MicroRNA‐210 regulates human trophoblast cell line HTR‐8/SVneo function by attenuating Notch1 expression: Implications for the role of microRNA‐210 in pre‐eclampsia

Successful pregnancy depends on the precise regulation of extravillous trophoblast cell invasion ability. MicroRNA‐210‐3p (miR‐210), which is increased in the placenta of pre‐eclampsia. Furthermore, miR‐210 could inhibit trophoblasts invasion and might act as a serum biomarker for pre‐eclampsia. Previous studies have demonstrated that miR‐210 regulates HUVEC (human umbilical vein endothelial cell)‐mediated angiogenesis by regulating the NOTCH1 signaling pathway. Studies by our group have previously identified that NOTCH1 plays a positive role in regulating trophoblast functions. However, the miR‐210/NOTCH1 signaling pathway in the regulation of trophoblasts and pre‐eclampsia has not been characterized. Therefore, this study was conducted to investigate the role of miR‐210 and its relationship with NOTCH1 in trophoblasts. We first examined the expression levels of miR‐210 and NOTCH1 in pre‐eclamptic and normals placentas. Next, the expression and location of miR‐210 and NOTCH1 in the first‐trimester villi, maternal decidua, and placenta of late pregnancy were shown via in situ hybridization and immunohistochemistry. The trophoblast cell line HTR‐8/SVneo was used to investigate the effects of miR‐210 on the expression of NOTCH1 and cell bioactivity by upregulation and downregulation strategies. The results showed that miR‐210 expression was increased, whereas NOTCH1 expression was decreased in pre‐eclamptic placenta compared with controls. Upregulation of miR‐210 decreased NOTCH1 expression, impaired HTR‐8/SVneo proliferation, migration, invasion, and tube‐like formation capabilities, and promoted apoptosis. In contrast, downregulation of miR‐210 resulted in the opposite effects. These findings suggested that miR‐210 might act as a contributor to trophoblast dysfunction by attenuating NOTCH1 expression.     

Hypoxia‐induced downregulation of XIAP in trophoblasts mediates apoptosis via interaction with IMUP‐2: Implications for placental development during pre‐eclampsia

The regulation of trophoblast apoptosis is essential for normal placentation, and increased placental trophoblast cell apoptosis is the cause of pathologies such as intrauterine growth retardation (IUGR) and pre‐eclampsia. X‐linked inhibitor of apoptosis (XIAP) is expressed in trophoblasts, but little is known about the role of XIAP in placental development. In the present study, the function of XIAP in the placenta and in HTR‐8/SVneo trophoblasts under hypoxic conditions was examined. In addition, the correlation between XIAP and immortalization‐upregulated protein‐2 (IMUP‐2) was demonstrated in HTR‐8/SVneo trophoblasts under hypoxia, based on a previous study showing that increased IMUP‐2 induces trophoblast apoptosis and pre‐eclampsia. XIAP was downregulated in pre‐eclamptic placentas (P < 0.05). In HTR‐8/SVneo trophoblasts, XIAP expression was decreased and the expression of apoptosis‐related genes was increased in response to hypoxia. Ectopic expression of hypoxia inducible factor (HIF)‐1α in HRT‐8 SV/neo cells induced the nuclear translocation of XIAP and alterations of XIAP protein stability. Furthermore, hypoxia induced nuclear translocated XIAP co‐localized with upregulated IMUP‐2 in trophoblast nuclei, and the interaction between XIAP and IMUP‐2 induced apoptosis in HRT‐8 SV/neo cells. The present results suggest that hypoxia‐induced down‐regulation of XIAP mediates apoptosis in trophoblasts through interaction with increased IMUP‐2, and that this mechanism underlies the pathogenesis of pre‐eclampsia. J. Cell. Biochem. 114: 89–98, 2012. © 2012 Wiley Periodicals, Inc.     

