Role Of HOXB7in Regulation Of Progression And Metastasis Of Human Lung Adenocarcinoma

IntroductionLung cancer is one of the most frequent malignant tumors in the world, and theincidence of lung adenocarcinoma (LAC) is the first one in all types of lung cancer, havingbeen reached about40%.Many clinical datas indicated that fast progress, high rate ofrecurrence and metastasis ability of LAC is the main reason for the clinical treatment failureand poor prognosis of LAC patients, about90%of LAC patients died of multi-organmetastasis. So far, the risk factors for LAC incidence have been identified, but themechanisms about its rapid progression and metastasis are still unclear. Therefore, findingout the mechanisms of regulation of invasion and metastasis about LAC is very importantfor improving the survival rate of LAC patients.Some studies have confirmed that some homeobox molecules such as Oct4, Nanog areinvolved in the regulation of lung cancer metastasis. And the homeobox gene family (HOX)have been paid more and more attention. HOX gene family is consists of39highlyconserved homeodomain genes,which are divided into four clusters HOXA, HOXB, HOXCand HOXD. HOX gene family is mainly responsible for the regulation of vertebrateanimals and human embryonic organ development. As the master genes for cellproliferation and differentiation, its target protein are usually some membrane proteinreceptors, adhesion molecules and growth factors.Among all the HOX genes, HOXB7is an important gene during these years. HOXB7is a gene which belongs to the B clusters of HOX genes family and locates on chromosome17. During the period of embryonic development, HOXB7is primarily responsible for theregulation of proliferation,differentiation and migration of lung epithelial cells. Currently,some studies found that overexpression of HOXB7has been observed in colon cancer andoral cancer,which promotes the aberrant tumorigenesis and metastases.In breast cancer,abnormally high expression of HOXB7induced EMT and affected tumor metastasis. But there are no report on the expression of HOXB7gene in LAC, and whether its expressionparticipate in the regulation of progression and metastasis of LAC cells.In this study, we frist detected the expression of HOXB7in surgical specimens fromLAC patients and LAC cell lines.Then we compared its expression with the clinicalpathological variables and found out the significance of HOXB7expression in LAC.Futhermore,we used the RNA interference silence HOXB7gene expression and investigated therole of HOXB7in regulation of progression and metastasis of LAC through a series ofexperiment in vivo and in vitro.Methods1.The expression of HOXB7in surgical specimens of LAC patients and LAC celllines,and its correlation with clinical pathological variables.①A total of75pairs of primary LAC tumor tissues and corresponding normal lungepithelium tissues were obtained from the LAC patients who underwent a surgical resection.All the samples were used to construct a tissue microarray.Using Immunohistochemicalstaining analyze HOXB7protein expression and its correlation with clinical pathologicalvariables.②Using RT-PCR and western blot detected the expression of HOXB7in normallung bronchial epithelial cell line HBE and4LAC cell lines A549, H1975, H1650, H322.2. Investigate the role of HOXB7in regulation of proliferation capacity of LAC cellsin vitro and in vivo.In A549cells, silenced HOXB7gene expression with retrovirus-mediated RNAinterference and do the following research:①Using colony formation assay to detect thedifferences of proliferation capacity between the control group and HOXB7RNAi group.②Using flow cytometry assay to check the differences of cell cycle distribution betweenthe control group and HOXB7RNAi group.③Control group and HOXB7RNAi groupcells were subcutaneously injected into6-week-old female nude mice (five mice eachgroup).All mice were kept under specific pathogen-free conditions.Tumor volumes weremeasured every3days and calculated as [larger diameter*(smaller diameter)2]/2. After30days, all the mice were sacrificed.Xenograft tumors were fixed, embedded and stained withhaematoxylin and eosin or immunohistochemistry stained with antibodies against HOXB7and Ki-67. 