BACKGROUND: The transcription factor POU2F1 regulates the expression levels of microRNAs in neoplasia. However, the miR-29b1/a cluster modulated by POU2F1 in gastric cancer (GC) remains unknown. METHODS: Gene expression in GC cells was evaluated using reverse-transcription polymerase chain reaction (PCR), western blotting, immunohistochemistry, and RNA in situ hybridization. Co-immunoprecipitation was performed to evaluate protein interactions. Transwell migration and invasion assays were performed to investigate the biological behavior of GC cells. MiR-29b1/a cluster promoter analysis and luciferase activity assay for the 3'-UTR study were performed in GC cells. In vivo tumor metastasis was evaluated in nude mice. RESULTS: POU2F1 is overexpressed in GC cell lines and binds to the miR-29b1/a cluster promoter. POU2F1 is upregulated, whereas mature miR-29b-3p and miR-29a-3p are downregulated in GC tissues. POU2F1 promotes GC metastasis by inhibiting miR-29b-3p or miR-29a-3p expression in vitro and in vivo . Furthermore, PIK3R1 and/or PIK3R3 are direct targets of miR-29b-3p and/or miR-29a-3p , and the ectopic expression of PIK3R1 or PIK3R3 reverses the suppressive effect of mature miR-29b-3p and/or miR-29a-3p on GC cell metastasis and invasion. Additionally, the interaction of PIK3R1 with PIK3R3 promotes migration and invasion, and miR-29b-3p , miR-29a-3p , PIK3R1 , and PIK3R3 regulate migration and invasion via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway in GC cells. In addition, POU2F1 , PIK3R1 , and PIK3R3 expression levels negatively correlated with miR-29b-3p and miR-29a-3p expression levels in GC tissue samples. CONCLUSIONS The POU2F1 - miR-29b-3p / miR-29a-3p-PIK3R1 / PIK3R1 signaling axis regulates tumor progression and may be a promising therapeutic target for GC.
MicroRNAs/metabolism* ; Humans ; Stomach Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement/physiology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Animals ; Mice ; Octamer Transcription Factor-1/metabolism* ; Mice, Nude ; Class Ia Phosphatidylinositol 3-Kinase/metabolism* ; Neoplasm Invasiveness ; Gene Expression Regulation, Neoplastic/genetics* ; Male ; Immunohistochemistry ; Female

OBJECTIVES: Esophageal squamous cell carcinoma (ESCC) is characterized by complex pathogenesis and poor prognosis. In recent years, epithelial-mesenchymal transition (EMT) in tumor initiation and progression has attracted increasing attention. Enhancer of zeste homolog 2 (EZH2), which is aberrantly expressed in various tumors, may be closely related to the EMT process. This study aims to examine the expression and correlation of EZH2 and EMT markers in ESCC cells and tissues, evaluate the effects of EZH2 knockdown on ESCC cell proliferation, invasion, and migration, and explore how EZH2 contributes to the malignant biological behavior of ESCC. METHODS: Bioinformatics analyses were used to assess EZH2 expression levels in ESCC. Small interfering RNA was used to knock down EZH2 in ESCC cell lines EC109 and EC9706. Cell proliferation, invasion, and migration were evaluated using cell counting kit-8 (CCK-8), wound healing, and Transwell assays. Protein and mRNA expression levels of EZH2, E-cadherin (E-cad), and vimentin (Vim) were detected by Western blotting and real time fluorogenic quantitative PCR (RT-qPCR), respectively. Immunohistochemical (IHC) staining was performed on 70 ESCC tissue samples and 40 paired adjacent normal tissues collected from the First Affiliated Hospital of Shihezi University between 2010 and 2016 to assess the expression of EZH2, E-cad, and Vim, and to analyze their associations with clinicopathological feature and patient prognosis. RESULTS: Bioinformatics analysis showed that EZH2 was highly expressed in ESCC (P<0.001), and high EZH2 expression was associated with worse prognosis (P<0.001). CCK-8, wound healing, and Transwell assays demonstrated that EZH2 knockdown significantly suppressed the proliferation, invasion, and migration of ESCC cells (P<0.001). In addition, Vim expression was significantly reduced, while E-cad expression was significantly increased at both protein and mRNA levels in EZH2-silenced cells (all P<0.05). IHC staining analysis revealed higher expression of EZH2 and Vim and lower expression of E-cad in ESCC tissues compared to adjacent normal tissues. Kaplan-Meier survival analysis showed that low expression of EZH2 and Vim and high expression of E-cad were associated with longer survival (all P<0.05). CONCLUSIONS EZH2 promotes malignant biological behavior in ESCC by mediating EMT. Elevated EZH2 expression is associated with poor prognosis in ESCC patients.
Humans ; Enhancer of Zeste Homolog 2 Protein/physiology* ; Esophageal Squamous Cell Carcinoma/pathology* ; Epithelial-Mesenchymal Transition/genetics* ; Esophageal Neoplasms/metabolism* ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement ; Cadherins/genetics* ; Vimentin/genetics* ; Male ; Female ; Middle Aged ; Neoplasm Invasiveness ; Prognosis ; RNA, Small Interfering/genetics* ; Gene Expression Regulation, Neoplastic

