The predictive value of VEGF, SCCAg and miRNA let-7a in the metastasis and recurrence of laryngeal carcinoma / 中国医师杂志

Objective:To investigate the predictive value of serum vascular endothelial growth factor (VEGF), squamous cell carcinoma-associated antigen (SCCAg) and miRNA let-7a in lymph node metastasis and postoperative recurrence in patients with laryngeal cancer.Methods:A total of 82 patients with laryngeal cancer in the Second Central Hospital of Baoding from November 2017 to October 2019 were selected as the research subjects, including 18 cases of lymph node metastasis (metastasis group) and 64 cases of non metastasis (non metastasis group). The blood routine was tested before operation, and the baseline data, serum VEGF, SCCAg and miRNA let-7a levels were compared between the two groups. Logistic regression was used to analyze the related influencing factors of lymph node metastasis in patients with laryngeal cancer. The correlation between serum VEGF, SCCAg, miRNA let-7a levels and clinicopathological characteristics was analyzed. The receiver operating characteristic (ROC)curve was used to analyze the value of each index and the combined diagnosis of lymph node metastasis in patients with laryngeal cancer. After 1 year of follow-up, the serum VEGF, SCCAg and miRNA let-7a levels of patients with or without recurrent laryngeal cancer were compared. ROC curve was used to evaluate the value of VEGF, SCCAg, and miRNA let-7a in predicting the recurrence of laryngeal cancer.Results:There were statistically significant differences in tumor node metastasis (TNM) stage, degree of infiltration, degree of differentiation, serum VEGF, SCCAg, and miRNA let-7a levels between the metastasis group and non metastasis group (all P<0.05). Serum VEGF, SCCAg, miRNA let-7a levels in patients with laryngeal cancer were related to TNM stage, degree of infiltration and degree of differentiation (all P<0.05). The combined diagnosis of serum VEGF, SCCAg and miRNA let-7a levels in the diagnosis of lymph node metastasis in patients with laryngeal cancer showed that the diagnostic sensitivity and specificity were 88.89% and 70.31%, respectively. The serum VEGF and SCCAg levels of patients with recurrence after operation were higher than those without recurrence, and the level of miRNA let-7a was lower than those without recurrence (all P<0.05). The sensitivity and specificity of combined serum VEGF, SCCAg and miRNA LET-7a levels in predicting postoperative recurrence of laryngeal cancer were 72.97% and 91.11%, respectively. Conclusions:VEGF, SCCAg, miRNA let-7a in patients with laryngeal cancer have a certain correlation with clinicopathological characteristics, which can assist in the diagnosis of lymph node metastasis and help clinical prediction of postoperative recurrence in patients with laryngeal cancer, and provide a reference for the formulation of clinical treatment plans.

Effect of Compound Phylanthus urinaria Ⅱ in Regulating Expression of lncRNA CCAT1 in Hepatocellular Carcinoma HepG2 Cells / 中国实验方剂学杂志

