Study On The Dual Regulation Mechanism Of Anti-tumor Drug PEITC On Heat Shock Response

BackgroundHeat shock response?HSR?is a protective response that activates the expression of heat shock proteins?Heat shock proteins,HSPs?to maintain homeostasis under certain stimulited situation?such as high fever,oxidative stress,heavy metals,et al?.Highly expressed HSPs?e.g.Hsp70,Hsp40,etc.?help other proteins fold and transport correctly to prevent protein from accumulating in cells after denaturation,thus destroying cell homeostasis.A lot of studies have shown that heat shock transcription factor 1?HSF1?is highly expressed in many tumor cells and is closely related to tumor progression and poor prognosis.The main mechanism of action is by enhancing its own 326 locus.Phosphorylation of serine?S326?up-regulates the expression of Hsp70,Hsp27,etc.,and promotes the activation of its downstream target genes c-myc,Ras,c-fos,etc.,resulting in malignant proliferation and apoptosis resistance of cells.A large body of evidence suggests that HSF1can be a good anti-tumor target.Recent studies have shown that HSF1 regulates HSP transcriptional expression and is also regulated by HSP feedback.Its relative molecular mechanism is not fully understood.Among them,Hsp70 is widely studied.Heat shock protein 70?Hsp70?is one of the HSPs family which is obviously regulated by HSF1 transcription.It is widely expressed in eukaryotic cells and highly conserved.Stress-induced Hsp70 is encoded by three very closely related paralogs,HSPA1A,HSPA1B and HsSPA1L.It is a member of the Hsp70 family and is highly expressed by cells in response to hyperthermia,oxidative stress,and pH changes.Studies have shown that the transcriptional activity of HSF1 is mainly regulated by the feedback regulation of HspA1A.However,the precise molecular mechanism of it is far from clear.Phenethyl isothiocyanate?PEITC?is a naturally occurring isothiocyanate in cruciferous vegetables.PEITC has been researched to prevent a variety of cancers,as well as to protect against neurodegenerative diseases,cardiovascular diseases,and bacterial infections.The study found that PEITC can activate a variety of cytoprotective pathways,such as HSF1-mediated heat shock response,and can cause changes in cell conversion factors.Therefore,we designed and synthesized a pair of gRNA?guide RNA?that targeting to the HSPA1A gene.After transfecting to the cells,the Cas9 protein was digested at a specific site of the HSPA1A genome and the GFP nucleic acid sequence was introduced in front of the ATG of the HSPA1A gene.Making the transcription level of the GFP gene controlled by the activity of the HSPA1A promoter.The stop codon TAA at the end of the GFP nucleic acid sequence blocked the translation of HSPA1A.A large number of transfected and enriched cells were screened to obtain a recombinant monoclonal cell line with the correct sequence of the recombinant genome.The recombinant cells were used as a model to screen and modulate HSF1/Hsp70.In view of the close correlation between HSF1 and malignant biological behaviors such as drug resistance and metastasis,it is particularly important to systematically and accurately screen drugs that regulate heat shock response.This project aims to enrich the library of compounds that can regulate HSF1 and improve related molecular mechanisms.PurposeThe use of CRISPR/Cas9 gene editing technology to introduce the GFP nucleic acid sequence at the ATG pre-site of the HSPA1A gene exon of cervical cancer cells?Hela?allows the transcription level of the GFP gene to be controlled by the activity of the HSPA1A promoter.The translation of HSPA1A was blocked in advance by adding the termination codon TAA at the 3'end of the GFP coding gene.The recombinant cell Hela/HSPA1A^-/-/GFP was used as a model to screen for drugs that regulate heat shock response and to explore relevant regulatory mechanisms.Methods and results1.A CRISPR/Cas9 gRNA plasmid and a HSPA1A^-/-/GFP recombinant plasmid for human HSPA1A gene were successfully constructed.To improve the efficiency of gRNA cleavage,we designed two gRNA sites upstream and downstream of the initiation codon of the HSPA1A gene.2.The gRNA plasmid and the recombinant plasmid were co-transfected into the cervical cancer cell line?Hela?,and the fluorescent recombinant monoclonal cells were screened by a high-speed flow cytometric sorting system?FACS?.The results of genetic recombination were verified by PCR,WB and FACS at gene,protein and cell levels.3.To further validate the heat shock stress system of recombinant cells,we used different drugs to treat cells.We unexpectedly found that PEITC and heat shock treatment not only had no synergistic effect,but also counteracted heat shock-mediated stress.4.The phosphorylation activity of serine 326 site of HSF1 after PEITC treatment was detected by WB.The results of it showed that PEITC enhanced the activation of HSF1during heat stress.5.In order to rule out the effect of knockoutHSPA1A on the results of PEITC treatment of 3#recombinant cells,WB and RT-PCR results were compared after Hela cells were treated under the same grouping and treatment conditions.The results show thatHSPA1A feedback regulates the phosphorylation status of HSF1.6.RNA immunoprecipitation?RIP?experiments have found and demonstrated that HSF1can directly bind to Hsp70 mRNA for translational regulation;Co-IP experiments found and demonstrated that HSF1 is involved in the regulation of ribosome assembly under stress;PEITC affects the interaction of HSF1 with mRNA or ribosomes under stress.ConclusionPEITC is an activator of HSF1;PEITC continuously activates HSF1 during heat stress and inhibits translation of downstream target proteins;HspA1A negative feedback regulates the phosphorylation status of HSF1,and HSF1 participates in the regulation of ribosome assembly and translation of Hsp70 mRNA under stress.