Effects of WNT5B over-expression on viability and apoptosis of human breast cancer cell line MCF-7

AIM: To detect the expression of WNT5B in normal breast epithelial cells and different breast cancer cell lines, and to investigate the effects of WNT5B over-expression on the viability and apoptosis of human breast cell line MCF-7.METHODS: The mRNA expression of WNT5B was detected by RT-PCR in different breast cancer cells. MCF-7 cells were transfected with plasmid pcDNA3.1/WNT5B or pcDNA3.1, and the expression of WNT5B at mRNA and protein levels was examined in the 2 groups by real-time PCR and Western blotting, respectively. Subsequently, the changes of cell viability and cell apoptosis were analyzed by CCK-8 assay and flow cytometry, respectively. RESULTS: The expression of WNT5B in the breast cancer cell lines was lower than that in MCF10A cells. The WNT5B expression in the MCF-7 cells in experimental group was significantly higher than that in vector group (P<0.05). However, the cell viability in experimental group decreased significantly as compared with vector group (P<0.05). The number of the cells in S-phase obviously increased, while the percentage of the cells in G₁-phase and G₂/M-phase decreased compared with vector group. The number of apoptotic cells in WNT5B group was significantly higher than that in vector group.CONCLUSION: The expression of WNT5B is decreased in breast cancer cells. WNT5B over-expression significantly inhibits the cell growth and promotes the cell apoptosis in breast cancer MCF7 cells.     

F-box domain is involved in regulating the proliferation of MCF-7 cells by FBXO39 protein

AIM: To investigate the effect of F-box domain on the regulation of MCF-7 cell proliferation by FBXO39 protein. METHODS: The effect of F-box domain on the localization of FBXO39 protein in the MCF-7 cells was investigated. MCF-7 cell cDNA library was used as the template resource. The full-length cDNA sequence of FBXO39 was amplified by PCR method and subcloned into eukaryotic expression vector pEGFP-C2. The pEGFP-FBXO39ΔF (F-box domain deletion mutation) plasmid was successfully constructed with the template resource of pEGFP-FBXO39 plasmid. The recombinant plasmids were transfected into the MCF-7 cells, and then the expression of FBXO39 and FBXO39ΔF were determined by Western blot. The cellular localization of FBXO39 and FBXO39ΔF were observed by confocal microscopy. The localization of endogenous FBXO39 in the MCF-7 cells was detected by immunofluorescence staining. In addition, MTT and EdU assays were used to measure the cell proliferation, flow cytometry was used to measure the cell cycle distribution, and immunohistochemical staining was used to observe the expression of FBXO39 in the breast cancer and para-carcinoma tissues. RESULTS: The eukaryotic expression vector pEGFP-FBXO39 and pEGFP-FBXO39ΔF were constructed successfully. F-box domain had no effect on the cell localization of FBXO39. FBXO39 promoted MCF-7 cell proliferation but FBXO39ΔF did not. FBXO39 was highly expressed in the breast cancer tissues. CONCLUSION: F-box domain had no effect on the cellular localization of FBXO39 protein. However, it plays an important role in the biological function of FBXO39. FBXO39 may be related to breast cancer tumorigenesis.     

mTOR kinase inhibitor CC-223 suppresses human breast cancer cell viability

AIM: To investigate the inhibitory effect of CC-223, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on the viability of human breast cancer cells and its mechanism. METHODS: The inhibitory effect of CC-223 on the viability of MCF-7 cells and MDA-MB-231 cells was measured by CCK-8 assay. The cell cycle distribution of breast cancer cells was examined by flow cytometry. The expression of cell cycle-related proteins and oncoproteins c-Myc and survivin was analyzed by Western blot. RESULTS: CC-223 significantly inhibited the viability of both MCF-7 and MDA-MB-231 cells (P<0.05). CC-223 induced cell cycle arrest in both G₁ phase and G₂/M phase in the MCF-7 cells (P<0.05). However, low concentration of CC-223 treatment resulted in the accumulation of MDA-MB-231 cell cycle in the G₂/M phase, and the cell number in G₁ phase was unaffected. Treatment with CC-223 for 24 h clearly inhibited the protein levels of cyclin B1, cyclin D1 and phosphorylated cell division cycle protein 2 in the breast cancer cells (P<0.05). CC-223 suppressed the expression of c-Myc and survivin in both MCF-7 cells and MDA-MB-231 cells (P<0.05). CONCLUSION: CC-223 inhibits cell viability by blocking cell cycle progression and down-regulating expression of c-Myc and survivin in both MCF-7 and MDA-MB-231 cells.     

