miR-125b靶向MCL1抑制胃癌 MGC-803细胞增殖

探讨mi R-125b对胃癌MGC-803细胞增殖的影响及机制,为阐明胃癌发病的分子机制提供实验依据.采用q RT-PCR和原位杂交,检测mi R-125b在正常胃黏膜(NGM)和胃癌(GAC)组织中的表达.将mi R-125b导入胃癌MGC-803细胞,观察mi R-125b高表达对MGC-803细胞增殖的影响.利用Targetscan 6.2软件及荧光素酶报告基因检测,分析mi R-125b对MCL1基因的靶向性作用.构建MCL1干扰载体,观察干扰MCL1基因表达对MGC-803细胞增殖的影响.结果发现,mi R-125b在胃癌组织中低表达,其表达与胃癌的分化程度及患者预后呈正相关,与TNM分期、淋巴结转移呈负相关(P0.01).mi R-125b高表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01);mi R-125b与MCL1基因的3′UTR(2 613~2 620)结合,抑制MCL1的m RNA及蛋白质表达(P0.01);沉默MCL1基因表达后MGC-803细胞的增殖降低、凋亡率增加、裂解caspase-3与裂解PARP表达增加(P0.01).从而得出结论,mi R-125b在胃癌组织中低表达,其表达与胃癌组织分化程度、TNM分期、淋巴结转移及患者预后密切相关;mi R-125b靶向抑制MCL1基因表达,活化caspase-3信号通路,抑制MGC-803细胞增殖.     

The lncRNA small nucleolar RNA host gene 5 regulates trophoblast cell proliferation,invasion, and migration via modulating miR-26a-5p/N-cadherin axis

Pre-eclampsia (PE) is a pregnancy-specific disease characterized by the occurrence of hypertension and proteinuria after two weeks of gestation. Long noncoding RNAs (lncRNAs) are emerging as key regulators in PE development. This study aims to investigate the role of lncRNA, small nucleolar RNA host gene 5 (SNHG5), in the pathogenesis of PE. The expression of SNHG5 was significantly downregulated in placental tissues from patients with severe PE compared normal controls. Overexpression of SNHG5 promoted trophoblast (HTR-8/SVneo) cell proliferation, invasion, and migration, and flow cytometry results showed that SNHG5 overexpression inhibited apoptosis and caused a decrease of cell population at the G ₀/G ₁ phase and an increase of cell population at the S phase, while knockdown of SNHG5 had the opposite effects. The interaction between SNHG5 and miR-26a-5p was predicted by bioinformatics analysis and confirmed by luciferase reporter assay and RNA immunoprecipitation, and miR-26a-5p was negatively regulated by SNHG5; miR-26a-5p expression was upregulated in PE placental tissues and was inversely correlated with SNHG5 expression. Furthermore, miR-26a-5p was predicted to target the 3′ untranslated region of N-cadherin, which was confirmed by luciferase reporter assay, and miR-26a-5p overexpression suppressed N-cadherin expression in HTR-8/SVneo cells. N-cadherin mRNA expression was downregulated in PE placental tissues and was positively correlated with SNHG5 expression. Both overexpression of miR-26a-5p and knockdown of N-cadherin suppressed HTR-8/SVneo cell invasion and migration, and also attenuated the effects of SNHG5 on the cellular functions of HTR-8/SVneo cells. In conclusion, our study suggested that SNHG5 promotes trophoblast cell proliferation, invasion, and migration at least partly via regulating the miR-26a-5p/N-cadherin axis.     

A potential regulatory network among WDR86-AS1, miR-10b-3p,and LITAF is possibly involved in preeclampsia pathogenesis

