Model establishment and biological behaviour observation of mouse blastocyst co-cultured with hepatocarcinoma cell lines with differently invasive and metastatic potential in vitro

AIM: To explore interaction and biological behaviour changes of two kinds of cells-blastocysts and hepatocarcinoma cells in the same microenvironment. METHODS:The models of mouse blastocysts co-cultured with human hepatocarcinoma cell lines were established, then biological behaviours and mutual effects of the two kinds of cells in co-culture system were observed. RESULTS: Compared with control group, hepatocarcinoma cells with differently invasive and metastatic potential significantly enhanced the rates of blastocyst hatchment , attachment and outgrowth(P＜0.05). There was no significant difference in those among hepatocarcinoma cells co-cultured groups (P＞0.05). The blastocyst hatched and attached to hepatocarcinoma cells with differently invasive and metastatic potential. Then, differential trophoblasts invaded hepatocarcinoma cells. The clear-cut interfaces were gradually formed between both sides. Hepatocarcinoma cells on interface showed changes of growth direction and cell shapes and did not invade blastocysts. CONCLUSIONS: Hepatocarcinoma cells promoted blastocyst development. Blastocysts implanted and invaded hepatocarcinoma cells with differently invasive and metastatic potential in vitro, which indicate that blastocyst implantation in vitro does not relate with the kinds and differential level of interactional cells and the low selectivity maybe relate with high adaptability of early life.     

Expression of ERK5 in colorectal cancer tissues and its correlation with distant metastasis in colorectal cancer patients

AIM: To investigate the expression of extracellular signal-regulated kinase 5 (ERK5) in primary colorectal cancer (CRC) and adjacent normal mucosa, and to analyze the relationship between ERK5 expression and clinicopathological parameters for exploring the functions of ERK5 in the occurrence and development of CRC. METHODS: The expression of ERK5 in carcinoma tissues and normal mucosa was examined by a set of tissue microarrays and the method of immunohistochemistry. The potential relationship between ERK5 expression and clinicopathological features was also analyzed. RESULTS: ERK5 expression was significantly higher in CRC tissues (134/338, 39.6%) than that in normal tissues (21/80, 26.2%; P<0.05). Overexpression of ERK5 in CRC tissues was significantly correlated with distant metastasis (P<0.05). However, no correlation between ERK5 expression and age at surgery, sex, tumor location, the depth of invasion, lymph node metastasis, TNM staging or differentiation grade was found (P>0.05). According to the Kaplan-Meier analysis, there is no significant difference in 5-year overall survival between the patients with ERK5 expression at high level and at low level. CONCLUSION: ERK5 protein is highly expressed in CRC with distant metastasis. This may be a promotive factor in the process of distant metastasis.     

ER-α36 and miR-143 involved in invasion of gastric cancer cells

AIM: To investigate the relationship between the expression of estrogen receptor α36 (ER-α36) and the invasion of gastric cancer cells. METHODS: SGC7901 cells were treated with 17β-estradiol at high or low concentration. The invasion of gastric cancer cells and the expression of ER-α36 were detected. The SGC7901 cells with the characteristics of stable low expression and high expression of ER-α36 were constructed. The invasion ability and microRNA sequences were determined in above-mentioned recombinant cells. RESULTS: The decreased invasion ability and ER-α36 expression were detected in SGC7901 cells treated with low concentration of 17β-estradiol. The situation was the opposite in the cells treated with high concentration of 17β-estradiol. The expression of miR-143 was significantly decreased in the SGC7901 cells with stable high expression of ER-α36 and was increased in the SGC7901 cells with stable low expression of ER-α36. CONCLUSION: The expression of ER-α36 is positively related to the invasion of gastric cancer cells. It is possible that miR-143 plays an important role in the regulation of gastric cancer invasion.     

Effect of long non-coding RNA MEG3 on invasion and migration of colorectal cancer cells

AIM: To investigate the expression of long non-coding RNA maternally expressed gene 3(MEG3) in colorectal cancer(CRC) cells, and to observe the effect of MEG3 on the invasion and migration of CRC cells. METHODS: The levels of MEG3 in human normal colon cell NCM460 and CRC cells SW48 and LoVo were detected by real- time PCR. MEG3 was over-expressed by plasmid transfection, and the effects of MEG3 on the invasion and migration of SW48 and LoVo cells were analyzed by Transwell assay and wound healing assay. The expression of matrix metalloproteinase(MMP) family proteins was determined by Western blotting. RESULTS: The level of MEG3 was down-regulated in CRC cells compared with normal colon cell NCM460. The invasion and migration of CRC cells were reduced after MEG3 over-expression. Transwell invasion and migration assays showed that the numbers of transmembrane SW48 and LoVo cells were smaller in MEG3 over-expression group than control group(CONCLUSION: The expression of MEG3 is down-regulated in CRC cells. Over-expression of MEG3 inhibits the invasion and migration of CRC cells. TIMP-2, MMP-2 and MMP-9 might play an important role in this regulation.     

