[F10 expressions in cervical cancer tissues].

OBJECTIVE: To detect the expression of F10 at both mRNA and protein levels in cervical cancer tissues and explore its role in the occurrence and progression of cervical cancer. METHODS: F10 expressions at mRNA and protein levels were detected in 30 pairs of cervical cancer tissues and adjacent tissues using RT-PCR and immunohistochemistry. RESULTS: The mRNA and protein expressions of F10 were significantly higher in cervical cancer tissues than in the adjacent normal tissues (P<0.05). F10 expression was significantly higher in poorly differentiated cervical cancer than in well differentiated cancer tissues, and was also lower in patients with preoperative chemotherapy than in those without chemotherapy. CONCLUSION: F10 expression level is inversely correlated with the differentiation of cervical cancer and possible plays a role in the tumorigenesis and progression of cervical cancer.

[Effect of F10 gene silencing and over-expression on cell cycle of choriocarcinoma cell line JAR and the mechanisms].

OBJECTIVE: To explore the role of F10 gene in regulating cell cycles of choriocarcinoma cells and the underlying mechanisms. METHODS: Using untreated cells as the control, JAR cells with F10 gene silencing or stable F10 over-expression were examined for cell cycle changes by flow cytometry (FCM) and for expressions of cyclin and cyclin-dependent kinase (CDKs) with Western blotting and immunofluorescence technique. RESULTS: JAR cells over-expressing F10 gene showed reduced duration of cell cycle compared with untreated and with cells after F10 gene silencing. In F10-over-expressing cells, Western blotting revealed significantly up-regulated expressions of cyclin A2, B1, D1, E and CDK2, 6, and 7, but not CDK4, as compared with the control cells and cells with F10 gene silencing (P<0.05), and these results were consistent with those by immunofluorescence assay. CONCLUSION: F10 gene may accelerate cell cycle progression and promote cell proliferation by up-regulating the expressions of cyclin A2, B1, D1, E and CDK 2, 4, 6, 7 in choriocarcinoma cells.

Regulation of trophoblast invasion: the role of matrix metalloproteinases.

Pregnancy success is determined by a complex progress that includes trophoblast invasion and placentation. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are metal-dependent endopeptidases capable of degrading extracellular matrix, and appear to play a critical role in trophoblast invasion. This article reviews in detail the role of MMPs, TIMPs, and their regulators in the mechanism of trophoblast invasion in early human pregnancy.

Interleukin-10 may participate in regulating trophoblast invasion in human placentae throughout gestation.

PROBLEM: A successful human pregnancy requires cytotrophoblasts from the fetal portion of the placenta to adopt tumor-like properties. But unlike tumor metastasis, cytotrophoblast invasion is highly regulated both spatially and temporally. The mechanisms that regulate human trophoblast invasion are understood poorly. METHOD OF STUDY: With a view to obtain some findings on the mechanisms that regulate human trophoblast invasion, we applied the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method to compare the expression of invasion-associated genes in cytotrophoblasts isolated from first- and third-trimester placental tissues. RESULTS: In trophoblast cells of first-trimester pregnancy, the mRNA contents of matrix metalloproteinase (MMP)-9 and urokinase-type plasminogen activator (uPA) were higher than that in the third-trimester cytotrophoblasts, while no difference of MMP-2 mRNA expression level was found between trophoblastic cells of different gestational ages. The expression level of plasminogen activator inhibitors-1 mRNA in first-trimester cytotrophoblasts was shown to be much lower than that in trophoblast cells prepared from third-trimester placental tissues. Furthermore, expression of both tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in cytotrophoblasts were significantly up-regulated in third-trimester when compared with the first-trimester of pregnancy. To further investigate the factors that caused the change of invasion-associated genes expression in trophoblast cells, we found that interleukin-10 (IL-10) could decrease the content of MMP-9 mRNA in cytotrophoblasts of first-trimester gestation, and the magnitude of suppression increased with increasing IL-10 concentration. CONCLUSION: The gradually reduced trophoblast invasion with gestational weeks might be on account of the change of proteolytic enzymes/activator/inhibitor genes expression. IL-10 could be one of the factors participating in the regulation of trophoblast invasion during gestational process.