Preeclampsia (PE), a pregnancy-specific disorder, is a leading cause of perinatal maternal and fetal mortality and morbidity. Impaired migration and invasion of trophoblastic cells and an imbalanced systemic maternal inflammatory response have been proposed as possible causes of pathogenesis of PE. Comparative analysis of PE-affected placentas and healthy placentas has uncovered differentially expressed long noncoding RNAs, microRNAs, and mRNAs. This study was conducted to investigate the effect of a regulatory network among these RNAs on PE pathogenesis. Long noncoding RNA WDR86-AS1, microRNA miR-10b-3p, and mRNA of protein LITAF were identified by screening of genes in existing databases with aberrant expression in PE-affected placentas and potential mutual interactions as revealed by TargetScan, miRanda, and PicTar analyses. This study identified their expression in PE-affected and healthy placentas by RT-PCR. An in vitro experiment was performed on human trophoblast HTR-8/SVneo cells cultured under normoxic or hypoxic conditions. MiR-10b-3p targets were identified in luciferase reporter assays and RNA pull-down assays. The mouse model of PE was set up using a soluble form of FLT-1 for in vivo testing. Lower levels of miR-10b-3p but higher expression of WDR86-AS1 and LITAF were observed in PE-affected placentas and trophoblast cells under hypoxia. WDR86-AS1 and LITAF mRNA were confirmed as targets of miR-10b-3p. WDR86-AS1 downregulated miR-10b-3p but promoted LITAF expression. Microarray analyses revealed that LITAF controlled the inflammatory responses and migration and proliferation of HTR-8/SVneo cells under hypoxia. Indeed, knockdown of WDR86-AS1 and LITAF or overexpression of miR-10b-3p attenuated the hypoxia-induced inhibition of cellular viability, migration, and invasion. Moreover, miR-10b-3p overexpression attenuated the pathological symptoms caused by soluble FLT-1 in vivo. In summary, the WDR86-AS1/miR-10b-3p/LITAF network is probably involved in PE pathogenesis.     

Silencing lncRNA-DGCR5 increased trophoblast cell migration,invasion and tube formation,and inhibited cell apoptosis via targeting miR-454-3p/GADD45A axis

Long noncoding RNA (lncRNA)-DGCR5 has been recognized as a potential tumor progression regulator, while its expression and specific functions in preeclampsia (PE) development remain unveiled. The expressions of miR-454-3p, lncRNA-DiGeorge syndrome critical region gene 5 (DGCR5) and growth arrest and DNA damage protein-inducible 45A (GADD45A) in placental tissues from PE patients or HTR-8/SVneo cells were assessed by Western blot or qRT-PCR. Dual-luciferase reporter assay determined the binding relations between miR-454-3p and GADD45A and between miR-454-3p and lncRNA-DGCR5. The viability, apoptosis, migration, invasiveness and tube formation of HTR-8/SVneo cell were evaluated using cell counting kit (CCK)-8, Annexin-V/Propidium iodide staining, wound healing, transwell and tube formation assays, respectively. miR-454-3p was low-expressed in PE tissue, and upregulation of miR-454-3p increased viability and promoted migration, invasion and tube formation in HTR-8/SVneo cells while inhibiting apoptosis. Then, miR-454-3p was found to directly target GADD45A which was high-expressed in PE tissues. Overexpressing GADD45A decreased the viability and inhibited the migration, invasion and tube formation of HTR-8/SVneo cells while enhancing apoptosis, and it neutralized the effect of miR-454-3p upregulation. In turn, miR-454-3p upregulation reversed the effect of GADD45A overexpression. Meanwhile, miR-454-3p could also target lncRNA-DGCR5. Silencing lncRNA-DGCR5 increased miR-454-3p expression and cell viability and promoted migration, invasion and tube formation in HTR-8/SVneo cells while inhibiting apoptosis, and it counteracted the effect of miR-454-3p downregulation. As usual, miR-454-3p downregulation reversed the effect of lncRNA-DGCR5 silencing. To conclude, silencing lncRNA-DGCR5 increased viability, promoted migration, invasion and tube formation, and inhibited apoptosis in HTR-8/SVneo cells by rescuing the inhibition of GADD45A expression caused by miR-454-3p.