OBJECTIVE: To explore the effect of miR-143 regulating matrix metalloproteinase(MMP)-13 expression on migration and invasion of osteosarcoma cells. METHODS: The mouse osteosarcoma cell line 143B cells were cultured in 96-well plates, and blank group, negative group, positive group, and intervention group were set up. Then, the blank group did no treatment 50 μg miR-143 mimic was added to positive group, negative group added equal mimic NC (control sequence of miR-143 mimic), the intervention group was added 50 μg miR-143 mimic and 10 μg MMP-13 protein, all groups continued to culture for 3 to 6 hours, and finally the serum was aspirated to treat for half an hour. The protein expressions of miR-143 and MMP-13 in each group were measured by fluorescence quantitative PCR experiment and Western blot experiment, respectively, and the invasion and migration abilities of cells were measured by Transwell and scratch experiments. RESULTS: The expression of MMP-13 protein in the positive group and the intervention group was significantly lower than that in the blank group, and the positive group was lower than the intervention group (P<0.05);The mean numbers of invasive cells in blank group, negative group, positive group and intervention group were (1 000.01±44.77), (959.25±46.32), (245.04±4.33), (634.06±33.78) cells/field, respectively;the scratch healing rate of the positive group and the intervention group was significantly lower than that of the blank group, and the positive group was lower than the intervention group (P<0.05). CONCLUSION MMP-13 is a target of miR-143, which can reduce the migration and invasion ability of osteosarcoma cells by inhibiting the expression of MMP-13.
Osteosarcoma/pathology* ; MicroRNAs/genetics* ; Matrix Metalloproteinase 13/genetics* ; Neoplasm Invasiveness ; Animals ; Mice ; Cell Line, Tumor ; Cell Movement

OBJECTIVE: To identify and characterize read-through RNAs and read-through circular RNAs (rt-circ-HS) derived from transcriptional read-through hypoxia inducible factor 1α (HIF1α) and small nuclear RNA activating complex polypeptide 1 (SNAPC1) the two adjacent genes located on chromosome 14q23, in renal carcinoma cells and renal carcinoma tissues, and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells. Tissue microarrays of 437 different types of renal cell carcinoma (RCC) were constructed, and chromogenic in situ hybridization (ISH) was used to investigate expression of rt-circ-HS in different RCC types. Small interference RNA (siRNA) and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation, migration and invasiveness by cell counting kit 8 (CCK8), EdU incorporation and Transwell cell migration and invasion assays. RT-PCR and Western blot were used to exa-mine expression of HIF1α and SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA, respectively. The binding of rt-circ-HS with microRNA 539 (miR-539), and miR-539 with HIF1α 3' untranslated region (3' UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays. RESULTS: We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 2-6 and SNAPC1 exon 2-4. Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells. ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5% (295/437), and the expression was different in different types of RCCs. Mechanistically, rt-circ-HS promoted renal carcinoma cell proliferation, migration and invasiveness by functioning as a competitive endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α. CONCLUSION The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.
Humans ; Carcinoma, Renal Cell/pathology* ; Cell Proliferation ; Hypoxia ; Kidney Neoplasms ; MicroRNAs/genetics* ; Neoplasm Invasiveness/genetics* ; RNA, Circular/metabolism* ; RNA, Small Interfering ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics*

Humans ; MicroRNAs/genetics* ; Cell Line, Tumor ; Osteosarcoma/pathology* ; Neoplasm Invasiveness ; Cell Proliferation ; Bone Neoplasms/pathology* ; Cell Movement ; Neoplasm Metastasis