Complement 5a‐mediated trophoblasts dysfunction is involved in the development of pre‐eclampsia

Pre‐eclampsia (PE) is a life‐threatening multisystem disorder leading to maternal and neonatal mortality and morbidity. Emerging evidence showed that activation of the complement system is implicated in the pathological processes of PE. However, little is known about the detailed cellular and molecular mechanism of complement activation in the development of PE. In this study, we reported that complement 5a (C5a) plays a pivotal role in aberrant placentation, which is essential for the onset of PE. We detected an elevated C5a deposition in macrophages and C5a receptor (C5aR) expression in trophoblasts of pre‐eclamptic placentas. Further study showed that C5a stimulated trophoblasts towards an anti‐angiogenic phenotype by mediating the imbalance of angiogenic factors such as soluble fms‐like tyrosine kinase 1 (sFlt1) and placental growth factor (PIGF). Additionally, C5a inhibited the migration and tube formation of trophoblasts, while, C5aR knockdown with siRNA rescued migration and tube formation abilities. We also found that maternal C5a serum level was increased in women with PE and was positively correlated with maternal blood pressure and arterial stiffness. These results demonstrated that the placental C5a/C5aR pathway contributed to the development of PE by regulating placental trophoblasts dysfunctions, suggesting that C5a may be a novel therapeutic possibility for the disease.     

Curcumin stimulates angiogenesis through VEGF and expression of HLA‐G in first‐trimester human placental trophoblasts

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real‐time polymerase chain reaction (qRT‐PCR), promoter DNA methylation, qRT‐PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG‐3 (trophoblast), and HMEC‐1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor‐2 and fatty acid‐binding protein‐4 (FABP4) in these cells. Curcumin‐stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA‐G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down‐regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA‐G by curcumin, hitherto unknown, is a novel finding since HLA‐G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.     

Resveratrol promotes trophoblast invasion in pre‐eclampsia by inducing epithelial‐mesenchymal transition

Impairment spiral arteries remodelling was considered to be the underlying cause of pathogenesis of pre‐eclampsia (PE). Resveratrol (RE) was reported that it could modulate cellar phenotype to ameliorate diverse human diseases. However, the biological function of RE in PE remains poorly understood. In this report, we investigated the effect of RE on trophoblast phenotype both in vivo and in vitro. We conducted MTT and transwell assays to explore cell proliferation and invasion events in HTR‐8/SVneo. In mice model, the clinical characteristics of PE were established through the injection of NG‐nitro‐l ‐arginine methyl ester (L‐NAME). Furthermore, related experiments were performed to detect cellar phenotype‐associated signalling pathway, including epithelial‐mesenchymal transition (EMT) and Wnt/β‐catenin. Cell assays indicated that RE could increase trophoblasts migration and invasion. In addition, hypertension and proteinuria were markedly ameliorated by RE compared with the controls in PE mice model. Moreover, treatment by RE in trophoblasts or in PE model, we found that RE activated EMT progress through the regulation of E‐cadherin, β‐catenin, N‐cadherin, vimentin expression, and further altered the WNT‐related gene expression, including WNT1, WNT3 and WNT5B. Our findings demonstrated that RE might stimulate the invasive capability of human trophoblasts by promoting EMT and mediating the Wnt/β‐catenin pathway in PE.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

Potential regulatory network in the PSG10P/miR‐19a‐3p/IL1RAP pathway is possibly involved in preeclampsia pathogenesis

Preeclampsia (PE), a pregnancy‐specific disorder, is a leading cause of perinatal maternal‐fetal mortality and morbidity. Impaired cell migration and invasion of trophoblastic cells and an imbalanced systemic maternal inflammatory response have been proposed as potential mechanisms of PE pathogenesis. Comparative analysis between PE placentas and normal placentas profiled differentially expressed miRNAs, lncRNAs, and mRNAs, including miR‐19a‐3p (miRNA), PSG10P (lncRNA), and IL1RAP (mRNA). This study was conducted to investigate their potential roles in PE pathogenesis. The expression of miR‐19a‐3p, PSG10P, and IL1RAP was examined in PE and normal placentas using RT‐qPCR. An in vitro experiment was performed in human trophoblast HET8/SVneo and TEV‐1 cells cultured in normoxic and hypoxic conditions. MiR‐19a‐3p targets were identified using Targetscan, miRanda, and PicTar analysis as well as luciferase reporter assays. The mouse model of PE was conducted using sFlt‐1 for in vivo tests. Lower levels of miR‐19a‐3p, but higher levels of PSG10P and IL1RAP were observed in PE placentas and the trophoblast cells in hypoxia. Luciferase reporter assays confirmed that PSG10P and IL1RAP were both direct targets of miR‐19a‐3p. Exposure to hypoxia inhibited cell viability, migration, and invasion of HET8/SVneo and TEV‐1 cells. Knocking out PSG10P and IL1RAP or overexpressing miR‐19a‐3p rescued the inhibition caused by hypoxia. In vivo experiments showed that IL1RAP promoted the expression of caspase‐3, a key apoptosis enzyme, but inhibited MMP9, which is responsible for degrading the extracellular matrix, suggesting a significant role of IL1RAP in cell proliferation, migration, and invasion. miR‐19a‐3p, PSG10P, and IL1RAP were all found to be involved in PE pathogenesis. With a common targeting region in their sequences, a regulatory network in the PSG10P/miR‐19a‐3p/IL1RAP pathway may contribute to PE pathogenesis during pregnancy.     

