Effects of Zuogui Pill on osteoclast activity and expression of miR-133b-3p/RhoA in postmenopausal osteoporosis rats / 国际中医中药杂志

Objective:To study the effects of Zuogui Pills on the expressions of miR-133b-3p and RhoA in osteoclasts of postmenopausal osteoporosis rats; To discuss its potential mechanism.Methods:SD female rats were randomly divided into normal group, model group, sham-operation group, and Zuogui Pills group using a random number table method, with 6 rats in each group. The model group and Zuogui Pills group were treated with oophorectomy to construct a rat model of osteoporosis. Zuogui Pills group was orally administered with Zuogui Pills decoction at a concentration of 10 g/kg for 12 consecutive weeks. Colorimetric method was used to measure the serum calcium and phosphorus levels of rats, and ELISA method was used to detect ALP levels. Bone density meter was used to measure the bone density of the femurs of rats in each group. The osteoclast of each group were cultured, and the expressions of RANKL and RUNX2 protein were detected by Western blot. MiRNA sequencing and differential expression analysis were performed on bone tissues of rats. Osteoclasts were treated with miR-133b-3p mimic and its negative control. The cell proliferation activity of osteoclasts was detected by cell counting kit-8 (CCK-8). The osteoclast differentiation activity was detected by the tartrate-resistant acid phosphatase staining. The dual-luciferase reporter assay was used to detect the relationship between miR-133b-3p and RhoA. The "rescue" experiment of miR-133b-3p mimic and RhoA co-expression were used to study the molecular regulatory mechanism of Zuogui Pills on osteoclast activity.Results:Compared with the model group, the bone mineral density of Zuogui Pills group significantly increased ( P<0.05, P<0.01), the levels of calcium and phosphorus in serum increased, the level of alkaline phosphatase ALP decreased ( P<0.05), the expression of RANKL protein decreased, and the expression of RUNX2 protein increased. Sequencing results showed that rno-miR-133b-3p was down-regulated in osteoclasts of postmenopausa osteoporosis rats treated with Zuogui Pills with the maximum difference ( P<0.01). Q-PCR results showed that the expression of miR-133b-3p in osteoclasts of Zuogui Pills group was significantly lower than that of the model group. The upregulation of miR-133b-3p could significantly promote the cell proliferation and differentiation of osteoclasts. RhoA overexpression could reverse the excessive proliferation and differentiation of osteoclasts caused by miR-133b-3p overexpression. Conclusions:RhoA is the target gene regulated by miR-133b-3p. Zuogui Pills can inhibit the activity of osteoclasts by regulating miR-133b-3p/RhoA axis, relieving the symptoms of osteoporosis.

MiR-133b regulates the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4 / 中南大学学报(医学版)

OBJECTIVES@#Bladder cancer is one of the most common urothelial tumors with high incidence and mortality rates. Although it has been reported that microRNA (miR)-133b can regulate tumorigenesis of bladder cancer, the mechanism remains unclear. Sex-determining region Y-box transcription factor 4 (SOX4) exhibits an important role in tumorigenesis, but it is unclear whether SOX4 and miR-133b are associated with regulation of pathogenesis of bladder cancer. This study aims to determine the expressions of SOX4 and miR-133b in bladder cancer tissues and cells, investigate their effects on the proliferation, colony formation, and invasion of bladder cancer cells, and to explore the association between miR-133b and SOX4 in regulating biological featurss of bladder cancer cells.@*METHODS@#The bladder cancer and adjacent tissue samples of 10 patients who underwent surgical resection in the Second Xiangya Hospital of Central South Universty from Januray to June 2015 were obtained. The levels of miR-133b were tested by real-time PCR, and the protein levels of SOX4 were evaluated using Western blotting in bladder cancer tissues, matched adjacent tissues, and cell lines. The correlation between miR-133b expression and SOX4 expression in bladder cancer tissues was analyzed. Using the online database TargetScan, the relationship between SOX4 and miR-133b was predicted. MiR-133b mimics, miR-133b inhibitor, and short hairpin RNA (shRNA)-SOX4 were transfected into T24 cells by Lipofectamine 2000. The relationship between miR-133b and SOX4 was also verified by a dual-luciferase reporter assay. The proliferation of T24 cells cultured for 0, 12, 48, 72, and 96 h was evaluated by cell counting kit-8 (CCK-8) assay. The colony formation capacity of bladder cancer cells was tested after 14-day culture, and cell invasion capacity was evaluated with Transwell invasion assay.@*RESULTS@#Bladder cancer tissue and bladder cancer cells had low level of miR-133b but high level of SOX4, compared with matched adjacent tissues and normal bladder epithelial cells. A negative correlation between miR-133b mRNA and SOX4 protein levels in bladder cancer tissues was also found (r=-0.84). The results of online database TargetScan showed that miR-133b targets at SOX4, and overexpression of miR-133b significantly attenuated the expression of SOX4 in T24 cells. Both overexpression of miR-133b and knockdown of SOX4 significantly inhibited the proliferation, colony formation, and invasion capacity of bladder cancer cells in vitro. SOX4 down-regulation restored the effects of miR-133b inhibitor on the proliferation, colony formation, and invasion capacity of T24 cells.@*CONCLUSIONS@#The up-regulation of SOX4 contributes to the progression of bladder cancer, and miR-133b can regulate the proliferation, colony formation, and invasion of bladder cancer cells via inhibiting SOX4.