Objective:To explore the anti-hepatoma effect of compound <italic>Phylanthus urinaria</italic> Ⅱ （ CPU Ⅱ） by inhibiting the expression of the long non-coding RNA （lncRNA） colon cancer associated transcript-1 （CCAT1） and restoring the expression of microRNA let-7a. Method:Real-time fluorescence quantitative polymerase chain reaction （PCR） was used to detect the expression of lncRNA CCAT1 in normal liver cells （LO2 cells） and hepatocellular carcinoma HepG2 cells， and the differences in expression between these two types of cells were compared. The methylthiazolyl tetrazolium（MTT） assay was used to detect the proliferation of HepG2 cells after treatment with different concentrations of CPU Ⅱ and 5-fluorouracil（5-FU） for 24， 48 and 72 h. Hepatocellular carcinoma HepG2 cells were cultured <italic>in vitro </italic>and set into three gropes： cell control group， CPU Ⅱ low-dose group （0.8 g·L^-1） and high-dose group （1.6 g·L^-1）. Real-time PCR was used to detect the mRNA expression of lncRNA CCAT1， microRNA let-7a and its target genes high mobility group protein A2（HMGA2）， and N-RAS in each grope. Western blot was used to detect the protein expression of HMGA2， and Cyclin D₁ in each grope. Result:As compared with LO2 cells， expression of lncRNA CCAT1 in HepG2 cells was significantly up-regulated （<italic>P</italic><0.05）. Results of MTT assay showed that the 50% inhibiting concentration（IC₅₀）of CPU Ⅱ and 5-FU on hepatocellular carcinoma HepG2 cells was 1.649， 0.044 648 g·L^-1respectively. As compared with the control group， CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L^-1） significantly inhibited the proliferation of HepG2 cells （<italic>P</italic><0.05）， and the effect was most remarkable in CPU Ⅱ high-dose group （<italic>P</italic><0.05）. The results of Real-time PCR showed that as compared with control group， the expression of lncRNA CCAT1 mRNA was significantly inhibited in CPU Ⅱ high-and low-dose groups （<italic>P</italic><0.05）， and the expression of microRNA let-7a mRNA was obviously up-regulated in high-dose group （<italic>P</italic><0.05）， but the expression of HMGA2 mRNA in CPU Ⅱ high-and low-dose groups as well as the expression of N-RAS mRNA in CPU Ⅱ low-dose group were down-regulated （<italic>P</italic><0.05）. Western blot results showed that as compared with the cell control group， the protein expression of HMGA2 and Cyclin D₁ in CPU Ⅱ high-and low-dose groups （1.6， 0.8 g·L^-1） was significantly down-regulated （<italic>P</italic><0.05）. Conclusion:CPU Ⅱ can inhibit the expression of lncRNA CCAT1， recover the expression of microRNA let-7a， and suppress the mRNA and protein expression of related downstream target genes in hepatoma cells line HepG2， thereby inhibiting the proliferation of hepatocellular carcinoma cells and exerting anti-hepatocellular carcinoma effect.

Let-7a-5p represses proliferation, migration, invasion and epithelial-mesenchymal transition by targeting Smad2 in TGF-β2-induced human lens epithelial cells

Transforming growth factor b2 (TGF-b2)/Smad signaling is widely accepted as a key inducer of proliferationand epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs), contributing to thedevelopment of posterior capsule opacification (PCO). Increasing evidence shows that microRNAs (miRNAs)play important roles in PCO pathogenesis. Herein, we aimed to explore the role and molecular mechanism oflet-7a-5p on TGF-b2-induced proliferation and EMT in LECs. qRT-PCR was performed to detect theexpression of let-7a-5p and Smad2 mRNA. Western blot was used to determine the Smad2 level and theinduction of EMT. The targeted correlation between let-7a-5p and Smad2 was confirmed using dual-luciferasereporter and RNA immunoprecipitation assays. CCK-8 assay was employed to determine cell proliferation, andtranswell assays were performed to assess cell migration and invasion. We found that TGF-b2 induced EMT ofLECs, and TGF-b2 upregulated Smad2 expression and reduced let-7a-5p expression in LECs. Smad2 was adirect target of let-7a-5p. Moreover, let-7a-5p upregulation repressed proliferation, migration, invasion andEMT in TGF-b2-induced LECs. But, Smad2 expression restoration abrogated the inhibitory effect of let-7a-5pupregulation. In conclusion, our data indicated that let-7a-5p upregulation repressed TGF-b2-induced proliferation, migration, invasion and EMT at least partly by targeting Smad2 in LECs, highlighting that let-7a-5pmight act as a promising therapeutic target to intervene to the progression of PCO.

