The Study Of Molecular Mechanism Of E3 Ubiquitin Ligase HRD1 On Regulating Tumorigenesis And Development

There are thousands of proteins in cells and they form a fine network.Their interactions in cells are strictly controlled.This imbalance of the networks will lead to various genetic diseases and cancer.Cancer can progress from stabilization of oncoproteins or destabilization of tumor suppressor genes.At the same time,cancer cells can maintain a high amount of proteolytic machinery and promote the degradation of tumor suppressor proteins or the activation of proto-oncogenes.Therefore,the study of the ubiquitin-proteasome system(UP-S)will be of great significance for the treatment of tumors,and there may be a large number of potential targets for targeted interventions.HRD1,also known as synoviolin,is an endoplasmic reticulum-associated protein degradation E3 ubiquitin ligase.HRD1 recognizes,ubiquitinates,and participates in endoplasmic reticulum-associated protein degradation pathways,and eliminates misfolded proteins in the endoplasmic reticulum to maintain protein homeostasis in the endoplasmic reticulum.At the same time,studies have shown that HRD1 can not only ubiquitinate misfolded proteins to mediate their degradation,but also participate in numerous cell biological processes including regulation of tumorigenesis and development by degrading other non-folding proteins.Therefore,the study of HRD1 and its potential target proteins has important significance for understanding the molecular mechanism of tumorigenesis and developing potential tumor therapeutic targets.This study is divided into two parts.The first part explores HRD1 acts as an E3 ubiquitin ligase and degrades PTEN by the proteasome pathway to promote the proliferation of hepatoma cells and the molecular mechanism of hepatocellular carcinoma progression.The second part mainly discusses the E3 ubiquitin ligase HRD1,which affects the stability of SIRT2 in lung cancer cells through ubiquitination-mediated degradation,and the effect of HRD1-SIRT2 interaction on the development of lung cancer.Part I: HRD1-mediated degradation of PTEN promotes cell proliferation and progression of hepatocellular carcinomaPTEN is a very important tumor suppressor,and mutation of this gene is a step in the development of many cancers.The proteomics showed that HRD1 is one of the interaction proteins of tumor suppressor PTEN,and previous studies have shown that HRD1 is up-regulated in HCC and PTEN is down-regulated in HCC.This study validated the interaction between HRD1 and PTEN,and further examined the regulatory role of HRD1-PTEN interaction in the development of HCC.The main findings are as follows: 1.HRD1 interacts with PTEN and mediates ubiquitination and degradation of PTEN through proteasome pathwayFirstly,the interaction between HRD1 and PTEN was verified by exogenous and endogenous protein-protein interaction analysis.It was further detected that HRD1 can positively regulate the ubiquitination level of PTEN.Overexpression of HRD1 specifically increases the ubiquitination level of PTEN protein.In addition,CHX assay showed that overexpression of HRD1 significantly shortened the half-life of PTEN protein,and knockdown of HRD1 in hepatoma cells increased the half-life of PTEN,moreover,the recovery of PTEN protein levels in HRD1 knockdown HCC cells treated with the proteasome inhibitor MG132 indicated that HRD1 was able to degrade PTEN via the ubiquitination-mediated proteasome pathway.2.The regulation of HRD1-PTEN interaction in the development of HCCWe established a series of cell lines include stable knockdown of PTEN,stable knockdown of HRD1,and stable knockdown of PTEN followed by knockdown of HRD1 in Hep3 B and MHCC97 H cells,also cell lines of high expression of PTEN,high expression of HRD1,and high expression of PTEN and HRD1 in Hep3 B and MHCC97 H cells.The effects of HRD1-PTEN on the proliferation of hepatoma cells were detected by MTS and clone formation experiments.The effect of HRD1-PTEN on the migration ability of hepatoma cells was detected by wound-healing assay,and the effect of invasion of HRD1-PTEN on hepatoma cells was detected by Transwell assay.The results showed that HRD1 promoted the proliferation,migration and invasion of HCC cells through regulating PTEN.At the same time,the effect of HRD1 on hepatocarcinogenesis was detected by using a nude mouse model.The results showed that the deletion of HRD1 could inhibit tumor growth.Further,a negative correlation between HRD1 and PTEN expression patterns was also shown in human HCC samples.Therefore,HRD1-mediated PTEN ubiquitination has a certain regulatory effect on the development of HCC.Part II: E3 ubiquitin ligase HRD1,affecting the stability of SIRT2 in lung cancer cells through ubiquitination-mediated degradationPrevious studies have shown that both SIRT2 and HRD1 play an important role in neurodegenerative diseases represented by Parkinson’s disease(PD).Most of SIRT2’s research focuses on its own deacetylation function,and there are few studies on post-translational modification.At the same time,more and more studies have shown that SIRT2 is also a tumor suppressor and is down-regulated in various cancers.Analysis of gene expression profiles also showed a negative correlation between HRD1 and SIRT2 expression in lung cancer.Therefore,we speculate that SIRT2 and HRD1 might have a corresponding relationship,and may play a certain role in the development of lung cancer.This study examined the interaction between HRD1 and SIRT2 and further analyzed the role of HRD1-SIRT2 in the development of lung cancer.The main findings are as follows: 1.HRD1 acts as an E3 ubiquitin ligase of SIRT2 to promote the degradation of SIRT2 The interaction between HRD1 and SIRT2 was verified by exogenous and endogenous protein-protein interaction analysis.Further ubiquitination experiments and CHX assay indicated that HRD1 is capable of mediating its ubiquitination and proteasomal degradation as an E3 ubiquitin ligase of SIRT2.2.The regulation of HRD1-SIRT2 interaction in the development of lung cancerWe established a series of cell lines include stable knockdown of SIRT2,stable knockdown of HRD1,and stable knockdown of SIRT2 followed by knockdown of HRD1 in A549 and NCI-H446 cells,also cell lines of high expression of SIRT2,high expression of HRD1,and high expression of SIRT2 and HRD1 in A549 and NCI-H446 cells.The effects of HRD1-SIRT2 on the proliferation of lung cancer cells were detected by MTS and clone formation experiments.The effect of HRD1-SIRT2 on the migration ability of lung cancer cells was detected by woulu-healing assay,and the impact of invasion of lung cancer cells by HRD1-SIRT2 was detected by Transwell assay.The results show that HRD1 promotes the proliferation,migration and invasion of lung cancer cells by regulating the protein level of SIRT2.At the same time,the nude mouse model was used to detect the effect of HRD1 on lung cancer in vivo.The results showed that the deletion of HRD1 could inhibit the tumor growth,and the abnormal expression of HRD1 could significantly promote the tumor growth.Further,a negative correlation between HRD1 and SIRT2 expression patterns was also shown in human lung cancer samples.3.The effect of drug treatment on SIRT2 protein levelsWe screened 13 drugs,including 8 Chinese medicines to treat A549 cells separately.The results showed that treatment with the antitumor drug etoposide increased the protein expression level of SIRT2 and decreased the protein expression level of HRD1.At the same time,knockdown of HRD1 makes lung cancer cells sensitive to apoptosis,while SIRT2 knockdown reduces the sensitivity of lung cancer cells to apoptosis.Therefore,the interaction study of HRD1-SIRT2 provides a certain molecular mechanism reference for the occurrence and development of lung cancer,and provides potential targets for tumor treatment.