| Backgroud:Bladder cancer is one of the most common malignant tumors of the urinary and reproductive system.About 430,000 people worldwide are diagnosed with bladder cancer each year.Such a large number of patients not only give severe challenges to clinical and basic workers in urology,oncology and other related fields,but also pose huge burden on economic and social development.Therefore,how to take earlier and more accurate diagnosis,more accurate and effective treatment has become a difficult problem for the basic and clinical workers of bladder cancer.The occurrence,development,therapeutic response and prognosis of bladder cancer are closely related to its molecular biological characteristics.At present,the molecular biology of bladder cancer has received a new stage of development,which is attributed to the continuous improvement of basic theories of bladder cancer genomics,transcriptome and proteomics and the rapid development of related experimental technologies.At present,the diagnosis of bladder cancer is still mainly dependent on cystoscopy.However,as an invasive examination,cystoscopy not only brings inevitable pain to the patient,increases the psychological burden of the patient,but also has the risk of urinary tract infection,bleeding and urethral injury.Therefore,a non-invasive examination method with low trauma,high sensitivity and specificity is urgently needed to assist in the diagnosis of bladder cancer.As a non-invasive diagnostic method,urine abscission cytology has been widely expected at the beginning.However,due to its low sensitivity and specificity,it has not been able to replace the dominant role of cystoscopy in the diagnosis of bladder cancer.With the continuous progress of bladder cancer molecular biology research,the research of molecular tumor markers in the noninvasive diagnosis of bladder cancer also changes with each passing day,but there is not a marker which is recommended that can replace cystoscopy,it still needs a more thorough basic and clinical research to search and select the ideal bladder cancer molecular markersFor patients with metastatic bladder cancer and other advanced diseases,the current clinical treatment is mainly systemic chemotherapy,but for more than 20 years,the first-line systemic chemotherapy drugs for bladder cancer are mainly based on cis-platinum,with no significant progress.With the development of molecular biology of bladder cancer,targeted therapy for bladder cancer has received a new stage of development.The target of targeted therapies is to interfere tumor-specific signaling pathways,and some targeted therapies have entered clinical trials.However,due to the diversity and individual differences of bladder cancer,only one or several targeted drugs cannot effectively treat all advanced bladder cancer.Therefore,more basic and translational studies are needed to provide more effective targeted drugs for clinical treatmentPNO1 gene is located in human chromosome 2p14 and consists of 7 exons and 6 introns.The PNO1 protein is a nucleolus protein.Ribosome is the place where cells synthesize proteins,known as the "protein factory of cells".It is composed of two subunits of size and many proteins are involved in the late stage of ribosome small subunits maturation,including PNO1 protein.At present,there are very few basic researches on PNO1 gene,and the expression and mechanism of PNO1 gene in human bladder cancer have not been studied in depth.This study aims to provide new and valuable theoretical basis and experimental basis for the diagnosis and targeted therapy of molecular tumor markers of bladder cancer by studying the expression and mechanism of PNO1 gene in bladder cancer.ObjectiveThis study intends to analyze the relationship between PNO1 gene expression and pathological grade and clinical stage of bladder cancer by detecting PNO1 gene expression in clinical specimens of human bladder cancer.Then,using RNA interference technology for target PNO1 gene and conducting experiments in vivo and in vitro to study its influence on the biological behavior of bladder cancer cells,and explore possible downstream genes and related pathways.Methods1.Examining the expression of PNO1 in clinical specimens and cell lines of human bladder cancer1.1 The method of immunohistochemistry was used to examine the expression of PNO1 in specimens of bladder cancerThe process of slicing,dewaxing,gradient hydration,adding antibody,rinsing and adding chromogenic agent was performed for the paraffin-embedded bladder cancer specimens.Finally,the tissue sections were observed under the microscope,and the view field photos were taken and collected.The protein expression was determined by the composition of dyeing scope and dyeing degree observed under the microscope.1.2 Detecting the expression of PNO1 in bladder cancer cell lines using real-time PCRFirstly,primers needed for PCR reaction were designed.Reverse transcription is performed to obtain cDNA.After the configuration of reaction system,real-time quantitative PCR was conducted.Finally,the instrument software was used for data analysis.2.Detecting the effects of interfering PNO1 gene expression on the biological behavior of