Effect of incRNA H19 up-regulation mediated by IL-6/STAT3 pathway in pathogenesis of ulcerative colitisassociated colorectal cancer in mice / 吉林大学学报(医学版)

Objetive: To detect the expressions of IncRNA H19 (H19) and IL-6/STAT3 pathway in the ulcerative colitis-associated colorectal cancer (CAC) tissue of the mice, and to explore its possible mechanism Methods: A total of 22 C57BL/6 mice were randomly divided into control group (n=10) and model group (n= 12). The CAC models were induced by azomethane (AOM) combined with dextran sodium sulfate (DSS) in the mice in model group. The mice were sacrificed on the 120th day, the disease activity index (DAI) of the mice was evaluated, the tumor formation rate was evaluated, the colon length was measured, and the pathomorphology of colon tissue of the mice was observed by HE staining. The serum IL-6 level of the mice was detected by ELISA. The expression levels of H19, let-7a, IL-6, STAT3 and c-Myc mRNA in colon tissue of the mice were detected by qPCR method. The expression levels of p-STAT3 and c-Myc proteins in colon tissue of the mice were detected by Western blotting method. Results: Compared with control group, the tumor formation rate of the mice in model group was 100%, the colon length was significantly shortened (P<0. 01), the DAI score was increased (P< 0.01), the colon tissue showed the intraepithelial neoplasia by HE staining, the expression levels of H19, IL-6, STAT3 and c-myc mRNA in colon tissue were significantly increased (P<0. 01), the expression level of let-7a mRNA in colon tissue was significantly decreased (P<0. 01), the serum IL-6 level was increased (P<0. 01), and the expression levels of p-STAT3 and c-Myc proteins in colon tissue were increased (P<0. 01). Conclusion: The pathogenesis of CAC in the mice may be related to the up-regulation of c-Myc and H19 and down-regulation of let-7a, which are mediated by IL-6/STAT3 pathway.

Panax japlcus var polysaccharide regulates proliferation and apoptosis of gastric cancer MKN45 cells by targeting let-7a/CDK6 molecular axis / 中国肿瘤生物治疗杂志

@#Objective: To investigate the effect of panax japlcus var polysaccharide (PJPS) on the proliferation and apoptosis of gastric cancer MKN45 cells and its regulatory mechanism. Methods: Human gastric cancer cell lines (HGC27, MGC803, MKN45) and gastric mucosal epithelial cell line GES-1 were selected for this study. Let-7a mimics and let-7a inhibitor were transfected into MKN45 cells; Gastric cancer cell lines were treated with 100 μg/ml PJPS and MKN45 was selected as the subsequent experimental cell line. MKN45 cells were cultured with 0, 10, 50, 100 and 120 μg/ml PJPS, respectively. The proliferation and apoptosis rate of MKN45 cells were detected by CCK-8 and flow cytometry, respectively. Expressions of cell cycle dependent kinase 6 (CDK6) and apoptosis-related proteins in MKN45 cells were detected by Western blotting, and the expression level of miRNAs regulating the proliferation of gastric cancer cells was detectedbyReal-timequantitativePCR(qPCR).TheDualluciferasereportergeneassaywasusedtovalidatethetargeting relationship between let-7a and CDK6. Results: Compared with other gastric cancer cells, 100 μg/ml PJPS significantly inhibited the proliferation of MKN45 cells (P<0.01). At the same time, 100 μg/ml PJPS significantly up-regulated the expression of let-7a in MKN45 cells (P<0.01). The Dual luciferase reporter gene assay confirmed that CDK6 was the target gene of let-7a. Furthermore, PJPS inhibited the expression of CDK6 by up-regulating let-7a, thereby inhibiting the proliferation and inducing apoptosis of MKN45 cells (all P<0.01). Conclusion: PJPS inhibits proliferation and induces apoptosis of gastric cancer MKN45 cells by regulating the let-7a/ CDK6 axis.

Exosome-mediated Let-7a inhibits malignant biological behaviors of triple-negative breast cancer cells by down-regulating MYC expression / 中国肿瘤生物治疗杂志

