat-producing genes, mitochondrial content, OCR and the expressions of SMAD7, p-SMAD2, and p-SMAD3 in brown adipocytes. Blocking miR-92a might promote brown adipocytes differentiation, mitochondrial oxidative respiration, and thermogenesis by targeting SMAD7 to inhibit the expressions of p-SMAD2 and p-SMAD3.     

GOLPH3 regulates the migration and invasion of glioma cells though RhoA

Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in the development of several human cancers. However, the biological significance of GOLPH3 in glioma progression remains largely unknown. In this study, we report, for the first time, that downregulation of GOLPH3 led to clear reductions in glioma cell migration and invasion. In addition, downregulation of GOLPH3 inhibited the expression of the small GTPase RhoA as well as cytoskeletal reorganization, which are both required for glioma cell migration. Furthermore, we found that the observed reductions in glioma cell migration and RhoA level could be rescued by RhoA overexpression. Taken together, these results show that GOLPH3 contributes to the motility of glioma cells by regulating the expression of RhoA.     

MiR-18b suppresses high-glucose-induced proliferation in HRECs by targeting IGF-1/IGF1R signaling pathways

MicroRNAs (miRNAs) are important for the proliferation of endothelial cells and have been shown to be involved in diabetic retinopathy (DR). In previous study, we found that miRNAs might play a critical role in hyperglycemia-induced endothelial cell proliferation based on miRNA expression profiling. Here, the roles of microRNA-18b (miR-18b) in the proliferation of human retinal endothelial cells (HRECs) were investigated in an in vitro model of HRECs grown in high glucose. We identified that levels of miR-18b were decreased in high-glucose-induced HRECs, compared with those in cells incubated in normal glucose. However, the reduction of miR-18b up-regulated vascular endothelial growth factor (VEGF) secretion and promoted effects on in vitro proliferation of HRECs. Mechanistically, insulin growth factor-1 (IGF-1) was identified as a target of miR-18b. IGF-1 simulation could antagonize the effect induced by miR-18b up-regulation, promoting cell proliferation and increasing VEGF production. In contrast, the opposite results were observed with silencing IGF-1, which was consistent with the effects of miR-18b overexpression. MiR-18b exerted its function on VEGF synthesis and cell proliferation by suppressing the IGF-1/insulin growth factor-1 receptor (IGF1R) pathway, consequently inhibiting the downstream phosphorylation of Akt, MEK, and ERK. Hence, this may provide a new insight into understanding the mechanism of DR pathogenesis, as well as a potential therapeutic target for proliferative DR.     

MiR-92a inhibits porcine ovarian granulosa cell apoptosis by targeting Smad7 gene

Smad7 has a key role in apoptosis of mammalian ovarian granulosa cells (GCs), as it antagonizes and fine-tunes transforming growth factor β (TGFβ) signaling. This study demonstrates that miR-92a regulates GC apoptosis in pig ovaries by targeting Smad7 directly. The expression level of miR-92a was down-regulated in atretic porcine follicles, whereas miR-92a expression led to inhibition of GC apoptosis. The Smad7 gene was identified as a direct target of miR-92a using a dual-luciferase reporter assay. Transfection of GCs with miR-92a mimics decreased Smad7 mRNA and protein levels, whereas expression of an miR-92a inhibitor in GCs had the opposite effect. In addition, knockdown of Smad7 prevented GC apoptosis in cells that expressed the miR-92a inhibitor.