Overexpression of CLN3 gene in human ovarian carcinoma

AIM: To determine whether overexpression of CLN3 is involved in the tumorigenesis of ovarian cacinoma. METHODS: 10 specimens of ovarian carcinoma were screened for CLN3 gene expression level by semiquantitative RT-PCR and Western blotting. RESULTS: Overexpression of CLN3 mRNA and protein was found in all ovarian tumor tissues as compared with normal tissues. CONCLUSION: The overexpression of CLN3 may contribute to tumor development of ovarian cancer.     

Expression and distribution of C1C-3 in human glioma specimen

AIM: To investigate the expression of voltage-gated chloride channels (ClC)-3 protein and mRNA in human glioma specimen and its biological function. METHODS: The expression of C1C-3 was observed by immunohistochemical staining in 24 cases of human glioma, 4 cases of brain metastic cancer specimens and 5 cases of normal brain tissue as control; The C1C-3 mRNA expression were detected in the specimens with positive expression of ClC-3 protein by RT-PCR. RESULTS: ClC-3 protein was found negative in 4 cases of normal brain tissues and positive in 19 cases of human glioma and 4 cases of brain metastic cancer specimens. ClC-3 protein was mainly expressed in the membrane or cytoplasm of neoplastic cells and microvascular endothelial cells. The expression of ClC-3 mRNA was detected in 16 cases of human glioma and 4 cases of brain metastasis cancer specimens among the tissues with the positive expression of ClC-3 protein. The level of protein and RNA of ClC-3 in high malignant oligodendrogliomas was higher than that in low malignant ones. CONCLUSION: ClC-3 is generally expressed in human glioma and brain metastic cancer and is probably correlated with the classification of its pathological malignance.     

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.     

Effects of different oxygen concentrations on differentiation of marrow stroma cells into osteoblasts

AIM: To investigate the effect of different oxygen concentrations on the differentiation of marrow stroma cells into osteoblasts and to evaluate the expression of Cbfα1/Runx2, bone-morphogenesis protein 2 (BMP2) and peroxisome proliferator-activated receptor γ2 (PPAR-γ2) in bone marrow stromal cells. METHODS: The bone marrow stomal cells obtained from 4-month-old female SD rats were cultured in growth medium and were used between passages 3 to 5. The cells were divided randomly into 4 groups, each group has 8 samples. The cells in all 4 groups were used for the following experiments after cultured with different oxygen concentrations for 3 d in osteoblastic differentiation medium: total cellular RNA was isolated using total RNA kit; RT-PCR was performed to detect the mRNA expression of Cbfα1/Runx2, BMP2 and PPARγ2. The protein expression of Cbfα1/Runx2 and BMP2 was assayed by Western blotting. RESULTS: Compared to the cells in normoxia condition (20%), the mRNA and protein expressions of Runx2 were enhanced significantly, the mRNA expression of BMP2 was also enhanced significantly, the protein expression of BMP2 increased and the mRNA expression of PPARγ2 decreased significantly in the cells cultured with lower oxygen concentrations. The lower oxygen concentration was in the culture, the more Runx2 mRNA, BMP2 mRNA, BMP2 and Runx2 protein were expressed. On the contrary, hypoxia significantly decreased the expression of PPARγ2 mRNA in bone marrow stronmal cells and the lower the oxygen concentration was used, the less expression of PPARγ2 mRNA was achieved. CONCLUSION: Hypoxia promotes the mRNA and protein expressions of Runx2 and BMP2, also significantly decreases the expression of PPARγ2 mRNA in bone marrow stronmal cells in an oxygen concentration dependent manner, indicating that hypoxia significantly stimulates the differentiation of bone marrow stromal cells into osteoblasts instead of lipocytes.     

