Effects of inhibition of Cripto gene siRNA on vascular endothelial growth factor of colon cancer cell line LS-174T

AIM:To study the effects of Cripto gene on vascular endothelial growth factor (VEGF) of colon carcinoma cells.METHODS:Cripto siRNA was designed and constructed. Colon cancer LS-174T cells were divided into 4 groups: control group and different dose (3.125, 6.25 and 12.5 nmol/L) of siRNA groups. After transfected for 24, 48 and 72 h, colon cancer cells were harvested to carry on the next tests. Expression of Cripto mRNA was determined with real-time PCR, and immunofluorescence isothiocyanate (FITC) labeling assay and Northern blotting were performed to examine the expression of protein and mRNA of VEGF, respectively. The cells in control group and cells transfected with 12.5 nmol/L siRNA were inoculated into nude mice respectively. 30 days after inoculated, the mice of two groups were executed, and immunohistochemical (ICH) assay was used to evaluate the VEGF protein of mice tumor. RESULTS:siRNA down-regulated the Cripto mRNA in a dose and time dependent manner. Protein and mRNA of VEGF in transfected cells reduced in a dose and time dependent manner. Compared to control, the expression of VEGF protein from ICH assay was lowered significantly (P<0.05).CONCLUSION:Cripto gene might contribute to the regulation of angiogenesis of colon carcinoma. The down-regulation of Cripto gene by siRNA can suppress angiogenesis of human colon cancer.     

Priliminary analysis of microRNA expression profiles of side population cells in human colon cancer cell lines

AIM：To investigate the role of side population (SP) cells in multidrug resistance of colon cancer cells and microRNA biomarkers of SP cells in colon cancer cells. METHODS：SP cells in colon cancer cells were sorted by flow cytometry. The cell viability was measured by MTT method. MicroRNA expression profiles were detected by microRNA chip. MicroRNA expression was verified by real-time PCR. RESULTS：The ratios of SP cells in HCT-15, HT-29 and LoVo colon cancer cell lines were 16.75%, 13.02% and 9.52%, respectively. In all 3 colon cancer cell lines, IC50 of the antitumor drugs including 5-fluorouracil, oxaliplatin and adriamycin for the SP cells were significantly higher than those for non-SP cells (P<005). MicroRNA profiling showed that miR-5000-3p, miR-5009-3p and miR-552 were all up-regulated in the SP cells of all 3 colon cancer cell lines. This result was verified by real-time PCR. CONCLUSION：miR-5000-3p, miR-5009-3p and miR-552 are all up-regulated in the SP cells of colon cancer cell lines, and may be the potential microRNA biomarkers of SP cells in colon cancer.     

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.     

Expression of SAL-like 4 protein in human prostate cancer tissue/cell lines and its relationship with clinical outcome

AIM:To evaluate the expression of SAL-like 4 (SALL4) protein in human prostate cancer cell lines and tissues, and to analyze the relationship between SALL4 expression and the clinicopathological parameters. METHODS:Immunofluorescence, RT-PCR and Western blotting were performed to detect the expression of SALL4 at mRNA and protein levels in 3 common prostate cancer cell lines LNCaP, DU145 and PC-3. The normal prostate epithelial cell line RWPE-1 was used for control. The protein levels of SALL4 in the tissues of benign prostate hyperplasia and prostate cancer tissues were determined by the method of immunohistochemistry. RESULTS:The SALL4 protein was predominantly expressed in the cytoplasm of the cells. The protein levels of SALL4 in 3 common prostate cancer cell lines were significantly higher than that in RWPE-1 cells. However, the mRNA level of SALL4 had no obvious difference among the 4 cell lines. Immunohistochemistry results showed that the expression level of SALL4 in the cancerous tissues was significantly higher than that in noncancerous (benign and normal) prostatic tissues. In addition, we found that the expression level of SALL4 in prostate cancer was significantly correlated with the Gleason score, clinical stage, prognosis estimation and tissue prostate-specific antigen (PSA) expression, but not associated with age, the level of serum total PSA, prostate volume and the expression of androgen receptor in the tissues of the patients. CONCLUSION: The over-expression of SALL4 protein may play an important role in the pathogenesis and progression of prostate cancer, and provides some reference indexes for estimating the malignancy, progression and prognosis of prostate cancer.     

Enhancement of radiosensitivity and inhibition of p75NTR expression in esophageal cancer cell line Eca109 by a novel peptide P162 targeting to G3BP

AIM：To investigate the effect of a novel peptide P162 targeting to Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) on the radiosensitivity of esophageal cancer cells and the expression of p75 neurotrophin receptor (p75NTR) in esophageal cancer stem cells. METHODS：
The proliferation inhibitory rate was measured by CCK-8 assay. Colony formation assay was performed to determine the radiosensitizing effect of P162 on Eca-109 cell line. Single-hit multitarget mode was used to plot survival curves. The morphological changes of Eca109 cells were observed under inverted microscope. The expression of surface marker p75NTR in human esophageal cancer stem cells was analyzed by flow cytometry. RESULTS：The peptide P162 inhibited Eca109 cell proliferation in a time- and dose-dependent manner. The radiosensitization enhancement ratios (SERDq) of P162 at concentrations of 2.5, 5.0 and 10 μmol/L were 1.54, 2.35 and 2.33, respectively. With the increase in the irradiation dose, the expression level of surface marker p75NTR was increased. Compared with simple irradiation group, the expression level of p75NTR was obviously decreased in P162 treatment group. CONCLUSION：The peptide P162 increases the radiosensitivity of esophageal cancer cell line Eca109, and inhibits the expression of p75NTR in esophageal cancer stem cells. This sensitization effect may be related to inhibition of esophageal cancer stem cells.