Sphingosine-1-Phosphate Promotes Extravillous Trophoblast Cell Invasion by Activating MEK/ERK/MMP-2 Signaling Pathways via S1P/S1PR1 Axis Activation

Successful placentation depends on the proper invasion of extravillous trophoblast (EVT) cells into maternal tissues. Previous reports demonstrated that S1P receptors are expressed in the EVT cells and S1P could regulate migration and function of trophoblast cells via S1P receptors. However, little is known about roles of S1P in the invasion of EVT cells. Our study was performed to investigate S1P effect on the invasion of EVT cells. We used the extravillous trophoblast cell line HTR8/SVneo cells to evaluate the effect. In vitro invasion assay was employed to determine the invasion of HTR8/SVneo cells induced by S1P. MMP-2 enzyme activity and relative level in the supernatants of HTR8/SVneo was assessed by gelatin zymography and western blot. Based on the above, siRNA and specific inhibitors were used for the intervention and study of potential signal pathways, and Real-time qPCR and western blot were used to test the mRNA and protein level of potential signal targets. We found that S1P could promote HTR8/SVneo cell invasion and upregulates activity and level of MMP-2. The promotion requires activation of MEK-ERK and is dependent on the axis of S1P/S1PR1. Our investigation of S1P may provide new insights into the molecular mechanisms of EVT invasion.     

Epigenetic regulation of miR‐129‐2 and its effects on the proliferation and invasion in lung cancer cells

MicroRNAs (miRNAs) play a pivotal role in carcinogenesis. Dysregulation of miRNAs, both oncogenic miRNAs and tumour‐suppressive miRNAs, is closely associated with cancer development and progression. The levels of miRNAs could be changed epigenetically by DNA methylation in the 5′ untranslated region (UTR) of pre‐mature miRNAs. To investigate whether DNA methylation alters the expression of miR‐129 in lung cancer, we did DNA methylation assays and found that 5′ UTR region of miR‐129‐2 gene was absolutely methylated in both A549 and SPCA‐1 lung cancer cells, but totally un‐methylated in 95‐D cells. The expression of miR‐129 was restored by 5‐Aza‐2'‐deoxycytidine (DAC), a de‐methylation agent, in both A549 and SPCA‐1 cells, resulting in attenuated cell migration and invasion ability, and decreased protein level of NF‐κB, which indicates the involvement of NF‐κB pathway. To further illustrate the roles of miR‐129 in lung tumourigenesis, we overexpressed miR‐129 in lung cancer cells by transfection of miR‐129 mimics, and found arrested cell proliferation at G2/M phase of cell cycle and inhibited cell invasion. These findings strongly suggest that miR‐129 is a tumour suppressive miRNA, playing important roles in the development and progression of human lung cancer.     

miR‐126 reduces trastuzumab resistance by targeting PIK3R2 and regulating AKT/mTOR pathway in breast cancer cells

