The Effects And Mechanisms Of Pak4 On Proliferation,migration And Invasion And Of Esophageal Squamous Cancer Cells

Objective According to the 2017 China Cancer Registration Annual report,about 10,000 people are diagnosed with cancer every day in China,and about seven people are diagnosed with cancer every minute.The leading causes of death are lung cancer and digestive system cancers.Significant geographical distribution differences are prominent features of its epidemiology.Esophageal cancer is one of the six most common malignancies in the world.The incidence and mortality of esophageal cancer in China are the highest in the world,in which esophageal squamous cell carcinoma is the most common.Henan Province is a high incidence area of esophageal squamous cell carcinoma in China with high incidence and mortality.Esophageal squamous cell carcinoma is a serious threat to people’s life and health,so problems related to esophageal squamous cell carcinoma need to be resolved! There are many causes of esophageal squamous cell carcinoma,and their relationships are complicated.Many studies have found that it is associated with the activation of multiple signal pathways,but the mechanism of its occurrence and development is still unclear.It is well known that human life requires the co-regulation of many signaling pathways,including cell growth,differentiation,apoptosis,migration,cytoskeleton rearrangement,and so on.According to many studies,Paks locate at the junctions of these signaling pathways and have multiple biological functions.Paks are p21-activated serine/threonine protein kinases family that can be activated by Rac1 and Cdc42 in the small G protein Rho family.Paks consist of 6 members,Pak1-6.Among the six family members,Pak1 and Pak4 are most studied.According to lots of studies,Pak1 and Pak4 are found to be highly expressed in many tumor tissues,and Pak4 is the most closely associated member of the Paks with human tumors.As many as 78% of the more than 100 human tumor cells have been detected overexpressed Pak4.The human pak4 gene is located at chromosome 19q13.2 and is 3064 bp in length,encoding 591 amino acids.Its relative molecular mass is 72000 Da.The difference between the expression of Pak4 and other family members with tissue specificity is that Pak4 is widely expressed in embryonic and adult tissues.It can be phosphorylated by itself and activated by exogenous kinases through phosphorylation and methylation.Previous studies have shown that Pak4 may be involved in several signals,such as Pak4/MEK/ERK,Pak4/PI3K/AKT/m TOR,Pak4/LIMK1/confilin,etc.These pathways regulate the proliferation,migration and invasion of tumor cells,and affect the occurrence,development and prognosis of tumors.Multiple researches indicate that Pak4 plays an important role in regulating breast cancer,liver cancer,bladder cancer,prostate cancer,and other cancers.However,the roles and mechanisms of Pak4 in esophageal squamous cell carcinoma are not clear.This study will investigate the expression of Pak4 and p-Pak4 in esophageal squamous cell carcinoma tissues and esophageal squamous cancer cells by using human esophageal squamous cell carcinoma tissue microarrays and different types of human esophageal squamous cancer cells;by small RNA interference and c DNA transfections to study the effects of Pak4 on the proliferation,migration and invasion of esophageal squamous cancer cells;to further clarify the roles of Pak4 in the development of esophageal squamous cell carcinoma through nude mouse tumorigenesis in vivo;to explore the mechanisms of Pak4 on proliferation,migration and invasion of esophageal squamous cancer cells by western blotting.These results will provide a certain experimental and theoretical bases for Pak4 as a new target for the clinical treatment of patients with esophageal squamous cell carcinoma.Part one: Investigate the expression of Pak4 and p-Pak4(S474)in esophageal squamous cell carcinoma tissues and esophageal squamous cancer cells.Methods1.Immunohistochemistry was used to detect the expression levels of Pak4 and p-Pak4(S474)in esophageal squamous cell carcinoma tissues.2.Western blotting was used to detect the expression of Pak4 and p-Pak4(S474)in esophageal squamous cancer cell lines and normal esophageal