Graphical abstract

     

猪圆环病毒2型通过外泌体miR-125a-5p靶向Bcl-2诱导淋巴细胞凋亡

为研究miR-125a-5p在猪圆环病毒2型(porcine circovirus type 2,PCV2)诱导淋巴细胞凋亡中的作用及其作用机制,以PCV2感染PK-15细胞外泌体孵育的淋巴细胞为研究对象,采用流式细胞术、蛋白质免疫印迹试验(Western blotting)和实时荧光定量PCR,检测淋巴细胞凋亡率及凋亡相关miRNA表达;合成miR-125a-5p模拟物和抑制物转染PK-15细胞,检测miR-125a-5p过表达或抑制表达后细胞凋亡率;采用生物信息学方法预测miR-125a-5p的靶基因,双荧光素酶报告基因检测miR-125a-5p对靶基因的调控;Western blotting检测外泌体孵育淋巴细胞的线粒体凋亡信号通路相关蛋白Bcl-2、Bax、细胞色素C和caspase-3的表达。结果显示,感染PCV2的PK-15细胞分泌的外泌体极显著提高淋巴细胞凋亡率,在一定浓度范围内呈剂量依赖性;与PCV2诱导细胞凋亡相关的miRNA中,miR-125a-5p表达量极显著升高,miR-125a-5p模拟物转染细胞后极显著提高细胞凋亡率;利用TargetScan预测发现,miR-125a-5p与Bcl-2 3''UTR区有结合位点,miR-125a-5p模拟物极显著抑制pmir-Bcl-2 3''UTR-WT荧光素酶活性,对pmir-Bcl-2 3''UTR-MuT的荧光素酶活性无明显改变;外泌体孵育的淋巴细胞Bcl-2表达量显著降低,Bax、细胞色素C的释放和caspase-3表达量显著升高,Bcl-2/Bax的比值极显著降低。这表明,PCV2通过外泌体诱导淋巴细胞上调miR-125a-5p的表达,进而抑制Bcl-2 mRNA和蛋白表达,激活淋巴细胞线粒体凋亡通路诱导细胞凋亡。     

miR-582-5p Is Upregulated in Patients with Active Tuberculosis and Inhibits Apoptosis of Monocytes by Targeting FOXO1

Macrophage apoptosis is a host innate defense mechanism against tuberculosis (TB). In this study, we found that percentage of apoptotic cells in peripheral blood monocytes from patients with active TB was lower than that from healthy controls (p<0.001). To understand whether microRNAs can modulate apoptosis of monocytes, we investigated differentially expressed microRNAs in patients with active TB. miR-582-5p was mainly expressed in monocytes and was upregulated in patients with active TB. The apoptotic percentage of THP-1 cells transfected with miR-582-5p mimics was significantly lower than those transfected with negative control of microRNA mimics (p<0.001), suggesting that miR-582-5p could inhibit apoptosis of monocytes. To our knowledge, the role of miR-582-5p in regulating apoptosis of monocytes has not been reported so far. Systematic bioinformatics analysis indicated that FOXO1 might be a target gene for miR-582-5p and its 3′UTR contains potential binding sites for miR-582-5p. To determine whether miR-582-5p could influence FOXO1 expression, miR-582-5p mimics or negative control of microRNA mimics were transfected into THP-1 cells. RT-PCR and western blot analysis showed that the miR-582-5p could suppress both FOXO1 mRNA and protein expression. Co-transfection of miR-582-5p and FOXO1 3′UTR-luciferase reporter vector into cells demonstrated that significant decrease in luciferase activity was only found in reporter vector that contained a wild type sequence of FOXO1 3′UTR, suggesting that miR-582-5p could directly target FOXO1. In conclusion, miR-582-5p inhibited apoptosis of monocytes by down-regulating FOXO1 expression and might play an important role in regulating anti-M. tuberculosis directed immune responses.     

MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes

Introduction

Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA.

Methods

MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA).

Results

In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3''UTR abrogated the suppressive effect of miR125.

Conclusions

Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA.     

Haplotype-based association of two SNPs in miR-423 with unexplained recurrent pregnancy loss in a Chinese Han population