Objective: To study the effects of Homeobox C10 (HOXC10) on biological characteristics such as migration, invasion and proliferation of glioma cancer cells and to explore the role of HOXC10 gene in glioma microenvironment. Methods: The expression level of HOXC10 in high grade glioma (glioblastoma) and low grade glioma and its effect on patient survival were analyzed by using The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) database. Hoxc10-siRNA-1, HOXC10-siRNA-2 and siRNA negative control (NC) were transfected into U251 cells according to the operation instructions of HOXC10-siRNA transfection. 100 ng/ mL recombinant protein chemokine ligand 2 (reCCL2) was added into the transfection group, and was labeled as HOXC10-siRNA-1+ reCCL2 and HOXC10-siRNA-2+ reCCL2 groups. The expressions of HOXC10 mRNA and target protein in each group was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and western blot. The proliferation ability of cells in each group was detected by cell counting kit 8 (CCK8) method. The migration ability of cells was detected by Transwell assay and Nick assay, and cell apoptosis was detected by flow cytometry. The expression of chemokines in each group was detected by multiple factors. Co-incubation assays were performed to determine the role of HOXC10 and chemokine ligand 2 (CCL2) in recruiting and polarizing tumor-associated macrophages (M2-type macrophages). Results: The median expression level of HOXC10 in high grade gliomas was 8.51, higher than 1.00 in low grade gliomas (P<0.001) in TCGA database. The median expression level of HOXC10 in high grade gliomas was 0.83, higher than 0.00 in low grade gliomas (P=0.002) in CGGA database. The 5-year survival rate of patients with high HOXC10 expression in TCGA database was 28.2%, lower than 78.7% of those with low HOXC10 expression (P<0.001), and the 5-year survival rate of patients with high HOXC10 expression in CGGA database was 20.3%, lower than 58.0% of those with low HOXC10 expression (P<0.001). The numbers of cell migration in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (45±3) and (69±4) respectively, lower than (159±3) in NC group (P<0.05). The cell mobility of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group at 48 hours were (15±2)% and (28±4)% respectively, lower than (80±5)% of NC group (P<0.05). The expressions of vimentin in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (141 740.00±34 024.56) and (94 655.00±5 687.97), N-cadherin were (76 810.00±14.14) and (94 254.00±701.45), β-catenin were (75 786.50±789.84) and (107 296.50±9 614.53), lower than (233 768.50±34 114.37), (237 154.50±24 715.50) and (192 449.50±24 178.10) of NC group (P<0.05). The A value of HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.44±0.05) and (0.32±0.02) at 96 hours, lower than 0.92±0.12 of NC group (P<0.05). The apoptosis rates of HOXC10-siRNA-1 group and HOXC10 siRNA-2 group were (10.23±1.24)% and (13.81±2.16)%, higher than (4.60±0.07)% of NC group (P<0.05). The expression levels of CCL2 in U251 cells in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (271.63±44.27) and (371.66±50.21), lower than (933.93±29.84) in NC group (P<0.05). The expression levels of CCL5 (234.81±5.95 and 232.62±5.72), CXCL10 (544.13±48.14 and 500.87±15.65) and CXCL11 (215.75±15.30 and 176.18±16.49) in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were higher than those in NC group (9.98±0.71, 470.54±18.84 and 13.55±0.73, respectively, P<0.05). The recruited numbers of CD14(+) THP1 in HOXC10-siRNA-1 and HOXC10-siRNA-2 groups were (159.33±1.15) and (170.67±1.15), respectively, lower than (360.00±7.81) in NC group (P<0.05), while addition of reCCL2 promoted the recruitment of CD14(+) THP1 cells (287.00±3.61 and 280.67±2.31 in HOXC10-siRNA-1+ reCCL2 group and HOXC10-siRNA-2+ reCCL2 group, respectively, P<0.05). The expressions level of M2-type macrophage-related gene TGF-β in HOXC10-siRNA-1 group and HOXC10-siRNA-2 group were (0.30±0.02) and (0.28±0.02), respectively, lower than (1.06±0.10) in NC group (P<0.05). The expressions level of M1-related gene NOS2 in HOXC10-siRNA-1 and HOXC10-siRNA-2 were (11 413.95±1 911.85) and (5 894.00±945.21), respectively, higher than (13.39±4.32) in NC group (P<0.05). Conclusions: The expression of HOXC10 in glioma is high and positively correlated with the poor prognosis of glioma patients. Knockdown of HOXC10 can inhibit the proliferation, migration and metastasis of human glioma U251 cells. HOXC10 may play an immunosuppressive role in glioma microenvironment by promoting the expression of CCL2 and recruiting and polarizing tumor-associated macrophages (M2 macrophages).
Cell Line, Tumor ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Genes, Homeobox ; Glioma/pathology* ; Homeodomain Proteins/metabolism* ; Humans ; Neoplasm Invasiveness/genetics* ; Tumor Microenvironment