Knockdown of RRM2 gene by siRNA inhibits human breast cancer cell proliferation,migration and tumor growth in nude mice

AIM: To explore the effect of ribonucleotide reductase M2 (RRM2) gene knockdown by siRNA on the proliferation and migration of human breast cancer MCF-7 cells and the tumor growth in BALB/c nude mice. METHODS: The mRNA and protein expression leves of RRM2 in human breast cancer cell line MCF-7 and human normal breast cell line MCF-10A were determined by real-time PCR and Western blotting. siRNA-RRM2 was constructed and transfected into MCF-7 cells at different time points and different concentrations. The silencing efficiency of RRM2 gene was detected by real-time PCR. The cell proliferation was measured by CCK-8 assay. The migration was observed using Transwell cell migration system. The effect of siRNA-RRM2 on the tumor growth was determined in nude mice. RESULTS: The mRNA and protein levels of RRM2 were higher in MCF-7 cells than those in MCF-10A cells. siRNA-RRM2 down-regulated the expression of RRM2 in MCF-7 cells in a time-and concentration-dependent manner. The results of CCK-8 assay showed that siRNA-RRM2 inhibited the proliferation ability of MCF-7 cells, but not that of MCF-10A cells. The results of Transwell assay indicated that siRNA-RRM2 inhibited the migration ability of MCF-7 cells. siRNA-RRM2 also inhibited the tumor growth in nude mice. CONCLUSION: RRM2 overexpression is associated with the breast cancer proliferation and migration. Suppression of RRM2 function is a potential therapeutic strategy for treating breast cancer.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

MicroRNA-200a affects malignant biological behaviors of breast cancer cells by inhibiting epithelial-mesenchymal transition

AIM: To investigate the effect of microRNA-200a (miR-200a) on the malignant biological beha-viors of breast cancer cells and its regulatory mechanism. METHODS: The expression of miR-200a in human breast can-cer cell lines MDA-MB-231, MDA-MB-468 and MCF-7, and normal human mammary epithelial cell line MCF-10A was detected by RT-qPCR. CCK-8 assay was used to detect the viability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. Flow cytometry method and Transwell assay were used to detect the apoptosis and invasive ability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. The expression of SIP1, E-cadherin, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was determined by RT-qPCR and Western blot. RESULTS: Compared with MCF-10A cells, the lowest expression of miR-200a was observed in the MDA-MB-231 cells (P<0.05). Over-expression of miR-200a attenuated the viability of MDA-MB-231 cells (P<0.05), increased apoptosis (P<0.05) and decreased the invasion ability (P<0.05). The expression of SIP1, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was also significantly down-regulated, while the mRNA and protein expression of E-cadherin was significantly increased (P<0.05). Transfection with miR-200a inhibitor reversed the above results. CONCLUSION: Up-regulation of miR-200a inhibits the viability and invasion ability of MDA-MB-231 cells and promotes the apoptosis of MDA-MB-231 cells. miR-200a may regulate the biological behaviors of breast cancer by inhibiting epithelial-mesenchymal transition.     

PCDH10 inhibits proliferation of breast cancer MDA-MB-231 cells by NF-κB/cyclin D1 signaling pathway

AIM To investigate the inhibitory effect of protocadherin 10 （PCDH10） on proliferation of human breast cancer cells and its possible mechanism. METHODS RT-PCR was used to measure the mRNA expression levels of PCDH10 in 4 human breast cancer cell lines and normal mammary epithelial MCF-10A cells. The breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed by recombinant lentivirus （pLV-PCDH10） infection, and blank control （blank） group and negative control （pLV-NC） group were also set up. The cell proliferation ability was detected using methyl thiazolyl tetrazolium （MTT） and colony formation experiments. The cell cycle was detected by flow cytometry. Western blot was used to determine the expression of cyclin D1, cyclin-dependent kinase 4 （CDK4）, nucear factor-κB p65 subunit （NF-κB p65） and NF-κB inhibitor α （IκBα）. RESULTS The mRNA expression levels of PCDH10 in 4 breast cancer cell lines were lower than that in normal mammary epithelial MCF-10A cells （P<0.05）. A breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed successfully. Compared with negative control group, PCDH10 overexpression significantly inhibited cell proliferation, induced cell cycle arrest at G₁ phase, down-regulated the expression of cyclin D1 and CDK4, and decreased phosphorylation of NF-κB p65 and IκBα（P<0.05）. CONCLUSION PCDH10 inhibits the proliferation and blocks cell cycle progression of breast cancer cells by targeting NF-κB/cyclin D1 signaling pathway.     

