| Part ?:Effects of doxycycline on the proliferation,apoptosis and metabolism of cervical cancer cellsBackgroundCervical cancer is a kind of cancer,which is very harmful to women health.About 310000 women died of cervical cancer around the world in each year.The course of cervical cancer is complicated,the incidence rate is very high,and the incidence population is getting younger and younger.Cervical cancer continues to pose an unsettled health issue worldwide.Surgery,chemotherapeutic agents and radiotherapy are in clinical use but they only improve clinical outcome for early stage of cervical cancer.Novel therapeutic strategies which can either independently or in combination with current clinically available chemo drugs,are required for the management of the recurrent and metastasis of cervical cancer,because of the insensitivity to radiotherapy and drug tolerance seriously.However,the discovery and development of novel compounds can be a long and laborious process.Drug repositioning may circumvent this process and facilitate the rapid translation of hypothesis-driven science into the clinics.Antibiotics have been reported to be a novel promising therapeutic strategy for the treatment of cancer.One FDA-approved antibiotic,tigecycline,selectively kills acute myeloid leukemia(AML)stem cells.Another antibiotic azithromycin inhibits tumor-sphere formation of cancer cell lines across multiple tumor types,including breast,ovarian,prostate and lung.Doxycycline,which is an antibiotic approved by FDA in 1967,belongs to tetracycline class and mainly used in the treatment of mycoplasma,chlamydia and other infections.In recent years,doxycycline has been found to have other biological effects besides antibacterial effects in clinical and biomedical studies.Reports have expanded upon these findings to determine potential confounding effects of the tetracyclines,particularly doxycycline--the predominantly used compound,are widely used in biomedical research as mediators of inducible gene expression systems.Researchers found that these concentrations of drugs can significantly alter the metabolic profile of the cell,as well as reduce the proliferative rate.Due to its small side effects,good pharmacokinetics and active therapeutic effect in some tumors,relocalization of doxycycline's clinical utility for the treatment of tumors can be considered to solve the difficulties in the development of new drugs for tumors.ObjectiveTo research biological effects of doxycycline,apoptosis and cell metabolism by using cell model experiments and animal model experiments.Methods1.MTS assay:Among all the high-risk types of HPV,we selected HPV type 16 and 18-positive cervical cancer cell lines because they are most prevalent.Six cervical cancer cell lines including two HPV 18 positive cells(HeLa and CaLo),two HPV 16 positive cells(CaSki and SiHa)and two HPV negative cells(ViBo and C-33A)were tested.First,we planted these 6 types of cervical cancer cell lines into 96-well plates.After 72 hours of treatment with 5?M,10?M,20?M and 40?M concentrations of doxycycline,the light absorption value of the cells under the wavelength of 490nm was detected by MTS cell proliferation experiment,and the influence of doxycycline on the proliferation of cervical cancer cells was quantitatively analyzed.2.Annexin V-FITC/PI assay:First,we planted these 6 types of cervical cancer cell lines into 6-well plates.After 72 hours of treatment with S?M,10?M,20?M and 40?M concentrations of doxycycline,tumor volume reached 200 mm3,the mice were treated with vehicle control or intraperitoneal doxycycline 100 mg/kg twice daily.Tumor volume was monitored over a period of 2 weeks after initiation of treatment..The effect of doxycycline on this kind of cancer cells was measured at 24 h,48 h and 72 h after treatment with 40?M concentration of doxycycline.3.Western Blot:After 24 hours of treatment with 40?M concentrations of doxycycline,HeLa cells were collected and protein were extracted,Protein levels of the cleaved Caspase-3 were detected by Western Blot.4.Cell oxygen consumption rate and extracellular acidification rate and ATP level were measured:The effects of cell oxygen consumption rates,extracellular acidification rates and ATP levels were measured at 24 h,48 h and 72 h after treatment with doxycycline at 40?M concentration.5.Xenograft mouse model:5×106 HeLa cells were subcutaneously injected into the left flank of SCID mice.When the tumor volume reached 200 mm3,the mice were treated with vehicle control or intraperitoneal doxycycline 100 mg/kg twice daily.Tumor volume was monitored over a period of 2 weeks after initiation of treatment.Cell apoptosis was detected by PCNA marker and TUNEL by immunofluorescence.Results1.The effects of doxycycline on proliferation of cervical cancer cells:After 72 hours of treatment with different concentrations of doxycycline,the proliferation ability of cervical cancer cells gradually decreased with the increasing concentration of doxycycline,the cell proliferation ability of the 10?M doxycycline treatment group decreased to 70%-50%,and 40?M treatment the proliferation