Hsa_circ_0000670 promoted the progression of gastric cancer through the miR-515-5p/SIX1 molecular axis / 中华肿瘤杂志

Objective: To explore whether hsa_circ_0000670 promotes the progression of gastric cancer by regulating the miR-515-5p/SIX1 molecular axis. Methods: The gastric cancer and adjacent normal tissues of 35 gastric cancer patients admitted to Rugao Hospital Affiliated to Nantong University from 2014 to 2015 were collected. The expression levels of circ_0000670, miR-515-5p and Sine oculis homeobox 1 (SIX1) in gastric cancer tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The correlations between circ_0000670 and miR-515-5p, miR-515-5p and SIX1, circ_0000670 and SIX1 were analyzed by the Pearson method. Patients were divided into low circ_0000670 expression group (17 cases) and high circ_0000670 expression group (18 cases) based on the median of circ_0000670 expression level, and Kaplan-Meier was used to analyze the 5-year survival of patients. Cell proliferation was assessed via clone formation assay. Cell cycle and apoptosis were detected by flow cytometry. Wound healing and Transwell assays were used to detect cell migration and invasion ability. The targeting relationship between miR-515-5p and circ_0000670 or SIX1 was confirmed by the dual luciferase reporter assay. Nude mice were injected into HGC-27 cells transfected with sh-NC or sh-circ_0000670, and the volume and weight of the transplanted tumor were measured, also, the levels of circ_0000670, miR-515-5p and SIX1 in the transplanted tumor tissue were detected. Results: The expression levels of circ_0000670 and SIX1 in gastric cancer tissues and cell lines were significantly increased (P<0.05), while the expression levels of miR-515-5p were significantly decreased (P<0.05). The survival rate of patients in the low circ_0000670 expression group (82.4%) was significantly higher than that in the high circ_0000670 expression group (28.7%, P=0.034). Circ_0000670 was negatively correlated with miR-515-5p (r=-0.846, P<0.001), and miR-515-5p was negatively correlated with SIX1 (r=-0.615, P<0.001), but circ_0000670 was positively correlated with SIX1 (r=0.814, P<0.001). Transfection of si-circ_0000670 or miR-515-5p mimic could significantly reduce the number of clone-forming cells, migration distance, migration and invasion cells (P<0.05), and increase the ratio of G(0)/G(1) phase cells, apoptosis rate and the protein level of E-cadherin (P<0.05), decreased the proportion of S-phase cells and the protein level of Vimentin (P<0.05). The dual luciferase report assay confirmed that circ_0000670 could target miR-515-5p, and miR-515-5p could bind to SIX1. Co-transfection of si-circ_0000670 and miR-515-5p inhibitor could significantly attenuate the effects of si-circ_0000670 on cell proliferation, migration, invasion, cell cycle and apoptosis (P<0.05). Co-transfection of miR-515-5p mimic and pcDNA-SIX1 could significantly reduce the effects of miR-515-5p mimic on cell proliferation, migration, invasion, cell cycle and apoptosis (P<0.05). Compared with the sh-NC group [volume=(596.20±125.46) mm(3) and weight=(538.00±114.39) g], the volume and weight of transplanted tumors in the sh-circ_0000670 group [volume=(299.20±47.58) mm 3 and weight=(289.80±48.73 g)] were significantly reduced (P<0.05), the expression levels of circ_0000670 and SIX1 were significantly reduced (P<0.05), and the expression level of miR-515-5p was significantly increased (P<0.05). Conclusion: Knockdown of circ_0000670 could inhibit cell proliferation, migration, invasion of gastric cancer cells, induce cell cycle arrest in G(0)/G(1) phase and promote cell apoptosis by regulating the miR-515-5p/SIX1 axis.

Genetic research progress in branchio-oto syndrome/ branchio-oto-renal syndrome / 中南大学学报(医学版)