3. Investigate the role of HOXB7in regulation metastasis capacity of LAC cells invitro and in vivo.①Using Western blot to detect the expression of epithelial markers such as E-cadherin and mesenchymal markers such as vimentin and Fibronectin between the controlgroup and HOXB7RNAi group.②Using immunofluorescence staining to detect theexpression of epithelial markers such as E-cadherin and mesenchymal markers such asvimentin and Fibronectin between the control group and HOXB7RNAi group.③Using thescratch wound healing and transwell migration assay to detect migration ability betweenthe control group and HOXB7RNAi group in vitro.④Control group and HOXB7RNAigroup cells were intraperitoneally (i.p.) injected into6-week-old female nude mice (10miceeach group). After35days, the mice were sacrificed and dissected. The metastatic statewere analyzed.Results1.mRNA and protein levels of HOXB7in all four LAC cell lines were significantlyhigher than in HBE cells.Among these cell lines, including A549, H1975,H1650, and H322,the highest expression of HOXB7protein was observed in A549cells.2. Out of the75tissue samples, HOXB7was highly expressed in54(72.0%) tumortissues,but only in seven (9.3%) adjacent normal tissues. Increasing HOXB7expression wasobserved with the progression of the tumor. Statistical analysis revealed that HOXB7protein expression was correlated with the tumor status (P=0.028),nodal status (P=0.012),and the tumor stage (P=0.029) in LAC. Kaplan–Meier survival analysis showed thatincreased expression of HOXB7was associated with poor clinical outcomes in LACpatients.3. Lentivirus encoding short hairpin RNAs(shRNA) targeting HOXB7was used toinfect A549cells, which has the highest expression of HOXB7of the four LAC cell lines.HOXB7RNAi group cells grew much slower and formed less and smaller colonies than thecontrol group cells. Flow cytometry analysis revealed that compared to the control groupcells, the number of HOXB7RNAi group cells at the G1phase was increased,whereas thatnumber at the S phase decreased,respectively. These results suggested that HOXB7plays acritical role in regulation of G1/S cell cycle checkpoint, which controls proliferation of LAC cells.4.The size of tumors formed from HOXB7RNAi group cells was much smaller thanthe tumors formed from control group cells in nude mice at30days post-injection.IHCanalysis of the dissected xenograft tumors revealed that the expressions of HOXB7and Ki-67, a cellular marker for proliferation,in tumors form HOXB7RNAi group cells were lowerthan their expression in tumors form control group cells.5. HOXB7RNAi group cells had a spindle-like mesenchymal morphology, controlgroup cells had a cobblestone-like epithelial morphology. HOXB7RNAi group cells alsohad a smaller cell size and formed a highly clustered pattern at48h after seeding. Theexpression of epithelial marker E-cadherin was increased, the expressions of mesenchymalmarkers fibronectin and vimentin were markedly reduced in HOXB7RNAi group cells. Thechanges of EMT markers were confirmed by Immunofluorescence staining followed byconfocal microscopy analysis.The HOXB7RNAi group cells healed the wound-scratchmuch slower than the control group cells. Less HOXB7RNAi group cells than controlgroup cells migrated from the upper chamber to the bottom surface of boyden chamber at24h post-seeding. These results suggested that HOXB7regulates the migratory ability ofLAC cells in vitro.6. The mice injected (i.p.) with HOXB7RNAi group cells had much less ascitic fluidin the peritoneal cavity than the ones injected (i.p.) with control group cells. The incidencesof metastases of organs in mice injected with HOXB7RNAi group cells were much fewerthan that in the mice injected with control group cells.Conclusion1. HOXB7expression might play a critical role in regulation of malignantprogression and metastasis of LAC.2. HOXB7plays a critical role in regulation of G1/S cell cycle checkpoint, whichcontrols proliferation of LAC cells in vitro and in vivo.3. HOXB7regulates the migratory ability of LAC cells in vitro and in vivo.4. HOXB7could be an independent prognostic factor for LAC and an additionalmarker for determination of LAC malignant progression.