Long non-coding RNA KCNQ1 overlapping transcript 1 promotes the progression of esophageal squamous cell carcinoma by adsorbing microRNA-133b

OBJECTIVE: The long non-coding RNA (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) exerts vital regulatory functions in diverse tumors. However, the biological function of KCNQ1OT1 in esophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: KCNQ1OT1 expression was detected in ESCC tissues using quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis, migration, and invasion were detected by the CCK-8 assay, EdU assay, flow cytometry analysis, and Transwell experiments, respectively. Bioinformatics analysis, luciferase reporter experiments, and RNA immunoprecipitation assays were used to predict and validate the regulatory relationships between KCNQ1OT1, microRNA-133b (miR-133b) and epidermal growth factor receptor (EGFR). RESULTS: KCNQ1OT1 expression was remarkably upregulated in ESCC tissues and cell lines. Overexpression of KCNQ1OT1 markedly promoted ESCC cell proliferation, migration, and invasion and enhanced the expression of N-cadherin, MMP-2, and MMP-9, but inhibited apoptosis and E-cadherin expression in ESCC cell lines; KCNQ1OT1 knockdown exerted the opposite effects. KCNQ1OT1 could directly bind to miR-133b and suppress its expression, and miR-133b reversed the effects of KCNQ1OT1 overexpression in ESCC cells. MiR-133b reduced the expression of epidermal growth factor receptor (EGFR); further, KCNQ1OT1 activated the phosphatidylinositol 3-kinase/AKT serine/threonine kinase 1 (PI3K/AKT) signaling pathway by repressing miR-133b repression and indirectly upregulating EGFR. KCNQ1OT1 expression was positively correlated with EGFR mRNA expression and negatively correlated with miR-133b expression. CONCLUSION: KCNQ1OT1 facilitates ESCC progression by sponging miR-133b and activating the EGFR/PI3K/AKT pathway.

miR-133b inhibits myocardial ischemia-reperfusion-induced cardiomyocyte apoptosis and accumulation of reactive oxygen species in rats by targeting YES1 / 南方医科大学学报

OBJECTIVE@#To investigate the effect of miR-133b on cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion (I/R) and explore the mechanism.@*METHODS@#Thirty-six adult SD rats were randomized into sham-operated group, I/R group, AdmiR-NC group and AdmiR-133b group, and rat models of myocardial I/R were established in the latter 3 groups with myocardial injections of saline or recombinant adenoviruses in the left ventricle. The expression of MiR-133b was detected using RT-qPCR, and cardiac function of the rats was determined using FDP 1 HRV and BRS analysis system. Serum CK-MB and cTnI levels were determined by ELISA, myocardial injury was evaluated with HE staining, cardiomocyte apoptosis was detected by flow cytometry, and ROS content was determined using a DCFH-DA probe. In the in vitro experiment, H9C2 myocardial cells with hypoxia/reoxygenation (H/R) treatment were transfected with Mir-NC or MiR-133b mimic, and the cellular expression of MiR-133b, cell apoptosis, and ROS content were determined. Dual luciferase reporter assay was performed to verify the targeting relationship between miR-133b and YES1. The effects of pc-YES1 or miR-133b mimic transfection on YES1 expression, apoptosis, and ROS content in H9C2 cells were evaluated.@*RESULTS@#Compared with those in I/R group, miR-133b expression was obviously up-regulated, LVEDP, cTnI and CK-MB levels were significantly decreased, and LVSP, +dp/dt, -dp/dt, HR and CF levels were increased in admiR-133b group (@*CONCLUSIONS@#miR-133b can inhibit I/R-induced myocardial cell apoptosis and ROS accumulation by targeting YES1 to reduce myocardial I/R injury in rats.

