ACTL8 expression and its correlation with clinicopathological features and prognosis in breast cancer

AIM: To investigate the actin-like protein 8 (ACTL8) expression and its relationship with clinicopathological features and prognosis in breast cancer.METHODS: The expression of ACTL8 in human normal mammary epithelial cell line MCF-10A and 5 breast cancer cell lines was detected by Western blot. The expression of ACTL8 was also investigated by immunohistochemistry in 6 cases of breast cancer specimens with adjacent normal tissues. The data in 488 cases of breast specimens from TCGA dataset were downloaded, and the relationship between the mRNA expression of ACTL8 and the clinicopathological features and prognosis was analyzed.RESULTS: The expression of ACTL8 in 4 breast cancer cell lines was significantly higher than that in breast epithelial cell line MCF-10A.The level of the ACTL8 expression in breast tumors was significantly higher than that in the corresponding adjacent normal breast tissues. The mRNA expression of ACTL8 was correlated with age, tumor size, clinical TNM stage and lymph node metastasis of breast cancer patients (P < 0.05). The high expression level of ACTL8 mRNA indicated a poor prognosis of breast cancer patients. CONCLUSION: ACTL8 protein is highly expressed in breast cancer specimens and is closely correlated with the clinicopathological features and prognosis, suggesting that ACTL8 is a prognostic marker for breast cancer or a potential new target for treatment of breast cancer.     

Effect of curcumin on expression of p21 and CD44V6 in human breast cancer xenografts

AIM:To study the effect of curcumin on the expression of p21 and CD44V₆ in breast carcinoma in nude mice.METHODS:Nude mice were xenografted with human breast cancer cell line MCF-7 and randomly divided into 2 groups (n=4 in each group): control group and curcumin group. In latent period,the percentage of tumor development was observed. Tumors were measured and the surface areas were calculated. RT-PCR was performed to detect the expression level of cyclin D1,p21 and CD44V₆ mRNA. RESULTS:The tumor surface areas in the curcumin group were significantly lower than those in control group. In curcumin treatment group,the expression of p21 was up-regulated while cyclin D1 was nearly not changed. The expression of CD44V₆ was significantly down-regulated in curcumin group.CONCLUSION:Curcumin inhibits the expression of CD44V₆ and up-regulates the expression of p21 in nude mice bearing human breast cancer cell line MCF-7.     

Role of miR-183 in breast cancer progression

AIM:To investigate the expression of miR-183 in breast cell lines and tissue specimens and its effects on the biological behaviors of breast cancer cells. METHODS:Human breast cell lines and clinical tissue specimens of breast diseases were used in the study. The expression of miR-183 was determined by real-time PCR. The characteristics of cell proliferation, invasion and migration were examined after miR-183 was transfected by lipofection. RESULTS:Altered expression of miR-183 was found in the highly invasive breast cancer cells as compared with weakly invasive breast cells. The expression of miR-183 was also significantly decreased in the breast cancer tissues as compared with that in the benign breast disease tissues. The capacities of breast cancer cell invasion and migration were significantly increased after transfection of miR-183 inhibitor and were decreased after transfection of miR-183 mimic. No significant change of cell proliferation was observed after miR-183 transfection. CONCLUSION:miR-183 may play an important role in breast cancer progression, especially in the cell invasion and migration.     

Long noncoding RNA MALAT1 regulates Rac1b expression and is associated with colorectal cancer metastasis

AIM: To investigate the role of MALAT1 in colorectal cancer metastasis.METHODS: The mRNA expression levels of MALAT1 and Rac1b in the tumor and adjacent normal tissues were examined by real-time PCR. MALAT1 was knocked down by siRNA in colorectal cancer cell lines. The expression of Rac1b and the epithelial-mesenchymal transition markers was examined by Western blot. Cell proliferation was determined by EdU analysis. The effects of MALAT1 on the cell migration and invasion were examined by Transwell assay. RESULTS: The expression of MALAT1 was down-regulated in colorectal cancer. Down-regulation of MALAT1 induced Rac1b overexpression, which in turn increased the expression levels of E-cadherin and β-catenin. Furthermore, down-regulation of MALAT1 promoted the cell proliferation, invasion and migration. CONCLUSION: MALAT1 is associated with metastasis of colorectal cancer through regulating the expression of Rac1b and the downstream factors.     

