MiR-26b regulates invasion and migration of lung cancer cells through targeting hENTI depending on RhoA/ROCK-I pathway / 中南大学学报(医学版)

Objective:To investigate the effect of miR-26b on the invasion and migration of lung cancer cell and to explore its mechanism.Methods:qPCR was used to detect the expression of miR-26b in lung cancer.Luciferase reporter gene was used to detect interaction between miR-26b and hENT1.Transwell assay was used to detect invasion ability after treatment of miR-26b mimics.Scratch assay was used to detect migration ability after treatment of miR-26b mimics.The expressions of hENT1,ROCK-I and RhoA were detected by Western blot.The changes of cytoskeleton after miR-26b mimics treatment with phalloidin were observed.The effect of miR-26b mimics on the tumor size and volume of lung cancer was determined by subcutaneous tumor formation in nude mice.Results:MiR-26b expression was significantly reduced in lung cancer.With the progress of lung cancer,the expression of miR-26b was reduced.With the progress in differentiation of lung cancer,the expression of miR-26b was decreased.Decrease of miR-26b was associated with lung cancer lymph node metastasis.HENT1 was the direct target of miR-26b;miR-26b regulated the invasion and migration ability of human lung carcinoma A549 cells.MiR-26b regulated the expression of hENT1,ROCK-1 and RhoA.After the treatment with miR-26b mimics,the F-actin staining was significantly reduced,whereas the formation of wrinkles and the formation of pseudopodia were significantly reduced.Subcutaneous tumor formation in nude mice showed that miR-26b mimics treatment significantly reduced the tumor size and mass.Conclusion:MiR-26b plays a role in tumor suppression in lung cancer.miR-26b can regulate the invasion and migration ability of lung carcinoma A549 cells by targeting hENT1 depending on the RhoA/ROCK-1 pathway.

Effects of Chinese FormulaXuetongling on MDA-MB-231 Human Breast Cancer Cell Invasion and MMP-9 Protein Expression / 中国中医药信息杂志

Objective To research the action mechanism of Chinese FormulaXuetongling on MDA-MB-231 human breast cancer cell invasion.Methods Transwell Invasion assay was applied to investigate the inhibitory effects on cancer cell invasion ofXuetongling extract in different concentrations;MMP-9 secretion activity was detected by zymography assay after the treatment of XTL extract in MDA-MB-231;Western blot was used to detect the effect of XTL extract on MMP-9 protein expression in MDA-MB-231.ResultsXuetongling extract in different concentrations significantly suppressed the cell invasion, MMP-9 secretion and MMP-9 protein expression in dose dependent manner.Conclusion The inhibitory effect ofXuetongling on MDA-MB-231 cell invasion may be due to the down-regulation of both MMP-9 secretion and MMP-9 protein expression.

Comparison of an ELISA assay for the detection of adhesive/invasive Neospora caninum tachyzoites / Comparação de um ensaio de ELISA para a detecção de taquizoítas adesivos/invasivos de Neospora caninum

Neospora caninum belongs to the phylum Apicomplexa, the causative agent of neosporosis, which leads to economic impacts on cattle production. A common feature among apicomplexan parasites is the invasive process driven mostly by the parasite. As a first evaluation of candidate molecules that play a possible role by interfering in this invasive process, the in vitro invasion assay is a fast and direct way to screen future agonists or antagonists. This work involved the development of a new cell culture ELISA and transient β-galactosidase activity applied to the semi-quantitative detection of N. caninum in Vero cell culture. Cell culture ELISA is based on histochemistry and immunology, resulting in a colorimetric reaction. The β-galactosidase activity was obtained by the transient transfection of the lacZ gene under control of RPS13 promoter of N. caninum. These methods were used to evaluate the effects of temperature (37°C and 85°C) on the invasion and adhesion of tachyzoites. The three tested methods (real time PCR, β-galactosidase activity and ELISA) showed a similar pattern, indicating that different methods may be complementary.

Neospora caninum, parasita do filo Apicomplexa, é causador da neosporose, doença responsável por perdas econômicas importantes na pecuária. Um fator comum entre os apicomplexas é o processo de invasão majoritariamente dirigido pelo parasita. Dentre as primeiras avaliações de moléculas candidatas, que possivelmente interferem no processo de invasão, o ensaio de invasão in vitro é um meio rápido e direto de selecionar futuros agonistas ou antagonistas. Este trabalho desenvolveu um novo ELISA baseado em cultura (Cell-culture ELISA) e um ensaio que mede a atividade transiente de β-galactosidase, aplicados para a detecção semiquantitativa de N. caninum em células Vero. Cell-culture ELISA é baseado em histoquímica e imunologia, resultando em uma reação colorimétrica. A atividade da β-galactosidase foi obtida pela transfecção transiente do gene LacZ sob controle do promotor RPS13 de N. caninum. Esses métodos avaliaram os efeitos da temperatura (37°C e 85°C) sobre a invasão e adesão. Os três métodos testados (real time PCR, atividade de β-galactosidase e ELISA) mostraram um padrão similar, indicando que diferentes métodos podem ser complementares. Adicionalmente, esse ELISA é adequado para aplicação em laboratórios carentes de uma complexa estrutura para métodos de detecção moleculares.

