miR-497 inhibits the growth and metastasis of SGC-7901 human gastric cancer anoikis resistant cells via blocking Wnt/β-catenin signaling pathway / 细胞与分子免疫学杂志

Objective To investigate the effects of microRNA497 (miR-497) on the metastasis of gastric cancer and its possible molecular mechanism. Methods SGC-7901 gastric cancer parent cells were cultured in an ultra-low adhesion environment, and the anoikis resistance model of SGC-7901 cells was created after re-adhesion. Clone formation assay, flow cytometry, TranswellTM test and scratch healing test were used to detect the differences of biological behavior compared with their parent cells. Fluorescence quantitative PCR was performed to detect the expression of miR-497. Western blot analysis was used to detect the changes of key proteins of Wnt/β-catenin signaling pathway and epithelial mesenchymal transformation (EMT) related proteins such as vimentin and E-cadherin. Parent cells and anoikis resistant SGC-7901 cells were transfected with miR-497 inhibitor or miR-497 mimic, and CCK-8 assay was used to detect the proliferation activity. TranswellTM invasion assay was performed to detect the invasion ability of cells. TranswellTM migration test and scratch healing assay was used to determine the migration ability. Western blot analysis was used to detect the expressions of Wnt1, β-catenin, vimentin and E-cadherin. By transfecting miR-497 mimic into the anoikis resistance SGC-7901 cells and inoculating them subcutaneously in nude mice, the changes in the volume and mass of tumor tissues were measured and recorded. Western blot analysis was used to determine the expressions of Wnt1, β-catenin, vimentin and E-cadherin of tumor tissues. Results Compared with the parent cells, the anoikis resistance SGC-7901 gastric cancer cells had faster proliferation rate, stronger colony formation, lower apoptosis rate, stronger invasion and migration ability. The expression of miR-497 was significantly decreased. After down-regulation of miR-497, the proliferation ability, invasion and migration ability were significantly enhanced. The expressions of Wnt1, β-catenin and vimentin increased significantly, while E-cadherin decreased notably. The results of up-regulation miR-497 were the opposite. The tumor growth rate, tumor volume and mass of miR-497 overexpression group were significantly lower than those of control group. The expressions of Wnt1, β-catenin and vimentin decreased significantly, while the expression of E-cadherin increased significantly. Conclusion The expression of miR-497 is low in the anoikis resistance SGC-7901 cells. miR-497 can inhibit the growth and metastasis of gastric cancer cells by blocking Wnt/β-catenin signaling pathway and EMT.

MiR-3653 blocks autophagy to inhibit epithelial-mesenchymal transition in breast cancer cells by targeting the autophagy-regulatory genes ATG12 and AMBRA1 / 中华医学杂志(英文版)

BACKGROUND@#Metastasis is the main cause of tumor-associated death and mainly responsible for treatment failure of breast cancer. Autophagy accelerates tumor metastasis. In our work, we aimed to investigate the possibility of microRNAs (miRNAs) which participate in the regulation of autophagy to inhibit tumor metastasis.@*METHODS@#MiRNA array and comprehensive analysis were performed to identify miRNAs which participated in the regulation of autophagy to inhibit tumor metastasis. The expression levels of miR-3653 in breast cancer tissues and cells were detected by quantitative real-time polymerase chain reaction. In vivo and in vitro assays were conducted to determine the function of miR-3653. The target genes of miR-3653 were detected by a dual luciferase reporter activity assay and Western blot. The relationship between miR-3653 and epithelial-mesenchymal transition (EMT) was assessed by Western blot. Student's t -test was used to analyze the difference between any two groups, and the difference among multiple groups was analyzed with one-way analysis of variance and a Bonferroni post hoc test.@*RESULTS@#miR-3653 was downregulated in breast cancer cells with high metastatic ability, and high expression of miR-3653 blocked autophagic flux in breast cancer cells. Clinically, low expression of miR-3653 in breast cancer tissues (0.054 ± 0.013 vs . 0.131 ± 0.028, t  = 2.475, P  = 0.014) was positively correlated with lymph node metastasis (0.015 ± 0.004 vs . 0.078 ± 0.020, t  = 2.319, P  = 0.023) and poor prognosis ( P  < 0.001). miR-3653 ameliorated the malignant phenotypes of breast cancer cells, including proliferation, migration (MDA-MB-231: 0.353 ± 0.013 vs . 1.000 ± 0.038, t  = 16.290, P  < 0.001; MDA-MB-468: 0.200 ± 0.014 vs . 1.000 ± 0.043, t  = 17.530, P  < 0.001), invasion (MDA-MB-231: 0.723 ± 0.056 vs . 1.000 ± 0.035, t  = 4.223, P  = 0.013; MDA-MB-468: 0.222 ± 0.016 vs . 1.000 ± 0.019, t  = 31.050, P  < 0.001), and colony formation (MDA-MB-231: 0.472 ± 0.022 vs . 1.000 ± 0.022, t  = 16.620, P  < 0.001; MDA-MB-468: 0.650 ± 0.040 vs . 1.000 ± 0.098, t  = 3.297, P  = 0.030). The autophagy-associated genes autophagy-related gene 12 ( ATG12 ) and activating molecule in beclin 1-regulated autophagy protein 1 ( AMBRA1 ) are target genes of miR-3653. Further studies showed that miR-3653 inhibited EMT by targeting ATG12 and AMBRA1 .@*CONCLUSIONS@#Our findings suggested that miR-3653 inhibits the autophagy process by targeting ATG12 and AMBRA1 , thereby inhibiting EMT, and provided a new idea and target for the metastasis of breast cancer.

