The Mechanism Of UBXN4 Gene And Different Stress Environments On The Development Of Prostate Cancer

background and purpose:Prostate cancer(PCa),as a male-specific urogenital tumor,ranks among the top men in the world for tumor incidence.Among global new male cancers in 2018,prostate cancer ranked second with an incidence of 13.5%.Cancer mortality ranks fifth(6.7%).In addition,Among the 2019 new male cancers announced by the United States,the incidence of prostate cancer has surpassed that of lung cancer,ranking first,and the mortality rate of cancer ranks second in male cancer [1].Prostate cancer is increasingly becoming the number one killer of men's health worldwide.The occurrence,development,invasion and metastasis of tumor is a very complicated process.Although in recent years,with the continuous deepening of research on prostate cancer,there has been a deeper understanding of the genes and epigenetic aspects of the occurrence,development,invasion and metastasis of prostate cancer.But until now,the core functional genes and their related molecular mechanisms in the development of prostate cancer have not been clear.With the popularization of next-generation genome sequencing and the development of single-cell sequencing technology,rapid and high-throughput discovery of tumor mutation genes and fusion genes has become possible,and it has become a commonly used tumor research method [2].However,due to the high-throughput and big-data nature of sequencing,these research methods will generate a lot of redundant information.How to better abandon redundant information to select and conduct research on key genes and epigenetic levels is particularly important.CRISPR-Cas9 gene editing technology has attracted more and more attention from scientific researchers since its introduction.Based on it,a series of derivative technologies have been developed to make efficient and reliable gene intervention technology a reality [3].The discovery and application of a CRISPR library screening strategy based on the CRISPRCas9 gene editing technology in 2015 has enabled quantitative screening and validation of gene functions [4].However,there are currently no specific high-frequency mutant genes specifically targeted for prostate cancer,especially the CRISPR library screening of the fusion gene's parent gene,which makes genes that are really meaningful for prostate cancer easy to be ignored [5].UBXN4,also known as Erasin,was originally discovered to interact with VCP / P97 through its UBX domain to participate in the process of endoplasmic reticulum-related protein degradation(ERAD)[6].Although there are reports in the literature that the UBX domain-containing protein family is involved in tumorigenesis and progression,the role of UBXN4 in tumors has not been much reported.The interaction between tumors and tumor microenvironment is crucial to the occurrence,development and evolution of tumors.The impact of tumor microenvironment on tumor cells mainly includes two categories: one is biochemical signals,and the other is mechanical and physical signals in microenvironments.There have been many previous studies on biochemical signals in the microenvironment.But little is known about mechanophysical signals,such as how tumor cells respond to the mechanical stiffness of tissues in the microenvironment [7]This project mainly combines CRISPR library screening strategies and next-generation sequencing from clinical samples,High throughput functional verification was performed for the high frequency mutant genes and fusion genes obtained from sequencing.According to the proliferation and migration phenotype,the dominant clones for proliferation were selected.The dominant clones for the final focus were the gene UBXN4 inactive mutants.The effect of UBXN4 mutation on the proliferation and migration phenotype of prostate cancer cell lines was further verified in vivo and in vitro.Based on this,we hope to analyze other deeper antitumor mechanisms besides participating in ERAD.Since this experiment involves four prostate cancer cell lines derived from different metastatic sites,and the mechano-physical properties in the microenvironment where they originated are very different,this paper also preliminary explored the cell lines of different metastatic sites of prostate cancer under different mechanical hardness.Culture on the bottom,and compare the benign and malignant transformation tendency of different cell lines during the in vitro culture.Based on this,the heterogeneity of the PC3 and LNCap cell lines' response to the mechanical environment such as soft and hard substrates in vitro culture is explained.Part I The role of UBXN4 gene in the development of prostate cancer and its mechanismMethods: 1.On the basis of next-generation sequencing of prostate cancer and adjacent tissues,construct a CRISPR library for high-frequency mutant genes and fusion genes for functional gene screening and select proliferating dominant cell clones(named Clone6).2.Clone6 proliferation,migration and tumor formation phenotype experiments,then sg RNA identification of Clone6 cells,and finally identified the key mutant gene(UBXN4).3.Transfection of prostate cancer cell lines(DU145,LNCa P,PC3,and C42B)with small interfering RNA,and using si NC as a