Reduced expression of SIRT2 in serous ovarian carcinoma promotes cell proliferation,migration and invasion

AIM: To compare the expression of SIRT2 in ovarian surface epithelial (OSE) cell line and serous ovarian carcinoma (SOC) cell lines, and to investigate the effects of SIRT2 on the cell proliferation, migration and invasion. METHODS: The expression levels of SIRT2 in the OSE cell line and the SOC cell lines were determined by Western blot. The SIRT2 siRNAs and overexpression construct were designed and verified. Transient transfection of SIRT2 siRNAs or overexpression construct was performed, and the effect of SIRT2 on the cell proliferation, migration and invasion was evaluated. RESULTS: SIRT2 levels in the 5 strains of SOC cell lines were significantly lower than that in the OSE cell line. SIRT2 knockdown in HOSEpiC cells significantly enhanced the ability of cell colony formation and accelerated the cell growth rate. On the contrary, overexpression of SIRT2 in HO8910 cells dramatically repressed the number of cell colonies and cell activity. SIRT2 significantly changed the ability of ovarian cell migration. Knockdown of SIRT2 facilitated the cell invasion. CONCLUSION: The expression of SIRT2 in the SOC cells is significantly down-regulated. In the OSE cells, SIRT2 acts as a tumor suppressor and mediates the inhibition of cell proliferation, migration and invasion.     

Interaction of polymorphisms of ICAM-1 gene K469E and MCP-1 gene -2518A/G in invasion and metastasis of gastric carcinoma

AIM: To investigate the interaction of polymorphisms of intercellular adhesion molecule-1 (ICAM-1) gene K469E and monocyte chemoattractant protein-1 (MCP-1) gene -2518A/G in the invasion and metastasis of gastric carcinoma. METHODS: Based on TNM classification, 4 500 patients with confirmed gastric carcinoma from the First Affiliated Hospital of Xinxiang Medical University in China from December 2009 to November 2014 were divided into stageⅠ group, stage Ⅱgroup, stage Ⅲ group, stage Ⅳ group, and stage 0 group, with 900 cases in each group. No significant difference among the 5 groups in age, gender, ethnicity, birthplace and living habit was observed. The genetic polymorphisms of ICAM-1 gene K469E and MCP-1 gene -2518A/G were analyzed by the technique of polymorphism-polymerase chain reaction (PCR) in peripheral blood leukocytes of above-mentioned cases. RESULTS: Statistical tests showed signi-ficant differences in the frequencies of K469E (EE) and -2518A/G (GG) among each group (P<0.01). The risk of the invasion and metastasis of gastric carcinoma significantly increased in subjects with K469E (EE) genotype and in those with -2518A/G (GG) genotype. Combined analysis of the polymorphisms showed that distribution frequency of K469E (EE)/-2518A/G (GG) in stage Ⅰ group, stage Ⅱ group, stage Ⅲ group, stage Ⅳ group and stage 0 group was 39.22%, 53.22%, 59.22, 65.44% and 12.11%, respectively (P<0.01). The people who carried with K469E (EE)/-2518A/G (GG) had a high risk of the invasion and metastasis of gastric carcinoma, and statistical analysis suggested a positive interaction in a super-multiplicative model between K469E (EE) and -2518A/G (GG) in increasing the risk of the invasion and metastasis of gastric carcinoma. CONCLUSION: ICAM-1 gene K469E (EE) and MCP-1 gene -2518A/G (GG) are the risk factors in the invasion and metastasis of gastric carcinoma, and significant interactions between genetic polymorphisms of K469E and -2518A/G added the risk of the invasion and metastasis of gastric carcinoma.     