USP25 promotes hepatocellular carcinoma progression by interacting with TRIM21 via the Wnt/β-catenin signaling pathway / 中华医学杂志(英文版)

BACKGROUND@#Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The ubiquitin-specific peptidase 25 (USP25) protein has been reported to participate in the development of several cancers. However, few studies have reported its association with HCC. In this study, we aimed to investigate the function and mechanism of USP25 in the progression of HCC.@*METHODS@#We analyzed USP25 protein expression in HCC based on The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) database cohorts. Then, we constructed USP25-overexpressing and USP25-knockdown HepG2, MHCC97H, and L-O2 cells. We detected the biological function of USP25 by performing a series of assays, such as Cell Counting Kit-8 (CCK-8), colony formation, transwell, and wound healing assays. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed to detect the interaction between USP25 and the Wnt/β-catenin signaling pathway. The relationship between USP25 and tripartite motif-containing 21 (TRIM21) was assessed through mass spectrometry and co-immunoprecipitation (Co-IP) analysis. Finally, we constructed a mouse liver cancer model with the USP25 gene deletion to verify in vivo role of USP25.@*RESULTS@#USP25 was highly expressed in HCC tissue and HCC cell lines. Importantly, high expression of USP25 in tissues was closely related to a poor prognosis. USP25 knockdown markedly reduced the proliferation, migration, and invasion of HepG2 and MHCC97H cells, whereas USP25 overexpression led to the opposite effects. In addition, we demonstrated that USP25 interacts with TRIM21 to regulate the expression of proteins related to epithelial-mesenchymal transition (EMT; E-cadherin, N-cadherin, and Snail) and the Wnt/β-catenin pathway (β-catenin, Adenomatous polyposis coli, Axin2 and Glycogen synthase kinase 3 beta) and those of their downstream proteins (C-myc and Cyclin D1). Finally, we verified that knocking out USP25 inhibited tumor growth and distant metastasis in vivo .@*CONCLUSIONS@#In summary, our data showed that USP25 was overexpressed in HCC. USP25 promoted the proliferation, migration, invasion, and EMT of HCC cells by interacting with TRIM21 to activate the β-catenin signaling pathway.

lncRNA AC005224.4/miR-140-3p/SNAI2 regulating axis facilitates the invasion and metastasis of ovarian cancer through epithelial-mesenchymal transition / 中华医学杂志(英文版)