MicroRNAs (miRNAs) have been found to play a key role in drug resistance. In the current study, we aimed to explore the potential role of miR‐126 in trastuzumab resistance in breast cancer cells. We found that the trastuzumab‐resistant cell lines SKBR3/TR and BT474/TR had low expression of miR‐126 and increased ability to migrate and invade. The resistance, invasion and mobilization abilities of the cells resistant to trastuzumab were reduced by ectopic expression of miR‐126 mimics. In comparison, inhibition of miR‐126 in SKBR3 parental cells had the opposite effect of an increased resistance to trastuzumab as well as invasion and migration. It was also found that miR‐126 directly targets PIK3R2 in breast cancer cells. PIK3R2‐knockdown cells showed decreased resistance to trastuzumab, while overexpression of PIK3R2 increased trastuzumab resistance. In addition, our finding showed that overexpression of miR‐126 reduced resistance to trastuzumab in the trastuzumab‐resistant cells and that inhibition of the PIK3R2/PI3K/AKT/mTOR signalling pathway was involved in this effect. SKBR3/TR cells also showed increased sensitivity to trastuzumab mediated by miR‐126 in vivo. In conclusion, the above findings demonstrated that overexpression of miR‐126 or down‐regulation of its target gene may be a potential approach to overcome trastuzumab resistance in breast cancer cells.     

MicroRNA‐194 protects against chronic hepatitis B‐related liver damage by promoting hepatocyte growth via ACVR2B

Persistent infection with the hepatitis B virus leads to liver cirrhosis and hepatocellular carcinoma. MicroRNAs (miRNAs) play an important role in a variety of biological processes; however, the role of miRNAs in chronic hepatitis B (CHB)‐induced liver damage remains poorly understood. Here, we investigated the role of miRNAs in CHB‐related liver damage. Microarray analysis of the expression of miRNAs in 22 CHB patients and 33 healthy individuals identified miR‐194 as one of six differentially expressed miRNAs. miR‐194 was up‐regulated in correlation with increased liver damage in the plasma or liver tissues of CHB patients. In mice subjected to 2/3 partial hepatectomy, miR‐194 was up‐regulated in liver tissues in correlation with hepatocyte growth and in parallel with the down‐regulation of the activin receptor ACVR2B. Overexpression of miR‐194 in human liver HL7702 cells down‐regulated ACVR2B mRNA and protein expression, promoted cell proliferation, acceleratedG1 to S cell cycle transition, and inhibited apoptosis, whereas knockdown of miR‐194 had the opposite effects. Luciferase reporter assays confirmed that ACVR2B is a direct target of miR‐194, and overexpression of ACVR2B significantly repressed cell proliferation and G1 to S phase transition and induced cell apoptosis. ACVR2B overexpression abolished the effect of miR‐194, indicating that miR‐194 promotes hepatocyte proliferation and inhibits apoptosis by down‐regulating ACVR2B. Taken together, these results indicate that miR‐194 plays a crucial role in hepatocyte proliferation and liver regeneration by targeting ACVR2B and may represent a novel therapeutic target for the treatment of CHB‐related liver damage.     

microRNA‐128‐3p overexpression inhibits breast cancer stem cell characteristics through suppression of Wnt signalling pathway by down‐regulating NEK2

Emerging evidence has reported that dysregulation of microRNAs (miRNAs) participated in the development of diverse types of cancers. Our initial microarray‐based analysis identified differentially expressed NEK2 related to breast cancer and predicted the regulatory microRNA‐128‐3p (miR‐128‐3p). Herein, this study aimed to characterize the tumour‐suppressive role of miR‐128‐3p in regulating the biological characteristics of breast cancer stem cells (BCSCs). CD44^＋CD24^?/low cells were selected for subsequent experiments. After verification of the target relationship between miR‐128‐3p and NEK2, the relationship among miR‐128‐3p, NEK2 and BCSCs was further investigated with the involvement of the Wnt signalling pathway. The regulatory effects of miR‐128‐3p on proliferation, migration, invasion and self‐renewal in vitro as well as tumorigenicity in vivo of BCSCs were examined via gain‐ and loss‐of‐function approaches. Highly expressed NEK2 was found in breast cancer based on GSE61304 expression profile. Breast cancer stem cells and breast cancer cells showed a down‐regulation of miR‐128‐3p. Overexpression of miR‐128‐3p was found to inhibit proliferation, migration, invasion, self‐renewal in vitro and tumorigenicity in vivo of BCSCs, which was further validated to be achieved through inhibition of Wnt signalling pathway by down‐regulating NEK2. In summary, this study indicates that miR‐128‐3p inhibits the stem‐like cell features of BCSCs via inhibition of the Wnt signalling pathway by down‐regulating NEK2, which provides a new target for breast cancer treatment.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

