Aberrantly Expressed MicroRNAs in Cancer-Associated Fibroblasts and Their Target Oncogenic Signatures in Hepatocellular Carcinoma.

Cancer-associated fibroblasts (CAFs) contribute to tumor progression, and microRNAs (miRs) play an important role in regulating the tumor-promoting properties of CAFs. The objectives of this study were to clarify the specific miR expression profile in CAFs of hepatocellular carcinoma (HCC) and identify its target gene signatures. Small-RNA-sequencing data were generated from nine pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively. Bioinformatic analyses were performed to identify the HCC-CAF-specific miR expression profile and the target gene signatures of the deregulated miRs in CAFs. Clinical and immunological implications of the target gene signatures were evaluated in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA_LIHC) database using Cox regression and TIMER analysis. The expressions of hsa-miR-101-3p and hsa-miR-490-3p were significantly downregulated in HCC-CAFs. Their expression in HCC tissue gradually decreased as HCC stage progressed in the clinical staging analysis. Bioinformatic network analysis using miRWalks, miRDB, and miRTarBase databases pointed to TGFBR1 as a common target gene of hsa-miR-101-3p and hsa-miR-490-3p. TGFBR1 expression was negatively correlated with miR-101-3p and miR-490-3p expression in HCC tissues and was also decreased by ectopic miR-101-3p and miR-490-3p expression. HCC patients with TGFBR1 overexpression and downregulated hsa-miR-101-3p and hsa-miR-490-3p demonstrated a significantly poorer prognosis in TCGA_LIHC. TGFBR1 expression was positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and M2 macrophages in a TIMER analysis. In conclusion, hsa-miR-101-3p and hsa-miR-490-3p were substantially downregulated miRs in CAFs of HCC, and their common target gene was TGFBR1. The downregulation of hsa-miR-101-3p and hsa-miR-490-3p, as well as high TGFBR1 expression, was associated with poor clinical outcome in HCC patients. In addition, TGFBR1 expression was correlated with the infiltration of immunosuppressive immune cells.

MicroRNA-101-3p inhibits nasopharyngeal carcinoma cell proliferation and cisplatin resistance through ZIC5 down-regulation by targeting SOX2.

This study aims to explore the mechanism of microRNA (miR)-101-3p-mediated SOX2/ZIC5 axis in the progression of cisplatin resistance of nasopharyngeal carcinoma (NPC). ZIC5 expression was analyzed with a bioinformatics database and detected in NPC cell lines. Cisplatin-resistant cells (HNE-1/DDP and C666-1/DDP) were transfected with sh-ZIC5, sh-SOX2, sh-SOX2 + pcDNA3.1-ZIC5, or miR-101-3p Agomir + pcDNA3.1-SOX2. MiR-101-3p, SOX2, and ZIC5 expression was assessed after transfection, and cancer associated phenotypes were evaluated after cisplatin treatment. The potential relationships among miR-101-3p, SOX2, and ZIC5 were analyzed. A xenograft mouse model of NPC was established with HNE-1 cells stably transfected or not transfected with oe-ZIC5 and subjected to tail vein injection of miR-101-3p Agomir and intraperitoneal injection of cisplatin. Overexpression of ZIC5 was found in cisplatin-resistant NPC cells. Downregulating ZIC5 in NPC cells decreased cell viability, promoted apoptosis, and reduced cisplatin resistance. SOX2 had a binding site on ZIC5, and SOX2 promoted proliferation, migration, and cisplatin resistance and inhibited cell apoptosis by up-regulating ZIC5. Mechanistically, miR-101-3p was decreased in cisplatin-resistant NPC cells and negatively targeted SOX2. Overexpression of miR-101-3p inhibited tumor growth and cisplatin resistance in xenograft mouse model, which was reversed by ZIC5 overexpression. In conclusion, the miR-101-3p/SOX2/ZIC5 axis was implicated in cancer associated phenotypes and cisplatin resistance in NPC.

miR-101-3p contributes to the progression of preeclampsia by suppressing WDR5-mediated proliferation and invasion of trophoblast.