[Function of F10 gene, a novel hydatidiform mole-related gene: a preliminary study].

OBJECTIVE: To study the function of F10 gene, a novel hydaditiform mole-related gene. METHODS: A549 cell line was transfected with the F10 gene of forward or reverse sequence or with the empty vector, respectively. The cellular mRNA was extracted after 24 h of transfection to screen for the differentially expressed genes among the 3 transfected and the control cells using differential display-polymerase chain reaction (ddPCR). RESULTS: The bands representing differentially expressed genes were amplified from the cells, and the products were linked to T-Vector for sequence analysis. Several genes were screened by Blasting and their expressions were confirmed by fluorescent quantitative PCR. CONCLUSION: F10 gene is functionally related to cell proliferation and apoptosis.

[Construction and identification of a stable eukaryotic expression system for F10 gene].

OBJECTIVE: To detect the transcriptional level of a novel gene F10 associated with the pathogenesis of hydatidiform mole in human cell lines and screen the cell lines with low F10 expression to construct a stable eukaryotic expression system for F10 gene. METHODS: The expression level of F10 mRNA was detected with fluorescent quantitative PCR in A549, 16HBE, Bel7402, HIC, HepG2, 293, PC and MGC cell lines. A549 cell line was transfected with plasmid pRc-CMV2-F10 via electroporation to allow stable F10 expression, and the positive cell clones were selected by G418. The insertion and expression of F10 gene in the A549 cells was analyzed using fluorescent quantitative PCR. RESULTS: F10 mRNA was expressed differentially in these cells lines, and the Bel7402 cells, PC and MGC cells showed the highest F10 mRNA expression, followed by HepG2 and HIC cells and further by 293 cells, and 16HBE and A594 cells had the lowest expression. After transfection, A594 cells showed genomic integration of F10 gene and high expression level of F10 mRNA. CONCLUSION: The pulmonary carcinoma cell line A549 with stable expression of F10 gene has been established, which may facilitate further study of the biological functions of F10 gene.

[Preliminary study of gene expression profiling in human type I and II endometrial carcinoma].

OBJECTIVE: To study gene expression profiling in human type I and II endometrial carcinoma. METHODS: Six Affymetrix human genome genechips were utilized to investigate the differences in gene expression profiles between type I and II endometrial carcinoma with bioinformatic analysis. RESULTS: Many genes were highly expressed in estrogen-dependent endometrial carcinoma, and some of them were involved in the metabolism and conversion of estrogen, while some others in estrogen regulation. CYP2C9, for instance, was involved in the conversion of estrogen sulfate to 16-hydroxy sulfate metabolite, DDC in estrogen-dependent pathogenesis of endometrial carcinoma possibly by DDC interaction with AR to enhance steroid receptor transcription. CONCLUSION: High expression of these genes in estrogen-dependent endometrial carcinoma may provide insights into their roles in the pathogenesis and prognosis of this malignancy.

[Significance of pelvic lymph node human papillomavirus DNA detection in cervical cancer].

OBJECTIVE: To assess the significance of human papillomavirus (HPV) DNA detection in the pelvic lymph nodes in cervical cancer. METHODS: HPV L1 gene fragment was amplified by HPV-specific PCR with general consensus primers from cervical cancer tissues. The types of HPV were identified by sequencing of the PCR product. The relationship between HPV positivity and pathological findings in metastatic pelvic lymph nodes of cervical cancer was analyzed. RESULTS: The positivity rate of HPV DNA was 40% in the pelvic lymph nodes in 40 cases of cervical cancer. HPV DNA was detected in the pelvic lymphatic nodes of all the 10 cases with pathologically confirmed lymph node metastasis of cervical cancer. HPV18 was detected in both of cervical and pelvic lymph nodes in some cases. CONCLUSIONS: HPV DNA can be detected in the pelvic lymph nodes before pathological identification of lymph node metastasis and indicates early pelvic lymph node metastasis of cervical cancer. The presence of HPV18 often indicates high likeliness of lymph node metastasis and suggests poor prognosis of the patients.