@#Objective: To investigate the role of exosome (EXO) transporting Let-7a to regulate MYC gene in the malignant biological behaviors of triple negative breast cancer (TNBC) cell, and to explore the underlying mechanism. Methods: After the completion of cell culture, the gene and protein expressions of MYC and Let-7a in TNBC MDA-MB-231cells were detected by qPCR and WB, respectively. Recombinant lenti-virus vector carrying Let-7a and Crisper/Cas-9 system with MYC knockdown were transfected into MDA-MB-231 cells; MTT assay, Transwell assay and Scratch healing assay were performed to examine the proliferation, invasion and migration of MDA-MB-231 cells. Luciferase activity assay was performed to validate the binding between MYC and Let-7a. EXO was isolated and identified by transmission electron microscopy and WB assay in wild-type and Let-7a over-expressed MDA-MB-231 cells, respectively. After co-incubation of two types of EXO and MDA-MB-231 cells, the effects of Let-7a on biological behaviors of MDAMB-231 cells via EXO were detected by qPCR, WB, MTT and Transwell etc. Results: Let-7a was negatively correlated with MYC in breast cancer tissues and cell lines (all P<0.05); MYC promoted while Let-7a inhibited the proliferation, migration and invasion of breast cancer cells (all P<0.01); Let-7a silenced MYC by acting on 3'UTR of MYC gene, thereby reducing the expression of MYC protein (P<0.05); Let-7a was enveloped by EXO and transported to cancer cells, there by inhibiting the proliferation, migration and invasion of MDA-MB-231 cells. Conclusion: EXO some mediated Let-7a silences MYC gene by acting on its 3'UTR region, thus inhibiting the proliferation, migration and invasion of MDA-MB-231 cells.

Metformin induced apoptosis of nasopharyngeal carcinoma cell CNE-1 by regulating miR-let-7a and IGF-1R / 医学研究生学报

Objective research the effects of metformin on proliferation and apoptosis in nasopharyngeal carcinoma cell CNE-1 and investigate the role of miR-let-7a、IGF-1R in it. Methods The nasopharyngeal carcinoma cell CNE-1 was treated with different concentrations of metformin for 24h, then the proliferation activity of cell was detected by CCK8 method; the apoptosis rate of cell was measured by flow cytometry; the expression levels of bcl-2、bax and IGF-1R mRNA and miR-let-7a were detected by real-time quantitative PCR; the expression level of IGF-1R protein was detected by Western blot. Results Compared with the control group, the cell proliferation activity of metformin group decreased(P<0.05), and it gradually decreased along with the increase of metformin concentration. The cell apoptosis rate of metformin group increased(P<0.05 except for the 5 mmol/L group), and it gradually increased along with the increase of metformin concentration. The expression levels of bax mRNA and miR-let-7a were up-regulated in the metformin group(P<0.05), while the expression levels of bcl-2 mRNA,IGF-1R mRNA and IGF-1R protein were decreased(P<0.05). Conclusion Metformin could inhibit the proliferation and induce apoptosis of CNE-1. The mechanism maybe related to the up-regulation of miR-let-7a and down-regulation of IGF-1R.

Combined Let-7a and H19 Signature: A Prognostic Index of Progression-Free Survival in Primary Breast Cancer Patients / 한국유방암학회지

PURPOSE: The long non-coding RNA H19, a conservatively imprinted gene, acts as a molecular sponge for the let-7 family, which has been identified as a set of tumor suppressors. However, the combined prognostic value of H19 and let-7a signature in breast cancer patients remains unclear. METHODS: In this research we assessed the prognostic value of the combined H19 and let-7a signature in breast cancer patients by retrospectively reviewing that data of 79 patients who underwent neoadjuvant chemotherapy; we also investigated the expression and function of H19 in breast cancer cell lines in vitro. Survival data were calculated using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate survival analyses were conducted using the Cox proportional hazards regression method. As determined using X-tile, the optimal cutoff value for the risk score to assess progression-free survival (PFS) based on the combined signature was –0.1. RESULTS: Patients with an overall positive treatment response had higher let-7a and lower H19 levels. In addition, let-7a expression was negatively correlated with H19 expression. Patients with a risk score of >–0.1 had shorter overall survival and PFS. In vitro data showed that chemoresistant cell lines exhibit higher H19 and lower let-7a levels and knockdown H19 restores paclitaxel sensitivity. CONCLUSION: Our results suggest that the combined let-7a and H19 signature is a novel prognostic factor for breast cancer patients treated with neoadjuvant chemotherapy.