BACKGROUND: Decreased proliferation and invasion of trophoblast were proven to be involved in the pathogenesis of preeclampsia (PE). However, the regulatory network has not been clarified yet. This study aimed to explore the role of miR-101-3p in the progression of PE. METHODS: miR-101-3p expression in placentas of pregnant women with or without PE was analyzed by real-time quantitative PCR (RT-qPCR). Trophoblastic HTR-8/SVneo and HPT-8 cell lines were cultured and underwent hypoxia/reoxygenation (H/R) treatment to mimic PE in vitro. Cell proliferation and invasion were analyzed in gain-of and loss-of-function assays. Finally, we undertook in vivo studies to explore effects of miR-101-3p in the PE model. RESULTS: Compared to placentas from patients without PE, miR-101-3p expressed significantly higher in placentas from PE patients, and its level was positively correlated with the severity of patients. In vitro studies found that overexpression of miR-101-3p significantly suppressed cell proliferation and invasion, while knockdown of miR-101-3p reversed the impacts of H/R treatment. Further research showed that the expression of WD repeat domain 5 (WDR5) was significantly lower in placentas from patients with PE, and its level was negatively associated with the severity of patients. In vitro and in vivo studies confirmed that miR-101-3p promoted PE progression through the regulation of WD WDR5 expression. CONCLUSION: Increased expression of miR-101-3p in placenta contributes to the development of PE by suppressing WDR5-mediated proliferation and invasion of trophoblast.

Extracellular vesicles derived from M2-polarized tumor-associated macrophages promote immune escape in ovarian cancer through NEAT1/miR-101-3p/ZEB1/PD-L1 axis.

Evidence has been presented demonstrating that CD8+ T cells confer anti-cancer effects, which offers a promising approach to enhance immunotherapy. M2-polarized tumor-associated macrophages (TAMs) could transfer RNA to cancer cells by secreting extracellular vesicles (EVs) and stimulate immune escape of cancer cells. Thus, the current study aimed at exploring how EVs derived from M2-polarized TAMs (M2-TAMs) affected the proliferation of ovarian cancer (OC) cells and apoptosis of CD8+ T cells. M2-TAMs were observed in OC tissues, which promoted proliferation of OC cells and CD8+ T cell apoptosis by secreting EVs. OC-associated differentially expressed gene NEAT1 was screened by bioinformatics analysis. The in vitro and in vivo effects of TAM-EVs-NEAT1 and its regulatory mechanism were assessed using gain- and loss-of-function assays in co-culture systems of TAMs-derived EVs, OC cells, and CD8+ T cells and in tumor-bearing mice. NEAT1 was highly expressed in M2-derived EVs and OC cells co-cultured with M2-derived EVs. NEAT1 sponged miR-101-3p to increase ZEB1 and PD-L1 expression. In vitro and in vivo assays confirmed the tumor-supporting effects of NEAT1 delivered by M2-derived EVs on OC cell proliferation and CD8+ T cell apoptosis as well as tumor growth. Collectively, M2-derived EVs containing NEAT1 exerted a tumor-promoting role in OC via the miR-101-3p/ZEB1/PD-L1 axis.

Morinda officinalis polysaccharide enable suppression of osteoclastic differentiation by exosomes derived from rat mesenchymal stem cells.

CONTEXT: Morinda officinalis F.C. How. (MO) (Rubiaceae) can strengthen bone function. OBJECTIVE: To examine the functional mechanism and effect of MO polysaccharides (MOPs) in rats with glucocorticoid-induced osteoporosis (GIOP). MATERIALS AND METHODS: Rats with GIOP were treated with 5, 15 or 45 mL/kg of MOP [n = 15 for each dose, intraperitoneal (i.p.) injection every other day for 8 weeks]. The body weight of rats and histomorphology of bone tissues were examined. Bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (Exo) were collected and identified. Bone marrow-derived macrophages (BMMs) were induced to differentiate into osteoclasts and treated with BMSC-Exo for in vitro studies. RESULTS: MOP reduced the body weight (5, 15, or 45 mg/kg MOP vs. phosphate-buffered saline: 8%, 15% and 25%, p < 0.01), elevated the bone volume to tissue volume (BV/TV), mean trabecular thickness (Tb.Th), mean trabecular number (Tb.N) and mean connectivity density (Conn.D) (40-86%, p < 0.01), decreased the mean trabecular separation/spacing (Tb.Sp) (22-37%, p < 0.01), increased the cortical bone continuity (35-90%, p < 0.01) and elevated RUNX family transcription factor 2 and RANK levels (5-12%, p < 0.01), but suppressed matrix metallopeptidase 9 and cathepsin K levels (9-20%, p < 0.01) in femur tissues. BMSC-Exo from MOP-treated rats (MOP-Exo) suppressed osteoclastic differentiation and proliferation of BMMs. The downregulation of microRNA-101-3p (miR-101-3p) or the upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2) blocked the functions of MOP-Exo. DISCUSSION AND CONCLUSIONS: MOP inhibits osteoclastic differentiation and could potentially be used for osteoporosis management. This suppression may be enhanced by the upregulation of miR-101-3p or the inhibition of PTGS2.