BACKGROUND@#Ovarian cancer is one of the most widespread malignant diseases of the female reproductive system worldwide. The plurality of ovarian cancer is diagnosed with metastasis in the abdominal cavity. Epithelial-mesenchymal transition (EMT) exerts a vital role in tumor cell metastasis. However, it remains unclear whether long non-coding RNA (lncRNA) are implicated in EMT and influence ovarian cancer cell invasion and metastasis. This study was designed to investigate the impacts of lncRNA AC005224.4 on ovarian cancer.@*METHODS@#LncRNA AC005224.4, miR-140-3p, and snail family transcriptional repressor 2 ( SNAI2 ) expression levels in ovarian cancer and normal ovarian tissues were determined using real-time quantitative polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) and Transwell (migration and invasion) assays were conducted to measure SKOV3 and CAOV-3 cell proliferation and metastasis. E-cadherin, N-cadherin, Snail, and Vimentin contents were detected using Western blot. Nude mouse xenograft assay was utilized to validate AC005224.4 effects in vivo . Dual-luciferase reporter gene assay confirmed the targeted relationship between miR-140-3p and AC005224.4 or SNAI2 .@*RESULTS@#AC005224.4 and SNAI2 upregulation and miR-140-3p downregulation were observed in ovarian cancer tissues and cells. Silencing of AC005224.4 observably moderated SKOV3 and CAOV-3 cell proliferation, migration, invasion, and EMT process in vitro and impaired the tumorigenesis in vivo . miR-140-3p was a target of AC005224.4 and its reduced expression level was mediated by AC005224.4. miR-140-3p mimics decreased the proliferation, migration, and invasion of ovarian cancer cells. SNAI2 was identified as a novel target of miR-140-3p and its expression level was promoted by either AC005224.4 overexpression or miR-140-3p knockdown. Overexpression of SNAI2 also facilitated ovarian cancer cell viability and metastasis.@*CONCLUSION@#AC005224.4 was confirmed as an oncogene via sponging miR-140-3p and promoted SNAI2 expression, contributing to better understanding of ovarian cancer pathogenesis and shedding light on exploiting the novel lncRNA-directed therapy against ovarian cancer.

Effect of long non-coding RNA 114227 on gastric cancer cell proliferation and migration / 中南大学学报(医学版)

OBJECTIVES@#Gastric cancer is a common cancer of the digestive system. Long non-coding RNA (lncRNA) plays an important role in the formation and development of gastric cancer. This study aims to investigate the effect of long non-coding lncRNA 114227 on biologic behaviors in gastric cancer cells.@*METHODS@#The experiment was divided into 4 groups: a negative control (NC) group, a lncRNA 114227 small interference (si-lncRNA 114227) group, an empty vector (Vector) group, and an overexpression vector (OE-lncRNA 114227) group. The expressions of lncRNA 114227 in gastric mucosa and gastric cancer tissues, gastric mucosal epithelial cells and different gastric cancer strains were determined by real-time reverse transcription PCR (real-time RT-PCR).The proliferation were detected by CCK-8 assay in gastric cancer cells. The epithelial-mesenchymal transformation (EMT) was utilized by Transwell assay, scratch healing assay, and Western blotting in gastric cancer cells. The effect of lncRNA 114227 on proliferation of gastric cancer cells was detected by tumor bearing experiment in nude mice in vivo.@*RESULTS@#The expression level of lncRNA 114227 in the gastric cancer tissues was significantly lower than that in the gastric mucosa tissues, and in 4 kinds of gastric cancer strains was all significantly lower than that in gastric mucosal epithelial cells (all P<0.01). In vitro, the proliferation and migration abilities of gastric cells were significantly reduced after overexpressing lncRNA 114227, and cell proliferation and migration were enhanced after silencing lncRNA 114227 (all P<0.05). The results of in vivo subcutaneous tumorigenesis in nude mice showed that the tumorigenic volume of the tumor-bearing mice in the OE-lncRNA 114227 group was significantly smaller than that of the Vector group, and the tumorigenic quality was lower than that of the Vector group (P<0.05), indicating that lncRNA 114227 inhibited tumorigenesis.@*CONCLUSIONS@#The expression of lncRNA 114227 is downregulated in gastric cancer gastric cancer tissues and cell lines. LncRNA 114227 may inhibit the proliferation and migration of gastric cancer cells through EMT process.