Effect of PARP-1 on cisplatin resistance in breast cancer MCF-7 cells

AIM: To study the effect of poly(ADP-ribose) polymerase-1 (PARP-1) on cisplatin resistance of human breast cancer MCF-7 cells and its possible mechanisms.METHODS: The expression of PARP-1 at mRNA and protein levels in MCF-7 cells and MCF-7/DDP cells was determined by real-time PCR and Western blot. The expression of PARP-1 in the MCF-7/DDP cells was blocked by PARP-1 siRNA. The cell viability and apoptosis were detected by the CCK-8 assay and flow cytometry analysis, respectively. Furthermore, the protein levels of PARP-1, Bcl-2, Bax, cleaved caspase-3, caspase-3, cytochrome C (Cyto-C), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) were detected by Western blot.RESULTS: The expression of PARP-1 at both mRNA and protein levels was significantly up-regulated in the MCF-7/DDP cells. The expression of PARP-1 was increased in the MCF-7 cells treated with cisplatin. Knockdown of PARP-1 induced the apoptosis of MCF-7/DDP cells with an increased sensitivity to cisplatin. Meanwhile, knockdown of PARP-1 down-regulated the protein levels of Bcl-2/Bax and p-ERK, but up-regulated the protein levels of cleaved caspase-3 and Cyto-C. After incubated with a specific ERK inhibitor U0126, the cell viability in PARP-1 siRNA group was down-regulated significantly.CONCLUSION: Knockdown of PARP-1 increases the sensitivity of MCF-7/DDP cells to cisplatin, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of ERK signaling pathway by inhibiting phosphorylation of ERK.     

Effect of HC-1119 on biological behaviors of triple-negative breast cancer BT549 cells

AIM: To explore the effect of new artificially synthesized androgen receptor (AR) antagonist HC-1119 on the biological function of triple-negative breast cancer (TNBC) BT549 cells and the molecular mechanism. METHODS: The AR expression was assessed in different human breast cancer cell lines MDA-MB-231, T47D, MCF-7, SKBR3 and BT549 by Western blot. The TNBC BT549 cells with AR positive expression were treated with HC-1119. The cell viability was measured by CCK-8 assay. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. The migration and invasion abilities were detected by Transwell assay in vitro. The protein expression of E-cadherin, vimentin and P21 was determined by Western blot. RESULTS: AR was positively expressed in BT549 cells. HC-1119 inhibited the cell viability in a time-and dose-dependent manner (P<0.05), increased the percentage of apoptotic cells and the percentage of S-phase cells significantly, repressed the migration and invasion abilities (P<0.05), and decreased P21 expression at protein level (P<0.01). No influence on the expression of E-cadherin and vimentin in the BT549 cells was observed. CONCLUSION: AR antagonist HC-1119 decreases the viability, migration ability and invasion ability, enhances the apoptosis, and arrests the cell cycle distribution of TNBC BT549 cells. HC-1119 represses the viability of BT549 cells by down-regulating P21 expression, while the process of epithelial-mesenchymal transition is not involved in the inhibition of cell migration.     

