| Backgroud and ObjectionHOXA6 gene is a member of A cluster of HOX family,located in the 7 q15.2.It contains two exons and mainly presents in the normal endometrium tissue,kidney tissue and adipose tissue(https://www.ncbi.nlm.nih.gov/gene/3203).It is important transcription factors and its coding protein presents in the three-dimensional configuration,spiral Angle-spiral.Its main channel of DNA double helix plays a regulatory role in the process of cell DNA transcription.HOXA6 gene participates in important roles in blood cancer,colorectal cancer and many other cancer oncogene's role.However,the function of HOXA6 in the development of gastric cancer remains unclear.As a consequence,further research on this aspect is needed.We found that HOXA6 protein may interact with PBX2 to play a vital role as a carcinogenic factor using bioinformatics method.PBX2 also is a transcription factor.Numerous studies have shown that PBX2 is significantly higher than the corresponding normal tissue,and it can promote the proliferation,invasion and metastasis of tumors.Whether HOXA6 can interact with PBX2 or not,the molecular mechanism of HOXA6 and PBX2 in gastric cancer metastasis remains unclear.The study aims to clarify the role of HOXA6 in Gastric Cancer cells,biological function of HOXA6 in the invasion and metastasis potential of gastric cancer cells.How HOXA6 acts on PBX2 through the probable pathway?The study may find specific molecular mechanisms in the invasion and migration of gastric cancer cells and provide new important clues and possible drug targets for the diagnosis and treatment of gastric cancer.Methods:1.The expression of HOXA6 in gastric cancer was observed:10 pairs of gastric cancer and adjacent normal gastric mucosa tissues from the same patient were collected;Gastric cancer cell lines(N87,SGC7901,AGS,BGC823,KATOIII,AGS,Snul and MKN28)and control cells(GES-1)were used to extract total proteins,and the expression of HOXA6 was detected by Western blot.2.The relationship between HOXA6 expression and clinicopathological characteristics of gastric cancer patients:The expression of HOXA6 in normal tissues and primary gastric cancer tissues was observed by immunohistochemical staining with tissue microarray.Clinicopathological data were collected to analyze the relationship between the low and high expression of HOXA6 in gastric cancer tissues and the clinicopathological characteristics.The relationship between HOXA6 expression and patient prognosis:Immunohistochemical staining and score were performed,and Kaplan-Meier survival curve was used to observe the survival status of patients with primary gastric cancer with HOXA6(+)or high expression/HOXA6(-)or low expression,so as to reveal the relationship between HOXA6 expression and patient prognosis.3.The influence of HOXA6 expression on cell proliferation:EDU staining,CCK8 method and plate clone formation assay were used to detect HOXA6 expression and cell proliferation of gastric cancer cells.The effects of HOXA6 on the morphology of gastric cancer cells:cell morphology was observed under inverted or electron microscopy;The distribution of F-actin filaments was observed under fluorescence microscopy or confocal laser scanning microscopy.The effects of HOXA6 expression on tumor cell adhesion,invasion and metastasis were evaluated scratch test,Transwell and Matrigel invasion chamber were used to observe whether HOXA6 could promote the invasion and metastasis of gastric cancer cells.4.Construct and identify the lentiviral vectors with overexpression and shRNA of HOXA6 gene:The overexpression of HOXA6 gene or RNA interference lentiviral vectors were constructed and transfected into gastric cancer cells to establish stable transfected cell lines;The expression of target protein was determined by Western blotting.The effects of HOXA6 on invasion and metastasis in vivo:First,the tail vein lung metastasis model of nude mice was established to observe the size and number of metastatic tumour formation in vivo with HOXA6 siRNA.The expressions of HOXA6 and E-cadherin were observed by Q-RT-PCR.At the same time,paraffin specimens were made and the expressions of HOXA6 and E-cadherin were observed by immunohistochemistry.5.The binding interaction of HOXA6 and PBX2 in cells:(1)Bioinformatics(https://string-db.org/cgi/input.pl)found that the binding effect of HOXA6 and PBX2 in cells,the interaction between HOXA6 and PBX2 in cells was observed by immunoprecipitation,and the expression of HA(PBX2)or FLAG(HOXA6)was detected by Western blot.PBX2 affects the stability of HOXA6,and the changes in the protein level of overexpression of PBX2 or interference efficiency of HOXA6 were detected.To evaluate the correlation between the expression of PBX2 and HOXA6 in gastric cancer:(1)After protein extraction from various gastric cancer cell lines,the expression levels of PBX2 and HOXA6 were detected by Western blot;(2)Expression of PBX2 and HOXA6 was detected by immunohistochemistry.Two proteins were stained in gastric mucosa and gastric cancer tissues of 20 patients with adjacent cancer,and Spearman test was used for correlation analysis.6.To describe the effect of low expression of PBX2 on HOXA6-induced invasion and metastasis:transient transfection of PBX2-siRNA or Scrambled-siRNA in HOXA6-expressing stable cell lines,and transient transfection of HOXA6-siRNA or Scrambled-siRNA in PBX2-expressing stable cell lines were conducted to observe the change of invasion and metastasis ability of tumour cells by invasion and wound healing experiments,respectively.7.The effect of HOXA6 expression on PBX2-induced metastases:PBX2 shRNA or control shRNA were transfected into stable cell lines with overexpression of HOXA6 to construct stable cell lines with high expression of HOXA6 and low expression of PBX2.The model of lung metastasis in the tail vein of nude mice was established,and the expressions of HOXA6,PBX2 and E-cadherin were observed by Q-RT-PCR method.Meanwhile,the expression of HOXA6,PBX2 and E-cadherin were observed by immunohistochemistry.Results:1.The expression of HOXA6 protein in gastric cancer tissues was higher than that in adjacent normal tissues,and the expression of HOXA6 protein in gastric cancer cell lines was significantly higher than that in normal gastric mucosa cell lines,2.The up-regulation of HOXA6 expression was closely related to differentiation,lymph node metastasis,AJCC stage,TNM stage and adverse survival outcome in gastric cancer patients,but was independent of age and gender.3.Overexpression of HOXA6 can promote the proliferation,migration and invasion of tumour cells;On the contrary,HOXA6 silencing inhibited the proliferation,migration and invasion of tumour cells.4.HOXA6 can interact with PBX2 and mutually stabilize.The expressions of HOXA6 and PBX2 were positively correlated in gastric cancer cells and tissues.5.HOXA6 and PBX2 have co-in the invasion and metastasis of gastric cancer cells.Silencing HOXA6 and PBX2 can both lead to the decrease of in vitro migration and invasion ability.6.In vivo,HOXA6 can promote the invasion and metastasis of the lung of gastric cancer cells,while silencing PBX2 can inhibit its effect.CONCLUSIONS:The expression of transcription factor HOXA6 is significantly up-regulated in gastric cancer.Patients with high HOXA6 expression are closely related to adverse survival outcomes.HOXA6 plays a role as an oncogene.Express HOXA6 can make gastric cancer cells show more phenotype of interstitial cells,promote cell proliferation,migration and invasion,and HOXA6 PBX2 synergy and make its stability,HOXA6-PBX2 shaft may be an important target in gastric cancer development,may offers a new important clues for diagnosis and treatment of gastric cancer and possible targets for drug treatment. |
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