Expression of Wip1 in non small cell lung cancer tissue and cells

AIM: To detect the expression level of wip1 in lung cancer tissue and three lung cancer cell lines, and to explore the relations between the expression level of Wip1 in lung cancer and various clinical and pathological features. METHODS: Real-time PCR was employed to detect the expression of Wip1 mRNA in 44 specimens of non small cell lung cancer tissues and normal tissues. The relations between the expression of Wip1 mRNA and various clinical and pathological features were analyzed. Real-time PCR was also employed to detect the expression of Wip1 mRNA in A549, NCI-1299, NCI-H460 and HBE for relative quantitative analysis.RESULTS: In the 44 specimens, the expressions of Wip1 mRNA in both cancer tissues and normal lung tissues were positive. Wip1 gene was over-expressed in 17 specimens in 44 non small cell lung cancer specimens. The rate was 38.6%. The relative level of Wip1 mRNA in NSCLC tissues was significantly higher than that in normal lung tissues (ratio=2.1644±1.3940, P<0.01). The expression of Wip1 mRNA was also correlated with pathological grading (P<0.05). The relative level of Wip1 mRNA in three kinds of lung cancer cells was significantly higher than that in HBE cells. The difference was statistically significant (P<0.05).CONCLUSION: The Wip1 mRNA is over-expressed in non small cell lung cancer, indicating that Wip1 is related to the tumorigenesis and may become the new target of non small cell lung cancer gene therapy. The expression of Wip1 mRNA is related to tumor cell differentiation and may use for the molecular biological reference index to estimate the malignant degree of cancer.     

Protein and mRNA expression of KAI1 metastasis suppressor gene and its clinical significance in cervical carcinoma

AIM: To study the mRNA and protein expression of Kang ai1 (KAI1) tumor suppressor gene and to determine the relationship between KAI1 and invasiveness and metastasis of cervical cancer. METHODS: The expression of KAI1 metastasis suppressor was detected by immunohistochemistry in paraffin slides and by real-time quantitative polymerase chain reaction (RT-PCR) in fresh tissue. The samples included 20 cases of normal cervical tissues, 20 cases of cervical intraepithelial neoplasia (CIN) and 40 cases of cervical carcinoma. The results of the gene expression combined with the pathological and clinical data were also analyzed. RESULTS: The expression of KAI1 protein and mRNA was related to the tissue differentiation of cervix. The positive rates of KAI1 expression were the highest in the normal cervical tissue, the middle in CIN and the lowest in cervical carcinoma with significant difference among three groups (P<0.01). The expression of KAI1 protein was not related with the grade of CIN (P>0.05). However, both mRNA and protein expression of KAI1 were related to the differentiation and the clinical stages of cervical cancer (P<0.01) and also related to the metastasis of the cancer. The positive rates between the non-lymphatic metastasis and lymphatic metastasis (P<0.05) were significant different. Cox regression and logistic regression showed that the tissue differentiation, clinical stages, lymphatic metastasis and expression of KAI1 were all related factors with recurrence and prognosis of cervical cancer. CONCLUSION: The down-regulation of KAI1 tumor suppressor gene at both mRNA and protein levels is related to the differentiation, clinical stages and metastasis of cervical cancer, indicating that the expression of KAI1 is a prognostic factor for cervical cancer.     

Expression of Glypican-3 gene and its regulating mechanism in hepatocellular carcinoma

AIM:To investigate the expression of Glypican-3 gene and its regulating mechanism in hepatocellular carcinoma. METHODS:The expression of GPC3 mRNA and its gene mutation in 48 hepatocellular carcinoma tissues, 39 paracarcinomatous tissues and 31 normal liver tissues were detected by using RT-PCR and PCR-SSCP. The expressions of GPC3 protein, P53 and PCNA protein were detected by using immunohistochemistry S-P. RESULTS:The positive expressive rate of GPC3 mRNA was 77.1% in hepatocellular carcinoma tissue. No expression of GPC3 mRNA in paracarcinomatous tissues and normal liver tissues was observed. No gene mutation of GPC3 in hepatocellular carcinoma tissue was found. No correlation was found between GPC3 and P53 (r=-0.12574, P>0.05). The mean index of proliferating cell nuclear antigen(PCNA) in positive and negative GPC3 expression were (46.32±27.54)% and (39.83±21.47)%, respectively (P>0.05). CONCLUSION:The high expression of GPC3 mRNA in hepatocellular carcinoma is independent to the gene mutation, and to the expression of P53 and PCNA protein.     