control,confirm the inhibitory effect of gene UBXN4 on the proliferation of prostate cancer cell lines.4.In the DU145 cell line,a CRISPR-Cas9 gene editing system was constructed again for another exon of the gene UBXN4,and a stable mutant cell line(DU145-KOUBXN4-mix)was selected and the proliferation and migration phenotype was identified.5.Construction of UBXN4 overexpression virus,overexpression of UBXN4 in WTDU145 and identification of the proliferation phenotype.Overexpression rescue experiments were performed in the UBXN4 knockout DU145 cell line.6.Based on co-immunoprecipitation combined mass spectrometry analysis,the regulatory mechanism of the gene UBXN4 is predicted to play a role in suppressing cancer,and a series of experimental verifications are performed.Results: 1.High-throughput sequencing of prostate cancer and adjacent tissues,and packaging of sg RNA specific to each high-frequency mutant gene and the fusion gene's parent gene and virus packaging to form a CRISPR lentivirus library.The library was transfected in prostate cancer cell line DU145 and infected with the appropriate concentration of Cas9 virus after drug screening.A significantly larger cell population was selected as the dominant clone group(named Clone6).2.Compared with wild DU145,cells in Clone6 group have stronger proliferation,migration and tumorigenicity.The sg RNA backbone sequencing,genomic PCR and sequencing,and Western Blot experiments on the Clone6 group of cells can indicate that the sg RNA entered in the predominant clone Clone6 cells is targeted to the second exon of the gene UBXN4 and causes a mutation in the gene UBXN4.3.Design the small interfering RNA of the gene UBXN4(si-UBXN4-1,2,3),and transfect small cell line DU145,LNCa P,PC3 and C4-2B with si NC as the control,and then CCK8 proliferation experiment,Ed U proliferation experiment,Transwell migration experiment and scratch test were performed.The results showed that compared with the si NC group,the si-UBXN4 group showed stronger proliferation and migration ability.4.Constructed the CRISPR-Cas9 gene editing system again for the exon 12 of the gene UBXN4.Sequence sequencing at the gene level and Western Blot at the protein level verified that the DU145 artificial gene UBXN4 mutation was successful,named DU145-KOUBXN4-mix,and selected a monoclonal cell line named DU145-KOUBXN4-1.Then the cells DU145-KOUBXN4-mix and DU145-KOUBXN4-1 were subjected to proliferation and migration phenotype experiments.The results showed that compared with the wild DU145 group,the DU145-KOUBXN4-mix and DU145-KOUBXN4-1 groups showed stronger proliferation and migration ability.5.Construction of UBXN4 overexpression plasmid and overexpression virus packaging,wild DU145 infection of overexpressed virus,screening of puromycin,RT-PCR and Western Blot experiments to verify overexpression efficiency,and CCK8 proliferation experiment and Ed U proliferation experiment.The results showed that compared with the wild DU145 group,the over-expressed UBXN4 group showed a slower proliferation trend.Overexpression recovery experiments were performed on Clone6 and DU145-KOUBXN4-mix and DU145-KOUBXN4-1 cell lines.It was found that overexpression of UBXN4 inhibited the proliferative effect caused by UBXN4 mutation.6.In the process of co-immunoprecipitation and mass spectrometry analysis using UBXN4 antibodies,it was found that the protein UBXN4 can bind to iron death related proteins(TFRC and SLC3A2)and protein Rab10,and iron death related experiments will be verified later.Conclusion: Based on the existing results,it can be concluded that the mutation of the gene UBXN4 can be screened on the basis of next-generation sequencing in combination with the CRISPR library,which can increase the proliferation and migration ability of prostate cancer cell lines and show stronger malignant transformation,suggesting that UBXN4 is a very strong Tumor suppressor genes.The subsequent design and transfection of small interfering RNA,overexpression of UBXN4 virus packaging and infection,and artificial mutation of different sites of the gene UBXN4,and other measures,the results of proliferation and migration experiments have shown that decreased expression of UBXN4 will promote the proliferation and migration of prostate cancer cells Overexpression of UBXN4 can inhibit the proliferation and migration ability of prostate cancer cells.Overexpression of UBXN4 rescue experiments can alleviate the malignant transformation caused by UBXN4 mutations.There are various indications that the gene UBXN4 can be used as a powerful tumor suppressor gene,thereby gaining a deeper understanding of the gene UBXN4,and a deeper understanding of the relationship between gene mutations and malignant transformation of prostate cancer.Genetic diagnosis and treatment provide important references.Part II Heterogeneity of Different Prostate Cancer Cell Lines to External Mechanical ForcesMethods: 1.According to the existing literature,the preparation and physical hardness characterization of PDMS substrates with different hardness cell culture were performed.2.Culture prostate cancer cell lines(PC3,LNCa P,DU145,and C42B)on different soft and hard substrates,and detect the malignant phenotypes of different cell lines,such as proliferation and