Effects of miR-9-5p on the migration, invasion and epithelial-mesenchymal transition process in cervical squamous cell carcinoma / 中南大学学报(医学版)

OBJECTIVES@#Cervical squamous cell carcinoma is the most common cancer in female reproductive system. This study aims to explore the effect of microRNA-9-5p (miR-9-5p) on the migration, invasion, and epithelial-mesenchymal transition (EMT) process of cervical squamous cells.@*METHODS@#Bioinformatics were used to predict the miRNAs that could bind to E-cadherin (E-cad). The Cancer Genome Atlas (TCGA) database was used to analyze and extract significantly differentially expressed miRNAs from part of cervical squamous cell carcinoma tissues and normal cervical tissues, and miR-9-5p was selected as the main research target. The translated regions (UTR) of wild-type E-cad (E-cad-WT 3'-UTR) or the 3'-UTR of mutant E-cad (E-Cad-MUT 3'-UTR) was transfected with miR-9-5p mimic normal control (NC), and miR-9-5p mimic was co-transfected human embryonic kidney cells (293T). The relationship between miR-9-5p and E-cad was detected by double luciferase assay. The expression of miR-9-5p in normal cervical epithelial cell lines (H8) and cervical squamous cell lines (C33A, siha, caski and Me180) were detected by quantitative real-time PCR. Then, the experiments were divided into groups as follows: a block control group, an overexpression control group (mimic-NC group), a miR-95p overexpression group (mimic group), an inhibitory expression control group (inhibitor-NC group), and a miR-9-5p inhibitory expression group (inhibitor group). The changes of migration ability were detected by scratch assay. Transwell invasion assay was used to analyze the changes of invasion ability, and the mRNA and protein changes of E-cad and vimentin were detected by quantitative real-time PCR and Western blotting.@*RESULTS@#MiR-9-5p had a targeting binding relationship with E-cad. Compared with the normal cervical tissue H8 cell line, the miR-9-5p was highly expressed in cervical cancer cell lines (C33A, siha, caski and Me180) (all P<0.05). The luciferase activity of E-cad-MUT was increased compared with that of E-cad-WT in miR-9-5p mimic cells (P<0.05). Compared with the blank control group, the protein and mRNA expressions of E-cad were decreased in the miR-9-5p mimic group (both P<0.05), which were increased in the miR-9-5p inhibitor group (both P<0.05). Compared with H8 cell line, the miR-9-5p was highly expressed in the cervical squamous cell lines (all P<0.05). Compared with the mimic-NC group, the distance of wound healing, the number of caski and Me180 cells invaded below the membrane, and the mRNA and protein expressions of vimentin were all increased in the miR-9-5p mimic group (all P<0.05), while the mRNA and protein of E-cad were decreased (both P<0.05). Compared with the inhibitor-NC group, the distance of wound healing, the number of caski and Me180 cells invading the membrane, and the mRNA and protein expressions of vimentin were decreased in the miR-9-5p inhibitor group (all P<0.05), but the mRNA and protein expressions of E-cad were increased (both P<0.05).@*CONCLUSIONS@#The miR-9-5p is highly expressed in cervical squamous cell carcinoma, which can increase the migration and invasion ability, and promote the EMT process of cancer cells.

Knockdown of ACC1 promotes migration of esophageal cancer cell / 中华肿瘤杂志

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.

The mechanism of S100A7 inducing the migration and invasion in cervical cancer cells / 中华肿瘤杂志

Objective: To investigate the mechanism of S100A7 inducing the migration and invasion in cervical cancers. Methods: Tissue samples of 5 cases of cervical squamous cell carcinoma and 3 cases of adenocarcinoma were collected from May 2007 to December 2007 in the Department of Gynecology of the Affiliated Hospital of Qingdao University. Immunohistochemistry was performed to evaluate the expression of S100A7 in cervical carcinoma tissues. S100A7-overexpressing HeLa and C33A cells were established with lentiviral systems as the experimental group. Immunofluorescence assay was performed to observe the cell morphology. Transwell assay was taken to detect the effect of S100A7-overexpression on the migration and invasion of cervical cancer cells. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to examine the mRNA expressions of E-cadherin, N-cadherin, vimentin and fibronectin. The expression of extracellular S100A7 in conditioned medium of cervical cancer cell was detected by western blot. Conditioned medium was added into Transwell lower compartment to detect cell motility. Exosomes were isolated and extracted from the culture supernatant of cervical cancer cell, the expressions of S100A7, CD81 and TSG101 were detected by western blot. Transwell assay was taken to detect the effect of exosomes on the migration and invasion of cervical cancer cells. Results: S100A7 expression was positively expressed in cervical squamous carcinoma and negative expression in adenocarcinoma. Stable S100A7-overexpressing HeLa and C33A cells were successfully constructed. C33A cells in the experimental group were spindle shaped while those in the control group tended to be polygonal epithelioid cells. The number of S100A7-overexpressed HeLa cells passing through the Transwell membrane assay was increased significantly in migration and invasion assay (152.00±39.22 vs 105.13±15.75, P<0.05; 115.38±34.57 vs 79.50±13.68, P<0.05). RT-qPCR indicated that the mRNA expressions of E-cadherin in S100A7-overexpressed HeLa and C33A cells decreased (P<0.05) while the mRNA expressions of N-cadherin and fibronectin in HeLa cells and fibronectin in C33A cells increased (P<0.05). Western blot showed that extracellular S100A7 was detected in culture supernatant of cervical cancer cells. HeLa cells of the experimental group passing through transwell membrane in migration and invasion assays were increased significantly (192.60±24.41 vs 98.80±47.24, P<0.05; 105.40±27.38 vs 84.50±13.51, P<0.05) when the conditional medium was added into the lower compartment of Transwell. Exosomes from C33A cell culture supernatant were extracted successfully, and S100A7 expression was positive. The number of transmembrane C33A cells incubated with exosomes extracted from cells of the experimental group was increased significantly (251.00±49.82 vs 143.00±30.85, P<0.05; 524.60±52.74 vs 389.00±63.23, P<0.05). Conclusion: S100A7 may promote the migration and invasion of cervical cancer cells by epithelial-mesenchymal transition and exosome secretion.