Antiproliferative effect of urolithin A, the ellagic acid-derived colonic metabolite, on hepatocellular carcinoma HepG2. 2. 15 cells by targeting Lin28a/let-7a axis

An abnormality in the Lin28/let-7a axis is relevant to the progression of hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC), which could be a novel therapeutic target for this malignant tumor. The present study aimed to investigate the antiproliferative and anti-invasive effects of urolithin A in a stable full-length HBV gene integrated cell line HepG2.2.15 using CCK-8 and transwell assays. The RNA and protein expressions of targets were assessed by quantitative PCR and western blot, respectively. Results revealed that urolithin A induced cytotoxicity in HepG2.2.15 cells, which was accompanied by the cleavage of caspase-3 protein and down-regulation of Bcl-2/Bax ratio. Moreover, urolithin A suppressed the protein expressions of Sp-1, Lin28a, and Zcchc11, and elevated the expression of microRNA let-7a. Importantly, urolithin A also regulated the Lin28a/let-7a axis in transient HBx-transfected HCC HepG2 cells. Furthermore, urolithin A decelerated the HepG2.2.15 cell invasion, which was involved in suppressing the let-7a downstream factors HMGA2 and K-ras. These findings indicated that urolithin A exerted the antiproliferative effect by regulating the Lin28a/let-7a axis and may be a potential supplement for HBV-infected HCC therapy.

Selenium compound induces apoptosis of cervical carcinoma cells of high risk HPV subtypes / 基础医学与临床

Objective To investigate the inducing effects and related pathways of selenium dioxide ( SeO2 ) on the apoptosis in 2 human cervical carcinoma cell lines of high risk HPV subtypes .Methods HeLa (HPV-18-positive) and Caski (HPV-16-positive) cells were incubated with different concentrations of SeO 2 for 24 h respectively.Mor-phological changes of HeLa and Caski cells were observed under inverted optical microscope ;cell proliferation and activity were examined by MTT assay;flow cytometry was employed to detect the cell apoptosis;the expressions of apoptosis-related proteins caspase-3 and p53 in cervical carcinoma cell lines were determined by Western blot analysis;the effects of SeO 2 on apoptosis-related miRNA LET-7a expression was detected by stem-loop reverse tran-scription-polymerase chain reaction (RT-PCR).Results Cell morphology was obviously changed in vitro.Cells be-came rounded and shrunken .SeO2 markedly inhibited cell proliferation and viability in a dose-dependent manner in both cell lines; In HeLa cells the inhibitory effects induced by 7.5-30 μmol/L of SeO 2 were significant ( P<0.05);The inhibitory effects on Caski were statistical significant (P<0.05) even with low concentrations of SeO 2. The apoptosis induced by SeO 2 increased dose-dependently in cervical carcinoma cell lines , which were higher in Caski.SeO2 up-regulated the apoptosis-related proteins in cervical carcinoma cell lines .The expressions of caspase-3 and p53 in HeLa cells both significantly increased as compared with control group (P<0.05), and peaked at the concentration of 7.5 μmol/L.Treated with higher concentrations ( 7.5-30 μmol/L ) of SeO 2 , the expression on Caski cells increased significantly ( P<0.05 ) .SeO 2 induced of expression of apoptosis-related miRNA LET-7a both in HeLa cells and Caski cells with statistical meanings ( P<0.05 );the effects reached its peak at the concentration of 7.5 μmol/L bothly.Conclusions SeO2 shows anti-tumor properties via apoptosis pathway by up-regulating the expressions of apoptosis-related proteins caspase-3, the mechanisms of can be potentially explained by p 53 and LET-7a in cervical cancer cell lines.The apoptosis-inducing effect of SeO2 is more sensitive in HPV16+cell line compared with HPV18+cell line.