MiR-101-3p Suppresses Progression of Cervical Squamous Cell Carcinoma by Targeting and Down-Regulating KPNA2.

Objective We explored mechanism of microRNA-101-3p/Karyopherin α2 (KPNA2) axis in cervical squamous cell carcinoma. Methods: Bioinformatics methods were applied to identify genes for the study. Cell functional assays were implemented to examine the role of the genes in malignant progression of cervical squamous cell carcinoma. Targeting relationship between genes was verified by dual-luciferase assay. Results: MicroRNA-101-3p was lowly expressed in cervical squamous cell carcinoma, while KPNA2 was highly expressed. Dual-luciferase assay identified direct targeting relationship between microRNA-101-3p and KPNA2. Functional assays manifested that highly expressed microRNA-101-3p suppressed cervical squamous cell carcinoma cell growth by targeting KPNA2. Conclusion: Overall, microRNA-101-3p/KPNA2 axis can play an important part in progression of cervical squamous cell carcinoma.

miR­101­3p negatively regulates inflammation in systemic lupus erythematosus via MAPK1 targeting and inhibition of the NF­κB pathway.

Systemic lupus erythematosus (SLE) is an autoimmune disease often used as a model in genomics research. The downregulation of microRNA­101­3p (miR­101­3p) participates in the progression of SLE, although the underlying mechanisms remain to be elucidated. The present study aimed to evaluate the specific roles of miR­101­3p in the SLE inflammatory response and its potential mechanisms. Reverse transcription­quantitative (RT­q) PCR was used to profile miR­101­3p expression in the peripheral blood mononuclear cells (PBMCs) from 40 female patients with SLE and 20 female healthy volunteers. The interactions between miR­101­3p and MAPK1 were identified and evaluated using dual­luciferase reporter and RNA pull­down assays. The levels of IL­10 and IFN­Î³ were evaluated by enzyme­linked immunosorbent assay. The expression of NF­κB p65 and phosphorylated IκBα were evaluated using western blotting. miR­101­3p expression was demonstrated to be downregulated in SLE PBMCs. miR­101­3p negatively regulated IL­10 and IFN­Î³ expression in SLE samples and was demonstrated to target MAPK1. Increases in MAPK1 expression eliminated miR­101­3p inhibition of IL­10 and IFN­Î³. MAPK1 activated the NF­κB pathway in SLE PBMCs and this activation was inhibited when miR­101­3p was overexpressed. In addition, treatment with BAY11­7085 (NF­κB activator) was demonstrated to reverse the inhibitory effects of miR­101­3p expression on both IL­10 and IFN­Î³ in SLE PBMCs. BAY11­7082 also markedly reduced MAPK1­induced increases in IL­10 and IFN­Î³ in SLE PBMCs. miR­101­3p overexpression attenuated the inflammatory response in SLE PBMCs by inhibiting the expression of MAPK1 and blocking the NF­κB pathway. The results revealed a novel regulatory mechanism in SLE inflammation and offer a new direction for the development of SLE treatments.

Knockdown of lncRNA HOTTIP Inhibits Retinoblastoma Progression by Modulating the miR-101-3p/STC1 Axis.

