| Background and objectiveCholangiocarcinoma is a malignant tumor arising from the biliary epithelial system.Although with a low overall incidence,its incidence has increased progressively worldwide especially in Southeast Asian countries and China over the past decades.Cholangiocarcinoma is an aggressive tumor with late diagnosis and poor prognosis.Most patients have advanced-stage disease at presentation and there is a high relapse rate in the minority of patients who are amenable to undergo potentially curative surgery.Therefore,it is of great significance to study on the etiology and the mechanism of recurrence and metastasis of cholangiocarcinoma.PTEN is a tumor suppressor gene discovered in 1997.It has been found that PTEN mutations and deletions exist in various malignancies,such as prostate cancer,bladder cancer and lung cancer.And the decreased expression of PTEN contributes to tumor initiation and progression due to its role in PI3K-AKT-m TOR signal pathway.In this study,we have found that there is a certain proportion of PTEN mutations and deletions in cholangiocarcinoma which is related to the recurrence and metastasis.Exosomes,which are biological nano-vesicles of about 30-100 nm secreted by most of cells,play an irreplaceable role in cell-cell communication by delivering functional cargos such as proteins,lipids,polysaccharides,DNA and RNAs.It has been reported that tumor-derived exosomes participate in formation and progression of various cancers including glioblastoma,breast cancer,leukemia,nasopharyngeal carcinoma and pancreatic cancer.Exosomes released by tumor cells cause upregulation of angiogenesis relevant genes,which lead to the enhancement of endothelial cell proliferation,metastasis,and sprouting.Exosomes derived from highly metastatic tumor cells can mediate the EMT process of target cells.Moreover,Exosomes play a vital role in the formation of pre-metastatic niche and the colonization process of disseminated tumor cells through long-distance signal transduction.However,the role of exosomes in the metastasis of cholangiocarcinoma is still unclear.It has been found that the function of PTEN loss in tumor metastasis is highly coincident with the biological characteristics of exosomes,which prompts us to think about the possible relationship between them.Clarifying the relationship between PTEN expression and the secretion of exosomes in cholangiocarcinoma cells is very important.Exosome biogenesis originates in the endocytic pathway.It has been reported that lysosomal function can impact exosome biogenesis.AKT and m TOR can affect the genesisand function of lysosomes by regulating Mi T-TFE gene.Whether PTEN,as a signal molecule upstream of PI3K-AKT-m TORC1,can also regulate the function of lysosome and whether this regulation can affect the secretion of exosome have not been studied.The answer of this problem will help us understand the role of PTEN in the recurrence and metastasis of cholangiocarcinoma.Based on the differential expression of PTEN in cholangiocarcinoma,we will explore the effect of PTEN on the function of lysosome and exosome biogenesis in the process of metastasis by establishing cholangiocarcinoma cells with differential expression of PTEN.The results may provide new ideas for the diagnosis and treatment of cholangiocarcinoma.Research methods1.Retrospective analysis was used to study the relationship between PTEN expression and the clinical outcomes,recurrence and metastasis of cholangiocarcinoma in clinical samples.2.Establish PTEN overexpressed,deleted or control cholangiocarcinoma cells by lentivirus and adenovirus infection or plasmid and si RNA transfection.3.Detect the effect of PTEN on the biological behavior of cholangiocarcinoma cells by transwell cell migration assays,plate clone formation assays,soft agar clone formation assays and sphere formation tests.4.Establish animal models of lung metastasis through caudal vein and liver metastasis through spleen to verify the role of PTEN in the metastasis of cholangiocarcinoma in vivo.5.Separate exosomes using ultrahigh speed centrifugation and detect the effect of PTEN on the secretion of exosome in cholangiocarcinoma cells by electron microscope,NTA detection and Western-blot assays.6.Observe the effect of exosomes derived from cholangiocarcinoma cells on the migration and plate clone formation of cholangiocarcinoma cells.7.Retrospectively analyze the correlation between CD63 expression and PTEN expression;clarify the relationship between CD63 expression and the recurrence and metastasis of cholangiocarcinoma in clinical samples.8.Detect the expression of CD63 and HGS in PTEN differentially expressed cells by immunofluorescence technique to determine the effect of PTEN on the number of MVB in cholangiocarcinoma cells.9.Observe the structure and number of MVB and ILV in PTEN knockout and controlcholangiocarcinoma cells using electron microscope.10.Compare the lysosomes in cholangiocarcinoma cells by Lysosensor and Lysotracker staining.11.Western-blot assay and RT-PCR assay are used to detect the expression of cathepsins in PTEN differentially expressed cells.12.Detect the lysosomal p H in cholangiocarcinoma cells.13.RT-PCR assays are used to detect the expression of various components of V-ATPase in PTEN differentially expressed cells.14.Observe the distribution of TFEB in cholangiocarcinoma cells by immunofluorescence technique.Research results1.PTEN mutations and deletions exist in a certain proportion of cholangiocarcinoma,which is related to the poor clinical prognosis,and the relapse and metastasis of cholangiocarcinoma.2.Decreasing the expression of PTEN promotes the migration,plate clone formation,soft agar clone formation and sphere formation of cholangiocarcinoma cells.3.PTEN knockout promotes the metastasis of cholangiocarcinoma in vivo.4.Decreasing PTEN expression levels promotes exosome release and increases the number of MVBs in cholangiocarcinoma cells.5.Exosomes derived from cholangiocarcinoma cells with low expression of PTEN can better promote the growth and migration of cholangiocarcinoma.6.Lysosomal Impairment increases the number of MVB and promotes the secretion of exosome.7.PTEN promotes lysosome regeneration by mediating nuclear translocation of TFEB.8.PTEN promotes lysosome acidification by up-regulating the expression of ATP6V1 A,a subunit of V-ATPase.9.PTEN loss promotes the metastasis of cholangiocarcinoma cells by inhibiting the function of lysosome and increasing the release of exosomes.ConclusionIt is found that PTEN deletions or mutations exist in nearly half of clinical samples in our research,which showed low-level expression of PTEN;Downregulation of PTEN canpromote the progression,recurrence and metastasis of cholangiocarcinoma,and predict the poor prognosis;PTEN loss can not only inhibit nuclear translocation of TFEB to reduce the number of lysosome,but also down-regulate the expression of ATP6V1 A to impair the lysosomal acidification which is necessary for lysosome degradation function;The decrease of lysosomal numbers and the impairment of function lead to the increase of MVBs and its fusion with plasma membrane,and eventually promote exosomes release,which have impact on the survival,migration and invasion of cholangiocarcinoma cells.Collectively,this study reveals the relationship among PTEN expression,lysosome function and exosome secretion,and our findings elucidate the regulatory mechanism of PTEN in the metastasis of cholangiocarcinoma for the first time. |
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