High expression of BIRC5 enhances cell viability,inhibits apoptosis and is associated with poor prognosis in gastric cancer

AIM To investigate the expression of baculoviral inhibitor of apoptosis protein repeat-containing protein 5 （BIRC5） in gastric cancer tissue and its relationship with prognosis of gastric cancer patients, and to explore the effect of BIRC5 knock-down on the viability and apoptosis of gastric cancer cells. METHODS The expression of BIRC5 was detected by immunohistochemistry in 67 cases of gastric cancer tissues and paracancerous tissues for analyzing the relationships with clinicopathological characteristics. The mRNA and protein expression levels of BIRC5 in gastric carcinoma cell lines （AGS, MKN-1 and MGC-803） and normal gastric epithelial cell line GES-1 were detected by RT-qPCR and Western blot. The AGS cells were divided into blank group （no treatment）, Ctr-sh group （blank plasmid transfection） and BIRC5-sh group （BIRC5-shRNA plasmid transfection）. The interference efficiency of BIRC5-shRNA was evaluated by Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry, and the levels of apoptosis-related proteins cleaved caspase-3, Bax and Bcl-2 were determined by Western blot. RESULTS BIRC5 was mainly expressed in cytoplasm, and the positive expression rate of BIRC5 in the gastric cancer tissues was higher than that in the adjacent tissues （P<0.01）. The positive rates of BIRC5 in the gastric cancer patients at TNM Ⅲ~Ⅳ stages and with lymph node metastasis were higher than those in the patients at TNM Ⅰ~Ⅱ stages and without lymph node metastasis, respectively （P<0.05）. The survival time of the patients with positive BIRC5 expression was shorter than that of the patients with negative BIRC5 expression （P=0.011 2）. The cell viability in BIRC5-sh group was lower than that in blank group and Ctr-sh group at time points of 48, 72 and 96 h. The apoptotic rate in BIRC5-sh group was increased compared with blank group and Ctr-sh group. The protein levels of cleaved caspase-3 and Bax in BIRC5-sh group were higher than those in blank group and Ctr-sh group, while the protein expression of Bcl-2 in BIRC5-sh group was lower than that in blank group and Ctr-sh group （P<0.05）. CONCLUSION High expression of BIRC5 in gastric cancer indicates poor prognosis. BIRC5 promotes the growth of gastric cancer cells and inhibits apoptosis.     

Effects of p21-activated protein kinase 2 down-regulation on proliferation and apoptosis of human breast cancer cells

AIM：To study the effect of p21-activated protein kinase 2 (PAK2) knockdown by RNA interference on the proliferation and apoptosis of human breast cancer cells. METHODS：The short hairpin RNA (shRNA) targeting PAK2 gene was designed and used for packing lentivirus in 293T cells.Human breast cancer MCF-7 cells were infected by the virus particles and PAK2 knockdown stable cell line was established by puromycin selection. The knockdown efficiency was assessed by Western blotting. The proliferation ability of MCF-7 cells was evaluated by CellTiter 96 AQueous and anchorage-independent growth assays. The cell apoptosis induced by staurosporine was detected by flow cytometry. RESULTS：The protein level of PAK2 was significantly suppressed after silencing of PAK2 gene in MCF-7 cells (P<0.01). Furthermore, knockdown of PAK2 caused remarkable inhibition of the cell proliferation and colony formation (P<0.01). Staurosporine induced more apoptosis in the PAK2 knockdown cells compared with the control cells (P<0.01). CONCLUSION：Knockdown of PAK2 inhibits the proliferation of MCF-7 cells and increases the sensitivity of chemotherapeutic drug-induced cell apoptosis, suggesting that PAK2 might be a new therapeutic target in breast cancer treatment.     

miR-193b enhances cytotoxicity of doxorubicin by targeting Mcl-1 in breast cancer

AIM: To investigate the effect of microRNA(miR)-193b on doxorubicin therapy in breast cancer in vitro.METHODS: miR-193b level in plasma was detected by real-time PCR in the patients with breast cancer or the healthy controls. MTT assay was performed to measure the inhibitory effect of miR-193b plus doxorubicin on the growth of MDA-MB-231 cells. Bioinformatics, real-time PCR and Western blot were performed to determine whether the expression of Mcl-1 was regulated by miR-193b. Mcl-1 expression vector was constructed, and the role of Mcl-1 vector toward miR-193b plus doxorubicin-induced cytotoxicity in MDA-MB-231 cells was observed by MTT assay.RESULTS: Down-regulation of miR-193b was found in breast cancer patients. The miR-193b plus doxorubicin group showed a higher growth inhibition than cisplation group in MDA-MB-231 cells. The expression of Mcl-1 at both mRNA and protein levels was down-regulated after miR-193b transfection. The growth inhibition of MDA-MB-231 cells treated with miR-193b plus doxorubicin was significantly decreased after the transfection of Mcl-1 expression vector.CONCLUSION: miR-193b sensitizes doxorubicin-induced cytotoxicity by targeting Mcl-1 in breast cancer.     