Vasohibin-2 promotes proliferation and metastasis of cervical cancer cells by regulating epithelial-mesenchymal transition / 南方医科大学学报

OBJECTIVE@#To explore the role of vasohibin-2 (VASH2) in regulation of proliferation and metastasis of cervical cancer cells.@*METHODS@#We analyzed the differentially expressed genes between cervical cancer cells with flotillin-1 overexpression and knockdown by RNA-seq combined with analysis of public databases. The expression levels of VASH2 were examined in normal cervical epithelial cells (HcerEpic), cervical cancer cell lines (HeLa, C-33A, Ca ski, SiHa and MS751) and fresh cervical cancer tissues with different lymph node metastasis status. We further tested the effects of lentivirus-mediated overexpression and interference of VASH2 on proliferation, migration, invasion and lymphatic vessel formation of the cervical cancer cells and detected the expression levels of key epithelial-mesenchymal transition (EMT) markers and TGF-β mRNA.@*RESULTS@#RNA-seq and analysis of public databases showed that VASH2 expression was significantly upregulated in cervical cancer cells exogenously overexpressing flotillin-1 (P < 0.05) and downregulated in cells with flotillin-1 knockdown (P < 0.05), and was significantly higher in cervical cancer tissues with lymph node metastasis than in those without lymph node metastasis (P < 0.01). In cervical cancer cell lines Ca Ski, SiHa, and MS751 and cervical cancer tissue specimens with lymph node metastasis, VASH2 expression was also significantly upregulated as compared with HcerEpic cells and cervical cancer tissues without lymph node metastasis (P < 0.05). Exogenous overexpression of VASH2 significantly promoted proliferation, migration, invasion and lymphatic vessel formation of cervical cancer cells, whereas these abilities were significantly inhibited in cells with VASH2 knockdown (P < 0.05). The cervical cancer cells overexpressing VASH2 showed significant down- regulation of e-cadherin and up- regulation of N-cadherin, Vimentin and VEGF-C, while the reverse changes were detected in cells with VASH2 knockdown (P < 0.05). TGF-β mRNA expression was significantly up-regulated in cervical cancer cells overexpressing VASH2 and down-regulated in cells with VASH2 knockdown (P < 0.001).@*CONCLUSION@#Flotillin-1 may participate in TGF-β signaling pathway-mediated EMT through its down-stream target gene VASH2 to promote the proliferation, migration, invasion and lymphatic vessel formation of cervical cancer cells in vitro.

