miR-125a-3p调控人结肠癌细胞浸润转移的作用及机制研究

目的:探讨miR-125a-3p在结肠癌细胞浸润与转移中的作用及其可能机制。方法:通过qRT-PCR方法检测miR-125a-3p在结肠癌细胞及组织样本中的表达;在结肠癌细胞过表达或沉默miR-125a-3p后,通过平板克隆实验、MTT实验、划痕实验、Transwell实验检测结肠癌细胞增殖、迁移及侵袭能力的变化;采用Western blot方法检测miR-125a-3p过表达后相关标志分子的表达水平变化情况。结果:miR-125a-3p在结肠癌细胞及组织呈现异常低表达;过表达miR-125a-3p抑制结肠癌细胞HCT116及SW480的增殖能力;过表达或沉默miR-125a-3p分别抑制或增强结肠癌细胞的迁移与侵袭能力;过表达miR-125a-3p在mRNA及蛋白水平均能够显著抑制Snail、N-cadherin及Vimentin的表达,而增加E-cadherin的表达。结论:miR-125a-3p参与调节结肠癌细胞浸润与转移,其机制可能是通过调控上皮间质转化途径介导的。     

miR-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为研究

目的:探讨mi R-199a-3p负调控CBX7影响肺癌细胞NCI-H460的生物学行为。方法:qRT-PCR法检测并比较肺癌组织、癌旁正常组织、肺癌细胞、正常肺上皮细胞中的mi R-199a-3p m RNA相对表达量。比较远处转移肺癌组织、未转移肺癌组织中mi R-199a-3p m RNA相对表达量。qRT-PCR法、Western Blot法检测并比较肺癌组织、癌旁正常组织中的CBX7 m RNA及蛋白的表达水平。荧光素酶活性法检测mi R-199a-3p与靶基因CBX7的结合。比较mi R-199a-3p模拟物转染组与阴性对照组的肺癌细胞中的CBX7 m RNA相对表达量及CBX7蛋白表达水平。CCK8实验检测mi R-199a-3p对肺癌细胞增殖的促进作用。Tranwell实验检测mi R-199a-3p对肺癌细胞侵袭与迁移能力的影响。结果:肺癌组织中mi R-199a-3p明显高于癌旁正常组织,发生远处转移的肺癌组织中mi R-199a-3p m RNA的表达量明显高于未发生转移的肺癌组织,差异有统计学意义(P<0.001)。肺癌组织中CBX7m RNA、CBX7蛋白表达水平均明显低于癌旁正常组织,差异有统计学意义(P<0.001)。荧光素酶活性法证实mi R-199a-3p可与靶基因CBX7结合抑制CBX7的表达。肺癌细胞中mi R-199a-3p m RNA的相对表达量明显高于正常肺上皮细胞,CBX7 m RNA相对表达量明显低于正常肺上皮细胞(P<0.05)。对于肺癌细胞,mi R-199a-3p模拟物转染组的CBX7 m RNA相对表达量及CBX7蛋白表达水平均明显低于阴性对照组(P<0.001)。CCK8实验证实mi R-199a-3p能够促进肺癌细胞的增殖,Tranwell实验证实mi R-199a-3p对肺癌细胞侵袭与迁移具有积极的促进作用。结论:mi R-199a-3p在肺癌的发生发展过程中发挥重要作用,能够通过抑制CBX7基因的表达,促进肺癌细胞的增殖、侵袭和转移。     

LncRNA SNHG12 promotes proliferation and epithelial mesenchymal transition in hepatocellular carcinoma through targeting HEG1 via miR-516a-5p