bladder cancer cells,and its downstream genes and regulatory pathways2.1 Design of interference target for PNO1 and construction of lentivirus vectorFirstly,PNO1 gene was used as the template to design interference target,and shRNA interference sequence was designed according to the interference target sequence.After the design,double-stranded DNA oligonucleotides were synthesized.Then,the construction of lentivirus vector with RNA interference sequence was carried out:firstly,the GV115 vector was linearized,and then the double-stranded DNA oligonucleotides were connected with the linearized vector.The connecting products were transformed into Ecoli receptive cells.Finally,plasmid extraction was carried out to obtain the lentivirus vector carrying the nucleotide sequence of RNA interference target.2.2 RNAi lentivirus packaging for PNO1 geneFirstly,plasmid transfection:293T cells at logarithmic growth stage were treated for the preparation of transfection.Each DNA solution and transfection reagent prepared were added to the sterilized centrifuge tube,then,mixed them evenly and slowly dropped into 293T cell culture medium for transfection.Then,lentivirus harvest and concentration:the supernatant of the transfected 293T cells was collected and packed in virus tubes as required and stored at-80 C.2.3 5637 and T24 human bladder cancer cells were infected by PNO1-shRNA lentivirusFirstly,5637 and T24 human bladder cancer cells were infected by PNO1-shRNA lentivirus particles labeled with GFP(green fluorescent protein)according to different multiplicity of infection(MOI).Then,5637 and T24 human bladder cancer cells at logarithmic growth stage were digested with trypsin,and cell suspensions were inoculated and cultured.When the cell fusion rate reached 30%,lentivirus PNO1-shRNA and control virus were added for infection.About 72h after infection,the expression of GFP was observed by fluorescence microscope,and the cell group with well cell status and qualified infection efficiency was selected for downstream detection.2.4 Detection of PNO1 gene expression in 5637 and T24 human bladder cancer cells interfered by PNO1 gene(1)The knockdown efficiency of PNO1 gene at mRNA level was detected by real-time PCRHuman bladder cancer cells 5637 and T24 were divided into two experimental groups:shCtrl group(negative control virus infected cell group)and shPNO1 group(PNO1 gene shRNA infected cell group).Then,real-time PCR was used to detect themRNA expression of PNO1 gene.(2)The protein expression level of PNO1 gene after interference was detected by Western BlottingCell samples were cleaved and total proteins were extracted,followed by SDS-PAGE electrophoresis,immunoblotting(membrane transfer),antibody hybridization,and finally X-ray development.2.5 Effect of PNO1 gene knockdown on the growth and proliferation of 5637 and T24 cell lines(1)Detection of cell growth by Celigo cell countingThe shCtrl and shPNO1 cells at logarithmic phase were inoculated in the ceilings after trypsin digestion.Performing the Celigo cell counting every day,and accurately calculating the number of cells with green fluorescence in each scan orifice.The growth curves were made based on cell count and cell proliferation ratio respectively,according to the cell count and point-in-time.(2)Cell activity testMTT method was used to detect cell vitality of shCtrl group and shPNO1 group bladder cancer cells:firstly,cells at logarithmic phase were inoculated in the ceilings after trypsin digestion,and then the thiazole blue(MTT)was added in the wells of culture plate.Fully absorbing culture medium 4h later,and dimethyl sulfoxide(DMSO)was added to dissolve the formazan particles.Then,the microplate reader was used to detect the absorbance,and the data collected were analysed statistically.(3)Cell cloning formation detectionFirstly,shCtrl and shPNO1 cells at logarithmic phase were inoculated in the ceilings after trypsin digestion.Then cell culture was terminated while the cell number in most single clones was more than 50,and the cell clones were photographed under fluorescence microscope before the end of the experiment.Finally,the cells were fixed with paraformaldehyde,and stained with GIEMSA dye,then photographed with digital camera,and the number of clones was calculated finally.2.6 The influence of PNO1 gene interference on the apoptosis of bladder cancer cells was detected by flow cytometryFirstly,5637 and T24 bladder cancer cells were divided into two experimental:shCtrl and shPNO1 group.Then,each group was inoculated to a 6-well.After routinely culturing and inducing apoptosis,the cell suspension was centrifuged,and dyed with Annexin V-APC.Finally,the amount of apoptotic cells was detected to verify the relationship between PNO1 gene and cell apoptosis.2.7 The downstream genes of PNO1 gene were screened and verified by Western BlottingThe total RNA was extracted from each group respectively.The IVT reaction(In vitro reverse transcription)was performed to amplify RNA.IPA(Ingenuity Pathway Analysis)analysis software was then used to analyse the result,and screen the downstream genes of PNO1 gene preliminarily.Meanwhile,the map of gene network was made.Finally,the