The Role And Molecular Mechanism Of HOXB7 In Tumorigenesis And Progression Of Gastric Cancer

Gastric carcinoma(GC) is the fourth most frequent cancer worldwide and the second most frequent cause of cancer-related deaths in China. The number of Global new gastric cancer was about one million per year, China accounted for as much as 40%. More seriously, the domestic incidence and mortality were more than double of the average level of the world, and the age of patients tend to be younger in recent years.With the cognition to the standardization of diagnosis and treatment of gastric cancer and promotion, despite the improvement in surgical techniques, adjuvant therapy and patient management, this cancer is still highly lethal and little progress has been achieved in improving the overall survival of gastric carcinoma patients. The advances in suitable therapy to increase the survival rate have been limited because the pathophysiological mechanisms of tumorigenesis are not fully understood. Therefore, it is necessary to elucidate the molecular mechanism of GC to ensure early diagnosis and develop an effective therapy.HOXB7 is one member of the homeobox genes family, which are essential for the regulation of morphogenesis and differentiation, acting as a family of transcriptional factors. As a highly conserved subgroup of the homeobox superfamily, homeobox genes targets on adhesion molecules, membrane protein receptors and growth factors in charge of proliferation, survival, migration, and invasion of cells, depending on the cell type, and then produce huge impact on tumorigenesis and tumor progression. At the same time, the HOX genes can conduct regulating effect on the related characteristics of cancer stem cells such as self-renew, infinite proliferation malignant biological behavior. So far, it has been reported that there are abnormal expression of HOX genes in breast cancer, colorectal cancer and so on. HOXB7, located on 17 chromosome has been proved an oncogene. It has been proved to activate forms of both Ras and RhoA proteins via up-regulating basic basic fibroblast growth factor (bFGF) and then to cause epithelial-mesenchymal transition(EMT) and further metastases of breastcells. However, biological functions of HOXB7 in the control of GC tumorigenesis and progression have not been well characterized.In this study, we firstly determined the ectopic expression of HOXB7 in gastric cancer patient, and found out the clinical evaluating significance according to the expression degree combining with the clinical characteristics. Secondly, we knock down HOXB7 exprssion with siRNA and lentivirus to observe whether HOXB7 has tumor promoing effect in gastric cancer in vivo and in vitro. In addition, we also testify the potential downstream targets of HOXB7 by cDNA microarray assay and qPCR and western blot validation. The regulation of PI3K/AKT signaling pathways by HOXB7 was further investigated.Material and Methods1. The expression and its clinical significance of HOXB7 in gastric cancer① The expression of HOXB7 mRNA in 96 paired surgical samples from gastric cancer and normal stomach tissues were determined by QT-PCR, and IHC detect the HOXB7 in paired cancer tissue.② HOXB7 expression in a panel of gastric cancer cell lines were examined by QT-PCR and Western blot analysis.③ HOXB7 expression and the clinical characteristics of high and low group were analyzed.2. The fuction of HOXB7 in gastric cancerin vivo and in vitro.① The cell proliferation was confirmed by MTS and Edu, cell apoptosis and cell-cycle were assessed by flow cytometry. Cell migration and invasion were investigated in the 24-transwell cell migration and collagen-based cell invasion system, while expression of HOXB7 knocked down with siRNA.② Xenografted gastric tumor model was used to validate the tumor suppressing effect by ectopic expression of HOXB7 in vivo with lentivirus.3. Screening and validation of the downstream targets of HOXB7① cDNA microarray and QT-PCRvalidation analysis were performed in BGC-823 and SGC-7901 cells transfected with siRNA.② p21, cyclinD1, PIK3R3, AKT were further determined by western blot, and we infer HOXB7 plays its role through PI3K/AKT signaling pathway in gastric caner.Results1. HOXB7 expression was significantly upregulated in tumor tissues relative to normal gastric tissues, and had its own clinical significance.① HOXB7 expression was obviously upregulated in tumor tissues relative to paired normal gastric tissue(P<0.01).② HOXB7 expression was upregulated in gastic cell lines, such as MKN45, BGC-823, MGC-803, SGC-7901, while downregulated in th AGS and HGC-27 cell lines.③ HOXB7 expression was relevant to tumor diffenrentiation(P=0.02), TNM stage(P=0.02), with exception of sex, age, volume and location of tumor. There was no correlation statistically with patients survival time according to the data.2. H0XB7 played a critical roles in tumorigenesis and progression of gastric cancer.① HOXB7 knockdown in BGC-823 and SGC-7901 resulted in decreased migration and invasion with alteration of EMT proteins, and influenced proliferation, apoptosis and cell cycle.② HOXB7 could promote tumor growth with xenografted gastric tumor model.3. HOXB7 modulated multiple downstream genes and took part in PI3K/AKT signaling pathway.① There were more than 200 downstream genes changed up to 2 fold after HOXB7 knockdown, including 60 up-regulated genes and 158 down-regulated genes in SGC-7901, while 149 up-regulated genes and 231 down-regulated genes inBGC-823.② Several genes were testified with QT-PCR including NRXN3、NFAT5、KRAS、 Smad2、WNT5A, and the resultswereaccordant withcDNAmicroarray.③ p21、CyclinD1、PIK3R3、pAKT and AKT protein expression were testified with western blot. The ectopic expression of HOXB7 probabley activate PI3K/AKT signaling pathway.Conclusions1. HOXB7 is generally overexpressed in GC, associated with patient clinical characteristics, such as tumor differentiation and TNM stage, but there was no correlation statistically with survival analysis.2. HOXB7 knockdown in BGC-823 and SGC-7901 influenced proliferation, apoptosis and cell cycle, and resulted in decreased migration and invasion with alteration of EMT proteins. The knockdown expression also suppresses tumor growth in a mouse xenograft model. HOXB7 functions as a candidate oncogenein gastric cancer.3. The impact of HOXB7 on biological change of gastric cancer cells altered expression of p21, cyclinDl and other downstream genes through activation PI3K/Akt signaling pathways.