Prognostic prediction of PTEN and Her-2 expression in breast cancer

AIM:To study the prognositic value of PTEN and Her-2 expression in primary breast cancer. METHODS:81 breast cancer specimens with 15 years follow-up were obtained from 1989 to 2004. Immunohistochemical methods were used to detect the expression of PTEN and Her-2 in 81 paraffin-embedded specimens. The correlation between expression of PTEN and clinipathological factors was discussed with the Chi-square test. The survival rate analysis results were calculated with Kaplan-meier method.Long-rank test and Cox model by SPSS 10.0 software. 〖JP+1〗RESULTS:(1) PTEN expression significantly affects 5-year and 10-year survival rate of breast cancer (P<0.01 and P<0.05), and significantly negative correlation with the Her-2 expression was observed. (2) The patients with negative PTEN combined with positive Her-2 expression had worse prognosis. (3) Tumor sizes, postoperative therapy and PTEN expression had significant relation with the 5-year survival rate (P<0.05), and ER, Her2 and PTEN expression had significant relation with the 10-year survival rate (P<0.05). CONCLUSION:PTEN and Her-2 expression significantly affects 5-year and 10-year survival rate in patients with breast cancer, and may be biomarkers and important prognostic factors in breast cancer.     

Relationship between expression of somatostatin and DNA ploidy in breast cancer

AIM: To investigate the relationship between somatostatin and the pathologic type, estrogen receptor,DNA ploidy of nuclei in tumor cells of breast cancer.METHODS: 67 cases of primary breast cancer and 25 cases of benign breast tumor were examined by immunohistochemical stretomyces avidin peroxidase method. 26 cases of breast cancer selected at random were analyzed by flow cytometry.RESULTS: Somatostatin expressed significantly higher in low malignant breast cancer than that in high malignant breast cancer (P<0.05). Most of cancers with positive staining of somatostatin were diploidy,most of cancers with negative staining were aneuploidy,there had significant difference between two groups (P<0.05).CONCLUSION: Somatostatin may delay the progress of breast cancer,and somatostatin levels in cancer tissues may become a useful indicator for assessing prognosis of patients with breast cancer.     

Nucleostemin gene expression in human breast tumor tissues and its relation with clinical pathology

AIM: To study the expression of nucleostemin (NS) gene in human breast tumor tissues and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor.METHODS: Total RNA was isolated from human breast tumor tissue.The methods of electrophoresis and RT-PCR were used in measuring NS gene expression level,and the relations of NS gene expression level with histological grades,histological types and TNM stages of the tumor were analyzed.RESULTS: The results indicated that there was no NS gene expression detected in normal breast tissues,and NS gene expression in malignant breast tumor tissues (P<0.01) was higher than that in the benign breast tumor tissues.The higher histological grades of the breast cancer showed the stronger NS gene expression (P<0.01),the higher TNM stages of the breast cancer showed the stronger NS gene expression (P<0.01),and the level of NS gene expression had not correlation with the histological types (P>0.05).CONCLUSION: It is suggested that there is no relation of NS gene expression level with histological types of the breast cancer,but there is a marked correlation of NS gene expression level with the histological grades and TNM stages.     

Protein expression of EGFR and c-Kit is associated with primary tumor progression in nasopharyngeal carcinoma