Comparison between Let-7a and U6 as an internal reference for RT-qPCR of miRNAs in rat cartilage / 西安交通大学学报(医学版)

Objective To evaluate the stability of U6 and let-7a as internal reference genes of miRNAs in RTqPCR by using femoral head samples of cartilage tissue from inbred DA rats.Methods Total RNA was extracted from femoral head cartilage tissues of female DA rats at three different time points,i.e.at birth (D0),ablactation (D21) and maturation (D42).The expressions of different miRNAs (miR-1,-25,-26a,-140,-146a,-150,-181a,-195,-223 and-337) were detected by RT-qPCR using U6 or let-7a as the internal reference.The two sets of miR expression were compared with the results from Solexa sequencing in our pioneer work to evaluate the stability of the two internal references.Results The relative values of U6 (P =0.045) and let-7a (P =0.021 5) revealed significant difference in the D42 sample.Both in U6 and let-7a systems,miR-26a,-140,-223,and-337 showed a similar tendency in expression and quantification but miR-1 and-146a did not have significant differences.miR-25,-150,-181a and-195 differed significantly (P＜0.05).Comparison of absolute quantification results between the two generations' sequencing showed that let-7a is more stable than U6.Conclusion Let-7a is more suitable to be used as the internal reference gene in RT-qPCR for miRNAs in cartilage tissue.

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation

PURPOSE: Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. MATERIALS AND METHODS: This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. RESULTS: Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. CONCLUSION: Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

Suppression of MicroRNA let-7a Expression by Agmatine Regulates Neural Stem Cell Differentiation

PURPOSE: Neural stem cells (NSCs) effectively reverse some severe central nervous system (CNS) disorders, due to their ability to differentiate into neurons. Agmatine, a biogenic amine, has cellular protective effects and contributes to cellular proliferation and differentiation in the CNS. Recent studies have elucidated the function of microRNA let-7a (let-7a) as a regulator of cell differentiation with roles in regulating genes associated with CNS neurogenesis. MATERIALS AND METHODS: This study aimed to investigate whether agmatine modulates the expression of crucial regulators of NSC differentiation including DCX, TLX, c-Myc, and ERK by controlling let-7a expression. RESULTS: Our data suggest that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated expression of TLX and increased expression of ERK by negatively regulating let-7a. CONCLUSION: Our study therefore enhances the present understanding of the therapeutic potential of NSCs in CNS disorders.

Expression of microRNA-Let-7a in patients of Non-small cell lung cancer and anti-tumor mechanism / 中国免疫学杂志

Objective:To investigate the expression of microRNA-Let-7a in the serum and tumor tissue of non-small cell lung cancer( NSCLC) patients and the effects of cancer cell migration and proliferation.Methods: 50 cases of NSCLC patients in our hospital as the study group,50 healthy volunteers were used as control group,we used Real-time RCR to detect the expression of microRNA-Let-7a in the serum and tumor tissue of NSCLC patients.Using microRNA let-7a mimics transfected into A549,the level of cancer cell migration was observed by transwell,the level of cell proliferation was observed by CCK-8,the level of k-Ras was observed by Real-time RCR and Western blot.Results: The expression of microRNA-Let-7a in the serum and tumor tissue of NSCLC patients was significantly higher than the control group,the difference was statistically significant (P<0.05).After microRNA let-7a transfected into A549,the levels of cancer cell migration and proliferation were significantly decreased,the difference was statistically significant (P<0.05),the mRNA and protein levels of k-Ras were reduced inA549 cells,the difference was statistically significant (P<0.05). Conclusion:The expression of microRNA let-7a is low in the serum and tumor tissue of NSCLC patients,and may weaken the levels of cancer cell migration and proliferation through the Ras signaling pathway.

Involvement of miR-Let7A in inflammatory response and cell survival/apoptosis regulated by resveratrol in THP-1 macrophage