OBJECTIVE: Retinoblastoma (RB) is a frequent eye cancer in children. Long non-coding RNA (LncRNA) HOXA transcript at the distal tip (HOTTIP) is aberrantly expressed in cancer tissues. This study explores the underlying mechanism of lncRNA HOTTIP in RB. METHODS: HOTTIP expression in normal retinal cells and RB cell lines was detected using qRT-PCR. The proliferation of RB cells was measured using CCK-8 and EdU assays, and apoptosis was detected using flow cytometry and Western blotting after the transfection of si-HOTTIP into Y79 cells and pc-HOTTIP into HXO-RB-44 cells. The target relationships between HOTTIP and miR-101-3p, and miR-101-3p and STC1 were predicted by bioinformatics website and verified using dual-luciferase reporter gene assay. The binding of HOTTIP and miR-101-3p was verified using RNA pull-down assay. STC1 mRNA and protein in RB cells were measured using qRT-PCR and Western blotting. Moreover, si-HOTTIP and in-miR-101-3p/in-NC, and si-HOTTIP and pc-STC1/pcDNA were co-transfected into Y79 cells respectively to evaluate cell proliferation and apoptosis. Xenograft study was conducted, and Ki67-positive expression was detected using immunohistochemical staining. RESULTS: HOTTIP expression was promoted in RB tissues and cells. Downregulation of HOTTIP inhibited proliferation and promoted apoptosis of Y79 cells, while upregulation of HOTTIP promoted proliferation and inhibited apoptosis of HXO-RB-44 cells. There were target relationships between HOTTIP and miR-101-3p, and miR-101-3p and STC1. Inhibition of miR-101-3p or overexpression of STC1 reversed the effect of si-HOTTIP on the proliferation and apoptosis of RB cells. Xenograft study showed that knockdown of HOTTIP suppressed the growth of RB in vitro. CONCLUSION: It could be concluded that HOTTIP sponged miR-101-3p to upregulate STC1 expression, thereby promoting RB cell proliferation and inhibiting apoptosis.

Significances of the plasma expression of microRNA-101-3p and microRNA-141-3p in children with sepsis / 中华实用儿科临床杂志

Objective:To examine the plasma expression levels and clinical significances of microRNA(miR)-101-3p and miR-141-3p in children with sepsis.Methods:One hundred and fifty-three children with sepsis admitted in Sanya People′s Hospital from January 2016 to October 2019 were divided into sepsis without shock group (94 cases) and septic shock group (59 cases). In addition, they were further divided into survival group (107 cases) and death group (46 cases) according to the 28-day survival.Another 60 healthy children were selected as the healthy control group.Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was performed to detect plasma levels of miR-101-3p and miR-141-3p in all subjects.Receiver operating characteristic curve(ROC) were depicted to identify the diagnostic and prognostic potentials of plasma miR-101-3p, miR-141-3p and procalcitonin(PCT) in sepsis. Pearson′ s correlation analysis was performed to analyze the correlation between the expression levels of miR-101-3p, miR-141-3p and PCT with Acute Physiology and Chronic Health Evaluation Ⅱ(APACHE Ⅱ)score, Sequential Organ Failure Assessment(SOFA)score, leukocyte count and C-reactive protein level in children with sepsis. Results:Plasma levels of miR-101-3p, miR-141-3p and PCT in septic shock group and sepsis without shock group were significantly higher than those in the healthy control group (all P<0.001). Moreover, plasma levels of miR-101-3p (4.25±1.46 vs.1.86±0.75), miR-141-3p (3.17±1.08 vs.1.20±0.52) and PCT [(20.75±9.36) μg/L vs.(5.80±2.40) μg/L] in septic shock group were significantly higher than those in sepsis without shock group (all P<0.001). In addition, plasma levels of miR-101-3p, miR-141-3p and PCT in survival group and death group were significantly higher than those in the healthy control group (all P<0.001). Notably, plasma levels of miR-101-3p (4.83±1.62 vs.1.40±0.58), miR-141-3p (3.50±1.13 vs.0.96±0.47), and PCT [(26.30±11.72) μg/L vs.(3.25±2.16) μg/L] in death group were significantly higher than those in the survival group (all P<0.001). ROC curve analysis showed that the area under the curve (AUC) and 95% confidence interval (95% CI) of the combined diagnosis of sepsis with miR-101-3p, miR-141-3p and PCT were significantly higher than that of miR-101-3p, miR-141-3p or PCT alone [0.908 (0.850-0.970) vs.0.810 (0.748-0.873), 0.784 (0.723-0.844) and 0.825 (0.764-0.883), respectively; Z1=4.682, Z2=5.380 and Z3=4.417, all P<0.05]. The sensitivity and specificity of the combined diagnosis was 92.5% and 84.0%, respectively.The AUC and 95% CI of the combined prediction of miR-101-3p, miR-141-3p and PCT in the mortality of children with sepsis children with were significantly higher than those with miR-101-3p, miR-141-3p or PCT alone [0.930 (0.872-0.986) vs.0.848 (0.786-0.907), 0.792 (0.730-0.853) and 0.820 (0.762-0.878), respectively; Z1=4.537, Z2=5.728 and Z3=5.106, all P<0.05]. The sensitivity and specificity of the combined prediction in the mortality was 94.6%, and 87.0%, respectively.Correlation analysis showed that miR-101-3p and miR-141-3p levels were positively correlated with PCT ( r=0.804, 0.773, all P<0.001), APACHE Ⅱ score ( r=0.738, 0.695, P<0.001) and SOFA score ( r=0.752, 0.764, all P<0.001). Conclusions:Plasma levels of miR-101-3p and miR-141-3p in children with sepsis significantly increased, which are correlated with the severity of sepsis.A combination detection of miR-101-3p, miR-141-3p and PCT has high diagnostic and prognostic potentials in children with sepsis.