MiR-200b suppresses proliferation of glioma and its stem cells by targeting CD133 / 肿瘤

Objective: To explicit whether miR-200b can suppress the proliferation and invasion by targeting CD133, and to explore a molecular mechanism that miR-200b plays a role in tumor suppression in glioma. Methods: The CD133 3'-untranslated region (3'-UTR) mRNA-luciferase reporter vector was constructed and the dual-luciferase reporter gene assay was employed to examine the effect of miR-200b on activity of luciferase. U251 cells were transfected with miR-200b mimics and CD133-small interfering RNA (siRNA) (as a positive control) by LipofectAMINE 2000, and the expressions level of CD133 protein was detected by Western blotting. The inhibition effects of CD133 on cell proliferation and invasion were observed after CD133 siRNA were transfected into U251 cells. U251-SC cell mammosphere assay was performed after cotransfection with miR-200b mimics and CD133 siRNA. Results: miR-200b could bind to the 3'-UTR of CD133 and inhibit the activity of luciferase. CD133 protein expression was significantly down-regulated when miR-200b was overexpressed in U251 cells. Overexpression of miR-200b inhibited the invasion of U251 cells. Overexpression of miR-200b antagonized a role of proliferation and invasion in U251 cells. Overexpression of miR-200b reduced mammosphere number and the size of glioma stem cells. Conclusion: miR-200b can suppress cell proliferation and invasion by targeting CD133 in glioma. Copyright © 2014 by TUMOR.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.

Influence of Hypoxic Condition on Invasion of Cultured Trophoblast / 대한주산의학회잡지

OBJECTIVE: To investigated whether lowering oxygen tension affects invasion of cultured trophoblast. METHODS: Trophoblasts were isolated from the normal placenta in early pregnancy(6-10 weeks in gestation). Isolated trophoblasts were cultured under normoxic(5% CO2, 95% humid air in incubator) and hypoxic(MERCK, 1% O2, 99% CO2) conditions for 24, 48 and 72 hours, respectively. The proliferation ability was measured using [H3] thymidine assay. Total RNA was extracted from the cultured trophoblasts. The expressions of matrix metalloproteinase(MMP-2) and tissue inhibitor of metallo- proteinase(TIMP-2) were determined by reverse transcription- polymerase chain reaction(RT-PCR) and Northern blot analysis. The invasiveness of cultured trophoblast was observed using in vitro invasion assay. RESULTS: [H] thymidine assay indicated that cellular DNA synthesis was not affected by the culture condition. The expression of MMP-2 mRNA was decreased at 24 hours and then progressively increased in the time-dependent manner in each culture condition. The expression of TIMP-2 was decreased in the time-dependent manner under hypoxic condition. In vitro invasion assay revealed that the cultured trophoblasts under hypoxic condition has more invasive ability than them under normoxic condition. CONCLUSION: These data suggests that hypoxic condition may stimulates the invasion of trophoblast in the human placentation. And MMP-2 and TIMP-2 may be related to control their invasiveness under hypoxic condition.

Separation of invasive subpopulation from primary human renal cell carcinoma via in vitro invasion assay / 第二军医大学学报

Objective:To isolate the invasive and non-invasive cells from primary human renal cell carcinoma(RCC) in vitro.Methods: Fresh RCC surgical specimens from 32 primary RCC patients were primarily cultured following enzyme digestion or mechanical minimization in vitro.In vitro invasion assay using the Transwell cultures coating Matrigel was performed for separation and recovery of invasive and non-invasive cells from the primary culture of 3 RCC patients.The concentration of Matrigel,recovery time and trypsinization were subsequently optimized.Results: The successful rate of primary culture was(90.6%)(29/32).Recovery of invasive cells was performed ideally when matrigel(diluted into 1.0 mg/ml and 20 ?l) was coated onto the filter of the well;cell suspension was at a concentration of 5?10~(5)/ml and invasive cells were recovered on the 5th day of culture.The growth of non-invasive cells was scattered,while that of the invasive cells was focal.The doubling time of invasive cells was 36.1 h and that of non-invasive was 50.6 h.Conclusion: The in vitro invasion assay using the Transwell is able to separate and recover the highly invasive primary RCC cells.The primary cells represent intact subpopulation composition,but it can hardly get through the life span of human primary tumor cells.