AIM: To investigate the protein expression of 2 receptor tyrosine kinases,epidermal growth factor receptor(EGFR) and c-Kit, in non-keratinizing nasopharyngeal carcinoma (NPC) and to analyze the relationship of that with the clinicopathological parameters. METHODS: Ninety-five samples of stage II and III non-keratinizing NPC biopsies were collected from the Department of Pathology, Sun Yat-sen University Cancer Center. The 10% formalin-fixed paraffin-embedded biopsy blocks were re-sectioned. Besides HE routine staining, immunohistochemistry was performed for detecting the expression of EGFR, c-Kit, latent membrane protein 1(LMP1) and Ki-67. RESULTS: The protein expression rates of EGFR and c-Kit were 70.53% (67/95) and 63.16% (60/95), respectively. The expression score of EGFR was positively correlated with that of c-Kit protein (P<0.05). They were both correlated with T staging (EGFR,P<0.05; c-Kit, P<0.01). Furthermore, the staining intensities of EGFR and c-Kit proteins were also correlated with T staging(EGFR, P<0.05; c-Kit,P<0.01). CONCLUSION: The proteins of EGFR and c-Kit are usually expressed in non-keratinizing NPC cells. The immunoreactive scores of these 2 receptor tyrosine kinases are positively correlated with each other. Either the expression rate or the immunoreactive intensity of EGFR and c-Kit proteins is correlated with primary tumor progression. Immunohistochemical staining of EGFR or c-Kit protein in NPC biopsies could be recognized as an insight into further gene analysis for target therapy.     

Relationship of p21WAF1 gene polymorphisms with protein expression in breast carcinomas

AIM: To investigate the relationship between p21^WAF1gene polymorphisms and protein expression in breast carcinoma. METHODS: Polymerase chain reaction single-strand conformation polymorphisms technique (PCR-SSCP) and immunohistochemical assay of S-P immunostaining technique were used to study polymorphisms of p21^WAF1 and protein expression respectively on the specimen of paraffin-embedded tissues in 100 cases of breast carcinomas and 40 benign breast diseases as control. RESULTS: Two p21^WAF1 gene polymorphisms were found in 18% (18/100) of breast carcinomas and 5% (2/40) of control samples. The difference between the two groups was statistically significant (χ²=3.94, P＜0.05). The positive immunohistochemical reaction of p21^WAF1 protein were found in 50% (50/100) of breast carcinomas and 12.5% (5/40) of control samples. The difference between the two groups was statistically significant (χ²=16.84, P＜0.01). The positive immunohistochemical reaction of p21^WAF1 protein were found in 100% (18/18) of breast carcinomas with p21^WAF1 gene polymorphisms and 39% (32/82) of no p21^WAF1 gene polymorphisms. The difference between two groups was statistically significant (χ²=21.95, P＜0.01). The p21^WAF1 gene polymorphisms were correlated with the protein expression in breast carcinomas (r=0.576, P＜0.01). CONCLUSION: p21^WAF1 gene polymorphisms may create the different copies of mRNA and may make relevant protein molecules.     

Expression of chemokine receptor CCR7 and VEGF-C is associated with prognosis of breast carcinoma

AIM: To explore the protein levels of chemokine receptor 7 (CCR7) and vascular endothelial growth factor (VEGF)-C in breast carcinoma, and to investigate the effects of CCR7 and VEGF-C on prognosis of breast carcinoma. METHODS: The protein expression levels of CCR7 and VEGF-C in the breast carcinoma tissues and normal breast tissues were detected by the method of immunohistochemistry. At the same time, the relationship between clinicopathologic characteristics and the protein expression of CCR7 and VEGF-C in the breast carcinoma tissues was analyzed. The relationship between the protein expression of CCR7 and VEGF-C and survival time of the breast cancer patients was estimated by Kaplan-Meier method.RESULTS: The positive expression rates of CCR7 and VEGF-C in the breast carcinoma tissues were significantly higher than those in the normal breast tissues (P<0.01). A positive correlation was observed between the protein expression of CCR7 and the protein expression of VEGF-C in the breast carcinoma tissues (r=0.613, P<0.01). The protein expression of CCR7 and VEGF-C was correlated with lymph node metastasis and TNM stage (P<0.05), but both were not related to patients' age, primary tumor size, estrogen receptor and progesterone receptor. The survival time of the patients with CCR7 and VEGF-C positive expression was significantly shorter than that of the patients without the expression (P<0.05).CONCLUSION: The positive expression of CCR7 and VEGF-C proteins is associated with the prognosis of breast cancer, and combined detection of CCR7 and VEGF-C protein expression levels may be helpful to judge the prognosis of breast cancer.     