MicroRNA-133b suppresses cell proliferation and invasion of esophageal squamous cell carcinoma via downregulating TAGLN2 expression / 中华肿瘤杂志

Objective@#To investigate the expression of microRNA-133b (miR-133b) in esophageal squamous cell carcinoma (ESCC), and explore its effect and the underlying molecular mechanisms on cell proliferation and invasion.@*Methods@#Real-time quantitative PCR (qPCR) was used to examine miR-133b expression in 63 ESCC tissues and paired adjacent non-cancerous tissues, several ESCC cells (Eca109, EC9706, EC1, TE1, KYSE70) and normal esophageal epithelial cell Het-1A. MiR-133b mimic, inhibitor and negative control (NC) were transfected into TE1 cells. The effect of miR-133b on cell proliferation and invasion were determined by CCK-8 and Transwell assays, respectively. Subsequently, the target gene of miR-133b was predicted by online tools TargetScan and miRDB, which was verified by dual luciferase reporter assays. Finally, Western blot was utilized to detect the effects of miR-133b overexpression on expression of target gene TAGLN2 as well as EMT-related proteins E-cadherin, N-cadherin, Snail, Slug and Vimentin.@*Results@#Relative levels of miR-133b in ESCC tissues (0.295±0.040) were significantly lower than those in adjacent non-cancerous tissues (1.002±0.011, P<0.001). The expression of miR-133b was tightly associated with clinical staging, lymph node metastasis and prognosis. Moreover, relative levels of miR-133b in ESCC cells Eca109, EC9706, EC1, TE1 and KYSE70 (0.679±0.031, 0.391±0.008, 0.236±0.016, 0.031±0.005 and 0.099±0.020) were evidently lower than that in normal esophageal epithelial cell Het-1A (1.005±0.016, all P<0.001). In TE1 cells, miR-133b mimic significantly increased the level of miR-133b to 6.199±0.627, and suppressed cell proliferation and invasion, whereas miR-133b inhibitor obviously decreased its expression to 0.182±0.023, and promoted cell proliferation and invasion. Most notably, the relative luciferase activities of miR-133b-mimic group (0.320±0.018) in TE1 cells transfected with TAGLN-3′UTR-WT were markedly lower than that in NC group (1.010±0.036, P<0.001), whereas those in TAGLN-3′UTR-MUT transfection cells were 1.019±0.056 and 1.008±0.021, respectively, showing no significantly statistical difference (P>0.05). Furthermore, miR-133b overexpression markedly downregulated TAGLN2, N-cadherin, Snail, Slug and Vimentin levels, and increased E-cadherin expression.@*Conclusion@#MiR-133b plays an important role in the proliferation and invasion of ESCC cells by regulating TAGLN2 expression, and it may be a potential therapeutic target for ESCC patients.

Regulation of miR-133b on Methamphetamine-induced Neuronal Apoptosis in PC12 Cells / 中山大学学报(医学科学版)