ability of the group dropped from 10%to zero.2.The effects of doxycycline on apoptosis of cervical cancer cells:With the increasing concentration of tumor volume reached 200 mm3,the mice were treated with vehicle control or intraperitoneal doxycycline 100 mg/kg twice daily.Tumor volume was monitored over a period of 2 weeks after initiation of treatment.The percentage apoptosis of cervical cancer cells increased from 20%-45%to 40%-60%after 40?M doxycycline treated for 24 hours and 48 hours,and the percentage of apoptosis reached 60%-70%after 72 hours of treatment,(P<0.01)the difference was statistically significant.3.The effects of doxycycline on the metabolism of cervical cancer cells:After 40?M doxycycline treated cells for 24,48 and 72 hours,the oxygen consumption rate of the cells decreased to 40%,20%,and 10%,(P<0.01);the ATP level decreased to 55%,20%,and 10%,(P<0.01);the rate of extracellular acidification showed a trend of increasing first and then decreasing;the differences were statistically significant.4.The effect of doxycycline on the expression of Caspase-3:doxycycline can obviously increase mount of cleaved-Caspase-3 in this kind of cancer cells;after adding the specific inhibitor of Caspase,Z-VAD-FMK,apoptosis was significantly reduced,and has obvious different in these two groups5.The effects of doxycycline on tumor growth of cervical cancer in mice:During the treatment with doxycycline,no signs of cytotoxicity,weight loss,abnormal behavior or other clinical symptoms were observed.Two weeks later,the average tumor volume of the treatment group and the control group was 490±190mm3 and 910±170mm3,respectively,and the difference was statistically significant(p<0.01).Doxycycline significantly delayed tumor growth.In addition,we observed less proliferating and more apoptotic tumor cells in mice group treated with doxycycline compared to control.Conclusions1.Doxycycline inhibits the sequencing error rate and GC content distribution to obtain clean reads for subsequent analysis.Three parallel replicates of the three groups all obtained clean reads above 6.5G,and the GC content ratio was about 50%.2.Doxycycline significantly inhibits the ATP level and the extracellular acidification rate.3.This kind of material can induce apoptosis of cancer cells by caspase pathway.4.In the HeLa cell xenograft mouse model,doxycycline can significantly inhibit tumor growth.Taken together,we observed less proliferating and more apoptotic tumor cells in mice group treated with doxycycline compared to control,we believe that doxycycline may be repositioned and used to treat cervical cancer,and targeted energy metabolism may be a potential strategy for cervical cancer treatment.Part ?:Transcriptome Sequencing and Bioinformatics Analysis of HeLa cell under the intervention of DoxycyclineBackgroundBioinformatics is an interdisciplinary subject with a wide range of research fields,involving various omics such as genomics,transcriptomics,proteomics,and pharmacogenomics.,with the core is to solve biomedical problems by studying the important information contained in biomedical resources.The development of Next Generation Sequencing(NGS)technology has ushered a new era of human genomics research.It is also called High-Throughput Sequencing(HTS),can sequence millions of nucleic acid molecules at one time to obtain tens of billions of base sequences that can cover the entire genome or transcriptome by sequencing while synthesizing,and has been widely used in many biological fields such as whole genome sequencing,transcriptome sequencing,chromosome immunoprecipitation sequencing.Transcriptomics has developed rapidly and has become the most widely used research technique.Therefore,after the completion of the Human Genome Project,the research direction has shifted from gene sequencing to gene function annotation.The transcriptome,as the link between the genome that carries genetic information and the protein group of biological functions,has many unknown areas to explore.mRNA(messenger RNA)is currently the most studied type of RNA,which can encode proteins and is a direct manifestation of genetic information to biological functions.The basic steps of applying next-generation sequencing technology for mRNA transcriptome sequencing include RNA extraction and detection,sequencing library construction and quality inspection,Illumina sequencing,acquisition of off-machine data,identification of differentially expressed genes and enrichment analysis of differentially expressed genes.Such as GO(Gene Ontology)function enrichment analysis,KEGG(Kyoto encyclopedia of genes and genomes)pathway enrichment analysis,etc.The most studied type of RNA,which can encode proteins and is a direct manifestation of genetic information to biological functions.The basic steps of applying next-generation sequencing technology for mRNA transcriptome sequencing include RNA extraction and detection,sequencing library construction and quality inspection.However,at present,the specific mechanism of doxycycline inducing cervical cancer cell apoptosis through the Caspase-dependent pathway is not