KIF2C: a novel link between Wnt/β-catenin and mTORC1 signaling in the pathogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is the fourth-leading cause of cancer-related deaths worldwide. HCC is refractory to many standard cancer treatments and the prognosis is often poor, highlighting a pressing need to identify biomarkers of aggressiveness and potential targets for future treatments. Kinesin family member 2C (KIF2C) is reported to be highly expressed in several human tumors. Nevertheless, the molecular mechanisms underlying the role of KIF2C in tumor development and progression have not been investigated. In this study, we found that KIF2C expression was significantly upregulated in HCC, and that KIF2C up-regulation was associated with a poor prognosis. Utilizing both gain and loss of function assays, we showed that KIF2C promoted HCC cell proliferation, migration, invasion, and metastasis both in vitro and in vivo. Mechanistically, we identified TBC1D7 as a binding partner of KIF2C, and this interaction disrupts the formation of the TSC complex, resulting in the enhancement of mammalian target of rapamycin complex1 (mTORC1) signal transduction. Additionally, we found that KIF2C is a direct target of the Wnt/β-catenin pathway, and acts as a key factor in mediating the crosstalk between Wnt/β-catenin and mTORC1 signaling. Thus, the results of our study establish a link between Wnt/β-catenin and mTORC1 signaling, which highlights the potential of KIF2C as a therapeutic target for the treatment of HCC.

Circ_0049447 acts as a tumor suppressor in gastric cancer through reducing proliferation, migration, invasion, and epithelial-mesenchymal transition / 中华医学杂志(英文版)

BACKGROUND@#Although increasing abnormal expression of circular RNAs (circRNAs) has been revealed in various cancers, there were a small number of studies about circRNAs in gastric cancer (GC). Here, we explored the expression and function of a novel circRNA, circ_0049447, in GC.@*METHODS@#A total of 80 GC tissues and non-tumorous tissues were collected from the First Affiliated Hospital of China Medical University. And all cells were cultured with 10% fetal bovine serum and incubated at 37°C and 5% CO2. The expression of circ_0049447 was quantified by real-time polymerase chain reaction. The biological function of circ_0049447 on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) was evaluated by cell counting kit-8 (CCK-8), colony formation assay, transwell migration and invasion assay, and Western blotting. Luciferase report assay was used to verify the direct binding between circ_0049447 and predicted microRNA (miRNA). Furthermore, a xenograft mouse model was used to validate the function of circ_0049447 in vivo.@*RESULTS@#We demonstrated that circ_0049447 was downregulated in GC (P < 0.001). The area under the receiver operating characteristic curve reached 0.838, while sensitivity was 82.3% and specificity was 77.2%. CCK-8 and colony formation assay showed that overexpression of circ_0049447 could inhibit the proliferation (P < 0.05). Transwell migration and invasion assay showed upregulated circ_0049447 could impede migration in GC cells (P < 0.05). In addition, overexpression of circ_0049447 could impede GC cell EMT. Upregulation of miR-324-5p in GC specimens and direct binding between miR-324-5p with circ_0049447 proven by luciferase reporter assay indicated that circ_0049447 may inhibit GC by sponging certain miRNA.@*CONCLUSION@#Circ_0049447 acts as a tumor suppressor in GC through reducing proliferation, migration, invasion, and EMT, and it is a promising biomarker for diagnosis.

Extracellular signal regulated kinase 5 promotes cell migration, invasion and lung metastasis in a FAK-dependent manner

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

Silencing long non-coding RNA HIF1A-AS2 inhibits proliferation, invasion and migration of cervical cancer cells / 南方医科大学学报

OBJECTIVE@#To explore the inhibitory effects of silencing long non-coding RNA (LncRNA) HIF1A-AS2 on epithelialmesenchymal transition (EMT) and tumor stem cell-like phenotype in cervical cancer cells.@*METHODS@#We designed 3 shRNA constructs for silencing HIF1A-AS2 in CaSki cells, and the shRNA with the strongest interference effect was selected for subsequent experiment. CaSki cells were transfected with shRNA-NC or Sh-HIF1A-AS2, and the changes in cell viability, invasion ability, EMT, expressions of EMT-related proteins, formation of cell spheres and expressions of stem cell markers were detected.@*RESULTS@#Transfection with shRNA-NC and Sh-HIF1A-AS2 did not significantly affected the viability of CaSki cells (@*CONCLUSIONS@#Silencing HIF1A-AS2 can inhibit proliferation, invasion and migration of cervical cancer cells

PPP3CA silence regulates MET process, cell apoptosis, proliferation and migration in metanephric mesenchyme cells / 生物工程学报