OBJECTIVE: To investigate the expression of circPCSK5 in gastric cancer (GC) and its role in regulation of the proliferation, invasion and epithelial-mesenchymal transition (EMT) of GC cells. METHODS: High-throughput sequencing was performed in 3 pairs of GC and adjacent gastric mucosa tissues to obtain the differential expression profile of circRNA. The expression of circPCSK5 was detected in 62 patients undergoing radical surgery for GC using RT-qPCR, and the correlation between circPCSK5 expression level and clinicopathological data of the patients was analyzed. The overall survival and disease-free survival of the patients were assessed with Kaplan-Meier survival analysis, and the independent risk factors affecting the patients' prognosis were analyzed using Cox proportional hazards regression model. The stability and subcellular localization of circPCSK5 were assessed using RNase R and actinomycin D assays, fluorescence in situ hybridization and nucleocytoplasmic separation assay. CCK-8 assay, EdU assay and Transwell assay were employed to examine the changes in proliferation, migration and invasion of GC cells with circPCSK5 knockdown or overexpression; Western blotting and RT-qPCR assays were used to detect the expression levels of EMT markers in the transfected cells. RESULTS: The expression of circPCSK5 was significantly upregulated in GC tissues and cells (P < 0.001, P < 0.01). The expression level of circPCSK5 was positively correlated with tumor size, vascular invasion, lymph node metastasis and AJCC stage of GC (P < 0.05). The overall survival and disease-free survival were significantly lower in GC patients with high circPCSK5 expression than in those with low circPCSK5 expression (P < 0.001). High circPCSK5 expression was an independent risk factor for a poor prognosis of GC patients (P < 0.05). Knockdown of circPCSK5 significantly inhibited the proliferation, migration and invasion of HGC27 cells (P < 0.01), increased the expressions of E-cadherin, and decreased the expression of N-cadherin and vimentin (P < 0.01). CircPCSK5 overexpression promoted the proliferation, migration and invasion of MKN45 cells (P < 0.01), reduced E-cadherin expression and increased N-cadherin and vimentin expressions (P < 0.01). CONCLUSION CircPCSK5 is highly expressed in GC and promotes the proliferation, invasion and EMT of GC cells, suggesting its potential as a prognostic biomarker and therapeutic target for GC.
Humans ; Epithelial-Mesenchymal Transition/genetics* ; Stomach Neoplasms/pathology* ; Vimentin/metabolism* ; Neoplasm Invasiveness/genetics* ; In Situ Hybridization, Fluorescence ; Cell Movement/genetics* ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Cadherins/metabolism*

OBJECTIVE: To investigate the regulatory effect of miR-4324 on ankyrin 2(Talin2) expression and biological behaviors of breast cancer cells and the clinical implications of changes in miR-4324 and Talin2 expressions in breast cancer. METHODS: In breast cancer and adjacent tissues, the expressions of Talin2 and miR-4324 were examined with immunohistochemistry and qRT-PCR, respectively and the association of Talin2 expression levels with the prognosis and clinicopathological features of breast cancer patients was analyzed.The human breast cancer cell line SKBR-3 was transfected with miR-4324 mimic, miR-4324 inhibitor, si-Talin2, or both miR-4324 inhibitor and si-Talin2, and the changes in biological behaviors of the cells were examined; the cellular expression of Talin2at the mRNA and protein levels were detected with qRT-PCR and Western blotting.Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-4324 and Talin2.The effect of miR-4324-mediated regulation of Talin2 on SKBR-3 cell migration was assessed using Transwell assays. RESULTS: Talin2 expression was significantly higher in breast cancer tissues than in the adjacent tissues, and its expression level was correlated with lymph node metastasis and high HER-2 expression in breast cancer (P < 0.05) but not with the patient's age, clinical stage, histological grade or expressions of estrogen and progesterone receptors (P >0.05).The expression of miR-4324 was significantly reduced in breast cancer tissues as compared with the adjacent tissues (P < 0.01).In SKBR-3 cells, transfection with miR-4324 mimics significantly inhibited proliferation, migration and invasion (P < 0.05) and promoted apoptosis (P < 0.01) of the cells.Dual luciferase reporter gene assay confirmed that cotransfection with miR-4324 mimics significantly reduced luciferase activity of Talin2-3'-UTR WT reporter plasmid (P < 0.05).Transfection of the cells with miR-4324 mimics significantly reduced mRNA and protein expressions of Talin2(P < 0.05).Transwell migration assay showed that the migration ability of SKBR-3 cells was significantly enhanced after transfection with miR-4324 inhibitor (P < 0.01), lowered after transfection with si-Talin2(P < 0.01), and maintained at the intermediate level after co-transfection with miR-4324 inhibitor+si-Talin2 group (P < 0.05). CONCLUSIONS High expression of Talin2 is associated with lymph node metastasis and HER-2 overexpression in breast cancer patients.Down-regulation of miR-4324 inhibits the proliferation, invasion and migration and induces apoptosis of breast cancer cells, and the inhibitory effect of miR-4324 knockdown on breast cancer cell migration is mediated probably by targeted inhibition of Talin2 expression.
Female ; Humans ; Breast Neoplasms/pathology* ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic ; Luciferases/genetics* ; Lymphatic Metastasis ; MicroRNAs/metabolism* ; Neoplasm Invasiveness/genetics* ; RNA, Messenger