MicroRNAs (miRNAs) regulate diverse cellular processes such as cell differentiation, proliferation and apoptosis. Mutation in miRNAs results in various pathological conditions such as inflammation, viral infections, neurodegeneration, and autoimmunity. We have evaluated the association of miR-423 rs6505162C>A and rs8067576 A>T among patients with recurrent pregnancy loss (RPL) and controls from North China. Our study found that one SNP rs6505162C>A in miR-423 coding region was associated with the increase risk of humanunexplained RPL (URPL), but no differences were found in another SNP rs8067576 A>T. However, in two-locus haplotype analysis, miR-423-CC/TT haplotype was associated with an increased risk of URPL. The level of mature miR-423 was obviously down-regulated in cells transfected with miR-423-CC/TT haplotype. miR-423-CC/TT haplotype inhibited HTR-8/SVneo cells proliferation and migration and promoted cells apoptosis. Further experiments identified that mesoderm development candidate 1 (MESDC1) was a functionally relevant target of miR-423, and its expression was reversely regulated by miR-423. More importantly, dual-luciferase assay indicated miR-423-CC/TT haplotype decreasing miR-423 expression, could up-regulate MESDC1 expression. Collectively, our data suggest that miR-423-CC/TT haplotype in pre-miR-423 may aggravate the risk of developing URPL by influencing the level of mature miR-423 and its target gene MESDC1.     

MicroRNA-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of X-linked inhibitor of apoptotic protein in endothelial cells

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that have emerged as one of the central players of gene expression regulation. Endothelial cell apoptosis plays a fundamental role in the development of atherosclerosis. This study was designed to determine the effect of miR-513a-5p on apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) and miR-513a-5p expression levels were determined. MiR-513a-5p target gene indentification, validation, and signalling pathways were investigated. Treatment of HUVECs with TNF-α and LPS up-regulated miR-513a-5p expressions more than 2-fold compared to control (P < 0.05). Inhibition of miR-513a-5p by antisense (AS) miR-513a-5p reversed TNF-α and LPS induced apoptosis (P < 0.01). Transfection of HUVECs with miR-513a-5p mimics also induced apoptosis (P < 0.01). Treatment of HUVECs with TNF-α and LPS attenuated X-linked inhibitor of apoptosis (XIAP) while increased caspase-3 expression, poly ADP-ribose polymerase (PARP) cleavage, and p53 expression. These effects were reversed by inhibition of miR-513a-5p. Of those miR-513a-5p candidate target genes, we identified and validated XIAP as a miR-513a-5p target gene. Targeting of the XIAP 3′-untranslated region by miR-513a-5p using luciferase reporter assay resulted in attenuated luciferase activity. Transfection of HUVECs with AS miR-513a-5p increased XIAP protein expression while miR-513a-5p mimics attenuated XIAP expression. These results together suggest that miR-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of XIAP in HUVECs.     

Upregulated long noncoding RNA Linc00261 in pre-eclampsia and its effect on trophoblast invasion and migration via regulating miR-558/TIMP4 signaling pathway

Pre-eclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality but the exact underlying mechanisms of PE pathogenesis remain elusive. Accumulated data suggested that the long noncoding RNAs (lncRNAs) play important roles in the pathogenesis of PE. The present study identified the changes of lncRNA Linc00261 in PE and its effects on trophoblasts invasion and migration. Our results showed that the expression of Linc00261 was upregulated in placental tissues of PE women compared with those of healthy pregnant women. Overexpression of Linc00261 suppressed cell invasion and migration, induced cell apoptosis, and caused cell-cycle arrest at G₀/G₁ phase of HTR-8/SVneo cells; while knockdown of Linc00261 had the opposite effects on the HTR-8/SVneo cells. Mechanistic studies showed Linc00261 functioned as a competing endogenous RNA for miR-558 in HTR-8/SVneo cells, and miR-558 was negatively regulated by Linc00261. The expression level of miR-558 in the PE group was significantly lower than the control group, and the expression level of Linc00261 was negatively correlated with the expression level of miR-558 in the placental tissues of women with PE. Furthermore, miR-558 was found to negatively regulate the expression of TIMP metallopeptidase inhibitor 4 (TIMP4) via targeting the 3′ untranslated region in the HTR-8/SVneo cells. Overexpression of miR-558 increased HTR-8/SVneo cell invasion and migration, which was attenuated by TIMP4 overexpression. More importantly, both overexpression of miR-558 and knockdown of TIMP4 partially reversed the suppressive effects of Linc00261 overexpression on cell invasion and migration of HTR-8/SVneo cells. Collectively, our results for the first time showed the upregulation of Linc00261 in the placental tissues of severe PE patients. The mechanistic results indicated that Linc00261 exerted the suppressive effects on the trophoblast invasion and migration via targeting miR-558/TIMP4 axis, which may involve in the pathogenesis of PE.     