Novel mechanism of miRNA‐365‐regulated trophoblast apoptosis in recurrent miscarriage

Clinical pregnancies increasingly end in recurrent miscarriage (RM) during the first trimester, with genetic factors shouldering the main responsibility. MicroRNAs (miRNAs) regulate gene expression in a wide array of important biological processes. We examined the potential role of dysregulated miRNAs in RM pathogenesis and trophoblast development as an approach to elucidate the molecular mechanism behind RM. miRNA profiles from clinical specimens of RM and induced abortion (IA) were compared, and several miRNAs were found to be aberrantly expressed in RM samples. Among the miRNAs, miR‐365 was significantly differentially expressed in RM decidual tissues. Furthermore, our results demonstrate that miR‐365 functions as an upstream regulator of MDM2/p53 expression, cell cycle progression and apoptosis in trophoblasts. Bioinformatic prediction and experimental validation assays identified SGK1 as a direct target of miR‐365; consistently, its protein levels were low in decidual tissues. Additionally, functional studies revealed that SGK1 silencing elicits cell cycle arrest and apoptosis in trophoblasts and that SGK1 overexpression attenuates the effects of miR‐365 on apoptosis and MDM2/p53 expression. Collectively, our data provide evidence that the up‐regulation of miR‐365 may contribute to RM by decreasing SGK1 expression, which suggests its potential utility as a prognostic biomarker and therapeutic target for RM.     

LINC00667 promotes Wilms’ tumor metastasis and stemness by sponging miR‐200b/c/429 family to regulate IKK‐β

Wilms' tumor, also known as nephroblastoma, is a kind of pediatric renal cancer. Previous studies have indicated that microRNAs (miRNAs) regulate various cancers progression. However, whether miR‐200 family regulated Wilms' tumor progression remains to be elucidated. In our study, miR‐200b/c/429 expression was downregulated in Wilms' tumor tissue samples from 25 patients. And data from three independent analyses of quantitative real‐time polymerase chain reaction revealed that the expression of miR‐200b/c/429 was downregulated in Wilms' tumor cell lines. Functionally, Cell counting kit‐8 assay revealed that cell viability was reduced by overexpressing miR‐200b/c/429. Transwell assay manifested that cell migration and invasion was hindered by miR‐200b/c/429 overexpression. Sphere‐forming and western blot assays demonstrated that miR‐200b/c/429 overexpression suppressed the sphere formation ability. Mechanically, nuclear factor‐κB (NF‐κB) pathway was confirmed to be associated with Wilms' tumor progression; miR‐200b/c/429 overexpression inactivated NF‐κB pathway as miR‐200b/c/429 was identified to target IκB kinase β (IKK‐β), an NF‐κB pathway‐related gene. Moreover, miR‐200b/c/429 was sponged by LINC00667 in Wilms' tumor cells. LINC00667 competitively bound with miR‐200b/c/429 to regulate IKK‐β expression and then activated NF‐κB pathway in Wilms' tumor. Subsequently, rescue assays illustrated that silencing of IKK‐β could reverse the effect of miR‐200b/c/429 inhibition on the progression of sh‐LINC00667‐transfected Wilms' tumor cells. In summary, LINC00667 promoted Wilms' tumor progression by sponging miR‐200b/c/429 family to regulate IKK‐β.     

miR‐193b represses influenza A virus infection by inhibiting Wnt/β‐catenin signalling