Branchio-oto syndrome (BOS)/branchio-oto-renal syndrome (BORS) is a kind of autosomal dominant heterogeneous disorder. These diseases are mainly characterized by hearing impairment and abnormal phenotype of ears, accompanied by renal malformation and branchial cleft anomalies including cyst or fistula, with an incidence of 1/40 000 in human population. Otic anormalies are one of the most obvious clinical manifestations of BOS/BORS, including deformities of external, middle, inner ears and hearing loss with conductive, sensorineural or mix, ranging from mild to profound loss. Temporal bone imaging could assist in the diagnosis of middle ear and inner ear malformations for clinicians. Multiple methods including direct sequencing combined with next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), or array-based comparative genomic hybridization (aCGH) can effectively screen and identify pathogenic genes and/or variation types of BOS/BORS. About 40% of patients with BOS/BORS carry aberrations of EYA1 gene which is the most important cause of BOS/BORS. A total of 240 kinds of pathogenic variations of EYA1 have been reported in different populations so far, including frameshift, nonsense, missense, aberrant splicing, deletion and complex rearrangements. Human Endogenous Retroviral sequences (HERVs) may play an important role in mediating EYA1 chromosomal fragment deletion mutations caused by non-allelic homologous recombination. EYA1 encodes a phosphatase-transactivator cooperated with transcription factors of SIX1, participates in cranial sensory neurogenesis and development of branchial arch-derived organs, then regulates the morphological and functional differentiation of the outer ear, middle ear and inner ear toward normal tissues. In addition, pathogenic mutations of SIX1 and SIX5 genes can also cause BOS/BORS. Variations of these genes mentioned above may cause disease by destroying the bindings between SIX1-EYA1, SIX5-EYA1 or SIX1-DNA. However, the role of SIX5 gene in the pathogenesis of BORS needs further verification.

A study on the mechanism of mi-362 targeting six1 inhibiting proliferation and migration of cervical cancer cells / 实用肿瘤学杂志

Objective To investigate the mechanism of Mi-362 targeting Six1 inhibiting proliferation and migration of cer-vical cancer cells. Methods The expression levels of microRNA-362 in cervical cancer tissues and adjacent tissues were measured in 142 patients with cervical cancer in our hospital. At the same time,Hela cancer cell group,microRNA-362 mimics group and mi-croRNA-362 inhibitor group were set up to determine the viability of cancer cells,the number of monoclonal formation of cancer cells,the apoptotic rate of cancer cells,cell cycle,the number of perforations,and the levels of microRNA-362 and Six1 in cervical cancer fluid of Hela. Results The expression level of miR-362 in cervical cancer tissue was lower than that in adjacent tissue(P<0. 05). The higher the infiltration of lymphatic vessel space,pathological stage,TNM stage,lymph node metastasis and depth of infil-tration,the lower the expression rate of miR-362(P<0. 05). The OD value and survival rate in the miR-362 mimics group were lower than those in the Hela cancer cells group(P<0. 05),while the OD value and survival rate in the mir-362 inhibitor group were higher than those in the Hela cancer cells group and the miR-362 mimics group(P<0. 05). The number of clones formed in the miR-362 mimics group was lower than that in the Hela cancer cell group(P<0. 05),and the number of clones formed in the miR-362 inhibitor group was higher than that in the Hela cancer cell group and the miR-362 mimimics group(P<0. 05). The apoptotic rate of miR-362 mimics group was higher than that of Hela cancer cell group(P<0. 05),and that of miR-362 inhibitor group was lower than that of Hela cancer cell group and miR-362 mimics group(P<0. 05). The G1 phase in the miR-362 mimics group was higher than that in the Hela cancer cell group(P<0. 05),and the G1 phase in the miR-362 inhibitor group was lower than that in the Hela cancer cell group and the miR-362 mimimics group(P<0. 05). The number of cell membrane penetration in the miR-362 mimics group was lower than that in the Hela cancer group(P < 0. 05),and that in the miR-362 inhibitor group was higher than that in the Hela cancer group and the miR-362 mimimics group(P<0. 05). The expression level of miR-362 in the miR-362 mimics group was higher than that in the Hela cancer cell group(P<0. 05),and the expression level of miR-362 in the miR-362 inhibitor group was lower than that in the Hela cancer cell group and the miR-362 mimics group(P<0. 05). The expression level of Six1 in the miR-362 mimics group was lower than that in the Hela cancer cell group(P<0. 05),and that in the miR-362 inhibitor group was higher than that in the Hela cancer cell group and the miR-362 mimics group(P<0. 05). Conclusion miR-362 plays an important inhibition in the occurrence and development of cervical cancer,and its mechanism is related to the inhibition of prolifer-ation,migration and invasion of cancer cells by microRNA-362 through negative regulation of Six1.

