The Effects Of Homeobox Gene HOXB7 On The Proliferation And Invasion Of Ovarian Carcinomas

Ovarian cancer is the leading cause of death among gynecologicalmalignancies, mainly because of introperitoneal diffusion of cancer cells.The molecular mechanisms involved in the progression of ovarian cancerare unclear, which has hampered the development of an effective preventionand treatment.Homeodomain protein HOXB7 is a recently found transcriptional factor.It has been reported that HOXB7 plays an important role in oncogenesis,but little is known about the relationship between HOXB7 and ovariancarcinogenesis. In this article, we investigated theHOXB7 expression innormal ovarian tissue, benign and borderline ovarian serous cystadenoma,serous and clear cell ovarian adenocarcinoma. We interfered HOXB7 geneexpression by siRNA in ovarian cancer cell lines SKOV3 and ES-2, andsubsequently, Western blot, RT-PCR, immunohistochemistry, flowcytometric assessment, gelatin zymography, matrigel invasion assay andmotility assay were applied to explore the effects of HOXB7 on ovariancancer cells proliferation and invasion.These experiments are divided into 3 parts, as following: (1) Expressionof HOXB7 in ovarian tumors (2) The effects of HOXB7 on proliferation andapoptosis in ovarian cancer cells (3) The effects of HOXB7 on motilityand invasiveness in ovarian cancer cells.SectionⅠExpression of HOXB7 in ovarian tumorsObjective To examine the expression of HOXB7 in normal ovarian, benignand borderline ovarian serous cystadenoma, ovarian serousadenocarcinoma, ovarian clear cell adenocarcinoma tissues. The relationship between the expression level of HOXB7 and normal, benign andmalignant serous ovarian tumors was analyzed.Methods Western blot was used to detect protein expressions of HOXB7 genein normal ovarian, serous adenocarcinoma and clear cell adenocarcinomatissues. Immunochemistry was used to investigate the expression of HOXB?protein in benign and borderline ovarian serous cystadenoma, ovarianserous adenocarcinoma, ovarian clear cell adenocarcinoma tissues.Results (1) The expression of HOXB7 protein was higher in ovarian serousadenocarcinoma, ovarian clear cell adenocarcinoma than that of normalovarian tissue(P＜0.05). (2) The expression level of HOXB7 protein wassignificantly higher in ovarian serous adenocarcinoma and ovarian clearcell adenocarcinoma than that of benign,borderline serous cystadenoma(P＜0.05). (3) HOXB7 gene expression level was positively correlated withthe stage and grade of ovarian cancers.Conclusions The expression of HOXB7 protein was higher in ovarian serousadenocarcinoma, ovarian clear cell adenocarcinoma than that of normalovarian, benign,borderline serous cystadenoma. HOXB7 gene expressionlevel was positively correlated with the stage and grade of ovariancancers.Overexpression of HOXB7 may be relatd to the tumorigenesis ofepithelial ovarian cancer.SectionⅡThe effects of HOXB7 on proliferation and apoptosisof ovarian cancer cellsObjective To investigate the effects of HOXB7 on proliferation andapoptosis of ovarian cancer cells and related mechanism of HOXB7 onproliferation.Methods RT-PCR and Western blot wereused to detect the mRNA and proteinlevel expressions of HOXB7 gene in ES-2 and SKOV3 cell lines. Accordingto the sequence of HOXB7 mRNA, three HOXB7 specific double-strand oligonucleotide and non-target were designed to interfere the expressionof HOXB7, and were cloned into pRNAT plasmid vectors. The recombinants weretransfected into ES-2 and SKOV3 cells. Inhibition of HOXB7 expression bysiRNA was confirmed by semi-qutitative RT-PCR and Western blot. Cellproliferation and cell apoptosis of ES-2 and SKOV3 before and afterinterfering HOXB7 expression were evaluated by flow cytometry and BrdUincorporation. We further investigated the expression of cyclin D1,cyclinE, which were critical in regulating cell proliferation.Results (1) Both ES-2 and SKOV3 cell lines expressed HOXB7 