Long non-coding RNA SNHG6 promotes tumorigenesis in melanoma cells via the microRNA-101-3p/RAP2B axis.

Numerous studies have reported that the long non-coding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG6; ENSG00000245910) participates in the development of malignant tumors. However, the underlying mechanism of SNHG6 in the development of melanoma remains unknown. Thus, the present study aimed to investigate the biological role of SNHG6 in the progression of melanoma. SNHG6 expression in melanoma tissues and cells was assessed using a bioinformatics approach and reverse transcription-quantitative PCR analysis. Cell viability was determined using the Cell Counting Kit-8 and colony formation assays. The correlation between microRNA (miR)-101-3p, SNHG6 and RAP2B expression levels was assessed using Pearson's correlation analysis. Bioinformatic analysis and luciferase reporter assay were utilized to confirm the interaction between miR-101-3p and SNHG6 or RAP2B. The Transwell assay was conducted to examine the migratory and invasive activities of melanoma cells. In the present study, SNHG6 expression was upregulated in melanoma tissues and cell lines, and SNHG6 silencing suppressed melanoma cell viability, migration and invasion. SNHG6 was directly bound to miR-101-3p, which interacted with RAP2B. In addition, miR-101-3p expression was negatively correlated with SNHG6 or RAP2B expression. miR-101-3p silencing partially abrogated the suppressive effect of SNHG6-knockdown on RAP2B expression. Moreover, the data demonstrated that RAP2B overexpression reversed the inhibitory effects on melanoma cell proliferation, migration and invasion induced by SNHG6 silencing. In conclusion, the present study identified that SNHG6 accelerated melanoma progression via regulating the miR-101-3p/RAP2B axis. Thus, the SNHG6/miR-101-3p/RAP2B signaling pathway may be a novel therapeutic target for melanoma.

Long Noncoding RNA RC3H2 Facilitates Cell Proliferation and Invasion by Targeting MicroRNA-101-3p/EZH2 Axis in OSCC.

In our previous studies, enhancer of zeste homolog 2 (EZH2) has been proven to be a key oncogenic driver in oral squamous cell carcinoma (OSCC). However, the regulatory mechanisms on EZH2 remain poorly understood in OSCC. Here, through multi-transcriptomics, bioinformatics analysis, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the co-expression network of long noncoding RNA RC3H2 (RC3H2), microRNA-101-3p (miR-101-3p), and EZH2 were screened and validated as a competing endogenous RNA (ceRNA) mechanism in OSCC. Silencing of RC3H2 inhibited OSCC cell proliferation, colony formation, migration, and invasion in vitro and reduced the expression of EZH2 and H3K27Me3, whereas RC3H2 overexpression significantly promoted OSCC cell growth, colony formation, migration, invasion, and xenograft tumor growth in vivo and increased the expression of EZH2 and H3K27Me3. A fluorescence in situ hybridization (FISH) assay verified that RC3H2 was predominately localized to the cytoplasm. RNA pull-down and luciferase activity assays showed that miR-101-3p was physically bound to RC3H2 as well as EZH2, and its inhibitor reversed the inhibitory effect of RC3H2 knockdown on progression of OSCC. Taken together, our findings demonstrate that RC3H2 as completive endogenous RNA sponging miR-101-3p targets EZH2 and facilitates OSCC cells' malignant behavior.

MicroRNA-101-3p inhibits fibroblast-like synoviocyte proliferation and inflammation in rheumatoid arthritis by targeting PTGS2.