Effects of p21-activated kinase 6 on invasion and migration of human non-small-cell lung cancer A549 cells

AIM：To investigate the effects of p21-activated kinase 6 (PAK6) on the invasive and migratory abilities of human non-small-cell lung cancer A549 cells. METHODS：The expression of PAK6 mRNA in A549 cells, human bronchial epithelial (HBE) cells, non-small-cell lung cancer tissues and paired adjacent non-tumor tissues was measured by real-time PCR. After A549 cells were transfected with siRNA-PAK6 (siPAK6) or negative control (NC) for 48 h, the expression of PAK6 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively. The invasion and migration of A549 cells were detected by Matrigel invasion assay and Transwell migration assay. The cytoskeletal changes were observed with FITC-phalloidin staining under confocal microscope. RESULTS：The level of PAK6 mRNA in A549 cells was higher than that in HBE cells (3.50±1.16 vs 1.12±0.42, P<0.05). The level of PAK6 mRNA in non-small-cell lung cancer tissues was higher than that in paired adjacent non-tumor tissues (5.13±1.33 vs 1.08±0.37, P<0.05). The expression of PAK6 protein decreased by 72% in A549 cells transfected with siPAK6 (P<0.05), and the level of PAK6 mRNA significantly decreased in A549 cells transfected with siPAK6 (3.72±0.75 vs 0.69±0.21, P<0.05). Matrigel invasion assay and Transwell migration assay demonstrated that knockdown of PAK6 markedly attenuated the invasion and migration of A549 cells (P<0.05). The cytoskeletal actin remodeling and reduction of stress fibers in A549 cells transfected with siPAK6 were observed under confocal microscope. CONCLUSION：PAK6 may affect the invasive and migratory abilities of non-small-cell lung cancer cells by cytoskeletal actin remodeling.     

Role of ACSL3 expression in suppressing prostate cancer cell metastasis

AIM:To analyze the difference of long-chain acyl-CoA synthetase 3 (ACSL3) expression between normal prostate epithelial cells and prostate cancer cells.METHODS:ACLS3 mRNA expression between normal prostate epithelial cells and prostate cancer cells was compared using RT-PCR. Meanwhile, ACSL3 gene was amplified from prostate cancer cells, and the eukaryotic expression plasmid pCDNA3.1(+)-Flag-ACLS3 and lentivirus Lenti-ACLS3 were constructed. After transfection of ACSL3-plasmid and lentivirus into the prostate cancer cells, ACSL3 expressive was detected by RT-PCR and Western blotting, and then Matrigel invasion assay was performed to investigate the alteration of the invasive ability of the prostate cancer cells with over-expression of ACSL3. RESULTS:A significant difference of ACSL3 mRNA level between normal prostate epithelial cells and prostate cancer cells was observed. ACSL3 was highly expressed in localized prostate cancer cells compared to metastatic prostate cancer cells, while ACSL3 expression was higher in androgen-dependent prostate cancer cells than that in androgen-independent prostate cancer cells. Furthermore, the eukaryotic expression plasmid and lentivirus containing ACLS3 gene were successfully constructed. The prostate cancer cell line which stably over-expressed ACLS3 was established. Up-regulation of ACSL3 inhibited the invasive ability of prostate cancer cells. CONCLUSION:ACSL3 plays an antagonistic role in invasiveness of prostate cancer.     

Inhibitory effect of AuNPs-Endostar on melanoma lung metastasis in mice

AIM: To observe the influence of gold nanoparticles combined with Endostar (AuNPs-Endostar) on the melanoma lung metastasis of mice and the underlying mechanism. METHODS: C57BL/6 mice (n=24) were selected for constructing the model of spontaneous lung metastasis of melanoma B16-F10 cells. Subsequently, the mice were randomly divided into Endostar group, AuNPs group, AuNPs-Endostar group and model group. After the formation of melanoma, the mice in each group were injected with different drugs through tail vein for 0.1 mL daily. After 9 d, the mice were narcotized for cutting the tumors in situ. After the operation, they were raised for 2 weeks before killed for obtaining the lung tissues to observe the situation of the metastasis. HE staining was utilized for observing the necrosis status of the tumors in situ, while immunostaining was applied for testing the expression of CD31, carbonic anhydrase-IX (CA-IX), vimentin and zonula occludens-1 (ZO-1) in the tumors. RESULTS: Compared with model group, the pulmonary metastasis in the groups with medical treatment was obviously reduced. In AuNPs-Endostar group, the metastasis inhibition rate was the highest, and the tumor necrosis was also decreased obviously, with the significant reduction of CD31, CA-IX and vimentin expression in the tumors and significant increase in ZO-1 expression. CONCLUSION: Compared with using Endostar or AuNPs alone, the combination of AuNPs with Endostar significantly improves the curative effect of inhibiting the pulmonary metastasis of melanoma in the mice. The mechanism may be related to reducing the tumor angiogenesis, norma-lizing the blood vessels and improving tumor hypoxia, thus inhibiting the tumor epithelial-mesenchymal transition, increasing the tight junctions between tumor cells and decreasing the invasiveness.