Hepatocellular carcinoma (HCC) is the most common cancer and its prognosis is poor due to metastasis and recurrence. EMT is associated with metastasis. A deep understanding of regulatory mechanism of EMT is critical. LncRNA is involved in regulation of various biological processes including EMT. This study aimed to investigate the regulatory signal axis among lncRNA SNHG12, miR-516a-5p and the target gene HEG1 during EMT. Cell cycle and apoptosis were analyzed by flow cytometry. Tumorigenesis was analyzed by clone formation assay. Wound healing assay and transwell assay was performed to detect migration and invasion, respectively. Interaction among SNHG12, miR-516a-5p and HEG1 were analyzed by dual luciferase assay and RIP assay. We also detected expression of RNA and protein by QPCR and western blotting. Finally, tumor growth was analyzed by tumorigenesis assay in vivo. Ki-67 and HEG1 level in tumor tissues was analyzed by IHC. SNHG12 and HEG1 were upregulated, miR-516a-5p was downregulated in HCC cell lines. SNHG12 could interact with and inhibit miR-516a-5p. MiR-516a-5p could interact with HEG1 and inhibit HEG1 expression. Knock down SNHG12 inhibited proliferation, migration, invasion, EMT and promoted apoptosis of HCC cells. Such effects were antagonized by inhibiting miR-516a-5p. SNHG12 overexpression lead to opposite results. Similar results were observed in mice. SNHG12 could promote EMT in HCC through targeting and inhibiting miR-516a-5p, which eventually upregulated HEG1 expression, in both cell and mice.     

In vitro and in vivo anti-metastatic effect of the alkaliod matrine from Sophora flavecens on hepatocellular carcinoma and its mechanisms

BackgroundsHepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer with high metastasis and recurrence rates. Hypoxia-induced miRNAs and HIF-1α are demonstrated to play essential roles in tumor metastasis. Matrine (C₁₅H₂₄N₂O), an alkaloid extracted from Sophora flavescens Aiton, has been used as adjuvant therapy for liver cancer in China. The anti-metastasis effects of matrine on HCC and the underlying mechanisms remain poorly understood.PurposeWe aimed to investigate the effects of matrine on metastasis of HCC both in vitro and in vivo, and explored whether miR-199a-5p and HIF-1α are involved in the action of matrine.MethodsMTT method, colony formation, wound healing and matrigel transwell assays were performed to evaluate the effects of matrine on cell proliferation, migration and invasion. Nude mice xenograft model and immunohistochemistry (IHC) assay were employed to investigate the anti-metastatic action of matrine in vivo. Quantitative real-time PCR, western blot and dual luciferase reporter assay were conducted to determine the underlying mechanisms of matrine.ResultsMatrine exerted stronger anti-proliferative action on Bel7402 and SMMC-7721 cells under hypoxia than that in normoxia. Both matrine and miR-199a-5p exhibited significant inhibitory effects on migration, invasion and EMT in Bel7402 and SMMC-7721 cells under hypoxia. Further study showed that miR-199a-5p was downregulated in HCC cell lines, and this microRNA was identified to directly target HIF-1α, resulting in decreased HIF-1α expression. Matrine induced miR-199a-5p expression, decreased HIF-1α expression and inhibited metastasis of Bel7402 and SMMC-7721 cells, while miR-199a-5p knockdown reversed the inhibitory effects of matrine on cell migration, invasion, EMT and HIF-1α expression. In vivo, matrine showed significant anti-metastatic activity in the nude mouse xenograft model. H&E and IHC analysis indicated that lung and liver metastasis nodules were reduced, and the protein expression of HIF-1α and Vimentin were significantly decreased by i.p injection of matrine.ConclusionsMatrine exhibits significant anti-metastatic effect on HCC, which is attributed to enhanced miR-199a-5p expression and subsequently impaired HIF-1α signaling and EMT. These findings suggest that miR-199a-5p is a potential therapeutic target of HCC, and matrine may represent a promising anti-metastatic medication for HCC therapy.     