Chronic hypoxia enhances aggressiveness of MCF-7 breast cancer cells through EMT

AIM: To investigate the effects of chronic hypoxia on the aggressiveness of MCF-7, a human breast cancer cell line, and the underlying mechanisms.METHODS: MCF-7 cells were cultured under hypoxia (1% O₂, 5% CO₂ and 94% N₂) or control (95% O₂ and 5% CO₂) condition. The viability, proliferation, and invasion and migration abilities of the MCF-7 cells were determined by MTT assay, CCK-8 assay, cell counting, and cell invasion and migration assays. Anchorage-independent growth and the alteration of cellular polarization of the MCF-7 cells were tested by soft agar colony formation assay and Matrigel-3D culture assay, respectively. The effects of chronic hypoxia on the growth and metastasis of MCF-7 cells in vivo were investigated by xenograft in nude mice. The morphological changes of the MCF-7 cells were observed under an inverted microscope. Hypoxia-induced alterations in the levels of hypoxia inducible factor-1 (HIF-1) and phosphorylated glycogen synthase kinase-3β (p-GSK-3β) as well as epithelial-mesenchymal transition (EMT) molecules, such as E-cadherin, N-cadherin, vimentin, matrix metalloproteinase (MMP)-3 and MMP-9, were determined by Western blot.RESULTS: Chronic hypoxia significantly increased the viability, proliferation, and invasion and migration abilities of MCF-7 cells in vitro, enhanced the anchorage-independent growth, facilitated cellular polarization alteration in Matrigel-3D culture, and promoted cancer metastasis in vivo. Hypoxia up-regulated HIF-1, activated GSK-3β, down-regulated E-cadherin and increased the protein levels of N-cadherin, vimentin, MMP-3 and MMP-9. CONCLUSION: Chronic hypoxia enhances the aggressiveness of breast cancer cells probably through EMT.     

Effect of di-indolyl thiozoline on proliferation of lung cancer A549 cells

AIM: To investigate the effect of di-indolyl thiozoline (DIIT) on the proliferation of human lung cancer A549 cells. METHODS: The effects of DIIT on the proliferation of human lung cancer A549 cell line were determined by CCK-8 assay and EdU assay. The effects of DIIT on the expression of cyclin-dependent kinase 4 (CDK4), cyclin D1, and the phosphorylation of Akt and mTOR were determined by Western blot. RESULTS: After the A549 cells were treated with DIIT at 12.5, 25, 50 and 100 mg/L, the cell viability detected by CCK-8 assay was decreased by 12%, 27% (P<0.01), 33% (P<0.01) and 52% (P<0.01), respectively, compared with DMSO control group. The EdU positive cell number determined by EdU assay was decreased by 10%, 21% (P<0.05), 26% (P<0.05) and 34% (P<0.01), respectively, compared with DMSO control group. Compared with DMSO control group, DIIT inhibited the phosphorylation of Akt and mTOR and the expression of cyclin CDK4 and cyclin D1 (P<0.05). CONCLUSION: Di-indolyl thiozoline inhibits the proliferation of A549 cells, which may be related to the decreases in phosphorylation levels of Akt and mTOR and the inhibition of cell cycle-related protein expression.     

Effect of nerve growth factor on proliferation and survival of breast cancer cell line MDA-MB-231 and MCF-7

AIM: To study the effect of nerve growth factor (NGF) on the proliferation and survival of breast cancer cell lines MDA-MB-231 and MCF-7. METHODS: Two breast cancer cell lines MDA-MB-231 and MCF-7 were used in the experiment. The expression of NGF and its receptor tyrosine kinase A (TrkA) was determined by the method of immunofluorescence. The NGF autocrine was detected by the method of enzyme linked immunosorbent assay (ELISA). The expression of TrkA was measured by Western blotting. The effect of NGF blocking agent Ro 08-2750 on the proliferation of the cells was evaluated by MTT assay (mono-nuclear cell direct cytotoxicity assay). The cell apoptosis and the change of the cell cycle after exposed to Ro 08-2750 were observed by flow cytometry. RESULTS: Both cell lines expressed NGF and TrkA. Ro 08-2750 inhibited the proliferation of both cell lines in a dose-dependent manner. According to the results of flow cytometry, the S-phase cells in both cell lines increased but the G₂/M-phase cells decreased after treated with Ro 08-2750. An apoptotic peak occurred in MDA-MB-231 cells. CONCLUSION: The results suggest that proliferation of MDA-MB-231 and MCF-7 cell lines is dependent on NGF. NGF promotes the survival of MDA-MB-231 cells.     