Hypoxia-inducible microRNA-218 inhibits trophoblast invasion by targeting LASP1: Implications for preeclampsia development

Preeclampsia (PE) is a major contributor to maternal morbidity and mortality. However, the molecular mechanisms underlying PE progression are not well characterized. Here, we investigated the role of miR-218 in PE development. The expression of miR-218 and its host genes SLIT2 and SLIT3 was up-regulated in preeclamptic placentae compared to normal placentae. miR-218 expression was induced by hypoxia and decreased after knockdown of HIF-1α in an extravillous trophoblast cell line (HTR-8/SVneo). Chromatin immunoprecipitation assays showed direct binding of HIF-1α to the promoters of SLIT2 and SLIT3. Bioinformatics analysis identified LASP1 as a direct target of miR-218. Overexpression of miR-218 repressed the expression of LASP1 at both the mRNA and protein level. Meanwhile, miR-218 repressed the activity of a luciferase reporter containing the 3′-untranslated region of the LASP1 gene. Furthermore, expression of LASP1 rescued the inhibitory effect of miR-218 on HTR-8/SVneo cell invasion. Together, these results indicated that miR-218 contributes to PE by targeting LASP1 to inhibit trophoblast invasion.     

Over-expression of miR-146b and its regulatory role in intestinal epithelial cell viability,proliferation, and apoptosis in piglets

Background

Weaning stress affects the small intestine of piglets. MiR-146b is differentially expressed in suckling and weaned piglets. In this study, we evaluated the effects of miR-146b on cell viability, proliferation, and apoptosis in IPEC-J2 cells.

Results

Transfection with miR-146b mimics successfully increased miR-146b levels by 1000× (P?<?0.001). The over-expression of miR-146b significantly promoted the apoptosis (P?<?0.01) of IPEC-J2 cells, with no significant effects on cell viability or proliferation. MiR-146b suppressed the luciferase activity of the miR-TLR4-wt by 57% compared with the negative control, while mutation of the miR-146b binding site significantly blocked the suppressive effect (P?<?0.05). Western blot results showed that TLR4 levels decreased in IPEC-J2 cells transfected with miR-146b mimics (P?<?0.05).

Conclusions

The over-expression of miR-146b promotes IPEC-J2 cell apoptosis. TLR4 is a direct target of miR-146b in IPEC-J2 cells.

Reviewers

This article was reviewed by Eugene Berezikov and Jan B Hoek.

     

MicroRNA-210 alleviates oxidative stress-associated cardiomyocyte apoptosis by regulating BNIP3

Oxidative stress-induced myocardial apoptosis and necrosis are involved in ischemia/reperfusion (I/R) injury. This study was performed to investigate microRNA (miR)-210’s role in oxidative stress-related myocardial damage. The expression of miR-210 was upregulated in myocardial tissues of I/R rats, while that of Bcl-2 adenovirus E1B 19kDa-interacting protein 3 (BNIP3) was downregulated. To simulate in vivo oxidative stress, H9c2 cells were treated with H₂O₂ for 48 h. MiR-210 level was increased upon H₂O₂ stimulation, peaked at 8 h, and then decreased. An opposite expression pattern of BNIP3 was observed. BNIP3 was demonstrated as a direct target of miR-210 via luciferase reporter assay. H₂O₂-induced cell apoptosis was attenuated by miR-210 mimics, whereas aggravated by miR-210 inhibitor. MiR-210 knockdown-induced cell apoptosis in presence of H₂O₂ was attenuated by BNIP3 siRNA. Our work demonstrates that miR-210 plays a protective role in H₂O₂-induced cardiomyocyte apoptosis at least by regulating the pro-apoptotic BNIP3.     