Relationship between expression level of TIMP-3 and invasion/metastasis of hepatocellular carcinoma

AIM: To detect the levels of tissue inhibitor of metalloproteinase-3 (TIMP-3) in both plasma and the tissue of hepatocellular carcinoma (HCC), and to elucidate their association with clinical features.METHODS: Plasma protein levels of TIMP-3 in 56 HCC patients and 30 cases of controls were detected by ELISA.The mRNA and protein levels of TIMP-3 in 30 HCC tissue samples with their portal vein tumor embolus and lymphatic metastasis tissues, and in normal liver tissues from 30 controls were detected by RT-PCR and Western blotting.The relationship between mRNA and protein levels and their clinic-pathological data were analyzed.RESULTS: The plasma TIMP-3 protein levels in the extrahepatic metastasis patients were obviously lower than those in the non-extrahepatic metastasis patients (P<0.05).The mRNA levels of TIMP-3 in normal liver, carcinoma in situ, portal vein tumor embolus and lymphatic metastasis tissues were 0.78±0.09, 0.52±0.09, 0.42±0.07 and 0.40±0.08, respectively, with significant differences among them (P<0.05).The protein levels of TIMP-3 in these 4 kinds of tissues were 115.08±8.60, 77.04±8.83, 64.43±3.80 and 62.80±3.73, respectively, also with significant differences among them (P<0.05).CONCLUSION: The expression of TIMP-3 significantly decreases in the carcinoma in situ tissues of HCC patients, and decreases more obviously in the portal vein tumor embolus and lymphatic metastasis tissues, indicating that low expression of TIMP-3 may play an important role in HCC invasiveness and metastasis.     

Clinical significance of KLF15 expression in human lung adenocarcinoma

AIM: To explore the clinical significance of Krüpple-like factor 15 (KLF15) protein expression in the patients with lung adenocarcinoma for exploring the therapeutic and prognositic biomarkers of lung cancer. METHODS: Four cases of lung adenocarcinoma tissues and matched adjacent tissues were collected from our hospital, and the expression of KLF15 protein in these tissues was analyzed by Western blot. At the same time, 72 cases of archived paraffin-embedded samples and clinical data of the patients with lung adenocarcinoma were also collected. The KLF15 protein expression in the archived paraffin-embedded lung adenocarcinoma samples was detected by immunohistochemical staining. The correlations between KLF15 protein expression and clinical characteristics of the patients including prognosis were also analyzed. In addition, the KLF15 protein was up-regulated in A549 cells, and then the effects of KLF15 protein on the viability of the cells were measured by CCK-8 assay. RESULTS: The protein expression of KLF15 in the 4 cases of lung adenocarcinoma tissues was significantly lower than that in matched paracancerous tissues. Fifty-three cases of lung adenocarcinoma specimens showed low expression or no expression of KLF15 protein in total 72 cases (73.6%). The 5-year survival rate of the patients with high expression of KLF15 protein in their specimens was higher than that of the patients with the low expression of KLF15 protein (P<0.01), and the expression of KLF15 protein was significantly correlated with the pathological staging (P<0.01) and T stage (P<0.01) of the patients with lung adenocarcinoma. Furthermore, the low expression of KLF15 protein was an important poor prognostic indicator of the patients. Up-regulation of KLF15 protein in the A549 cells significantly inhibited the growth of the cells. CONCLUSION: KLF15 inhibits the growth of lung adenocarcinoma cells. It could be used as a therapeutic target and a prognostic biomarker for the patients with lung adenocarcinoma.     

Effect of SMYD3 over-expression on miR-124 expression and proliferation ability in human intrahepatic cholangiocarcinoma cells

AIM：To explore the effect of SET and MYND domain-containing protein 3 (SMYD3) over-expression on miR-124 expression and proliferation ability of human intrahepatic cholangiocarcinoma cells. METHODS：Transient transfection of SMYD3 eukaryotic expression plasmid into human intrahepatic cholangiocarcinoma cell line HCCC-9810 were performed. The expression of SMYD3 at mRNA and protein levels was measured by qRT-PCR and Western blotting, respectively. The expression of miR-124 was detected by qRT-PCR, and the methylation status of miR-124 gene was determined by methylation-specific PCR. Cell proliferation was examined by CCK-8 assay and colony formation experiment. RESULTS：After transfected with SMYD3 eukaryotic expression plasmid, the over-expression of SMYD3 in HCCC-9810 cells was observed. Compared with the blank cells, the expression level of miR-124 was significantly decreased and miR-124 gene promoter methylation was significantly increased. In addition, SMYD3 over-expression significantly promoted the proliferation of HCCC-9810 cells. CONCLUSION：The transient transfection of SMYD3 plasmid increases the methylation of miR-124 gene promoter and induces under-expression of miR-124. Over-expression of SMYD3 promotes the proliferation of cholangiocarcinoma cells.     