Inhibitory effect of nvp-bez235 on proliferation and migration of hepatocellular carcinoma cell hepg2 and its mechanism / 中国药理学通报

Aim To evaluate the efficacy of NVP-BEZ235 on the proliferation and migration of HepG2 human hepatocellular carcinoma cell in vitro, and to investigate the molecular mechanism. Methods The cytotoxicity of NVP-BEZ235 on HepG2 cells treated with NVP-BEZ235(0, 25, 50, 100, 250, 500 nmol·L-1) was detected by MTT assay. The ability of NVP-BEZ235 to modulate HepG2 cells proliferation and apoptosis was detected by morphology assay and Hoechst 33342 staining. The motility of NVP-BEZ235 treatment on HepG2 cells was determined by wound healing assay and Transwell assay.The expression levels of the related protein including PI3K/Akt/mTOR signal pathway, epithelial-mesenchymal transition(EMT) process and Six1/Ezrin signal axis were detected by Western blot. The expression levels of EMT markers were also detected by immunofluorescence staining. Results The cell viability significantly decreased in HepG2 cells treated with NVP-BEZ235 compared with control group, which showed a dose-dependent manner. NVP-BEZ235 could induce apoptosis and inhibit HepG2 cell migration. E-cadherin protein expression significantly increased; however, the expressions of p-AktSer473, p-S6Thr389, p-4E-BP1Thr37/46, Vimentin, Snail, Six1 and p-EzrinTyr353 decreased in NVP-BEZ235 treated HepG2 cells compared with those in control group. Conclusions NVP-BEZ235 could effectively suppress the proliferation and migration of HepG2 cells, which may be related to the down-regulation of p-AktSer473, p-S6Thr389, p-4E-BP1Thr37/46 and modulating both Six1/Ezrin signal axis and the EMT process by NVP-BEZ235 treatment.

Effect of miR-145 targeting Six1 on invasion of gastric cancer and its molecular mechanism / 国际肿瘤学杂志

Objective To investigate the expressions of microRNA-145 (miR-145) and Six1 in gastric cancer tissues and gastric cancer cell lines,and to investigate the effect of miR-145 targeting Six1 on invasion of human gastric cancer cells and its molecular mechanism.Methods Sixty patients with advanced gastric cancer confirmed by gastroscopy and pathology in the Fourth Hospital of Changsha were selected,from January to November,2017.The expressions of miR-145 mRNA and Six1 in 60 cases of gastric cancer and their matched adjacent tissues were detected by real-time fluorescence quantitative PCR (qRT-PCR) and their correlations were analyzed.miR-145 mimics were transfected into MKN-45,BGC-823 and SGC-7901 gastric cancer cell lines.The relative levels of miR-145 mRNA in three gastric cancer cell lines were detected by qRT-PCR.The SGC-7901 gastric cancer cell lines with the highest expression level of miR-145 mRNA were selected for subsequent experiments.The mimic group (transfected with miR-145 mimic),NC group (transfected with miR-145 negative control) and empty carrier group (no addition of any oligonucleotides) were set up.The invasive ability of gastric cancer cells was detected by Transwell test.The ability of angiogenesis was verified by microtubule formation experiment.The expression levels of downstream proteins of the gastric cancer cell lines SGC-7901 such as Six1,E-cadherin and vimentin were detected by Western blotting method.The MirTrap system and the luciferase report test verified whether miR-145 targeted the Six1 gene.Results The expressions of miR-145 mRNA in gastric cancer tissues and in para cancerous tissues were 0.579 ± 0.086 and 1.009 ±0.121,respectively.The difference was statistically significant (t =-22.498,P ＜0.001).The expressions of Six1 were 2.516 ± 0.208 and 1.041 ± 0.227,respectively.The difference was statistically significant (t =37.119,P ＜ 0.001).The expressions of miR-145 mRNA and Six1 were negatively correlated (r =-0.728,P ＜0.001).After overexpression of miR-145,in the mimic,NC and empty carrier groups,the numbers of cells passing through the membrane were 48.550 ± 3.716,82.800 ± 3.797,87.467 ± 8.023,respectively.The difference was statistically significant (F =87.789,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜0.001).In the mimic,NC and empty carrier groups,the numbers of tube formation of human umbilical vein endothelial cells were 24.333 ± 1.211,34.167 ±2.041,36.500 ±3.209,respectively.The difference was statistically significant (F =47.103,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜ 0.001).In the mimic,NC and empty carrier groups,the expression levels of Six1 in gastric cancer SGC-7901 cells were 1.392 ±0.072,2.426 ±0.099,2.371 ±0.079,respectively.The difference was statistically significant (F =289.517,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜0.001).In the mimic,NC and empty carrier groups,the expression levels of E-cadherin were 4.001 ±0.132,2.714 ±0.181,2.653 ±0.218,respectively.The difference was statistically significant (F =106.572,P ＜0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜ 0.001).In the mimic,NC and empty carrier groups,the expression levels of vimentin were 1.634 ± 0.132,3.349 ± 0.102,3.501 ± 0.185,respectively.The difference was statistically significant (F =389.032,P ＜0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜ 0.001).MirTrap system verified that the target Six1 mRNA levels in the mimic,NC and empty carrier groups were 1.101 ± 0.097,0.582 ± 0.037,0.573 ± 0.032,respectively.The difference was statistically significant (F =138.922,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜0.001).Luciferase reporter assay showed that the double fluorescence ratios of Six1 wild type recombinant vector group were 7.324 ± 0.415,10.755 ± 0.481,10.430 ± 0.309 in the mimic,NC and empty carrier groups respectively.The difference was statistically significant (F =129.345,P ＜ 0.001).There were significant differences between the mimic and NC groups and between the mimic and empty carrier groups (both P ＜0.001).The double fluorescence ratios of Six1 mutant recombinant vector group were 10.938 ± 0.091,11.077 ±0.126,11.028 ±0.205 in the mimic,NC and empty carrier groups respectively,and the difference was not statistically significant (F =1.318,P =0.297).The MirTrap system and the luciferase report test showed that miR-145 was targeted to the Six1 gene.Conclusion The expressions of miR-145 mRNA and Six1 in gastric cancer and para cancerous tissues were different and negatively correlated.miR-145 inhibited the invasion,angiogenesis and epithelial mesenchymal transition of gastric cancer cells by targeting Six1 gene.

