The Mechanism Of PIWIL2 In The Occurrence And Development Of Esophageal Cancer And Isolation And Identification Of Adipose Mesenchymal Stem Cells

The incidence of malignant tumors is ever-increasing around the world and seriously affect the health of the residents in our country and lead to a huge burden on medical treatment.Esophageal cancer is one of the most lethal malignancies,the incidence of which ranks eighth among all cancers and the death rate is fifth.Chia is a country with high incidence of esophageal cancer.More than 90%of the pathological types are Esophageal squamous cell carcinoma(ESCC).Therefore,the study of esophageal squamous cell carcinoma is of great significance for the health of Chinese residents.The tumor cells often have epigenetic variation and change their protein's library.In recent years,it has been found that tumor cells frequently activate genes that are limited to germ cells.These genes are expressed only in germ cells,but are activated by cancer cells to promote proliferation,metastasis and apoptosis.Therefore,these genes are known as cancer/testis antigens(CTAs).PIWIL2 protein is also a cancer/testicular antigen.PIWIL2 is a member of the Argonaute protein family.It is specifically expressed in the testicular tissue and is used to maintain the development and function of the germline stem cells.In recent years,studies have shown that PIWIL2 is highly expressed in many human and mouse tumor tissues and is closely related to the development of cancer.However,there is no clear research about how PIWIL2 affects cancer progression in esophageal cancer.Therefore,we will study the mechanism of PIWIL2 in the occurrence and development of esophageal cancer.We detected the expression of PIWIL2 in esophageal squamous cell carcinoma samples and explored the clinical significance of PIWIL2 in esophageal squamous cell carcinoma.We identified the mechanism of PIWIL2 which effect the phenotype of esophageal squamous cell carcinoma in vivo and in vitro.The results showed that the expression of PIWIL2 in the tissues of 40 esophageal squamous cell carcinoma was significantly higher than the adjacent tissue.At the same time,we confirmed the expression of PIWIL2 protein gradually increased in rats esophageal carcinoma model from the normal esophagus to esophageal cancer throughout the development process of esophageal cancer,indicating that PIWIL2 may be an important indicator of the occurrence and development of esophageal cancer.In vitro cell experiments showed that overexpression of PIWIL2 could enhanced the proliferation,invasion,drug resistance,and increase the proportion of tumor stem cells.At the same time,the same confirmation was obtained in the inhibiton of PIWIL2.Next,we confirmed that PIWIL2 activated PI3K/Akt signaling pathway and promoting cell proliferation.Moreover,PIWIL2 activated TGF-beta/Smad signaling pathway and activated matrix metalloproteinase MMP14?MMP9 to induce epithelial cell mesenchymal transition(EMT)and promoted cell invasion and metastasis.We also found that PIWIL2 can increase the expression of Sox2,Oct4,Nanog and Bmi-1 of the sternness transcription factors and affect the self renewal of tumor stem cells.To sum up,PIWIL2 plays a role in promoting cancer in the progression of esophageal squamous cell carcinoma,and may be a prognostic indicator and therapeutic target for esophageal squamous cell carcinoma.Mesenchymal stem cells(MSCs)are one of the adult stem cells that can be obtained from multiple tissues.The adipose derived mesenchymal stem cells can be obtained from autologous adipose,because of convenient collection,content rich,high yield,and can meet the requirement of tissue engineering and disease treatment,so it is widely used in clinical and scientific research.At present,the separation of MSCs is mainly by adherence.However,they are promiscuous groups.Cells may have different abilities of proliferation,differentiation and hematopoiesis support.Different MSCs subpopulations can be identified by using specific surface or intracellular markers.However,the current studies of the MSCs subpopulations are mainly focused on bone marrow MSCs,so the subpopulations study of adipose MSCs needs to be further developed.Adipose mesenchymal stem cells were isolated from human adipose tissue Flow cytometry and immunofluorescence showed that CD29,CD44,CD73,CD90 and CD105 were positive expressed,while CD31,CD34 and CD45 were negative.The mRNA expression level of Sox2,Oct4 and Nanog of sternness genes in adipose derived mesenchymal stem cells were significantly higher than that of fibroblast,esophageal cancer cell line KYSE150 and cervical cancer cell line Hela.MSC had the ability to differentiate into adipose,osteogenic and chondrogenic.The positive rate of CD 105 decreased gradually,while the other surface markers did not change.Therefore,we used CD 105 to seperate MSCs and further studied the difference between CD105(+)MSC and CD105(-)MSC.After morphological observation,CD105(-)MSC cells became worse attachment ability.The proliferation of CD105(-)MSC was slowed down,the population doubling time was longer and cultured only within 20 generations,while CD105(+)MSC could continue to proliferate to 38 generation.The level of mRNA expression of Sox2,Oct4 and Nanog of CD105(+)MSC gene was higher than those of CD105(-)MSC.Both CD105(+)MSC and CD105(-)MSC can differentiate into adipose and osteogenic differentiation.The karyotype analysis of MSCs are normal karyotypes,and no chromosome variation is found,which ensures the safety of its clinical application.We selected CD105 subpopulations of MSCs and implement the culture more long-term stability,and the karyotype analysis examined the safety,provide seed cells for clinical application and scientific research.