Expression of Survivin and its relationship to prognosis in human hepatocellular carcinoma

AIM: To study the expression of Survivin and its relationship to prognosis in human hepatocellular carcinoma(HCC).METHODS: The expression of Survivin protein in 83 cases of HCC and their neighboring noncancerous tissues was detected by immunohistochemistry.The expression of Survivin mRNA in 11 cases of HCC and their neighboring noncancerous tissues was detected by semi-quantitative RT-PCR.RESULTS: Survivin protein expression was detected in 53 of 83 (63.9%) HCC tissues and 21 of 53 (39.6%) corresponding noncancerous tissues.22 of 53 Survivin-positive HCCs (41.5%) showed punctate nuclear staining in HCC cells,24 of 53 Survivin-positive HCCs (45.3%) showed cytoplasmic staining in HCC cells and the remaining 7 showed both nuclear and cytoplasmic staining.The positive rates of nuclear Survivin expression in cases of envelope invasion or tumor metastasis were significantly higher than those in cases without envelope invasion or tumor metastasis (P<0.05).The patients with positive nuclear Survivin expression had the higher chance of poor prognosis than those with negative nuclear Survivin expression (P<0.01).Survivin mRNA was detected in all the 11 HCC and 5 of 11 (45.5%) neighboring noncancerous tissues.CONCLUSION: There is a high expression of Survivin in HCC and positive nuclear Survivin may play an important role in malignant biological behavior and prognosis of HCC.     

Relationship between tumor metastasis suppressor gene KAI1 and the invasive and metastatic characteristics of osteosarcoma

AIM:To study the expression of metastasis suppressor gene KAI1 mRNA in osteosarcoma tissue and osteosarcoma cell lines,and the relationship between it and the biological behavior of the tumor cells.METHODS:RT-PCR was used to detect KAI1 mRNA in 18 cases of resected fresh osteosarcoma samples and three cultured osteosarcoma cell lines.The proliferative rate,the adhesive and invasive abilities of the 3 cell lines were detected.The results were treated by analysis system of images and analyzed with t test.RESULTS:The relative amount of KAI1 mRNA in osteosarcomas with lung metastasis was 0.80±0.50,while that was 1.48±0.64 in osteosarcomas without lung metastasis,the former was significantly lower than the latter (P<0.05).However,KAI1 mRNA had no corelation with the recurrence of osteosarcoma.The expression of KAI1 mRNA in U2-OS was highest (P<0.05),while the proliferation rate,the adhesive and invasive ability of U2-OS were the lowest among the 3 cell lines (P<0.01).CONCLUSION:The metastasis suppressor gene KAI1 might take part in influencing the lung metastasis of osteosarcoma,which might be caused by inhibiting the tumor cell proliferation,adhesion and invasion.     

Effect of BMP9 over-expression on migration and invasion of human lung squamous-cell carcinoma NCI-H520 cells

AIM: To investigate the effect of bone morphogenetic proteins 9(BMP9) on the migration and invasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism. METHODS: The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial (HBE) cells was detected by RT-PCR and Western blot. The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expression of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot. The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays. The mRNA and protein levels of the migration-related factor matrix metalloproteinase 2(MMP2) were detected by RT-PCR and Western blot. The level of phosphorylated Smad1/5(p-Smad1/5) was detected by Western blot. Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9. The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays. RESULTS: The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells. After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly decreased, and the mRNA and protein levels of MMP2 were decreased. Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability. CONCLUSION: Over-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells. The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.     

Relationship between FRAS1 protein and brain metastases of NSCLC

AIM: To explore the relationship between FRAS1 protein and brain metastases of non-small cell lung cancer (NSCLC).METHODS: The mRNA expression of FRAS1 in the brain metastatic tumor tissues and primary tumor tissues of NSCLC was detected by qPCR. The protein expression of FRAS1 in the tumor tissues and normal tissues adjacent to tumor tissues of NSCLC was measured by SP method of immunohistochemistry. The protein expression of FRAS1 in NSCLC primary tumor tissues with or without brain metastases was also determined.RESULTS: The mRNA expression of FRAS1 in the brain metastatic zone was nearly 10 times higher than that in the primary tumor tissues, and there was significant difference between the 2 groups (P<0.05). FRAS1 protein was expressed in the NSCLC primary tumor tissues, but was not found in the normal tissues adjacent to primary tumor tissues. The protein expression of FRAS1 in the NSCLC with brain metastases was significantly higher than that without brain metastases (P<0.01).CONCLUSION: FRAS1 protein may be associated with the occurrence of NSCLC. The over-expression of FRAS1 protein may be related to brain metastases with NSCLC.     