very clear.So in this study we intend to explore the possible mechanism of doxycycline inducing apoptosis of cervical cancer cells through Caspase-dependent pathway through transcriptome sequencing,differential gene expression analysis,KEGG pathway enrichment,and is benefit for the using of doxycycline in this kind of cancer.ObjectiveExplore the mechanism of doxycycline inducing apoptosis through Caspase-dependent pathway in cervical cancer.Methods1.Taking the cervical cancer HeLa cell line as the research object,set up solvent group(N),doxycycline group(DOX),doxycycline+Caspase inhibitor(Z-VAD-FMK)group(C3_DOX),each group with three repeating groups respectively.The DOX group was treated with 40 ?M doxycycline for 72 hours,and the C3_DOX group was treated with 40 ?M doxycycline for 72 hours,and then treated with 10?M Caspase inhibitor Z-VAD-FMK for 20 minutes.2.Transcriptome sequencing:RNA extraction and detection,library construction and quality inspection,and computer sequencing(Illumina)were performed on 9 groups of HeLa cells in three groups respectively.After the data were obtained,statistics and quality evaluation of data output were performed,and String Tie software was used to perform new transcript assembly and complete data annotation.3.Use the feature Counts tool in subread software to quantitatively analyze gene expression levels;use DESeq2 software to perform gene differential expression analysis.4.the differentially expressed genes are concentrated in the apoptotic signaling pathway and ribonucleoprotein complex(RNP)biologenesis in DOX group.5.The genes up-regulated in DOX group vs.N group and at the same time down-regulated in C3_DOX group vs.DOX group,and the genes down-regulated in DOX group vs.N group at the same time,up-regulated in C3 DOX group vs.DOX group,were merged for classical pathway analysis based on IPA.Results1.Filter the raw data obtained from transcriptome sequencing,check the sequencing error rate and GC content distribution to obtain clean reads for subsequent analysis.Three parallel replicates of the three groups all obtained clean reads above 6.0G,and the GC content ratio was about 50%.2.Gene differential expression:DOX group vs.N group obtained 5838(up-regulated 2947,down-regulated 2891)differentially expressed genes;C3_DOX group vs.N group obtained 5819(up-regulated 2939,down-regulated 2880)differentially expressed genes;C3_DOX group vs.DOX group obtained 1963(up-regulated 751,down-regulated 1212)differentially expressed genes.P<0.05 and |log2foldchange|>0.0 as significant differences Threshold of expression.3.GO function enrichment analysis:the differentially expressed genes are concentrated in the apoptotic signaling pathway and ribonucleoprotein complex(RNP)biologenesis in DOX group vs.N group and C3 DOX group vs.N group;and are mainly related to molecular functions such as cell adhesion junctions,anchoring junctions,and RNP biosynthesis.4.The expressed genes mainly enriched in endoplasmic reticulum.5.IPA classic pathway analysis:EIF2 Signaling is significantly activated,with a Z-score of 2.828.Conclusions1.Gene expressions showed significant different compared with the solvent group after interventions by pooling in Doxycycline and Caspase inhibitor(Z-VAD-FMK).Doxycycline played a role in the induction of gene expression,which can change the gene expression pattern at a certain drug concentration.2.GO and KEGG enrichment analysis suggest that doxycycline can affect cell energy metabolism and induce cell apoptosis by affecting the biological production of proteins in the endoplasmic reticulum of HeLa cells.3,IPA analysis suggests that doxycycline may activate the EIF2 Signaling to induce apoptosis through Caspase-dependent pathway in cervical cancer.In summary,this part of the research suggests that doxycycline can change the metabolism of HeLa cells by changing the expression of endoplasmic reticulum genes in HeLa cells;and doxycycline induces cell apoptosis in HeLa cells through the caspase pathway.Death may be mediated through activation of the EIF2 Signaling pathway.Part ?:PERK-eIF2?-ATF4 signaling pathway mediates doxycycline-induced apoptosis of cervical cancer HeLa cellsBackgroundThe endoplasmic reticulum is an important organelle in common cells.Many undesirable stimuli such as hypoxia,ischemia,viral infection,pH changes,nutritional deficiency(especially glucose deficiency caused by protein glycosylation disorder),rapid cell proliferation,and oxidation Changes in reduction homeostasis,increased protein synthesis levels,and decreased calcium ion levels can all lead to an imbalance in endoplasmic reticulum homeostasis,which manifests as unfolded or misfolded proteins accumulate in the endoplasmic reticulum and damage the endoplasmic reticulum,this phenomenon is called endoplasmic reticulum stress(ERS).Recent studies have shown that endoplasmic reticulum stress is closely related to the occurrence and development of tumors,which is mainly related to cell apoptosis mediated by endoplasmic reticulum stress.Glucose regulatory protein 78(GRP78),effect of Doxycycline on HeLa cell metabolism is mainly