Rab1A knockdown inhibits malignant biological behaviors of breast carcinoma cells by inhibiting phosphorylation of AKT

AIM: To investigate the role of Rab1A gene in the malignant biological behaviors of breast carcinoma cells. METHODS: The expression levels of Rab1A in breast carcinoma tissues and normal adjacent tissues, and the basic expression level of Rab1A in different breast carcinoma cell lines were measured by Western blot. Small interfering RNA (siRNA) targeting Rab1A was designed, synthetized and transfected into the breast carcinoma MDA-MB-231 cells. After validation of efficiency of Rab1A gene expression knock-down, the malignant biological behaviors of the MDA-MB-231 cells were measured by CCK-8 assay, wound healing assay, Transwell assay and flow cytometry. The protein levels were determined by Western blot. RESULTS: Rab1A was expressed in normal breast tissue and cells at low level, and at high level in the cancer tissues and cancer cells (P<0.05). Compare with control group, after knock-down of Rab1A expression, the viability of MDA-MB-231 cells was significantly inhibited (P<005), the abilities of migration and invasion were reduced (P<0.05), the apoptosis was decreased (P<0.05), the percentage of G²/M phase was increased, the protein levels of p53, Bax, cleaved caspase-3 and PTEN were significantly increased (P<0.05), and the protein levels of Bcl-2, cyclin D1, cyclin B1, matrix metalloproteinase 2 (MMP2), p-AKT and mTOR were significantly decreased (P<0.05). CONCLUSION: Rab1A modulates the breast carcinoma cell viability, inhibits the migration and invasion abilities, induces G² arrest and effectively regulates the cell growth-, cell cycle-and apoptosis-related proteins. Knock-down of Rab1A expression inhibits the evolution and development of breast cancer by inhibiting the phosphorylation of AKT pathway, and Rab1A may function as a potential target in breast carcinoma treatment.     

Effects of gonadotropin-releasing hormone analogue on sensitivity of breast cancer cells to 5-FU and epirubicin in vitro

AIM: To investigate whether gonadotropin-releasing hormone analogue (GnRHa) affect the sensitivity of breast cancer cells to 5-FU and epirubicin in vitro. METHODS: Two breast cancer cell lines (MCF-7 and MDA- MB-231) were treated with different concentrations of GnRH analogue, triptorelin acetate, or with a GnRHa+5-FU or GnRHa+epirubicin. The cellular growth profiles were determined by CCK-8. The mRNA levels of GnRH receptor, PCNA and MDR1 were measured by RT - PCR. RESULTS: Both cell lines had positive GnRH receptor mRNA expression detected by RT- PCR. GnRHa did not suppress cell growth after GnRHa exposure. IC50 of 5-FU and epirubicin was not changed in the presence of GnRHa. Suppression of cell growth by the exposure to 5-FU and epirubicin was not changed in the presence of GnRHa. GnRHa treatment up-regulated PCNA mRNA expression in MDA-MB-231 cells but not in MCF-7 cells. The expression of MDR1 mRNA was down-regulated by GnRHa in MCF-7 cell lines. No MDR1 mRNA expression in MDA-MB-231 cells was observed. CONCLUSION: The present data suggest that GnRH analogue (triptorelin acetate) does not affect the sensitivity of breast cancer cell lines MCF-7 and MDA-MB-231 to 5-FU and epirubicin. GnRHa may decrease the drug resistance by down-regulating MDR1 mRNA expression.     

Pulsatilla saponin A affects proliferation and radiosensitivity of breast cancer cells by regulating miR-24-3p/RNF2 expression

AIMTo investigate the effect of Pulsatilla saponin A on proliferation and radiosensitivity of breast cancer cells and its mechanism. METHODSHuman breast cancer MCF-7 cells were treated with Pulsatilla saponin A at concentrations of 0, 5, 10, 15 and 20 mg/L and transfected with microRNA-24-3p (miR-24-3p) over-expression vector or inhibitory expression vector. The proliferation and radiosensitivity of the MCF-7 cells were measured by MTT assay and colony formation assay. The miR-24-3p expression and ring finger protein 2 （RNF2） mRNA level were detected by RT-qPCR. The protein expression of RNF2 was determined by Western blot. The luciferase reporter assay was used to detect the targeting relationship between miR-24-3p and RNF2. RESULTSCompared with control group (0 mg/L), the proliferation inhibitory rate of the MCF-7 cells was significantly increased in 5, 10, 15 and 20 mg/L Pulsatilla saponin A groups (P<0.05). The survival score of the MCF-7 cells treated with Pulsatilla saponin A was significantly decreased after irradiation, and the expression of RNF2 was significantly decreased (P<0.05). miR-24-3p targeted RNF2 and negatively regulated its expression. When the MCF-7 cells were simultaneously treated with Pulsatilla saponin A and miR-24-3p, the cell survival curve significantly shifted down. Inhibition of miR-24-3p expression reversed the proliferation-inhibiting and radiation-sensitizing effects of Pulsatilla saponin A on the MCF-7 cells. CONCLUSION Pulsatilla saponin A may affect the proliferation and radiosensitivity of breast cancer cells through miR-24-3p/RNF2 signaling pathway.     