Kidney is one of the most important organs of the body and the mammalian kidney development is essential for kidney unit formation. The key process of kidney development is metanephric development, where mesenchymal-epithelial transition (MET) plays a crucial role. Here we investigated the biological function of PPP3CA in metanephric mesenchyme (MM) cells. qRT-PCR and Western blotting were used to detect PPP3CA and MET makers expression in mK3, mK4 cells respectively at mRNA and protein level. Subsequently, PPP3CA was stably knocked down via lentivirus infection in mK4 cells. Flow cytometry, EdU/CCK-8 assay, wound healing assay were conducted to clarify the regulation of PPP3CA on cell apoptosis, proliferation and migration respectively. PPP3CA was expressed higher in epithelial-like mK4 cells than mesenchyme-like mK3 cells. Thus, PPP3CA was silenced in mK4 cells and PPP3CA deficiency promoted E-cadherin expression, cell apoptosis. Moreover, PPP3CA knock down attenuated cell proliferation and cell migration in mK4 cell. The underlying mechanism was associated with the dephosphorylation of PPP3CA on ERK1/2. Taken together, our results indicated that PPP3CA mediated MET process and cell behaviors of MM cells, providing new foundation for analyzing potential regulator in kidney development process.

Astrocyte elevated gene-1 serves as a target of miR542 to promote glioblastoma proliferation and invasion / 中华医学杂志(英文版)

BACKGROUND@#Epithelial to mesenchymal transition (EMT) is strongly linked with tumor invasion and metastasis, which performs a vital role in carcinogenesis and cancer progression. Emerging evidence suggests that microRNAs (miRNAs) expression are closely associated to EMT by regulating targeted genes. MiR542 has been found to be involved in the EMT program and bound up with various cancers. However, the functions of miR542 and its underlying mechanism in glioblastoma multiforme (GBM) remain largely unknown. In the current study, we investigated the effect of astrocyte elevated gene-1 (AEG-1) on U251 cells aggressiveness, proliferation, apoptosis, and cell cycle.@*METHODS@#The screening of targeted miRNAs was performed, as well as the functional roles and mechanisms of miR542 were explored.@*RESULTS@#MiR542 was selected as the target because of the most significantly differential expression and this high level of expression negatively correlated with cell migration and proliferation, which suggested that miR542 could be a novel tumor suppressor. Moreover, we confirmed that AEG-1 was a direct targeted gene of miR542 by luciferase activity assay, reverse transcription-polymerase chain reaction, and immunoblotting analysis. Furthermore, miR542 suppressed the expression of AEG-1, which upgraded the level of E-cadherin and degraded Vimentin expression contributing to retraining EMT.@*CONCLUSION@#The in vitro findings demonstrated that miR542 inhibited the migration and proliferation of U251 cells and suppressed EMT through targeting AEG-1, indicating that miR542 may be a potential anti-cancer target for GBM.

Mir-30b-3p affects the migration and invasion function of ovarian cancer cells by targeting the CTHRC1 gene

BACKGROUND: The aim of this study was to investigate the effect role and mechanism of miR-30b-3p on ovarian cancer cells biological function. METHODS: The expression of miR-30b-3p was detected in ovarian cancer cell lines and normal ovarian epithelial cell line by qRT-PCR. Mir-30b-3p mimic was transfected into OVCAR3 cells. Cell-counting kit-8 (CCK-8) assay was conducted to explore the effect of mir-30b-3p on the OVCAR3 cells' proliferation. Cell cycle and apoptosis were detected by Flow cytometry. Cell invasion ability was detected by Transwell test. The regulation of putative target of miR-30b-3p was verified by double luciferase reporter assays and Western blot. RESULT: We found that miR-30b-3p was downregulated in OVCAR3 cells. Overexpression of miR-30b-3p suppressed proliferation, promoted apoptosis, slowed cell cycle and inhibited migration and invasion of OVCAR3 cells. Bioinformatics analysis identified 3'-untranslated region (3'UTR) of Collagen triple helix repeat-containing 1 (CTHRC1) as the presumed binding site for miR-30b-3p. Detection of double luciferase reporter and Western-Blot result confirmed that CTHRC1 was the target gene of miR-30b-3p. Furthermore, E-cadherin, ß-cadherin and Vimentin protein expression level were changed after transfection of miR-30b-3p. CONCLUSION: miR-30b-3p function as an anti-cancer gene. Overexpression of miR-30b-3p can inhibit the biological function of ovarian cancer cells. MiR-30b-3p targets CTHRC1 gene plays an important role in epithelial-mesenchymal transformation (EMT), and supports miR-30b-3p as a potential biological indicator for ovarian cancer in the future.