Upregulation of miR-199a-5p Protects Spinal Cord Against Ischemia/Reperfusion-Induced Injury via Downregulation of ECE1 in Rat

Ischemia–reperfusion (I/R)-induced spinal cord injury can cause apoptotic damage and subsequently act as a blood–spinal cord barrier damage. MicroRNAs (miRNAs) contributed to the process of I/R injury by regulating their target mRNAs. miR-199a-5p is involved in brain and heart I/R injury; however, its function in the spinal cord is not yet completely clarified. In this study, we investigated the role of miR-199a-5p on spinal cord I/R via the endothelin-converting enzyme 1, especially the apoptosis pathway. In the current study, the rat spinal cord I/R injury model was established, and the Basso Beattie Bresnahan scoring, Evans blue staining, HE staining, and TUNEL assay were used to assess the I/R-induced spinal cord injury. The differentially expressed miRNAs were screened using microarray. miR-199a-5p was selected by unsupervised hierarchical clustering analysis. The dual-luciferase reporter assay was used for detecting the regulatory effects of miR-199a-5p on ECE1. In addition, neuron expression was detected by immunostaining assay, while the expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were evaluated by Western blot. The results indicated the successful establishment of the I/R-induced spinal cord injury model; the I/R induced the damage to the lower limb motor. Furthermore, 18 differentially expressed miRNAs were detected in the I/R group compared to the sham group, and miR-199a-5p protected the rat spinal cord injury after I/R. Moreover, miR-199a-5p negatively regulated ECE1, and silencing the ECE1 gene also protected the rat spinal cord injury after I/R. miR-199a-5p or silencing of ECE1 also regulated the expressions of caspase-9, Bcl-2, p-JNK, p-ERK, and ECE1 in rat spinal cord injury after I/R. Therefore, we demonstrated that miR-199a-5p might protect the spinal cord against I/R-induced injury by negatively regulating the ECE1, which could aid in developing new therapeutic strategies for I/R-induced spinal cord injury.     

MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A

In this study, we aimed to explore the association between miR-99a-5p and CDC25A in breast cancer and the regulatory mechanisms of miR-99a-5p on breast cancer. The expressions of messenger RNA and microRNAs in breast cancer tissues and adjacent tissues were analyzed by the Cancer Genome Atlas microarray analysis. Quantitative real-time polymerase chain reaction was conducted to find out the expression levels of miR-99a-5p and CDC25A. The expression levels of proteins (CDC25A, ki67, cyclin D1, p21, BAX, BCL-2, BCL-XL, MMP2, and MMP9) were determined by Western blot analysis. The relationship between miR-99a-5p and CDC25A was predicted and verified by bioinformatics analysis and dual luciferase assay. After transfection, cell proliferation, invasion, and apoptosis of breast cancer tissues were, respectively, observed by cell counting kit-8 assay, transwell assay, and flow cytometry (FCM). Furthermore, the relationship among miR-99a-5p, CDC25A, and cell-cycle progression was determined by FCM assay. The nude mouse transplantation tumor experiment was performed to verify the influence of miR-99a-5p on breast cancer cell in vivo. The expression of miR-99a-5p in breast cancer tissues and cells was significantly downregulated, whereas CDC25A expression was upregulated. MiR-99a-5p targeted CDC25A and suppressed its expression in breast cancer cells. Overexpression of miR-99a-5p and decreased expression of CDC25A could suppress breast cancer cell proliferation and invasion and facilitate apoptosis. Cell-cycle progression was significantly activated by downregulated miR-99a-5p and upregulated CDC25A. Moreover, miR-99a-5p overexpression repressed the expressions of CDC25A, marker ki67, and Cyclin D1 proteins, whereas it upregulated the expression of p21 protein. MicroRNA-99a-5p suppresses breast cancer progression and cell-cycle pathway through downregulating CDC25A.     

microRNA-199a-5p suppresses glioma progression by inhibiting MAGT1

microRNAs (miRNAs) can function as a tumor suppressor or oncogenic genes in human cancers. Alternation expression of miR-199a-5p has been revealed in several human cancers. However, its expression pattern and biological roles in glioma remain unclear. Expression levels of miR-199a-5p in glioma were evaluated at first. The effects of miR-199a-5p expression on cell proliferation, migration, and invasion were investigated using the MTT assay, wound-healing assay, and transwell invasion assay. The expression of miR-199a-5p was found to be reduced in glioma cell lines. Overexpression of miR-199a-5p inhibits glioma cell proliferation, migration, and invasion in vitro. Furthermore, the target of miR-199a-5p was predicted by TargetScan and validated by luciferase activity reporter assay. We found magnesium transporter 1 (MAGT1) was a direct target of miR-199a-5p. Overexpression of MAGT1 reversed the effects of miR-199a-5p on glioma cell behaviors. Taken together, our study revealed that miR-199a-5p and MAGT1 have the potential to be used as a biomarker for glioma.     