OBJECTIVE: Rheumatoid arthritis (RA) is the most frequently occurring inflammatory arthritis. The present study was performed to characterize the role of microRNA-101-3p (miR-101-3p) and prostaglandin-endoperoxide synthase 2 (PTGS2) in inflammation and biological activities of fibroblast-like synoviocytes (FLSs) in RA. METHODS: Initially, miR-101-3p and PTGS2 expression in RA tissues of RA patients and RA rats was detected by qRT-PCR and Western blot analysis. Rat model of type II collagen-induced arthritis (CIA) was adopted to simulate RA, followed by injection of miR-101-3p mimics or siRNA against PTGS2. Next, the apoptosis in synovial tissue and the levels of tumor necrosis factor (TNF)-α, IL-1ß and IL-6 were identified. Subsequently, FLSs in RA (RA-FLSs) were isolated, after which in vitro experiments were conducted to analyze cell proliferation, apoptosis, migration and invasion upon treatment of up-regulated miR-101-3p and silenced PTGS2. Furthermore, the relationship of miR-101-3p and PTGS2 was determined by bioinformatics prediction and luciferase activity assay. RESULTS: We identified poorly expressed miR-101-3p and highly expressed PTGS2 in synovial tissues of RA patients and RA rats, which showed reduced synoviocyte apoptosis and enhanced inflammation. In response to miR-101-3p mimics and si-PTGS2, the RA-FLSs were observed with attenuated cell proliferation, migration and invasion, corresponding to promoted apoptosis. Down-regulation of PTGS2 could rescue the effect of inhibited miR-101-3p in synovial injury and phenotypic changes of FLS in RA rats. Notably, miR-101-3p was found to negatively regulate PTGS2. CONCLUSION: Taken together, miR-101-3p reduces the joint swelling and arthritis index in RA rats by down-regulating PTGS2, as evidenced by inhibited FLS proliferation and inflammation.

MicroRNA-101-3p regulates gastric cancer cell proliferation, invasion and apoptosis by targeting PIM 1 expression.

To investigate the effect of microRNA-101-3p (miRNA-101-3p) on proliferation, invasion and apoptosis of gastric cancer (GC) cells, and to explore its influence mechanism. Human GC cell line (AGS) and normal human gastric epithelial cell line (GES-1) were used in this study. The cells were transfected with proto-oncogene serine/threonine-protein kinase (PIM 1) overexpression plasmid, miRNA-101-3p mimics, or miRNA-101-3p non-homologous sequence using lipofectamine 2000. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to determine the expressions of miRNA-101-3p and PIM 1 in GC cells. Cell counting kit-8 (CCK-8) and Transwell assays were used to assess the effect of miRNA-101-3p on proliferation and invasion of GC cells. The regulatory effect of miRNA-101-3p on PIM 1 was assessed using bioinformatics analysis and luciferase reporter gene assay. The expression of miRNA-101-3p was significantly down-regulated in GC cells, relative to normal human gastric epithelial cells (p < 0.05). However, the expression of PIM 1 mRNA was significantly upregulated in GC cells, when compared with normal gastric epithelial cells (p < 0.05). The expression level of miRNA-101-3p was significantly higher in miRNA-101-3p mimic group than in miRNA-101-3p control and normal control groups (p < 0.05). Cell proliferation in miRNA-101-3p mimic group was significantly and time-dependently reduced, when compared with miRNA-101-3p control and normal control groups (p < 0.05). Transfection with miRNA-101-3p mimics significantly increased the invasiveness of AGS cells, and significantly promoted their apoptosis (p < 0.05). Results of qRT-PCR and Western blotting showed that increased miRNA-101-3p expression significantly reduced the expression of PIM 1, while decreased miRNA-101-3p expression promoted the expression of PIM 1 (p < 0.05). Results of bioinformatics showed that miRNA-101-3p had specific binding sequence in the 3'UTR region of PIM 1. Cloning of miRNA-101-3p sequence into the luciferase reporter plasmid led to significant inhibition of the expression of PIM 1 (p < 0.05), but had no inhibitory effect on mutated PIM 1 3'UTR (p > 0.05). Overexpression of PIM 1 significantly reversed the inhibition of proliferation and invasion, and promotion of apoptosis by miRNA-101-3p (p < 0.05). These results indicate that miRNA-101-3p inhibits the proliferation and invasion of GC cells, and promotes their apoptosis by regulation of the expression of PIM-1.

Zinc-induced protective effect for testicular ischemia-reperfusion injury by promoting antioxidation via microRNA-101-3p/Nrf2 pathway.