reflected in affecting the protein processing in the endoplasmic reticulum,and activating the EIF2 Signaling to induce HeLa cells to apoptosis through theof endoplasmic reticulum stress,the expression of GRP78 increases,which promotes the correct folding of misfolded or unfolded proteins,restores endoplasmic reticulum Ca2+transport,and maintains endoplasmic reticulum homeostasis to protect the normal function of cells.change the metabolic status of cells by changing the expression pattern of genes and reduce the cell Oxygen consumption rate and ATP level.Bioinformatics analysis suggests that the effect of Doxycycline on HeLa cell metabolism is mainly reflected in affecting the protein processing in the endoplasmic reticulum,and activating the EIF2 Signaling.When ERS persists,GRP78 increased protein synthesis levels,and decreased calcium ion levels can all lead to an imbalance in endoplasmic reticulum homeostasis,which manifests as unfolded or misfolded proteins accumulate in the endoplasmic,and initiates PERK-elF2?(true The nuclear transcription initiation factor 2?)pathway promotes the development of adaptive signaling pathways by inhibiting overall protein synthesis and activating the selective translation of transcription factor 4(ATF4).ATF4 is combined with the promoter of the enhancer-binding protein homologous protein(CHOP)gene,the increased expression of CHOP leads to an increase in the release of cytochrome C,initiates the Caspase apoptotic cascade,and finally leads to apoptosis.This group of previous studies has confirmed that doxycycline can effectively Bioinformatics analysis suggests that the effect of Doxycycline on HeLa cell metabolism is mainly reflected in affecting,and can change the metabolic status of cells by changing the expression pattern of genes and reduce the cell Oxygen consumption rate and ATP level.Bioinformatics analysis suggests that the effect of Doxycycline on HeLa cell metabolism is mainly reflected in affecting the protein processing in the endoplasmic reticulum,and activating the EIF2 Signaling to induce HeLa cells to apoptosis through the Caspase-dependent pathway.ObjectiveExplore the mechanism of PERK-eIF2?-ATF4 signaling pathway in HeLa cells under the intervention of doxycycline.Methods1.RT-qPCR:Taking the cervical cancer HeLa cell line as the research object,set up solvent group(N),doxycycline group(DOX),doxycycline+Caspase inhibitor(Z-VAD-FMK)group(C3_DOX),each group with three repeating groups respectively.The DOX group was treated with 40 ?M doxycycline for 72 hours,and the C3_DOX group was treated with 40 ?M doxycycline for 72 hours,and then treated with 10 ?M Caspase inhibitor Z-VAD-FMK for 20 minutes.Real-time quantitative PCR experiments were used to quantitatively analyze the gene transcription levels of endoplasmic reticulum stress-related protein molecules GRP78,eIF2?,PERK,ATF4,CHOP in HeLa cells treated with different methods.2.Western blot:this kind of method to analyze the express levels of ERS related protein,such as GRP78,eIF2?,PERK,ATF4,CHOP in the above three groups of HeLa cells.Results1.Doxycycline increases the transcription level of the HSPA5 gene and the protein expression level of GRP78/Bip.The abundance of HSPA5 gene expression in the doxycycline group and the doxycycline+inhibitor group were 2.031 and 1.284 times that of the solvent group,(p<0.01);the difference was statistically significant.2.Doxycycline increased the transcription levels of EIF2AK3 and EIF2S1,and the gene expression abundances were 1.629 times and 1.224 times that of the solvent group,(p<0.01),the difference was statistically significant;PERK and eIF2? protein expression levels did not show an increasing trend.3.Doxycycline obvious enhanced the gene and protein level of ATF4.The abundance of ATF4 gene expression in the doxycycline group and the doxycycline+inhibitor group were 1.901 times and 2.015 times that of the solvent group,(p<0.01),the difference was statistically significant.4.Doxycycline significantly increased the gene expression level and protein expression level of CHOP.The abundance of DDIT3 gene expression in the doxycycline group and the doxycycline+inhibitor group was divided into 5.407 times and 6.339 times that of the solvent group(p<0.01);and has obvious difference in the two groups?Conclusions1.Doxycycline triggers the endoplasmic reticulum stress response in HeLa cells.2.The EIF2 Signaling pathway related to endoplasmic reticulum stress is activated.3.Doxycycline induces HeLa cell apoptosis through the PERK-eIF2?-ATF4 signaling pathway.In summary,in this study we confirmed that Doxycycline triggers the endoplasmic reticulum stress response in HeLa cells,activates the EIF2 Signaling pathway,up-regulates the endoplasmic reticulum stress related proteins GRP78,eIF2?,PERK,ATF4,CHOP,and affects the endoplasmic reticulum processes such as protein biological production,and trigger the Caspase cascade reaction,lead to cell apoptosis.That is,Doxycycline induces apoptosis of cervical cancer HeLa cells through the PERK-eIF2?-ATF4 signaling pathway. |
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