The effect of zinc on human trophoblast proliferation and oxidative stress

Adequate Zinc (Zn) intake is required to prevent multiple teratogenic effects however deviations from adequate Zn intake, including high maternal Zn status, have been linked to increased incidence of pregnancy complications, including those associated with inadequate placentation. Using placental trophoblast HTR8/SVneo cells and first trimester human placental explants (n = 12), we assessed the effects of varying Zn concentrations on trophoblast proliferation, viability, apoptosis and oxidative stress. Compared to physiologically normal Zn levels (20 µM), HTR-8/SVneo cell proliferation index was significantly lower in the presence of physiologically elevated (40 µM; P = .020) and supra-physiological (80 µM; P = .007) Zn. The latter was also associated with reduced proliferation (P = .004) and viability (P < .0001) in cultured placental explants, but not apoptosis. Reactive oxygen species production in HTR8/SVneo cultures was significantly higher in the presence of 80 µM Zn compared to all physiologically relevant levels. Oxidative stress, induced by an oxidizing agent menadione, was further exacerbated by high (80 µM) Zn. Zn did not affect lipid peroxidation in either HTR8/SVneo cells or placental explants or antioxidant defense mechanisms that included glutathione reductase and superoxide dismutase. Further study should focus on elucidating mechanisms behind impaired trophoblast proliferation and increased oxidative stress as a result of elevated Zn levels.     

Dissimilar microRNA-21 functions and targets in trophoblastic cell lines of different origin

Trophoblast cells express a singular miRNA expression profile which varies during pregnancy and whose alteration may be associated with pregnancy complications. miR-21, a widely known oncomir, is highly expressed in human placenta but its role in regulating trophoblast cells remains unclear. The aim of this study was to investigate miR-21 functions and targets in HTR-8/SVneo immortalized trophoblast and JEG-3 choriocarcinoma cells, which are trophoblast cell models that differ in their cellular origin. Cells were transfected with miR-21-antagomir, -mimic or their respective controls. Following, cell proliferation (BrdU), migration (Transwell and scratch wound-healing assays), invasion (Matrigel assays) and apoptosis (flow cytometry, TUNEL assay and Western blotting) were assessed. Expression of the potential miR-21 targets phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) were analyzed by Western blotting. Inhibition of miR-21 decreased cell proliferation, migration, and invasion in JEG-3 and HTR-8/SVneo cells and additionally, induced apoptosis in JEG-3 cells. Silencing of miR-21 enhanced PDCD4 expression only in JEG-3 cells, and PTEN expression only in HTR-8/SVneo cells. Inhibition of miR-21 significantly increased phosphorylation of AKT in HTR-8/SVneo cells. In conclusion, miR-21 has cell-specific targets depending upon the origin of trophoblastic cells. Furthermore, miR-21 regulates major cellular processes including cell growth, migration, invasion and apoptosis suggesting that its impairment may lead to placental disorders.     

Bmo-miR-3377-5p down-regulates the Bombyx mori Sericin gene-1

MiRNAs are small non-coding molecules, which can regulate a huge number of genes. Based on bioinformatics analysis, we found a target site in the 3′UTR of BmSer-1 for binding bmo-miR-3377-5p. By using semi-quantitative RT-PCR, we detected that miR-3377-5p and BmSer-1 were both more highly expressed in the middle silk gland than in other tissues of 3-day-old fifth-instar Bombyx mori larvae, implying that there is a spatiotemporal condition for miR-3377-5p regulating on BmSer-1. To confirm this prediction, a BmSer-13′UTR recombinant luciferase reporter pGL3.0 [A3-luc-BmSer-1-3′UTR-SV40] and pri-bmo-miR-3377-5p expression pcDNA3.0 [ie1-egfp-pri-bmo-miR-3377-5p-SV40] were constructed and co-transfected into B. mori ovary cells (BmN cells). The results showed that miR-3377-5p suppressed the expression of BmSer-1 significantly (P < .001). When BmN cells were co-transfected by an artificial inhibitor together with a miR-3377-5p expression vector and a BmSer-1-3′UTR recombinant plasmid, BmSer-1 expression increased significantly (P < .05), indicating that the inhibitor was active against miR-3377-5p, and expression of BmSer-1 was recovered. Moreover, we injected miR-3377-5p expression plasmid and bmo-miR-3377-5p inhibitor into 3-day-old fifth-instar larvae. At 36 h post-injection, silk glands were collected for total RNA extraction. Quantitative RT-PCR analysis showed that miR-3377-5p down-regulated the expression of BmSer-1 in vivo, while there was no significant difference inhibitor treatment group compared with NC. Thus, we conclude that miR-3377-5p down-regulated the expression of BmSer-1. Our results provide insight for understanding the function of miRNAs and the regulation network of silk protein genes.