Expression of SIX1 in cervical cancer and relationship with cervical cancer cell proliferation / 临床与实验病理学杂志

Purpose To investigate the expression of SIX1 in cervical cancer and its biological function.Methods The expression of SIX1 and Ki-67 in 35 cervical cancer tissues,42 cervical intraepithelial neoplasia (CIN) tissues and 15 normal cervical tissues was detected by immunohistochemical of EliVision two-step staining.A plasmid vector expressing SIX1 siRNA was constructed and then stably transfected into Hela cells.The expression of SIX1 mRNA and protein was examined by RT-PCR and Western blot method respectively.The proliferation was exanined by MTT.Results The positive rate of SIX1 in cervical cancer was significantly higher than that of CIN and normal cervical tissues and was positively correlation with the proliferation index of Ki-67.After RNAi treatment,the mRNA and protein levels of SIX1 was down-regulated in Hela cells and the proliferation of Hela cells was inhibited.Conclusion SIX1 may play an important role in the occunence and development process of cervical cancer by promoting cervical cancer cell proliferation.

Effect of miR-185 targeting Six1 on epithelial-mesenchymal transition in lung cancer and its mechanism / 临床与实验病理学杂志

Purpose To study the molecular mechanism of miR-185 affecting the migration and invasion of human lung cancer cell.Methods MiR-185 overexpression was obtained by transfection of miR-185 mimic in lung squamous cell carcinoma cell line H520 and A549,transwell assay and cell scratch assay were used to detection of cell migration and invasion.The luciferase reporter assay confirmed that miR-185 targets the Six1 gene.qRT-PCR and Western blot were used to detect the impact of miR-185 cells Six1 gene expression.Western blot was used to detect the effect of miR-185 overexpression on the epithelialmesenchymal transition of lung cancer cells.Results miR-185overexpression reduced migration and invasion of lung cancer cells (P ＜ 0.05),increased epithelial cell marker E-cadherin expression (P ＜ 0.01),and decreased the expression of mesenchymal cell markers vimentin of (P ＜ 0.01).After overexpression of miR-185 in H520 cells,the expression level of Six1gene was reduced (P＜0.01).MiR-185 regulated the migration and invasion of lung cancer cells by targeting the Six1 gene.Conclusion MiR-185 targets the Six1 gene to regulate the EMT pathway of human lung cancer cells.