细胞周期蛋白依赖性激酶在miR-193a5p调控卵巢癌细胞增殖及上皮细胞间充质转变中的作用*

目的: 探讨miR-193a-5p靶向CDK14并调控卵巢癌细胞OVAC的增殖和上皮间充质转变(EMT)的作用。方法: 通过TargetScanHuman分析miR-193a-5p与CDK14的匹配情况,通过荧光素酶报告系统检测miR-193a-5p靶向CDK14情况;在miR-193a-5p mimics过表达或者miR-193a-5p inhibitor基因沉默miR-193a-5p的情况下,采用免疫印迹检测CDK14,EMT相关蛋白质E-cadherin、vimentin、fibronectin和N-cadherin的表达量,采用CCK-8检测卵巢癌细胞OVAC增殖情况, MMT检测卵巢癌细胞OVAC的细胞活力。结果: miR-193a-5p靶向CDK14的3‘UTR;过表达miR-193a-5后, CDK14的表达下降,EMT相关蛋白质E-cadherin的表达上升,vimentin、fibronectin和N-cadherin的表达下降,卵巢癌细胞OVAC的增殖和细胞活力均增加;同时,基因沉默miR-193a-5p后, CDK14的表达上升,EMT相关蛋白质E-cadherin的表达下降,vimentin、fibronectin和N-cadherin的表达量上升,卵巢癌细胞OVAC的增殖和细胞活力均减少。结论: miR-193a-5p通过靶向CDK14的3‘UTR降低卵巢癌细胞OVAC的增殖、细胞活力和EMT。     

CircATRNL1 increases acid-sensing ion channel 1 to advance epithelial-mesenchymal transition in endometriosis by binding to microRNA-103a-3p

Circular RNA ATRNL1 (circATRNL1) has been implicated in epithelial-mesenchymal transition (EMT) during endometriosis. Given the existing literature and our predictions through starBase in this research, it was assumed that circATRNL1 might orchestrate the microRNA (miR)? 103a-3p/acid-sensing ion channel 1 (ASIC1) axis to control EMT in endometriosis. To verify our hypothesis, we detect circATRNL1, miR-103a-3p, and ASIC1 expression in endometrial cancer cells (HEC-B, AN3-CA, KLE, HEC1-A, and Ishikawa). Ishikawa cells with the highest circATRNL1 level were selected as subjects, where circATRNL1, miR-103a-3p, or ASIC1 expression was knocked down. Scratch and Transwell assays were applied to assess cell migration and invasion, and CCK-8 and colony formation assays to detect cell proliferation. Western blot was used to measure E-cadherin, N-cadherin, Vimentin, and Slug expression to evaluate the EMT state. Furthermore, the binding of miR-103a-3p to circATRNL1 or ASIC1 was validated by luciferase reporter assay. CircATRNL1 and ASIC1 were upregulated but miR-103a-3p was downregulated in endometrial cancer cells. Mechanistically, circATRNL1 bound to miR-103a-3p to upregulate a target gene of miR-103a-3p, ASIC1. CircATRNL1 silencing contributed to the decline of proliferation, invasion, migration, and EMT in Ishikawa cells, while miR-103a-3p inhibitor reversed those changes. In addition, the EMT process was aggravated when miR-103a-3p was inhibited and this process was suppressed by silencing ASIC1 in the presence of downregulated miR-101a-3p. Our study supported that circATRNL1 might be a novel therapeutic candidate target for endometriosis treatment and provided unique insights into the molecular basis concerning the pathogenesis of endometriosis.     

miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines

Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid (HA) was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC specimens. The ability of miR-199a-3p to selectively kill CD44+ HCC may be a useful targeted therapy for CD44+ HCC.     