[Objective]To investigate the expression changes of miR-133b in methamphetamine(MA)-induced neuro-nal injury in PC12 cells and its regulative effects on cellular apoptosis.[Methods]PC12 cells were cultured and divided into control group and MA treated group.In MA treated group,PC12 cells were insulted with 800μmol/L MA in culture medium. The cellular injury of PC12 cells was observed under microscope. The cellular apoptosis was detected by Hoechst33342/PI double staining,and the expression level of miR-133b was examined by real-time quantitative PCR(RT-PCR). Further-more,miR-133b mimic and inhibitors were transfected into PC12 cells to analyze miR-133b's function in MA-induced cell apoptosis.[Results]The data showed that 800 μmol/L MA could induce obvious cellular injury,cause neurite shortened and increase the cell apoptosis. The RT-PCR data showed that the expression of miR-133b of PC12 cells treated with MA de-creased significantly.The apoptosis rate of PC12 cells decreased after transfection of miR-133b mimic,while increased after transfection of miR-133b inhibitors.[Conclusions]High concentration of MA causes neuron damage and induces neuronal apoptosis,and also decreases the levels of miR-133b expression. Whereas,overexpression of miR-133b can reduce the apoptosis of cultured PC12 cells.Thus,miR-133b plays a crucial role in MA mediated neurotoxicity.This study provides a theoretical basis for elucidating the mechanism of MA-induced neurotoxicity and may provide a new strategy for treating MA addiction.

The expression of miR-133b in the brain of methamphetamine dependent rats and its regulation on neuronal toxic injury / 中华行为医学与脑科学杂志

Objective To investigate the expression of miR-133b in the brain of methamphetamine(MA) dependent rats and its regulatory effects on neuronal toxic injury.MethodsThrough continuous intraperitoneal injection to rats with MA(10 mg/kg),the conditioned place preference(CPP) rats model was established,and the expression of miR-133b in the cerebral cortex of model rats was detected by real-time fluorescence quantitative PCR(RT-PCR).PC12 cells were cultured in vitro and treated with MA(800 μmol/L),and then miR-133b expression in cultured neurons was detected.miR-133b mimics and inhibitor were transfected to PC12 cells respectively to observe the effect of miR-133b on the mitochondrial membrane potential(MMP) of cultured neurons.ResultsAfter continuous intraperitoneal injection with MA for 14 days,the residence time of rats in the box with medicine((620.20±44.80)s) was significantly longer compared with the control group((341.80±25.12)s,P<0.01),which showed that MA dependent rats model was successfully established.The RT-PCR detection results showed that the expression of miR-133b in the cerebral cortex of model rats(0.36±0.05) significantly decreased compared with the control group(0.99±0.08,P<0.01).In the in vitro model,most of the neuronal cell bodies became round and the neuorites were withdrawn after MA treatment.Compared with the control group(1.00±0.02),the RT-PCR detection results showed that the expression of miR-133b in MA group(0.74±0.05) decreased(P<0.05).The JC-1 detection results showed that the MMP of the MA group(109.85±7.03) decreased significantly contrast to the control group(36.49±3.89,P<0.01),the MMP of the miR-133b mimics group(58.97±6.56) increased significantly contrast to the mimics control group(135.46±15.04,P<0.01) and the MMP of the miR-133b inhibitor group(162.84±14.15) decreased contrast to the inhibitor control group(139.81±12.26,P<0.05).ConclusionsThe expression of miR-133b in the cerebral cortex of MA dependent rats and in vitro neuron model treated with MA are significantly downregulated.By regulating the expression of miR-133b,the MMP damage of cultured neurons treated with MA is changed,indicating that miR-133b is not only involved in the nerve injury induced by MA,but also possiblely as a molecular target for intervention.

The expression and clinical significance of miR-146a/133b in Xinjiang women with cervical lesion / 重庆医学

Objective To explore the different expression and clinical significance of miR-146a/133b in cervical tissue in uy-ghur and Han women in Xinjiang.Methods The relative expression of miR-146a/133b in paraffin embedding tissues of cervicitis, CIN and cervical cancer was detected by the RT-qPCR.And analyzed the clinical significance in the development of cervical cancer. Results Compared with cervicitis,the expression of miR-146a/133b increased significantly in CIN and cervical cancer(P <0.05). With the cervical lesion was aggravating,the expression level increased.In cervical cancer tissue,the expression of miR-146a were different between Uyghur and Han women(P <0.05).Marriage age<20 years old,tumor diameter≥4 cm,with HPV infection in cervical cancer tissue,miR-146a/133b had high expression (P <0.05).Conclusion MiR-146a/133b are involved in incidence and development of cervical cancer,they may become new prognostic and evaluating molecular markers in cervical cancer.