Investigation Of OTUD5 Function In Regulating Triple-negative Breast Cancer Cell Proliferation Using CRISPR/Cas9

Backgrounds and purposesAccompanied by the progress of life sciences and medicine,most infectious diseases have been kept under control and Chinese standard of living is increasing remarkably.Along with the higher life expectancy and environmental influence,however,tumors have become the most life-threatening disease in China.Previous study shows that tumor cells often contain genomic instability,which contributes to their high variation and leads to the poor prognosis.It is estimated that the expense at tumor diagnosis and treatment is up to hundreds of billion yuan in China per year,which brings a heavy burden on family and society.To conquer tumors,discovering new drug targets is of great importance.Ubiquitin-proteasome system(UPS)is the main pathway functioning in specific degradation of intracellular proteins,and it takes part in many cellular processes,such as gene transcription,cell-cycle regulation,immunoreactions,tumorigenesis and inflammation.Deubquitinases can mediate the removal of ubiquitin from ubiquitinated proteins,which contributes to the dynamic balance of ubiquitination and deubiquitination in cells.Recently,deep insights have been gained into the role of UPS in tumorigenesis and tumor progress,thus leading to the research of new anti-tumor drugs targeting to the members of UPS.As the third generation of artificial endonuclease system,CRISPR/Cas9 gene-editing system has made its way in recent years.Compared with ZFN and TALEN systems,CRISPR/Cas9 system has many advantages,such as great convenience,high efficiency,low cost and large scope of applications.And it has become one of the most significant biotechnologies,which paves a way for plenteous research studies in life sciences and medicine.Based on the previous screening resu lts of growth-dependent genes related to the UPS in breast tumor cells,we chose OTUD5 to carry on our research in two aspects.Firstly,we determined whether OTUD5 is essential for cell proliferation in breast tumor cells,colorectal cancer cells and human normal liver cells,etc;Secondly,we studied the molecular mechanisms by which OTUD5 regulates tumor cell proliferation using BioID technology to screen out the proteins interacting with OTUD5,and these results will benefit the development of drugs targeted to OTUD5.Contents(1)We determined whether OTUD5 is essential for cell proliferation in MDA-MB-231 cells;(2)We determined whether OTUD5 is essential for proliferation of some specific kinds of tumor cells,including human cervical cancer cells,human colorectal cancer,human normal liver cells and several strains of cells derived from breast tumors.(3)We screened out the proteins interacting with OTUD5 through BioID technology.Furthermore,we studied the molecular mechanism behind the regulation of OTUD5 on tumor cell proliferation.Methods(1)Knock-down of OTUD5: First,we constructed a cell line stably expressing gRNA targeted to OTUD5 in MDA-MB-231 cells.After infection of adenovirus containing Cas9-GFP,we detected the GFP levels using a fluorescence microscope every 24 hours.After 96 hours,we detected the expression level of OTUD5;Next,we designed several different gRNA targeted to OTUD5.And through lentivirus vector system,we detected the expression level of OTUD5 96 hours after infection;Last,we constructed OTUD5 shRNA lentivirus vector,which aimed to knockdown OTUD5.(2)Determination of OTUD5 knockdown: There are two main methods determining whether OTUD5 is knocked down after CRISPR/Cas9 gene-editing system was applied.First in the genomic level,we designed primers targeted to the region of about 800 bp flanked the gRNA sequence in genome.After DNA amplification by PCR and anneal,the DNA fragment can form a region which can be recognized and resected by T7E1 endonuclease.Thus,the resection efficiency can reflect the knockdown level of OTUD5;Second in the protein level,we used Western Blot to detected the knockdown level of OTUD5.Also,we used Western Blot to detected knockdown level of OTUD5 in stable cell lines expressing shRNA targeted to OTUD5.(3)Determination of cell growth efficiency: To determine the cell growth efficiency,there are mainly two methods.One is MTS assays.MTS is a novel MTT analogue.On the condition that coupling regent PMS exists,MTS can be deoxidized by various dehydrogenases in mitochondria of living cells,and the products includes colored hydrosoluble formazan.As the OD values correlates with the cell numbers positively in a specific range,we can use the changes of OD values read by microplate readers to reflect the changes of cell numbers;the other is colony formation experiments.By comparing the numbers of colonies that different cells form in an identical time,we can determine their differences in cell growth efficiency.In addition,cell-cycle changes and apoptotic levels were determined by flow cytometry.M phase changes were determined by flow cytometry using PH3 staining.Cell-ageing levels were detected by SA-β-gal staining.(4)Study on the molecular mechanism by which OTUD5 regulates tumor cell proliferation: We screened out the proteins interacting with OTUD5 through BioID technology.Furthermore,we determined whether the candidate proteins in screening results interact directly with OTUD5 using immunoprecipitation and revealed the reason behind the regulation of OTUD5 on tumor cell growth.Results(1)We knocked down OTUD5 in MDA-MB-231 cells using CRISPR/Cas 9 system.And we found that the cell proliferation is inhibited after OTUD5 knockdown.Also,experiments using cells stably expressing shRNA targeted to OTUD5 showed similar results.Furthermore,to avoid off-target effects,several different gRNAs and shRNA targeted to OTUD5 were applied in MDA-MB-231,and the results were still consistent with the previous.(2)Using the gRNA targeted to OTUD5 from screening library of UPS-related genes,we found that knockdown of OTUD5 leads to the inhibition of cell proliferation in human cervical cancer cells,colorectal cancer cells,human normal liver cells and several cell strains derived from breast tumors.(3)Flow cytomtry experiments demonstrated that after OTUD5 knock-down,the apoptotic level of MDA-MB-231 cells showed little change,and G2/M phase were retardant.Of note,PH3 experiments showed that M phase was extended significantly after OTUD5 knock-down.SA-β-gal staining experiments showed that cell-ageing levels changed little after OTUD5 knock-down.(4)Using Bio ID technology,we screened out many candidate proteins interacting with OTUD5,and most parts of those candidate proteins are associated with RNA splicing and DNA damage repair biology pathways.However,the Co-IP experiment did not detect the presence of these proteins in the presence of OTUD5.Hence,we suggest that OTUD5 may just regulate the ubiquitination level of those candidate proteins,which remains to be further elucidated.Conclusions(1)CRISPR/Cas 9 gene editing system is an effective and convenient method in research,which can knock down the specific gene quickly and accurately.(2)OTUD5 is essential for tumor cell proliferation,and it can regulate cell proliferation through cell cycle regulation.Furthermore,this process does not involve level changes of cell apoptosis and cell ageing.(3)Bio ID technology is an effective method of screening out candidate proteins interacting with the target protein,and the candidate proteins from screening can bring new insights into further research.