Upregulation of miR-582-5p regulates cell proliferation and apoptosis by targeting AKT3 in human endometrial carcinoma

The human endometrial carcinoma is one of the most common female malignancies, and there is an urgent requirement to explore new therapeutic strategies. There is accumulating evidence that microRNAs (miRNAs) can serve as potential diagnostic and prognostic biomarkers for various types of cancer, but the significance of miR-582-5p still remains largely unknown in the endometrial carcinoma. The aims of this study were to understand and identify the influence of miR-582-5p on the proliferation and apoptosis of human endometrial carcinoma and its relevant mechanism. First, quantitative real-time PCR (qRT-PCR) was used to detect miR-582-5p and AKT3 expression in human tissue samples and cells. Then, CyQuant assay and 2D colony assay were employed to evaluate cell proliferation. Western blotting was used to determine protein expression. Subsequently, the luciferase reporter assay was used to identify the target of miR-582-5p. Finally, Annexin V assay was used to detect cell apoptosis. We found that miR-582-5p expression was significantly decreased in human endometrial carcinoma tissues, and miR-582-5p upregulation in human endometrial carcinoma cells inhibit cell proliferation and promote apoptosis. Moreover, AKT3 was validated as a target of miR-582-5p and AKT3 expression was inversely correlated with miR-582-5p expression. Besides, AKT3 upregulation efficiently abrogates the effect of miR-582-5p on the cells. These results demonstrated that miR-582-5p regulates cell proliferation and apoptosis in human endometrial carcinoma via AKT3. Thus, miR-582-5p represents a potential therapeutic target in human endometrial carcinoma meriting further investigation.     

Molecular Mechanisms of Regulation and Action of microRNA-199a in Testicular Germ Cell Tumor and Glioblastomas

MicroRNA-199a (miRNA-199a) has been shown to have comprehensive functions and behave differently in different systems and diseases. It is encoded by two loci in the human genome, miR-199a-1 in chromosome 19 and miR-199a-2 in chromosome 1. Both loci give rise to the same miRNAs (miR-199a-5p and miR-199a-3p). The cause of the diverse action of the miRNA in different systems is not clear. However, it is likely due to different regulation of the two genomic loci and variable targets of the miRNA in different cells and tissues. Here we studied promoter methylation of miR-199a in testicular germ cell tumors (TGCTs) and glioblastomas (gliomas) and discovered that hypermethylation in TGCTs of both miR-199a-1 and -2 resulted in its reduced expression, while hypomethylation of miR-199a-2 but not -1 in gliomas may be related to its elevated expression. We also identified a common regulator, REST, which preferentially bound to the methylated promoters of both miR-199a-1 and miR-199a-2. The action of miR-199a is dependent on its downstream targets. We identified MAFB as a putative target of miRNA-199a-5p in TGCTs and confirmed that the tumor suppression activity of the microRNA is mediated by its target MAFB. By studying the mechanisms that control the expressions of miR-199a and its various downstream targets, we hope to use miR-199a as a model to understand the complexity of miRNA biology.     

microRNA-199a-3p,DNMT3A,and aberrant DNA methylation in testicular cancer

It was previously demonstrated that miR-199a was downregulated in testicular germ cell tumor (TGCT), probably due to hypermethylation of its promoter. Further study found that re-expression of miR-199a in testicular cancer cells (NT2) led to suppression of cell growth, cancer migration, invasion and metastasis. More detailed analyses showed that these properties of miR-199a could be assigned to miR-199a-5p, one of its two derivatives. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this report, we identified DNA (cytosine-5)-methyltransferase 3A (DNMT3A), the de novo methyltransferase, as a direct target of miR-199a-3p using a 3′-UTR reporter assay. Transient expression of miR-199a-3p in NT2 cells led to decrease, while knocking down of miR-199a-3p in a normal human testicular cell line (HT) led to elevation, of DNMT3A2 (DNMT3A gene isoform 2) mRNA and protein levels. In clinical samples, DNMT3A2 was significantly overexpressed in malignant testicular tumor, and the expression of DNMT3A2 was inversely correlated with the expression of miR-199a-3p. However, DNMT3A did not affect miR-199a expression in NT2 cells. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation, partly through targeting DNMT3A. Overexpression of miR-199a-3p restored the expression of APC and MGMT tumor-suppressor genes in NT2 cells by affecting DNA methylation of their promoter regions. Our studies demonstrated the deregulation of miR-199a-3p expression in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potentially therapeutic use of synthetic miR-199a-3p oligonucleotides as effective hypomethylating compounds in the treatment of TGCT.     