[Sequence polymorphism of human papillomavirus type 16 E7 in cervical cancer].

OBJECTIVE: To explore the genetic polymorphism of E7 open-reading frame of human papillomavirus (HPV) type 16 in cervical cancer. METHODS: The types of HPV was identified by sequence analysis of the PCR product of HPV in cervical cancer tissues. HPV 16 gene fragment in the cervical cancer tissue was amplified by HPV-specific PCR with general consensus primers. RESULTS: The positivity rate of high-risk HPV types in 50 cases of cervical cancers was 78%. Mixed infection of HPV16 and HPV18 was found in 18 cases, and the infection with HPV16 alone occurred in 15 cases. HPV16 E7 was amplified from 25 out of the 34 cases positive for HPV16. A T-to- C change occurred in the 647th nucleotide in the viral nucleotide sequence, causing conversion of the Asn codon of E7 gene into a Ser codon. CONCLUSIONS: The most frequently observed substitution in HPV16 E7 open reading frame occurs in the discrete regions of 647-846, and some substitutions result in the same-sense mutation. The hot-spot mutation of HPV16 E7 in cervical cancers in Guangdong Province occurs at the nucleotide 647 and 846 in the DNA sequence.

[Association of the novel hydatidiform mole-related gene F10 with the invasiveness of trophoblastic tumor].

OBJECTIVE: To study the expressions of the novel gene F10 associated with hydatidiform mole in different trophoblastic tumors and explore the relation of F10 expression with the invasiveness of malignant trophoblastic tumor. METHODS: In situ hybridization was used to study the expression of F10 in 12 cases of hydatidiform mole, 6 cases of invasive mole, and 8 cases of choriocarcinoma. RESULTS: F10 mRNA was positive in all cases of hydatidiform mole, invasive mole, and choriocarcinoma, and the expression intensity significantly increased in the order of hydatidiform mole, invasive mole and choriocarcinoma (P<0.001). CONCLUSION: The expression of F10 gene may relate to the occurrence and invasiveness of trophoblastic tumor, with possible involvement in the invasion or malignant changes of trophoblastic cells.

[Detection of human papillomavirus DNA in cervical cancer tissue by PCR and direct sequencing].

OBJECTIVE: To explore an effective method for detecting human papillomavirus (HPV) DNA in cervical cancer tissue. METHODS: HPV L1 gene fragment in cervical cancer tissue was amplified by HPV-specific PCR with consensus primers, and typing of the HPV strains was performed on the basis of sequence analysis of the PCR product. RESULTS: The positivity rates of HPV DNA was 78% in the 50 cases of cervical cancer, and mixed infection with HPV16 and HPV18 strains was the most common, which accounted for 48% on the total infections. Infection with HPV58 was detected in one case. The sequencing results showed no difference in L1 sequence between the detected samples and the standard German HPV58 strain. CONCLUSION: PCR and direct sequencing approach is effective for detecting and typing of HPV DNA in cervical cancer tissue, through which rare HPV strain or mutants of known HPV strains may not escape detection.

[Effect of decidual cell conditioned media on invasion-related gene expression of ovarian tumor cell line COC1].