epithelial cells.Results Pak4 and p-Pak4 are highly expressed in esophageal squamous cell carcinoma tissues and esophageal squamous cancer cells compared with normal esophageal tissues and normal esophageal epithelial cells.Part two: Investigate the effects of reduced expression of Pak4 on proliferation,migration and invasion of esophageal squamous cancer cells.Methods 1.Seven PLKO.1 plasmid vectors that contain different sh RNA sequences of pak4 were constructed.2.Lentivius infection was used to knock down the expression level of Pak4 in esophageal squamous cancer cells,ECA109 and Kyse450,which have relatively high Pak4 expression.Western blotting was used to identify the efficiency of Pak4 knockdown in stable cell lines.3.Cell proliferation assay was used to investigate the changes of proliferation ability of esophageal squamous cancer cells after the decrease of Pak4 expression.4.Soft Agar clone formation assay was used to observe the changes of clone formation ability of esophageal squamous cancer cells after the decrease of Pak4 expression.5.Wound healing assay was used to investigate the changes of cell migration ability of esophageal squamous cancer cells after the decrease of Pak4 expression.6.Transwell invasion assay was used to check the changes of cell invasion ability of esophageal squamous cancer cells after the decrease of Pak4 expression.Results Pak4 is involved in the proliferation,migration and invasion of esophageal squamous cancer cells.Reduction of Pak4 expression will inhibit proliferation,clone formation,migration and invasion of esophageal squamous cancer cells.Part three: Investigate the effects of increased expression of Pak4 on the proliferation,migration and invasion of esophageal squamous cancer cells.Methods 1.The c DNA of pak4 was transfected into two kinds of esophageal squamous cancer cells,Kyse30 and Kyse150,which have relatively low Pak4 expression.Western blotting was used to identify the efficiency of Pak4 overexpression.2.Cell proliferation assay was used to investigate the changes of proliferation ability of esophageal squamous cancer cells after the increase of Pak4 expression.3.Soft Agar clone formation assay was used to observe the changes of clone formation ability of esophageal squamous cancer cells after the increase of Pak4 expression.4.Wound healing assay was used to investigate the changes of cell migration ability of esophageal squamous cancer cells after the increase of Pak4 expression.5.Transwell invasion assay was used to check the changes of cell invasion ability of esophageal squamous cancer cells after the increase of Pak4 expression.Results Overexpression of Pak4 will increase proliferation,clone formation,migration and invasion of esophageal squamous cancer cells.Pak4 is involved in the proliferation,migration and invasion of esophageal squamous cancer cells.Part four: The roles of Pak4 in esophageal squamous cell carcinoma were verified in vivo.Methods According to the previous results,three stable cell lines ECA109-shmock and ECA109-shpak4-1 and ECA109-shpak4-2 were selected for tumorigenesis in nude mice.The changes of tumor weight and tumor volume in nude mice after the decrease of Pak4 were observed.Results The tumorigenic time of ECA109-shmock is 4 days,the tumorigenic time of ECA109-shpak4-1 and ECA109-shpak4-2 are 9 days and 6 days respectively,and the tumor weight and volume of ECA109-shpak4-1 and ECA109-shpak4-2 were smaller than ECA109-shmock.The differences are statistically significant(P<0.05).Part five: Explore the mechanisms of Pak4 on proliferation,migration and invasion of esophageal squamous cancer cells.Methods Western blotting was used to detect the mechanisms of Pak4 on proliferation,migration and invasion of esophageal squamous cancer cells.Results Pak4 is involved in ERK,AKT and EMT signaling pathways in esophageal squamous cancer cells.Conclusions 1.Pak4 and p-Pak4 are highly expressed in esophageal squamous cell carcinoma tissues and esophageal squamous cancer cells compared with normal esophageal tissues and normal esophageal epithelial cells.2.Pak4 promotes the proliferation,migration and invasion of esophageal squamous carcinoma cells,and its effect may be achieved through the regulation of ERK,AKT and EMT signaling pathways.