The present study was performed to determine the protective effect of Zinc on the rat testicular ischemia-reperfusion (I/R) injury and its mechanism. In vivo, the pathological changes and the apoptosis index were significantly relieved in the rats with Low-dose Zinc pretreatment, compared to the I/R group. After Low-dose Zinc treatment, the levels of tissue Malondialdehyde (MDA) were significantly decreased, while tissue antioxidant indices were significantly increased. Meanwhile, the level of NF-κB was significantly lower compared to I/R group, while the levels of Nrf2-dependent antioxidant enzymes were significantly higher in Low-dose Zinc+I/R group. In vitro, Low-dose Zinc markedly increased Leydig cell (TM3) cell viability, and relieved testicular oxidative damage via down-regulating ROS. A total of 22 differently expressed microRNAs were screened out using microRNA microarray in rat testicular tissue caused by I/R injury, especially showing that miR-101-3p was selected as the target miRNA. Furthermore, the levels of Nrf2 and NF-κB were apparently increased/decreased in TM3 cells treated with Hypoxic/Reoxygenation (H/R) after miR-101-3p mimics/inhibitor. In addition, H/R-induced testicular oxidative damage was recovered in TM3 administrated with miR-101-3p inhibitor and si-Nrf2. Therefore, this study provided a novel insight for investigating protective effect of Zinc on testicular I/R injury by promoting antioxidation via miR-101-3p/Nrf2.

Exosomes derived from microRNA-101-3p-overexpressing human bone marrow mesenchymal stem cells suppress oral cancer cell proliferation, invasion, and migration.

Dysregulation of microRNAs (miRNAs) has been found to disrupt the progression of oral cancer. However, which miRNAs are most effective against oral cancer and how these miRNAs should be delivered are major unanswered problems. We aimed at investigating if human bone marrow mesenchymal stem cells (hBMSCs)-derived exosomes affect oral cancer development, and the potential regulatory mechanism associated with COL10A1 and miR-101-3p. COL10A1 was upregulated, while miR-101-3p was downregulated in oral cancer, and miR-101-3p targeted COL10A1 as verified by dual-luciferase reporter gene assay. Meanwhile, exosomes derived from hBMSCs were isolated and then co-cultured with oral cancer cells to identify the role of exosomes, and the results suggested that hBMSCs-derived exosomes overexpressing miR-101-3p inhibited oral cancer progression. Furthermore, tumorigenicity assay in nude mice further confirmed the inhibitory effects of hBMSCs-derived exosomes, loaded with miR-101-3p, on oral cancer, which provides a new theoretical basis in the treatment of oral cancer.

Downregulation of lncRNA NEAT1_2 radiosensitizes hepatocellular carcinoma cells through regulation of miR-101-3p/WEE1 axis.

Radioresistance is a major obstacle in hepatocellular carcinoma (HCC) radiotherapy. Aberrant expression of long non-coding RNA (lncRNA) has been postulated to be implicated in the development of HCC radioresistance. We investigated the role of lncRNA nuclear enriched abundant transcript 1_2 (NEAT1_2) in radioresistance of HCC and its molecular mechanism in this study. We found that NEAT1_2 and WEE1 were upregulated, and miR-101-3p was downregulated in HCC tissues, as well as HCC cell lines. Downregulation of WEE1 sensitized the radiosensitivity of HCC cells, as evidenced by decreased survival fractions of Huh7 and PLC5 cells and increased percentage of apoptotic cells. Also, knockdown of NEAT1_2 exerted a reinforcing effect on the radiosensitivity of HCC cells. In addition, WEE1 was confirmed as a direct target of miR-101-3p. Upregulation of miR-101-3p obviously decreased the mRNA and protein levels of WEE1 compared with that in the miR-NC group, while transfection of anta-miR-101-3p presented the opposite effects. In parallel, NEAT1_2 was identified to interact with miR-101-3p, and NEAT1_2 upregulated the expression of WEE1 in Huh7 cells through sponging miR-101-3p. Besides, the reinforcing effect of NEAT1_2 silencing could be attenuated by downregulation of miR-101-3p. To conclude, our results support the concept that downregulation of lncRNA NEAT1_2 radiosensitizes hepatocellular carcinoma cells through regulation of miR-101-3p/WEE1 axis.

Prognostic implications of decreased microRNA-101-3p expression in patients with non-small cell lung cancer.