genes. (2)Three recombinant plasmids of HOXB7 siRNA and one negative plasmid, whichwere named pRNAT-hoxb1, pRNAT-hoxb2, pRNAT-hoxb3 and pRNAT-neg, wereconstructed successfully. pRNAT-hoxb2 and pRNAT-hoxb3 knocked downexpression of HOXB7 specifically and effectively. Comparing with negativecontrol groups, the levels of HOXB7 mRNA in cells transfected withpRNAr-hoxb2 and pRNAT-hoxb3 were inhibited (83.95±2.14)%and(50.62±4.60)% in ES-2 cells, (80.93±2.88)%and(59.18±3.87)% in SKOV3 cells,respectively, (P＜0.05).And the levels of HOXB7 protein weredown-regulated (83.95±0.16)% and (46.47±2.29)% in ES-2cells, (71.25±0.86)% and (51.90±3.07)% in SKOV3 cells, respectively, (P＜0.05).(3) Comparing with negative control groups, knocking down HOXB7expression by siRNA inhibited proliferation of ES-2 and SKOV3 cells.Proliferating rates of ES-2 cell were control(50.13±3.19)%, pRNAT-neg(47.77±2.90)%, pRNAT-hoxb2(28.31±2.88)%,respectively. Proliferatingrates of SKOV3 cell were control(35.40±1.95)%, pRNAr-neg (33.13±2.19)%,pRNAr-hoxb2(22.78±2.08)%, respectively. The difference of cellproliferation rates between siRNA groups and negative control groups wassignificant (P＜0.05). (4) HOXB7 siRNA also arrested cell cycle in G1 phase.(5) The expression of cyclin D1 and cyclin E were down-regulated afterHOXB7 interference. (6) HOXB7 siRNA also induced ES-2 and SKOV3 cellapoptosis.Conclusions These results demonstrate that inhibition of HOXB7 couldinhibit proliferation of ES-2 and SKOV3 cells,the mechanism might be due to transcriptional regulation on cyclin D1 and cyclin E. Inhibition ofHOXB7 could induce apoptosis of ES-2 and SKOV3 cells.SectionⅢThe effects of HOXB7 on motility and invasiveness ofovarian cancer cellsObjective To investigate the effects of HOXB7 on motility andinvasiveness of ovarian cancer cells and its mechanism.Methods ES-2 cell lines with high metastasic ability was used. ES-2 cellswere divided into 3 groups: non-transfected control group, no-specificsiRNA group transfected with unrelated siRNA (pRNAT-neg) and HOXB7siRNAgroup transfected with HOXB7 siRNA (pRNAT-hoxb2). The recombinantplasmids of pRNAT-hoxb2 and pRNAT-neg which were designed and constructedin sectionⅡwere transfected into ES-2 cells. The clones stablyinterfering HOXB7 gene expression were selected by G418. RT-PCR, Westernblot were used to confirm the effect of HOXB7 siRNA. The motility andinvasiveness of 3 groups were evaluated by motility assay, matrigelinvasion assay. Gelatin zymography and Western blot were used to detectthe expressions of E-cadherin, MMP-2, SNAIL in 3 group cells, which werecritical in regulating cell motility and invasiveness.Results (1) HOXB7 expression was significantly inhibited in HOXB7 siRNAgroup than those in non-transfected control group and no-specific siRNAgroup. (2) The motility and invasiveness in ES-2 cells stably expressingpRNAT-hoxb2 were decreased comparing with cells stably expressingpRNAT-neg or non-transfected cells (P＜0.01). (3) MMP-2 protein level andactivity were lower, E-cadherin expression was higher and SNAIL expressionwas lower in HOXB7 siRNA groupthan those in non-transfected control groupand no-specific siRNA group, (P＜0.05)Conclusions Our results demonstrate that HOXB7 might promote motilityand invasiveness of ovarian cancer cells, which might be associated with upregulation of E-cadherin and downregulation of MMP-2, SNAIL. Theunregulated expression of HOXB7 might play an important role in invasionand metastasis of ovarian cancer.Our research showed that homeodomain protein HOXB7 is over-expressed inovarian cancer, and HOXB7 over-expression is related to proliferation andinvasion in ovarian cancer cells. In the present study, we also examinedthe change of gene expression involved in HOXB7 down-stream, and primarilyexplained the mechanisms that HOXB7 regulated proliferation and invasion.Our research provides a new path for further studying the tumorigenesisand progression of ovarian cancer.