MYH10 Combines with MYH9 to Recruit USP45 by Deubiquitinating Snail and Promotes Serous Ovarian Cancer Carcinogenesis, Progression, and Cisplatin Resistance.

The poor prognosis of serous ovarian cancer (SOC) is due to its high invasive capacity and cisplatin resistance of SOC cells, whereas the molecular mechanisms remain poorly understood. In the present study, the expression and function of non-muscle myosin heavy chain IIB (MYH10) in SOC are identified by immunohistochemistry, in vitro, and in vivo studies, respectively. The mechanism of MYH10 is demonstrated by co-immunoprecipitation, GST pull-down, confocal laser assays, and so on. The results show that the knockdown of MYH10 suppressed SOC cell proliferation, migration, invasion, metastasis, and cisplatin resistance both in vivo and in vitro. Further studies confirm that the MYH10 protein functional domain combines with non-muscle myosin heavy chain IIA (MYH9) to recruit the deubiquitinating enzyme Ubiquitin-specific proteases 45 and deubiquitinates snail to inhibit snail degradation, eventually promoting tumorigenesis, progression, and cisplatin resistance in SOC. In clinical samples, MYH10 expression is significantly elevated in SOC samples compared to the paratumor samples. And the expression of MYH10 is positively correlated with MYH9 expression. MYH10+/MYH9+ co-expression is an independent prognostic factor for predicting SOC patient survival. These findings uncover a key role of the MYH10-MYH9-snail axis in SOC carcinogenesis, progression, and cisplatin resistance, and provide potential novel therapeutic targets for SOC intervention.

[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.

[Role of hydatidiform mole-related gene F10 in the tumorigenicity of choriocarcinoma cell line JEG-3].

OBJECTIVE: To explore the role of the hydatidiform mole-related gene F10 in the tumorigenicity of choriocarcinoma cell lines JEG-3 in nude mice. METHODS: Choriocarcinoma JEG-3 cell lines with stable F10 gene over-expression and F10 gene silencing were established using cell transfection and RNA interference techniques, respectively. Thirty SPF nude mice (4-5 weeks old) were equally randomized into F10 over-expression group, control group, and F10 gene-silenced group for subcutaneous injection of 0.2 ml cell suspension (5 × 107 cells) of F10 gene over-expressing JEG-3 cells, non-treated JEG-3 cells, and F10 gene-silenced JEG-3 cells, respectively. The mice were observed and weighed every 3-4 days, and the tumor formation time was recorded to draw the tumor growth curve and calculate the tumor formation rate. RESULTS: The tumor formation rates were 100% in all the 3 groups. No significant difference was found in the tumor formation time among the F10 over-expression, F10-silenced and control groups (6.2 ± 0.78 vs 7 ± 2.49 vs 6.3 ± 0.67 days; F=0.781, P=0.468). A significantly greater tumor growth rate was noted in the F10 over-expression group compared with the other two groups (P<0.05), and the growth rate was significantly slower in F10-silenced group than in the control group (P<0.05). The subcutaneous tumor weight at 5 weeks after JEG-3 cell injection differed significantly among F10 over-expression, F10-silenced and control groups (571.1 ± 221.10 vs 136.2 ± 66.25 vs 354.5 ± 116.23 mg; F=21.199, P=0.000). CONCLUSION: F10 gene plays a role in the regulation of choriocarcinoma JEG-3 cell proliferation and might enhance its tumorigenicity in nude mice.

Dysregulated expression of matrix metalloproteinases and their inhibitors may participate in the pathogenesis of pre-eclampsia and fetal growth restriction.

BACKGROUND: Trophoblast invasion into the maternal endometrium serves an important function in human pregnancy. Dysregulation of the finely controlled process of trophoblast invasion can result in a wide spectrum of pregnancy abnormalities. AIMS: We aimed to elucidate the relationship between the expression of matrix metalloproteinases and pregnancy complication. STUDY DESIGN: The study group consisted of placental bed biopsy tissues obtained from normal vaginal deliveries (N=15), normal cesarean deliveries (N=15), pre-eclampsia (N=24) and fetal growth restriction (FGR) (N=10). We evaluated the expressions of MMP-2, -8, -9, -11, -19, -15 (MT2-MMP), -16 (MT3-MMP), and -24 (MT5-MMP), as well as TIMP-1 and -3, by applying Western blot and immunohistochemistry methods. SUBJECTS: Human placental tissues were used for this study. OUTCOME MEASURES: The expressions of MMP-2, -8, -9, -11, -19, -15 (MT2-MMP), -16 (MT3-MMP), and -24 (MT5-MMP), as well as TIMP-1 and -3 in human placenta tissues. RESULTS: Compared with those in normal pregnancies, the expression of MMP-2, -8, -9 and -11 was downregulated in villous tissues of pre-eclampsia and FGR cases (p<0.05). TIMP-1 and -3 were increased in pre-eclampsia and FGR (p<0.05). No significant difference was found between normal vaginal deliveries and cesarean deliveries. CONCLUSIONS: We speculate that the change in invasion-associated proteinase expression will affect placental development and may thus contribute to the development of complicated pregnancies.

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.

Overexpression of the hydatidiform mole-related gene F10 inhibits apoptosis in A549 cells through downregulation of BCL2-associated X protein and caspase-3.

The aim of this study was to investigate how the overexpression of the hydatidiform mole-related gene F10 affects apoptosis in human lung cancer A549 cells. A549 cells were transfected with pEGFP-N1-F10 (A549-F10) or pEGFP-N1 empty vector (A549-empty). Untransfected A549, A549-F10 or A549-empty cells were examined using the MTT cell proliferation assay and the TUNEL-FITC/Hoechst 33258 apoptosis assay. Western blotting was used to examine the expression levels of the pro-apoptotic genes, BCL2-associated X protein (BAX) and caspase-3. F10 was stably expressed in A549 cells. From 12 h, A549-F10 cells proliferated markedly faster than the untransfected and A549-empty cells. F10 overexpression also significantly inhibited apoptosis, as shown by the reduced number of TUNEL and Hoechst 33258 double-positive cells. This inhibition was likely due to an F10-induced reduction in the BAX and caspase-3 levels. The results of this study indicate that F10 overexpression inhibits apoptosis in A549 cells through the downregulation of the pro-apoptotic genes BAX and caspase-3.

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.