OBJECTIVE: To study the effect of decidual cell conditioned media (DCM) on the expression of the invasion-related gene of ovarian tumor cell line COC1. METHODS: After primary culture of the decidual cells of early and late pregnancy, DCM was extracted from the cell cultures for treatment of the ovarian tumor cell line COC1. Analysis of the invasion-related gene expression in COC1 cells was performed by way of reverse transcriptional (RT)-PCR. RESULTS: The COC1 expressed the mRNAs of matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase-2 (TIMP-2) and plasminogen activator inhibitor type 1 (PAI-1), but not those of MMP-9, TIMP-1 or urokinase-type plasminogen activator (u-PA). The DCM of the early- and late-pregnancy decidual cell cultures significantly down-regulated the MMP-2 expression, and up-regulated the expression of the TIMP-2 and PAI-1 mRNA in COC1 cells in comparison with the control cells (P<0.01). CONCLUSION: DCM can lower the invasive capacity of the ovarian tumor cell line COC1 by interrupting the balance between MMP-2 and TIMP-2 and between u-PA and PAI-1.

[Effects of exogenous tumor necrosis factor alpha on the invasiveness of human trophoblasts].

OBJECTIVE: To observe the effect of exogenous tumor necrosis factor alpha (TNF-alpha) and its polyclonal antibody (anti-TNF-alpha) on the invasiveness of human trophoblasts in vitro. METHODS: The effect of exogenous TNF-alpha and anti-TNF-alpha on the expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinases-2 (TIMP-2) in human trophoblastic cells was investigated with reverse transcription (RT)-PCR (RT-PCR). RESULTS: The trophoblasts cultured in vitro expressed MMP-2 but not TIMP-2. After incubation of the trophoblasts with 5 ng/ml TNF-alpha and TNF-alpha plus anti-TNF-alpha respectively for 48 h, significant elevation of MMP-2 expression was induced, while the reverse occurred following incubation of the cells with 50 ng/ml anti-TNF-alpha. None of the treatments induced TIMP-2 expression. CONCLUSION: Exogenous TNF-alpha may promote the invasiveness of the trophoblasts, which can be inhibited by anti-TNF-alpha.

DNA microarrays detect the expression of apoptosis-related genes in preeclamptic placentas.

AIMS: To investigate the expression of apoptosis-related genes in preeclamptic placentas and the possible mechanism of the regulation process. METHODS: Complementary DNA microarrays were employed to compare gene expression profiles of five preeclamptic and five normal placentas. RESULTS: Among the 368 genes detected over 35% showed an over 2-fold difference of expression between preeclamptic placentas and normal placentas. Many genes involved in cell cycle or apoptosis were more highly expressed in preeclamptic placentas than in normal placentas. The expression of many immune-activation genes in preeclamptic placentas was also higher than that in normal placentas. Additionally, many cytokine receptor/kinase genes were also induced in preeclamptic placentas. CONCLUSIONS: The change in expression of cell apoptosis-related genes in placentas might be involved in the pathogenesis of preeclampsia, while the activation of the immune system might be one cause of this change.

Comparative profiling of metabolism-related gene expression in pre-eclamptic and normal pregnancies.

Extensive endothelial dysfunction has been regarded as the central hallmark in the pathogenesis of pre-eclampsia, but the mechanisms leading to this dysfunction remain unclear. The levels of many metabolism substances, such as lipid peroxides, are changed in plasma of patients with pre-eclampsia. Some of those might be associated with the occurrence of pre-eclampsia. Our research was trying to reveal the source of metabolism substances which were elevated in plasma of preeclamptic patients. The expression of many metabolism-associated genes in 3 preeclamptic and strictly matched 3 normal placentas were compared by employing DNA chip representing 243 human hormone-associated genes. The results showed that, many redox metabolism-related genes (GenBank: U78168, X16699, D32143, AL035079, AF069668, X53463, J03746, X02317, X91247, etc) were found more highly expressed in preeclamptic placenta than that in normal placenta. Additionally, the mRNA levels of some genes which were related to other metabolism such as hormone (GenBank: NM_001718, Z22535, M38180, M76665, X75252, J03258, U56725), were also higher in placenta of patients with pre-eclampsia. Furthermore, many transcription factor genes were also up-regulated in pre-eclampsia. It suggested that, the change of genes expression in placenta was associated with the change of metabolism in patients with pre-eclampsia.