Hesperidin inhibits the epithelial to mesenchymal transition induced by transforming growth factor-ß1 in A549 cells through Smad signaling in the cytoplasm

Hesperidin, a natural compound, suppresses the epithelial-to-mesenchymal transition through the TGF-ß1/Smad signaling pathway. However, studies on the detailed effects and mechanisms of hesperidin are rare. The present study showed that, for A549 alveolar epithelial cells, the anti-proliferative effects of hesperidin occurred in a dose-dependent manner, with an IC50= 216.8 µM at 48 h. TGF-ß1 was used to activate the Smad signaling pathway and induce the epithelial to mesenchymal transition in cells. Treatment with hesperidin or SB431542 was used for antagonism of Smad pathway activation. Hesperidin inhibited the increase in ɑ-SMA and Col1ɑ-1 and the decrease in E-cadherin in a dose-dependent manner from concentration of 20 µM to 60 µM, as assessed by both ELISA and Western blotting assays; however, there was no significant effect on cellular morphological alterations. Moreover, the Western blotting assay showed that, in the cytoplasm, hesperidin and SB431542 had no significant effect on the protein expression of Smad 2, 3, 4, or 7 as well as 2/3. However, 60 µM hesperidin and SB431542 significantly decreased p-Smad2/3 protein expression. From the above results, it is concluded that hesperidin can partly inhibit the epithelial to mesenchymal transition in human alveolar epithelial cells; the effect accounts for the blockage of the phosphorylation of Smad2/3 in the cytoplasm rather than a change in Smad protein production in the cytoplasm

Long non-coding RNA CHCHD4P4 promotes epithelial-mesenchymal transition and inhibits cell proliferation in calcium oxalate-induced kidney damage

Kidney stone disease is a major cause of chronic renal insufficiency. The role of long non-coding RNAs (lncRNAs) in calcium oxalate-induced kidney damage is unclear. Therefore, we aimed to explore the roles of lncRNAs in glyoxylate-exposed and healthy mouse kidneys using microarray technology and bioinformatics analyses. A total 376 mouse lncRNAs were differentially expressed between the two groups. Using BLAST, 15 lncRNA homologs, including AU015836 and CHCHD4P4, were identified in mice and humans. The AU015836 expression in mice exposed to glyoxylate and the CHCHD4P4 expression in human proximal tubular epithelial (HK-2) cells exposed to calcium oxalate monohydrate were analyzed, and both lncRNAs were found to be upregulated in response to calcium oxalate. To further evaluate the effects of CHCHD4P4 on the cell behavior, we constructed stable CHCHD4P4-overexpressing and CHCHD4P4-knockdown HK-2 cells. The results showed that CHCHD4P4 inhibited cell proliferation and promoted the epithelial-mesenchymal transition in kidney damage and fibrosis caused by calcium oxalate crystallization and deposition. The silencing of CHCHD4P4 reduced the kidney damage and fibrosis and may thus be a potential molecular target for the treatment of kidney stones.

The transcription factor ZEB1 promotes an aggressive phenotype in prostate cancer cell lines / 亚洲男科学杂志(英文版)

It has been reported that one of the factors that promotes tumoral progression is the abnormal activation of the epithelial-mesenchymal transition program. This process is associated with tumoral cells acquiring invasive and malignant properties and has the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) as one of its main activators. However, the role of ZEB1 in promoting malignancy in prostate cancer (PCa) is still unclear. Here, we report that ZEB1 expression correlates with Gleason score in PCa samples and that expression of ZEB1 regulates epithelial-mesenchymal transition and malignant characteristics in PCa cell lines. The results showed that ZEB1 expression is higher in samples of higher malignancy and that overexpression of ZEB1 was able to induce epithelial-mesenchymal transition by upregulating the mesenchymal marker Vimentin and downregulating the epithelial marker E-Cadherin. On the contrary, ZEB1 silencing repressed Vimentin expression and upregulated E-Cadherin. ZEB1 expression conferred enhanced motility and invasiveness and a higher colony formation capacity to 22Rv1 cells whereas DU145 cells with ZEB1 silencing showed a decrease in those same properties. The results showed that ZEB1 could be a key promoter of tumoral progression toward advanced stages of PCa.