Expression of Six1,TGF-βand VEGF-C in patients with laryngeal squamous cell carcinoma and their correlation analysis / 重庆医学

Objective To detect the expressions of Six1 ,TGF‐βand their common induced VEGF‐C in human laryngeal squa‐mous cell carcinoma ,and to explore their significance in the occurrence ,development ,metastasis and prognosis of laryngeal squa‐mous cell carcinoma .Methods The clinical data and preserved paraffin samples of 96 patients with laryngeal squamous cell carci‐noma confirmed by postoperative pathology were collected .The protein expression of Six1 ,TGF‐βand VEGF‐C was determined by adopting immunohistochemistry and Western blotting .Results The positive expression rates of Six1 ,TGF‐β and VEGF‐C in the laryngel squamous cell carcinoma tissue samples were 84 .4% (81/96) ,89 .6% (86/96)and 91 .7% (88/96)respectively .The positive expression rate of Six1 was significantly higher in the patients with poor differentiation and lymph node metastasis ;the positive ex‐pression rate of TGF‐βwas significantly higher in the patients with lymph node metastasis and had no relationship with the degree of differentiation ;the positive expression rate of VEGF‐C was significantly higher in the patients with poor differentiation and lymph node metastasis ;there was a positive correlation between expression of Six 1 and VEGF‐C in the laryngel squamous cell canc‐er tissue .Conclusion The high expression of Six1 ,TGF‐βand VEGF‐C may promote lymphatic metastasis of laryngeal squamous cell carcinoma .Six1 expression may be one of the important influencing factors of the expression change of VEGF‐C .The combined detection of Six1 ,TGF‐βand VEGF‐C has an important clinical significance for judging the metastasis and prognosis of LSCC .

Expression and significance of Six1 in cervical cancer / 天津医药

Objective To investigate the clinical significance of protein expression of Six1 in cervical cancer. Meth?ods The immunohistochemical (IHC) staining was applied to detect the expression of Six1 protein in normal cervical tis?sues (n=32), cervical intraepithelial neoplasia (CIN) tissues (n=49) and cervical cancer tissues (n=123). The localization of Six1 protein was detected in vitro cultured HeLa cells using immunofluorescence (IF) staining. Results The positive rate of Six1was significantly higher in cervical cancer (72.3%) than that of CIN tissues (28.6%) and normal cervical tissues (15.6%,χ2=13.118 and 10.058 respectively, P<0.01). There were significance differences in expression levels of Six1 protein be?tween different tumor sizes and metastasis of cervical cancer (P < 0.01). The Six1 protein showed positive signals in cyto?plasm and nucleoli in HeLa cells. Conclusion Six1 expression is associated with cervical cancer, which may be a potential biomarker for invasion and metastasis of cervical cancer.

Molecular cloning and expression pattern of duck Six1 and its preliminary functional analysis in myoblasts transfected with eukaryotic expression vector.

Skeletal muscle development is regulated by Six1, an important myogenic transcription factor. However, the functional analysis of duck Six1 has not been reported. Here, we cloned the coding domain sequence (CDS) region of the duck Six1 gene using RT-PCR and RACE methods. Bioinformatics analysis revealed that duck Six1 CDS region comprised of 849 bp and encoded 282 amino acids and had a high degree of homology with other species, suggesting that the functions of duck Six1 gene are conserved among other animals. Real-time PCR used to determine the mRNA expression profiles of duck Six1 in different tissues and different developmental stages showed that Six1 was highly expressed in skeletal muscle and the embryonic stage. Furthermore, the eukaryotic expression vector pEGFP-duSix1 was constructed and transfected into the duck myoblasts; the MTT assay revealed an obvious increase of cell proliferation after transfection. The expression profiles of Six1, Myf5 and MyoD showed that their expression levels were significantly increased. These results together suggested that pEGFP-duSix1 vector was constructed successfully and overexpression of duck Six1 in the myoblasts could promote cell proliferation activity and significant up-regulate expression of Myf5 and MyoD.

Research advances of Six1 gene in breast cancer / 国际外科学杂志

Homeobox gene Six1,as a transcription factor has been proved to participate in most malignant tumors.Its overexpression is associated with the genesis and development of breast cancer intimately.Overexpression of Six1 in mammary cells is sufficient to induce activation of stem cells.Similarly,Six1-overexpressed tumors could activate the TGF-β pathway and Wnt/β-catenin pathway,lead to malignant transformation by inducing epithelial-mesenchymal transition and mammary tumorigenesis.Research on the role of sixl will improve understanding the mechanisms of breast cancer initiation and development.