27-Hydroxycholesterol modulates lung cancer cell proliferation by activation of LXR signaling pathway

AIM:To investigate the effect of cholesterol metabolite 27-hydroxycholesterol (27-OHC) on the proliferation of lung cancer cells. METHODS:Human lung cancer A549 cells were treated with 27-OHC at different concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5 and 10 μmol/L) for 24~48 h. The cell viability, cell cycle, cell prolife-ration, the intracellular cholesterol levels and cholesterol metabolism-related molecule expression were subsequently assessed by CCK-8 assay, flow cytometry, EdU staining, tissue total cholesterol detection kit, real-time PCR and Western blot. RESULTS:27-OHC decreased the viability of the A549 cells in a dose-and time-dependent manner (P<0.01) and inhibited the cell proliferation (P<0.05). The expression of typical liver X receptor (LXR) downstream target proteins including ATP-binding cassette transporter A1 (ABCA1), low-density lipoprotein receptor (LDLR), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CR) were modulated, which promoted the efflux of intracellular cholesterol, and reduced cholesterol influx and de novo synthesis, resulting in decreased intracellular cholesterol levels and cell viability. Furthermore, the inhibitory effect of 27-OHC on A549 cell viability was significantly attenuated after the LXR pathway was partially blocked by 5 μmol/L GSK2033 treatment (P<0.05). CONCLUSION:27-OHC inhibits A549 cell prolife-ration via activation of LXR signaling pathway.     

Effect of miR-21 over-expression on proliferation,migration and diffe-rentiation of c-Kit+ cardiac stem cells

AIM: To explore the effect of microRNA (miR)-21 on proliferation, migration and differentiation abilities of c-Kit⁺ cardiac stem cells (CSCs). METHODS: c-Kit⁺ CSCs were cultured and selected by the methods of enzyme digestion and magnetic bead separation. miR-21 mimics (50 nmol/L) and mimics negative control (MNC) were transfected into c-Kit⁺ CSCs with Lipofectamine^® 2000. The cells was divided into 3 groups:control group:c-Kit⁺ CSCs without any pretreatment; MNC group:the cells were transfected with MNC for 48 h; mimics group:the cells were transfected with miR-21 mimics for 48 h. qPCR was used to assess the expression of miR-21 in each group. CCK-8 and EdU assays were used to determine the cell proliferation. qPCR and immunofluorescence were used to detect the differentiation in each group. Scratch assay was adopted to explore the migration ability of the cells. RESULTS: The expression of c-Kit in the c-Kit⁺ CSCs were 90.8%, with 0.6% of CD45 and 0.5% of CD34. A significant increase in miR-21 expression was observed when the cells were transfected with miR-21 mimics for 48 h (P<0.05). CCK-8 and EdU assays showed that miR-21 significantly increased cell proliferation as compared with MNC group and control group (P<0.05). No difference in the expression of Nkx2.5, CD31 and α-SMA at mRNA and protein levels was observed, and no difference of the migration ability in 3 groups of the c-Kit⁺ CSCs was found. CONCLUSION: Over-expression of miR-21 significantly promotes the proliferation of c-Kit⁺ CSCs, without any effect on the cell migration and differentiation.     