miR-34a-5p靶向GMFB抑制细胞增殖对先天性巨结肠的治疗意义

目的探讨miR-34a-5p通过靶基因GMFB调控神经嵴细胞的增殖。 方法通过荧光定量PCR检测miR-34a-5p在先天性巨结肠症（HSCR）和正常结肠组织中的表达,并通过双荧光素酶报告基因检测miR-34a-5p的靶基因,SH-SY5Y细胞转染miR-34a-5p mimics及对照miR-NC,并转染GMFB表达载体,通过CCK8检测miR-34a-5p和GMFB对神经嵴细胞增殖的影响,并通过Western Blot检测miR-34a-5p对GMFB蛋白表达的影响。采用t检验和单因素方差分析。 结果荧光定量PCR结果显示,HSCR结肠组中miR-34a-5p的相对表达量为0.43±0.10,低于正常结肠组1.15±0.18,差异具有统计学意义（t = 3.50,P < 0.01）。CCK8结果显示,miR-34a-5p mimics组细胞在培养24?h和48?h后细胞A450值分别为0.53±0.03和0.87±0.04,低于miR-NC对照组0.87±0.03,1.42±0.04。双荧光素酶报告基因实验结果显示,miR-34a-5p mimics与GMFB 3'UTR WT载体共转染组荧光强度为0.44±0.03,低于对照miR-NC与WT载体共转染组1.02±0.06。CCK8结果所示,miR-34a-5p mimics+GMFB组细胞培养24?h和48?h后,A450值分别为0.99±0.02和1.50±0.03,高于miR-34a-5p mimics组0.53±0.03, 0.87±0.04,差异具有统计学意义（t?=?7.07,P < 0.01;t?=?9.14,P < 0.01）。Western Blot检测结果显示,miR-34a-5p mimics组细胞的GMFB蛋白表达量为0.25±0.01,低于miR-NC对照组0.90±0.03,差异具有统计学意义（t?=?35.60,P < 0.01）,miR-34a-5p mimics+GMFB组细胞GMFB蛋白表达量为1.03±0.03,高于miR-34a-5p mimics组0.25±0.01,差异具有统计学意义（t?=?42.74,P < 0.01）。 结论miR-34a-5p能够通过抑制靶基因GMFB的表达,抑制神经嵴细胞SH-SY5Y的增殖。     

miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*

目的: 探讨miR-34a-5p在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达,分析miR-34a-5p对TNBC细胞增殖、凋亡、迁移的作用,对TNBC荷瘤小鼠肿瘤生长的影响以及在TNBC中对B7-H1表达的影响。方法: 利用RT-qPCR、Western blot分析TNBC细胞中miR-34a-5p、B7-H1的表达,并利用Kaplan-Meier分析二者的表达与TNBC患者的生存关系;将miR-34a-5p转染TNBC细胞,通过CCK-8、流式细胞术及划痕实验检测miR-34a-5p对TNBC细胞增殖、凋亡、迁移的影响;利用RT-qPCR、Western blot检测miR-34a-5p、B7-H1表达水平的变化,双荧光素酶基因报告验证miR-34a-5p与B7-H1的相互作用;利用RT-qPCR、Western blot、IHC检测miR-34a-5p对MDA-MB-231荷瘤小鼠miR-34a、B7-H1表达的影响。结果: TNBC细胞中miR-34a-5p呈低表达,B7-H1呈高表达,二者均与TNBC患者的不良预后有关,差距具有统计学意义(P<0.01);miR-34a-5p抑制TNBC细胞增殖、侵袭,促进细胞凋亡,并且在TNBC细胞中靶向抑制B7-H1;miR-34a-5p agomir在体内抑制MDA-MB-231成瘤裸鼠的肿瘤生长和B7-H1表达。结论: miR-34a-5p在TNBC发生、发展中发挥着重要作用,靶向miR-34a-5p/B7-H1可能成为TNBC患者新的分子治疗策略。     