Significance of STC1 expression in peripheral blood and tissue of gastric carcinoma

AIM: To investigate stanniocalcin 1 (STC1) mRNA expression in the blood and tissue of gastric carcinoma. METHODS: The samples of peripheral blood and tumor tissues were collected from 74 patients with stage I-IV gastric carcinoma. STC1 mRNA was detected by RT-PCR, STC1 protein in tumor tissue was also detected by immunohistochemical method. RESULTS: The expression of STC1 mRNA in blood was significantly correlated with multiple histopathological prognostic factors, including primary tumor size, number of positive lymph nodes, and TNM stage. STC1 mRNA was not detected in the blood of volunteers without cancer, but detected in all gastric carcinoma tissue, and STC1 protein was expressed in all gastric carcinomas. CONCLUSION: STC1 is proposed as a novel, specific, and clinically useful molecular marker for detecting occult gastric carcinoma cells in blood. STC1 expression may play an important role in the progression of gastric carcinoma.     

Inhibitory effect of survivin antisense oligodeoxyribonucleotide combined with DDP in nude mice bearing human osteosarcoma xenograft

AIM:To investigate the feasibility and its mechanisms of improving therapeutic effect by antisense gene therapy combined with chemotherapy in osteosarcoma. METHODS:The human osteosarcoma implanted tumor model in the nude mice was established. By intratumoral injection and abdominal cavity administration, the tumor bearing mice were treated with survivin ASODN in combination with diamminedichloroplatinum (DDP) for a week. Comparison with each single-agent therapy and control group was performed in aspects such as tumor growth condition, pathological changes of tumor tissues;survivin protein expression in tumor tissues by immunohistochemistry, survivin mRNA expression levels by RT-PCR method and tumor apoptosis by Tdt-mediated dUTP nick end labeling (TUNEL). RESULTS:All nude mice survived the therapy. As compared with the control group, the antisense gene therapy group presented synchronous decrease in survivin mRNA and protein expression;all therapy group displayed tumor growth inhibition and cell apoptosis with different extent;while in contrast to single-agent therapy group, the combined therapy group showed stronger inhibition of tumor growth and abundant tumor cell apoptosis with the highest apoptotic rate. CONCLUSION:Synergistic effect was achieved by combination of DDP with ASODN that may overcome drug resistant of DDP and the combined strategy may shed new light on the cancer therapy.     

Expression and clinical significance of SCNM1 in hepatitis B-related hepatocellular carcinoma

AIM: To investigate the expression of sodium channel modifier 1 (SCNM1) in hepatitis B-related hepatocellular carcinoma (HCC) and its relationship with clinicopathological features and prognosis.METHODS: The specimens were collected from 108 patients with hepatitis B-related HCC who were treated in the Third Affiliated Hospital of Sun Yat-sen University from January 2013 to December 2015. All patients signed the informed consent and met the requirements of medical ethics. The mRNA expression level of SCNM1 in hepatitis B-related HCC tissues and tumor-adjacent tissues was detected by RT-qPCR, and the relationship between the mRNA expression of SCNM1 and the clinicopathological characteristics of hepatocellular carcinoma was analyzed. The relationship between SCNM1 expression and the prognosis of the patients was analyzed by Kaplan-Meier plotter.RESULTS: The data from TCGA database, Human Protein Atlas database and Oncomine database showed that the expression of SCNM1 in hepatocellular carcinoma tissues was significantly higher than that in the normal liver tissues (P<0.01). SCNM1 was mainly distributed in the nucleus. The results of RT-qPCR showed that the median mRNA expression of SCNM1 in hepatitis B-related HCC tissues was significantly higher than that in the matched tumor-adjacent tissues (t=8.082, P<0.01). The mRNA expression of SCNM1 was correlated with cirrhosis, alanine aminotransferase and tumor size (P<0.05), but not with sex, age and tumor envelope. The total survi-val time of the HCC patients with high expression of SCNM1 was shorter than that of the patients with low expression of SCNM1 (HR=1.53, P=0.016), and that of the patients with hepatitis B-related HCC was even shorter (HR=2.41, P=0.015).CONCLUSION: SCNM1 is highly expressed in hepatitis B-related HCC and may play an important role in the development of hepatitis B-related HCC.