migration,in order to explore the effects of different hardness substrates on tumor cells.3.PC3 and LNCa P were cultured on substrates with different mechanical hardness for 7 days,and then subcutaneous tumor-bearing experiments were performed in nude mice to explore the influence of different hardness substrates on the tumorigenic ability of tumor cell lines.4.Based on the results of the previous step and combined with previous literature,YAP / TAZ nuclear verification was performed on PC3 cells showing malignant phenotypic enhancement on a rigid substrate,to identify rigid substrates to promote the nuclear localization of YAP / TAZ to promote the proliferation and migration of PC3 cells.Results: 1.Two kinds of cell culture substrates were prepared according to existing literature methods,with hardnesses of 46.7KPa and 0.7KPa,respectively,to mimic the physiological hardness of bones and lymph nodes in the body,and cell culture was performed on the two hardness substrates.2.The following studies were performed with four prostate cancer cell lines(prostate cancer bone metastasis cell line PC3,prostate cancer lymph node metastasis cell line LNCa P,prostate cancer brain metastasis cell line DU145,and LNCa P bone metastasis cell line C4-2B).The results of Ed U cell proliferation experiments and Transwell cell invasion experiments showed that the PC3 cell line showed stronger proliferation and invasion ability on hard substrates,and the LNCa P cell line showed stronger proliferation and invasion ability on soft substrates.3.After PC3 and LNCa P cell lines were cultured on substrates of different hardness for 7 days,subcutaneous tumor-bearing experiments were performed in nude mice.The results showed that the PC3 cell line showed stronger in vivo tumorigenicity on rigid substrates.The LNCa P cell line shows stronger in vitro tumorigenicity on soft substrates.4.Aiming at the enhanced proliferation,invasion,and tumorigenic capacity of PC3 cells on hard substrates,the activation of YAP / TAZ pathways,which are the key pathways of mechanical response of hard substrates,was detected.Immunofluorescence results showed that,compared with LNCa P cells,PC3 cells showed obvious YAP nuclear localization on hard substrates,but this phenomenon did not occur on soft substrates.Western Blotting results showed that among PC3 and LNCa P cells cultured on the same rigid substrate,only PC3 cells showed dephosphorylation of YAP1,suggesting that they have nuclear translocation of YAP.After transfection with YAP / TAZ small interfering RNA,Ed U,invasion,and tumor-bearing experiments were performed on PC3 cells under rigid substrate conditions.The results showed that compared with the control group,the proliferation,invasion,and tumorigenicity of tumor cells in the small-interfering RNA group were significantly reduced.In summary,it is suggested that the mechanical response of the cells to the star pathway YAP / TAZ is involved in the enhanced malignant phenotypic changes of PC3 cells on hard substrates.Conclusion: According to the existing results,tumor cell lines derived from different metastatic sites of prostate cancer have different growth states on different hardness substrates.Prostate cancer bone metastasis cell line PC3 has stronger ability to proliferate,invade,and form tumors on a rigid substrate,and the acquisition of this malignant enhancement is closely related to the YAP / TAZ pathway and the dephosphorylation of YAP into the nuclear process.In addition,the LNCa P cell line of prostate cancer lymph node metastasis showed a stronger ability to proliferate,invade,and metastasize on the soft sink.Summary New generation sequencing combined with CRISPR library screening provides a good idea for large-scale effective discovery and verification of a large number of mutant genes and fusion genes in tumorigenesis and development.Based on this,it was found that the high-frequency mutation gene UBXN4 of prostate cancer,the mutation of the gene UBXN4 can enhance the proliferation,migration and tumorigenicity of prostate cancer cells,suggesting that the gene UBXN4 is a new tumor suppressor gene,a new functions beyond related protein degradation(ERAD).The correctness of the idea of second-generation sequencing combined with CRISPR library screening was verified,and a new tumor suppressor gene UBXN4 was discovered,which provided a more reliable basis for the genetic diagnosis and malignant identification of prostate cancer.The interaction between tumor cells and the tumor microenvironment has mechanophysical signals in addition to the biochemical signals that are most often of interest to researchers.This research experiment shows that prostate cancer cell lines derived from different metastatic sites show different proliferative invasion and tumorigenic capabilities in culture systems with different mechanical hardness substrates,indicating that different prostate cancer cell lines are responsible for extracellular mechanical physical signals.The heterogeneity of this response further provides a broader idea for understanding the organprostatic metastasis of tumors,that is,in addition to the occurrence of a series of biochemical signals,a series of mechanophysical signals also exist under the phenomenon of tumor proorgan metastasis.