This study was designed to evaluate ERK5 expression in lung cancer and malignant melanoma progression and to ascertain the involvement of ERK5 signaling in lung cancer and melanoma. We show that ERK5 expression is abundant in human lung cancer samples, and elevated ERK5 expression in lung cancer was linked to the acquisition of increased metastatic and invasive potential. Importantly, we observed a significant correlation between ERK5 activity and FAK expression and its phosphorylation at the Ser
A549 Cells ; Animals ; Cell Movement ; Epithelial-Mesenchymal Transition/genetics* ; Focal Adhesion Kinase 1/metabolism* ; Humans ; Lung Neoplasms/pathology* ; MAP Kinase Signaling System ; Mice ; Mitogen-Activated Protein Kinase 7/metabolism* ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Proteins/metabolism*

Objective To evaluate the effect of miR-145 on migration and invasion of ovarian cancer cells.Methods The effect of miR-145 overexpression on the expression levels of miR-145 and zeb-2 were detected with qRT-PCR and Western blotting.The changes of migration and invasion were examined using Transwell assay.Target genes of miR-145 were predicted by bioinformatics software.Dual-luciferase reporter assay were used to verify zeb-2 as a direct target of miR-145.zeb-2 siRNA was transiently transfected in SKOV3 and 3AO cells,Transwell was used to examine migration and invasion abilities.Results The migration and proliferation of SKOV3(=10.752,=0.000;=5.617,=0.005)and 3AO cells(=10.111,=0.001;=21.746,=0.000)decreased significantly after overexpression of miR-145.The results of dual-luciferase reporter assay showed that the relative luciferase activity of co-transfected miR-145 mimic and WT 3'UTR expression vectors was significantly lower than that of co-transfected mimic control and WT 3'UTR expression vectors(SKOV3:=4.572,=0.010;3AO:=3.528,=0.024).There was no significant difference in relative luciferase activity between co-transfected miR-145 mimic/MUT 3'UTR expression vector cells and co-transfected mimic control/MUT 3'UTR expression vector cells(SKOV3:=0.227,=0.831;3AO:=0.040,=0.970).Real-time quantitative PCR showed that the zeb-2 expressions in SKOV3(=1.490,=0.211)and 3AO cells(=0.114,=0.914)were not significantly different from negative control after 48 h of miR-145 overexpression.Western blot analysis showed that the expression of zeb-2 protein in SKOV3(=3.769,=0.020)and 3AO cells(=4.452,=0.011)decreased significantly compared with negative control after 72 h of miR-145 overexpression.Seventy-two hours after transfection of zeb-2 siRNA,Western blotting showed that the expression of zeb-2 protein in SKOV3(=4.660,=0.010)and 3AO cells(=4.594,=0.010)was significantly down-regulated.Transwell assay showed that the migration and invasion abilities of SKOV3(=18.655,=0.000;=18.026,=0.000)and 3AO cells(=5.500,=0.005;=8.780,=0.001)were significantly decreased.Conclusion miR-145 may inhibit the migration and invasion of ovarian cancer cells by targeting zeb-2.
Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Female ; Humans ; MicroRNAs ; genetics ; Neoplasm Invasiveness ; Ovarian Neoplasms ; pathology ; Zinc Finger E-box Binding Homeobox 2 ; genetics