To investigate the expression level of microRNA-101-3p (miR-101-3p) and its possible association with progression, prognosis and chemotherapy in patients with non-small cell lung cancer (NSCLC), the Gene Expression Omnibus (GEO) database was used. Quantitative polymerase chain reaction was used to verify the expression in 327 NSCLC and 42 adjacent normal lung tissues, of which 42 viable tissues were paired with nearby normal lung tissues. Based on the Cox regression model, univariate and multivariate analyses were used to address the factors that had effects on overall survival (OS) and disease-free survival (DFS) rate. Data from the GEO database demonstrated that the miR-101-3p expression in NSCLC was downregulated, compared with normal lung cancer. Survival analysis through univariate and multivariate models indicated that the miR-101-3p expression level was a crucial risk factor for OS and DFS in patients with NSCLC. A number of clinical parameters were determined to be associated with miR-101-3p expression, including tumor diameter, lymph node metastasis and tumor-node-metastasis stage. Adjuvant chemotherapy with high expression of miR-101-3p was determined to increase OS and DFS in patients with NSCLC, compared with patients with de novo or low expression of miR-101-3p. The present results demonstrated that miR-101-3p expression levels were associated with NSCLC progression and prognosis, which indicated that miR-101-3p may serve as a biomarker for patients with NSCLC who have received adjuvant chemotherapy.

MicroRNA-101-3p inhibits proliferation in retinoblastoma cells by targeting EZH2 and HDAC9.

Retinoblastoma is the most frequent intraocular malignant tumor type to occur in childhood. MicroRNA (miR)-101-3p has been reported to function as a tumor suppressor in various types of cancer. However, the biological function and underlying mechanisms of miR-101-3p in retinoblastoma are largely unknown. In the present study, it was identified that miR-101-3p was downregulated in retinoblastoma. MTT and flow cytometry assays demonstrated that ectopic overexpression of miR-101-3p significantly inhibited cell viability and cell cycle progression in WERI-Rb-1 and Y79 cells. In vivo mouse experiments further confirmed the anti-proliferative role of miR-101-3p in retinoblastoma. Additionally, predictions with TargetScan software indicated that the 3'-untranslated regions of enhancer of zeste homolog 2 (EZH2) and histone deacetylase (HDAC9) mRNAs are targeted by miR-101-3p. Accordingly, a dual luciferase reporter gene assay demonstrated that miR-101-3p directly targeted EZH2 and HDAC9 to suppress the proliferation of retinoblastoma cells. Meanwhile, the restoration of EZH2 or HDAC9 expression countered the anti-proliferative effect of miR-101-3p on WERI-Rb-1 and Y79 cells. Collectively, these data highlight the role of miR-101-3p in the tumorigenesis of retinoblastoma, and indicate its suitability as a novel therapeutic target.

Diagnostic value of strand-specific miRNA-101-3p and miRNA-101-5p for hepatocellular carcinoma and a bioinformatic analysis of their possible mechanism of action.

There is accumulating evidence that miRNA might serve as potential diagnostic and prognostic markers for various types of cancer. Hepatocellular carcinoma (HCC) is the most common type of malignant lesion but the significance of miRNAs in HCC remains largely unknown. The present study aimed to establish the diagnostic value of miR-101-3p/5p in HCC and then further investigate the prospective molecular mechanism via a bioinformatic analysis. First, the miR-101 expression profiles and parallel clinical parameters from 362 HCC patients and 50 adjacent non-HCC tissue samples were downloaded from The Cancer Genome Atlas (TCGA). Second, we aggregated all miR-101-3p/5p expression profiles collected from published literature and the Gene Expression Omnibus and TCGA databases. Subsequently, target genes of miR-101-3p and miR-101-5p were predicted by using the miRWalk database and then overlapped with the differentially expressed genes of HCC identified by natural language processing. Finally, bioinformatic analyses were conducted with the overlapping genes. The level of miR-101 was significantly lower in HCC tissues compared with adjacent non-HCC tissues (P < 0.001), and the area under the curve of the low miR-101 level for HCC diagnosis was 0.925 (P < 0.001). The pooled summary receiver operator characteristic (SROC) of miR-101-3p was 0.86, and the combined SROC curve of miR-101-5p was 0.80. Bioinformatic analysis showed that the target genes of both miR-101-3p and miR-101-5p are involved in several pathways that are associated with HCC. The hub genes for miR-101-3p and miR-101-5p were also found. Our results suggested that both miR-101-3p and miR-101-5p might be potential diagnostic markers in HCC, and that they exert their functions via targeting various prospective genes in the same pathways.