Expression of transforming growth factor-beta and insulin-like growth factor in molar and placental tissues.

The semiquantitative reverse transcription polymerase chain reaction was employed to detect the expression of transforming growth factor beta (TGF-beta) and insulin-like growth factor (IGF) in complete hydatidiform mole, normal first-trimester villi, the normal term placenta (after vaginal/abdominal deliver) and the preeclamptic placenta at term. The expression of IGF-I mRNA was seen in all five tissues, but its level was much lower in the term placental tissues with preeclampsia than in other tissues. The content of IGF-I mRNA in villous tissues from molar pregnancy was slightly higher than in normal first-trimester villi. IGF-II mRNA was detected at similar levels in all three sorts of term placental tissues. However, the expression level of IGF-II mRNA in tissues of complete molar pregnancy was significantly lower than in normal first-trimester villi. TGF-beta(3) was found expressed in all five tissues, while TGF-beta(1) and TGF-beta(2) mRNA were not detected. Compared to the normal first-trimester villi, the expression of TGF-beta(3) in complete hydatidiform molar tissues was comparatively higher. Furthermore, the expression levels of TGF-beta(3) in the preeclamptic placenta and the normal placenta after cesarean birth were higher than in the placenta after vaginal delivery. We concluded that, the change of TGF-beta and IGF expression in placental tissues might be involved in the development of trophoblastic diseases of pregnancy.

[Role of p38 pathway in PMA-induced in vitro invasion of JAR human choriocarcinoma cell line].

OBJECTIVE: To investigate the role of p38 mitogen-activated protein kinase (MAPK) signal transduction pathways in regulating the in vitro invasion of JAR human choriocarcinoma cells induced by phorbol 12-myristate 13-acetate (PMA). METHODS: ELISA was used to detect the kinase activity of the JAR cells in response to PMA stimulation, and the in vitro invasion capabilities of the stimulated cells were observed using transwell assay. Changes in the proliferation and activity of the JAR cells cultured in vitro following PMA treatment were also observed by MTT assay. RESULTS: p38 MAPK was activated dose-dependently in JAR cells upon the stimulation by PMA, which significantly enhanced the in vitro invasion of the JAR cells, while treatment of the cells with SB203580, a specific inhibitor of p38 MAPK, inhibited the invasion of the cells. The growth of the cells, as observed from the growth curves, was not affected by the treatment of PMA and/or SB203580. CONCLUSION: Activation of p38 MAPK signal transduction pathway may enhance the invasion capability of JAR cells, and p38 MAPK inhibition may therefore yield new possibility to control the invasion of choriocarcinoma.

Effect of polysaccharide Krestin on the up-regulation of macrophage colony-stimulating factor gene expression in protecting mouse peritoneal macrophages from oxidative injury.

Oxidative injury caused by oxidatively modified low density lipoprotein (Ox-LDL) plays an important role in the transformation of macrophages into foam cells and atherogenesis. Treatments to protect macrophages from oxidative injury will be effective in treating atherosclerosis. A macrophage-specific growth factor, macrophage colony-stimulating factor (M-CSF), was reported to be able to prevent the progression of atherosclerosis in Watanabe heritable hypercholesterolemic (WHHL) rabbits. A protein-bound polysaccharide, polysaccharide Krestin (PSK), was also proven to have effects in preventing atherosclerosis in our previous work. We proposed that, both M-CSF and PSK could protect macrophages from oxidative injury, and the effects of PSK were associated with its capability of inducing M-CSF expression. In our present results, M-CSF could alleviate the Ox-LDL- or tert-butyl hydroperoxide (tbOOH)-induced injury to mouse peritoneal macrophages, and PSK exhibited some similar effects. PSK treatment could induce M-CSF gene expression and secretion in mouse peritoneal macrophages. Furthermore actinomycin D and cycloheximide could attenuate that induction. We concluded that, maybe PSK exerted its effects on macrophages partly through the transcriptional induction of M-CSF in the cells.