Expression of CUG-binding protein 1 in breast cancer and its effect on cell viability and invasion ability

AIM: To investigate the expression of CUG-binding protein 1 (CUGBP1) in breast cancer tissues, and to explore the effect of CUGBP1 gene silencing on the viability and invasion ability of human breast cancer MCF-7 cells. METHODS: A total of 96 cases of patients with breast cancer undergoing surgical treatment were selected in the Second Affiliated Hospital of Zhengzhou University from March 2015 to September 2017. Immunohistochemical staining was used to detect the protein expression of CUGBP1 in the breast cancer and adjacent tissues. MCF-7 cells were cultured and divided into CUGBP1 interference sequence group, control sequence group and blank group. Western blot was used to detect the protein expression of CUGBP1, Twist, E-cadherin and vimentin in the cells. The cell viability was measured by MTT assay. The cell invasion ability was detected by Transwell assay. RESULTS: The positive expression rate of CUGBP1 protein in the breast cancer tissues was higher than that in the adjacent tissues (χ²=28.900, P<0.001). The differences of CUGBP1 protein expression in the breast cancer tissues among TNM staging, histological grading and lymph node metastasis were statistically significant (P<0.05). The relative protein expression levels of CUGBP1, Twist and vimentin in CUGBP1 interference sequence group were lower than those in control sequence group and blank group, while the relative protein expression of E-cadherin was higher than that in control sequence group and blank group (P<0.05). The cell viability at 24 h, 48 h, 72 h and 96 h in CUGBP1 interference sequence group was lower than that in control sequence group and blank group (〖P<0.05). The invasive cells in CUGBP1 interference sequence group were less than those in control sequence group and blank group (P<0.05). CONCLUSION: CUGBP1 protein is highly expressed in the breast cancer tissues. Specific silencing of 〖STBX〗CUGBP1〖STBZ〗 gene expression in breast cancer MCF-7 cells effectively inhibits the cell viability and invasiveness, and its mechanism may be related to inhibiting the process of epithelial-mesenchymal transition.     

Mechanism of AKT1 negatively regulating breast cancer cell migration

AIM: To investigate the molecular mechanism and downstream signaling pathway by which AKT1 inhibition regulates breast cancer cell migration. METHODS: RNA interference was used to knockdown the expression of AKT1. Western blot assay was performed to examine the expression of AKT1 total protein, β-catenin total protein and β-catenin nuclear protein. Immunofluorescence was used to examine the cellular localization of β-catenin. Transwell assay was used to investigate whether β-catenin nuclear accumulation as an alternative pathway was responsible for breast cancer metastasis induced by AKT1 inhibition. RESULTS: The total protein expression of AKT1 was decreased in MCF-7 and MDA-MB-231 cells treated with AKT1 siRNA. A significant increase in the protein expression of β-catenin was observed in MCF-7 cells and MDA-MB-231 cells treated with AKT1 siRNA. Immunofluorescence staining showed that MCF-7 cells and MDA-MB-231 cells displayed strong β-catenin staining in the cytoplasm and nucleus after knockdown of AKT1 expression. The ability of tumor cell migration increased dramatically after treated with AKT1 specific siRNA in the breast cancer MCF-7 cells and MDA-MB-231 cells in Transwell assay. XAV-939 reversed breast cancer cell migration induced by knockdown of AKT1 expression. CONCLUSION: β-catenin nuclear accumulation contributes to AKT1 inhibition-mediated breast cancer cell migration.     

Expression and function of circular RNA_0000231 in non-small-cell lung cancer

AIM: To investigate the expression and function of circular RNA_0000231 (circ_0000231) in non-small-cell lung cancer (NSCLC). METHODS: RT-qPCR was used to detect the expression of circ_0000231 in the NSCLC tissues and cell lines. circ_0000231 small interfering RNA (si-circ_0000231) or negative control siRNA of circ_0000231 (NC) was transfected into the NSCLC cells. The proliferation and apoptosis of the NSCLC cells were detected by CCK-8 assay, colony formation assay and flow cytometry, respectively. The expression of cyclin D1 (CCND1) and anti-apoptotic protein Bcl-2 were determined by RT-qPCR and Western blot. RESULTS: The expression of circ_0000231 in the NSCLC tissues and cell lines was significantly up-regulated compared with precancerous tissues and lung epithelial cells BEAS-2B (P<0.05). After transfection of NSCLC cells with si-circ_0000231, the cell viability, colony formation numbers were significantly decreased, and the apoptotic rate in si-circ_0000231 group was significantly increased as compared with NC group (P<0.01). In addition, the results of RT-qPCR and Western blot showed that transfection of si-circ_0000231 inhibited the expression of CCND1 and Bcl-2 (P<0.01). CONCLUSION: The expression of circ_0000231 is significantly increased in the NSCLC tissues and cells. Knock-down of circ_0000231 expression significantly inhibits the proliferation of NSCLC cells.