BACKGROUND/OBJECTIVES: Resveratrol, a natural polyphenol, has multiple functions in cellular responses including apoptosis, survival, and differentiation. It also participates in the regulation of inflammatory response and oxidative stress. MicroRNA-Let-7A (miR-Let7A), known as a tumor suppressor miRNA, was recently reported to play a crucial role in both inflammation and apoptosis. Therefore, we examined involvement of miR-Let7A in the modulation of inflammation and cell survival/apoptosis regulated by resveratrol. MATERIALS/METHODS: mRNA expression of pro-/anti-inflammatory cytokines and sirtuin 1 (SIRT1), and protein expression of apoptosis signal-regulating kinase 1 (ASK1), p-ASK1, and caspase-3 and cleaved caspase-3 were measured, and cell viability and Hoechst/PI staining for apoptosis were observed in Lipopolysaccharide (LPS)-stimulated human THP-1 macrophages with the treatment of resveratrol and/or miR-Let7A overexpression. RESULTS: Pre-treatment with resveratrol (25-200 µM) resulted in significant recovery of the reduced cell viabilities under LPS-induced inflammatory condition and in markedly increased expression of miR-Let7A in non-stimulated or LPS-stimulated cells. Increased mRNA levels of tumor necrosis factor-α and interleukin (IL)-6 induced by LPS were significantly attenuated, and decreased levels of IL-10 and brain-derived neurotrophic factor were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. Decreased expression of IL-4 mRNA by LPS stimulation was also significantly increased by miR-Let7A overexpression co-treated with resveratrol. In addition, decreased SIRT1 mRNA levels, and increased p-ASK1 levels and PI-positive cells by LPS stimulation were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. CONCLUSIONS: miR-Let7A may be involved in the inflammatory response and cell survival/apoptosis modulated by resveratrol in human THP-1 macrophages.

Methylation situation of let-7a-3 in chronic myeloid leukemia and its clinical significance / 重庆医学

Objective To investigate the methylation situation of let‐7a‐3 promoter in patients with chronic myeloid leukemia (CML) and its clinical significance .Methods The methylation level of let‐7a‐3 promoter in the bone marrow mononuclear cells of 52 CML patients and 25 controls was detected by using the real‐time quantitative methylation‐specific PCR (RQ‐PCR) .Results The non-hypomethylation of let‐7a‐3 promoter was positive in 31 cases(59 .6% ) of 52 CML patients ,while only 1 case(4% ,1/25) in the control group ,the difference between the two groups were statistically significant (P0 .05) .The non-hypomethylation level of let‐7a‐3 in chronic phase and accel‐erate phase was significantly higher than that in blastic crisis of CML .Conclusion The hypomethylation level of let‐7a‐3 promoter is decreased with disease progression .

miR-Let7A Modulates Autophagy Induction in LPS-Activated Microglia

Microglia regulate the secretion of various immunomediators in central nervous system diseases. Microglial autophagy is the crucial process for cell's survival and cytokine productions. Recent studies have reported that several microRNAs are involved in the autophagy system. miR-Let7A is such a microRNA that plays a role in various inflammation responses, and is magnified as a key modulator particularly in the autophagy system. In present study, we investigated whether miR-Let7A is involved in autophagy in activating microglia. Overexpression of miR-Let7A in LPS-stimulated BV2 microglial cells promoted the induction of the autophagy related factors such as LC3II, Beclin1, and ATG3. Our results suggest a potential role of miR-Let7A in the autophagy process of microglia during CNS inflammation.

The dysregulation of hippocampal serotonin receptor 4 and let-7a were associated with dual stress-induced depression in rats / 中华行为医学与脑科学杂志

Objective To study the effect of dual stress on the behaviors and the expression of hippocampal let-7a and serotonin receptor 4(HTR4) in rats.Methods Newborn SD rats were randomly divided into dual stress group (DS,n=6) and control group (C,n=6).The DS rats were deprived of the mother care 6 hours per day from postnatal day 1 to 14 and then were exposed to chronic mild stress for 21 days from 10 weeks old,while the rats from C group received no experimental handle but husbandry care.Open field test,forced swimmiug test and sucrose consumption test were conducted to evaluate rats' depression-like behaviors at the age of thirteen weeks.The let-7a level in hippocampus was detected by real-time Polymerase Chain Reaction and the HTR4 protein level was measured by Western Blotting.Results In the open filed test,the rearing times of DS rats was shorter than that of C group((7.50±2.35) vs (19.00±5.73),P＜0.05).In the forced swimming test,the floating time of DS rats was longer than that of C group ((110.17 ± 1.72)s vs (70.33± 1.16)s,P＜ 0.05).In the sucrose c onsumption test,DS rats consumed less sucrose than rats from C group did((0.80±0.73) vs (0.52±0.26),P＜ 0.05).The protein level of hippocampal HTR-4 in DS group was lower than that of C group((1.44±0.38) vs (0.46±0.29),P＜0.01).The let-7a level in DS group was higher than that of C group((0.04±0.01) vs (1.58±0.27),P＜0.01).The Pearson correlation analysis revealed that the sucrose preference rate of rats were negatively and positively correlated with hippocampal let-7a and HTR4 level respectively(r=-0.653,P＜0.05; r=0.774,P＜0.01),and hippocampal let-7a level showed negative association with HTR4 protein level (r=-0.803,P＜0.01).Conclusion Dual stress can induce the depressive behaviors of rats and affect the expression of let-7a and HTR4 in hippocampus.Hippocampal HTR4 and let-7a might be involved in determining individual ability to experience pleasure in rats;and hippocampal let-7a may be involved in the regulation of HTR4 gene expression in rats.