Effect of alpha-lipoic acid on expression of miR-21/Smad7 and fibrosis in kidney of diabetic rats

AIM: To investigate the protective effect of alpha-lipoic acid (ALA) on the kidney of the rats with diabetes mellitus (DM), and to discuss the mechanism. METHODS: The DM rats were divided into normal control (NC) group, DM group and ALA group. After treated with ALA for 6 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and the pathological changes of the kidney tissues were observed by HE staining and Masson staining. The protein levels of transforming growth factor-β1 (TGF-1), p-Smad2/3, Smad7, collagen I and collagen Ⅲ were determined by Western blot. In addition, the expression of microRNA-21 (miR-21) was detected by RT-qPCR. RESULTS: Compared with NC group, the kidney weight/body weight, blood glucose (BG), total cholesterol, triglyceride and 24-h urine protein were remarkably increased in DM group (P<0.05). The pathological observation of the kidney tissues showed fibrosis changes in DM group. The level of Smad7 was reduced in DM group, while the levels of TGF-β1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were increased (P<0.05). After treatment with ALA for 6 weeks, all the relevant biochemical parameters were reduced except BG, and the renal fibrosis lesions were obviously alleviated. Compared with DM group, the levels of TGF-1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were reduced in ALA group, while the level of Smad7 was increased (P<0.05).CONCLUSION: ALA may prevent the development of renal fibrosis in rats through restraining the expression of TGF-β1 and miR-21, increasing the levels of Smad7 protein, and reducing the deposition of extra cellular matrix.     

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.     

Regulation of estrogen on the expression of KLK6 mRNA and protein in human breast cancer cell line MCF-7

AIM:To investigate the effects of estrogen and tamoxifen on the expression of KLK6 mRNA and protein (hK6) in human breast cancer cell line MCF-7. METHODS:MCF-7 cells were incubated with 17-βE2 and tamoxifen at different concentrations for 72 hours, respectively. The expression levels of kallikrein 6 (KLK6) mRNA and protein were evaluated by fluorescence quantitative RT-PCR and flow cytometry, respectively. RESULTS:Compared with ethanol control, KLK6 mRNA expression levels were significantly decreased when 17-βE2 was added at concentrations of 10^-10 and 10^-8 mol/L (P<0.01). No statistical change was observed when 17-βE2 was at 10^-12 mol/L (P>0.05). Flow cytometry showed the same results. The average fluorescence intensity (AFI) that represents the level of hK6 was decreased after incubated with 17-βE2 (P<0.01). After incubation with tamoxifen, the levels of KLK6 mRNA and hK6 were increased (P<0.01). CONCLUSION:Estrogen down-regulates the expression levels of KLK6 mRNA and protein (hK6), while tamoxifen has an opposite effect.     

T0901317 induces apoptosis of human breast cancer MDA-MB-231 cells by up-regulating LXRα expression

AIM: To investigate the pro-apoptotic effect of T0901317, an artificial agonist of liver X receptor α (LXRα), on human breast cancer MDA-MB-231 cells and its mechanism. METHODS: MDA-MB-231 cells were treated with different concentrations (0, 10, 20 and 40 μmol/L) of T0901317 for different time (0, 12, 24 and 48 h). The cell apoptosis was determined by Annexin V/propidium iodide staining and Hoechst 33342 staining. The expression of apoptosis-related proteins, such as Bcl-2, caspase 3 and cleaved caspase-3, and LXRα was determined by Western blot. The mRNA expression of Bcl-2 and LXRα was analyzed by RT-qPCR. RESULTS: T0901317 induced the cell apoptosis in a dose-and time- dependent manner. The expression of cleaved caspase-3 and LXRα was up-regulated, but Bcl-2 was down-regulated by T0901317. The mRNA expression of Bcl-2 was down-regulated, while LXRα was up-regulated by T0901317.CONCLUSION: T0901317 up-regulates LXRα expression and induces the apoptosis of MDA-MB-231 cells.     

Inhibition of 293T cell proliferation by blocking miR-20 and miR-106 expression with antisense RNA

AIM: To inhibit 293T cell proliferation by reducing miR-20 and miR-106 expression with antisense RNA. METHODS: Antisense RNA (or antisense oligonucleotides, ASO) specific to miR-20 and miR-106 was synthesized, and the 293T cells were treated with these antisense RNA. Then, the cell proliferation derived from suppression of antisense RNA was studied by microscopy and fluorescence-activated cell sorter. The expression of miRNA and its target gene Rb were analyzed by real-time PCR and ELISA, respectively. RESULTS: Our results showed that ASO could inhibit the expression of miR-20 and miR-106 effectively and inhibit 293T cell proliferation. The results also demonstrated that ASO specific to miR-106 could up-regulate anti-oncogene Rb expression. CONCLUSION: ASO could inhibit 293T cell proliferation by inhibiting miRNA expression and up-regulating Rb expression.