MiR-192-5p inhibits proliferation,migration, and invasion in papillary thyroid carcinoma cells by regulation of SH3RF3

Background: The decreased level of miR-192-5p has been reported in several kinds of cancers, including bladder, colon, ovarian, and non-small cell lung cancer. However, the expression and function of miR-192-5p in papillary thyroid carcinoma/cancer (PTC) remains unknown.Objective: The present study aimed to explore the function and underlying mechanism of miR-192-5p in PTC development.Methods: PTC tissues and relative normal controls from PTC patients were collected. qRT-PCR analysis was performed to measure miR-192-5p and SH3RF3 mRNA level in PTC tissues and cell lines. CCK-8 method and FCM assay were used to test cell proliferation and apoptosis in TPC-1 cells, respectively. The abilities of cell migration and invasion were detected by wound healing and transwell assays, respectively. The protein expression was evaluated by Western blot. The interaction between miR-192-5p and Src homology 3 (SH3) domain containing ring finger 3 (SH3RF3) were confirmed by dual-luciferase reporter assay.Results: MiR-192-5p level was obviously decreased in PTC tissues and cell lines. Overexpression of miR-192-5p suppressed proliferation, migration, invasion, and EMT process, while induced apoptosis in TPC-1 cells. In addition, miR-192-5p negatively modulated SH3RF3 expression by binding to its 3′-untranslated region (3′UTR). Silencing SH3RF3 inhibited the migration, invasion, and EMT of TPC-1 cells. In the meantime, matrine, an alkaloid extracted from herb, exerted its anti-cancer effects in PTC cells dependent on increase in miR-192-5p expression and decrease in SH3RF3 expression.Conclusion: We firstly declared that miR-192-5p played a tumor suppressive role in PTC via targeting SH3RF3. Moreover, matrine exerted its anti-cancer effects in PTC via regulating miR-192-5p/SH3RF3 pathway.     

An antagonism between the AKT and beta-adrenergic signaling pathways mediated through their reciprocal effects on miR-199a-5p

We have recently reported that downregulation of miR-199a-5p is necessary and sufficient for inducing upregulation of its targets, including hypoxia-inducible factor-1alpha (Hif-1α) and Sirt1, during hypoxia preconditioning (HPC). Conversely, others and we have reported that miR-199a-5p is upregulated during cardiac hypertrophy. Thus, the objective of this study was to delineate the signaling pathways that regulate the expression of miR-199a-5p and its targets, and their role in myocyte survival during hypoxia. Since HPC is mediated through activation of the AKT pathway, we questioned if AKT is sufficient for inducing downregulation of miR-199a-5p. Our present study shows that overexpression of a constitutively active AKT (caAKT) induced 70% reduction in miR-199a-5p and was associated with a robust increase in HiF-1α (10 ± 2 fold) and Sirt1 (4 ± 0.8 fold) that was reversed by overexpression of miR-199a-5p. Similarly, insulin receptor-stimulated activation of the AKT pathway induced downregulation of miR-199a-5p and upregulation of its targets. In contrast, β-adrenergic receptor (βAR) activation in vitro and in vivo, induced 1.8–3.5-fold increase in miR-199a-5p. Accordingly, we predicted that βAR would antagonize AKT-induced, miR-199a-5p-dependent, upregulation of Hif-1α and Sirt1. Indeed, pre-treating the myocytes with isoproterenol before applying HPC, caAKT, or insulin resulted in 87 ± 3%, 75 ± 15%, and 100% reductions in Hif-1α expression, respectively, and sensitized the cells to hypoxic injury. Thus, activation of beta-adrenergic signaling counteracts the survival effects of the AKT pathway via upregulating miR-199a-5p.