Due to an increasing emergence of new and drug‐resistant strains of the influenza A virus (IAV), developing novel measures to combat influenza is necessary. We have previously shown that inhibiting Wnt/β‐catenin pathway reduces IAV infection. In this study, we aimed to identify antiviral human microRNAs (miRNAs) that target the Wnt/β‐catenin signalling pathway. Using a miRNA expression library, we identified 85 miRNAs that up‐regulated and 20 miRNAs that down‐regulated the Wnt/β‐catenin signalling pathway. Fifteen miRNAs were validated to up‐regulate and five miRNAs to down‐regulate the pathway. Overexpression of four selected miRNAs (miR‐193b, miR‐548f‐1, miR‐1‐1, and miR‐509‐1) that down‐regulated the Wnt/β‐catenin signalling pathway reduced viral mRNA, protein levels in A/PR/8/34‐infected HEK293 cells, and progeny virus production. Overexpression of miR‐193b in lung epithelial A549 cells also resulted in decreases of A/PR/8/34 infection. Furthermore, miR‐193b inhibited the replication of various strains, including H1N1 (A/PR/8/34, A/WSN/33, A/Oklahoma/3052/09) and H3N2 (A/Oklahoma/309/2006), as determined by a viral reporter luciferase assay. Further studies revealed that β‐catenin was a target of miR‐193b, and β‐catenin rescued miR‐193b‐mediated suppression of IAV infection. miR‐193b induced G0/G1 cell cycle arrest and delayed vRNP nuclear import. Finally, adenovirus‐mediated gene transfer of miR‐193b to the lung reduced viral load in mice challenged by a sublethal dose of A/PR/8/34. Collectively, our findings suggest that miR‐193b represses IAV infection by inhibiting Wnt/β‐catenin signalling.     

MicroRNA‐383 inhibits doxorubicin resistance in hepatocellular carcinoma by targeting eukaryotic translation initiation factor 5A2

Drug resistance occurs commonly in cancers, especially in hepatocellular carcinoma (HCC). Accumulating evidence has demonstrated that microRNAs (miRNAs) play a vital role in tumour chemoresistance. However, little is known about the role of miR‐383 in HCC chemoresistance. In the present study, RT‐PCR and western blotting were used to identify the expression profile of miR‐383 and eukaryotic translation initiation factor 5A2 (EIF5A2). The bioinformatics website Targetscan was used to predict the target genes of miR‐383. In vitro and in vivo loss‐ and gain‐of‐function studies were performed to reveal the effects and potential mechanism of the miR‐383/EIF5A2 axis in chemoresistance of HCC cells. The expression level of miR‐383 correlated negatively with doxorubicin (Dox) sensitivity. Overexpression of miR‐383 promoted HCC cells to undergo Dox‐induced cytotoxicity and apoptosis, whereas miR‐383 knockdown had the opposite effects. EIF5A2 was predicted as a target gene of miR‐383. EIF5A2 knockdown sensitized HCC cells to Dox. Moreover, miR‐383 inhibition‐mediated HCC Dox resistance could be reversed by silencing EIF5A2. Finally, we demonstrated that miR‐383 inhibition could enhance Dox sensitivity by targeting EIF5A2 in vivo. The results indicated that miR‐383 inhibited Dox resistance in HCC cells by targeting EIF5A2. Targeting the miR‐383/EIF5A2 axis might help to alleviate the chemoresistance of HCC cells.     

MiR‐133a‐3p Inhibits Oral Squamous Cell Carcinoma (OSCC) Proliferation and Invasion by Suppressing COL1A1

The aim of our study was to investigate the effects of miR‐133a‐3p on human oral squamous cell carcinoma (OSCC) cells by regulating gene COL1A1. OSCC tissues, adjacent tongue epithelial tissues, the immortalized oral epithelial cell line HIOEC, and OSCC cell lines (CAL‐27, TCA‐8113, SCC‐4, SCC‐9, and SCC‐15) were used in this research. Quantitative real‐time PCR (RT‐qPCR) was employed to determine the expression of miR‐133a‐3p and COL1A1. Dual luciferase reporter gene assay and Western blot were applied to verify the binding relationship between miR‐133a‐3p and COL1A1. Functional assays were also conducted in this study, including CCK‐8 assay, colony formation assay, flow cytometry analysis as well as Transwell assay. MiR‐133a‐3p was found low‐expressed both in OSCC tissues and cells lines compared with normal tissues and cell line, respectively, whereas COL1A1 was just the opposite. The over‐expression of miR‐133a‐3p or the down‐regulation of COL1A1 suppressed the proliferation, invasion, and mitosis of OSCC cells, whereas simultaneous down‐regulation of miR‐133a‐3p and up‐regulation of COL1A1 led to no significant alteration of cell activities. MiR‐133a‐3p could inhibit the proliferation and migration of OSCC cells through directly targeting COL1A1 and reducing its expression. J. Cell. Biochem. 119: 338–346, 2018. © 2017 Wiley Periodicals, Inc.