The effects of microRNA let-7a on biological behaviors of Ewing's sarcoma in vitro / 肿瘤

Objective: To investigate the effects of microRNA let-7a on the biological behaviors of Ewing's sarcoma cell lines A673 and SK-ES-1, and to explore the possible mechanisms. Methods: Has-miR-let-7a mimic was transfected into A673 and SK-ES-1 cells. Three groups were designed in this study, including let-7a (transfected with has-miR-let-7a mimic), control (transfected with has-miR-let-7a mimic-control) and the untreated (transfected with liposomes) groups. The expression levels of let-7a in A673 and SK-ES-1 cells after transfection with has-miR-let-7a mimic were examined by real-time fluorescence quantitative-PCR. The proliferation, migration and invasion abilities were detected by cell counting kit (CCK-8) and Transwell chamber assay, respectively. The cell cycle distribution and the apoptotic rates of A673 and SK-ES-1 cells were measured by flow cytometry. The expression levels of cyclin-dependent kinase 6 (CDK6), Rb and p-Rb proteins in A673 and SK-ES-1 cells were detected by Western blotting. Results: As compared with the control group and the untreated group, the expression levels of let-7a in A673 and SK-ES-1 cells after transfection with has-miR-let-7a mimic were up-regulated (P < 0.01), and the abilities of proliferation, migration and invasion were inhibited (all P < 0.01). The cell cycle was arrested at G0/G1-phase (P < 0.01) and the early apoptosis was increased (P < 0.01). The expression levels of CDK6 and p-Rb were down-regulated (P < 0.01), but the expression level of Rb had no change. Conclusion: Let-7a may inhibit the proliferation, migration and invasion abilities of Ewing's sarcoma A673 and SK-ES-1 cells and promote the apoptosis. This effect may be partially related to let-7a targeting the inhibition of CDK6 expression. Copyright © 2013 by TUMOR.

Expressions of lung cancer related genes and miRNA in peripheral blood of the residents surrounding hot springs with extremely high radon / 中华放射医学与防护杂志

Objective To investigate the expressions of lung cancer related genes and miRNA in peripheral blood of the residents surrounding the extremely high radon hot springs in Ruoergai County,Sichuan Province. Methods Peripheral blood samples were collected from the local residents.Expressions of lung cancer related genes (p53,k-ras) and miRNA (let-7a,miR-34a/b) were detected by real-time PCR and the protein expressions of p53 and k-ras were detected by Western blot.Results The expressions of p53 and k-ras mRNA of the residents in high radon area were 0.97 and 1.33 times of the control respectively (t =0.13,-1.12,P ＞0.05),and the p53 and k-ras protein levels were 0.70 and 1.23 times of the control respectively (t =0.72,0.46,P ＞ 0.05).The let-7a of the residents in high radon area was lower (t =1.63,P ＞ 0.05 ) while the miR-34a and miR-34b were significantly higher than those of the controls (t =- 3.20,- 3.32,P ＜ 0.05).Conclusions Based on the expressions of p53 and k-ras gene and miRNA,it can be concluded that the residents surrounding the high radon hot springs received radiation damage.