downstream genes of PNO1 gene screened were verified by Western Blotting.2.8 The downstream regulatory pathway of PNO1 gene was detectedIPA gene pathway analysis software was used to integrate and analyze the differently expressed genes analyzed by gene chip and the 800 signal and metabolic pathways collected by this software database,and the signal pathways with enriched differently expressed genes were preliminarily screened.Then,PathScan(?)Intracellular Signaling Antibody Array Kit(pathway protein Antibody chip)was used to detect and compare the changes of critical signal molecules of these Signaling pathways between shCtrl group and shPNO1 group of T24 human bladder cancer cells,to study the related regulatory pathways of PNO1 gene in bladder cancer.The experimental procedures included cell lysis,incubation,reaction with antibody detection solution,HRP and chemiluminescence reagent,development imaging and analysis.2.9 The mRNA expression of mTOR gene after PNO1 gene interference wasdetected by real-time PCRReal-time PCR was used to detect the mRNA expression of mTOR gene between shCtrl group and shPNO1 group.3.Tumorigenesis in animals:effects of PNO1 gene interference on the bladder cancer xenografts in nude mice3.1 Establishment of nude mice model with xenograft of bladder cancer Tumor cells at logarithmic growth stage were digested by trypsin,and made into cell suspension with complete medium respectively.10 nude mice in each group were injected by the tumor cell at the subcutaneous tissue of right armpit,and the nude mice were carefully fed and the growth status was observed.3.2 Volume and quality measurement and in vivo imaging of transplanted tumorsData of routine observational indexes were collected twice a week,including weight of nude mice,long and short diameter of tumor grafts,etc.36 days after the subcutaneous injection of tumorigenic cells,in vivo imaging detection was carried out.After that,nude mice were killed,the tumor was removed,photos of nude mice and transplanted tumor were taken,the tumors were weighed and measured.The data were collected.3.3 The expression of Ki-67 protein in transplanted tumor tissues was detected by immunohistochemistryThe tumor tissues were firstly embedded in paraffin,and then underwent the process of slicing,dewaxing and hydrating.Then the tumor sections were labeled with Ki67 antibody,and observed under microscope after color rendering and film sealing.Finally,collecting the data,photographing and analyzing were performed.3.4 The mRNA expression of Bcl-2 and Bax gene in the transplanted tumors was detected by real-time PCR Real-time PCR was used to compare the mRNA expression of Bcl-2 and Bax gene in the transplanted tumors between shCtrl group and shPNO1 group.3.5 The mRNA expression of mTOR gene in the transplanted tumors was detected by real-time PCRReal-time PCR was used to compare the mRNA expression of mTOR gene in the transplanted tumors between shCtrl group and shPNO1 group.4.Statistical analysesSPSS 25.0 was used for statistical analysis.ANOVA or t-test was used for statistical analysis of measurement data,while Fisher’s exact test or chi-square test was used for categorical data.P<0.05 was considered statistically significantResults1.1 PNO1 protein expression was increased in bladder cancer tissues and correlated with pathological grade and clinical stageImmunohistochemistry showed that PNO1 protein expression level was very low in normal bladder tissues,but increased in both low and high grade bladder cancer tissues and localized in the nucleus.The bladder cancer samples were divided into different groups according to clinical stage and pathological grade,and the results showed that the later the clinical stage was,the higher the pathological grade was,and the higher the PNO1 protein expression level was,which suggest that the PNO1 protein expression in bladder cancer tissues was related to the pathological grade and clinical stage of tumor.1.2 The mRNA expression of PNO1 gene in T24 and 5637 human bladder cancer cells was increasedThrough comparative analysis with human bladder epithelium immortalized cell SV-HUC-1,it is shown that PNO1 gene had high expression level of mRNA in both 5637 and T24 bladder cancer cell2.1 5637 and T24 human bladder cancer cell lines interfered of PNO1 gene were successfully constructedThe 5637 and T24 bladder cancer cell line models with low PNO1 expression level were successfully established.Observation results under fluorescence microscope 72 hours after lentivirus infection showed that the infection efficiency reached to 80%and the cell status was normal2.2 PNO1 gene expression was significantly down-regulated in 5637 and T24 human bladder cancer cells with PNO1 gene interference(1)The mRNA expression levels of 5637 and T24 human bladder cancer cells with PNO1 gene interference were significantly decreased using real-time PCRThe results showed that compared with shCtrl group,the expression of PNO1 gene in shPNO1 group was significantly inhibited by shRNA lentivirus infection,and the knockdown efficiency was 58.6%and 71.5%in 5637 and T24 cell lines respectively.(2)The protein expression of 5637 and T24 human bladder cancer cells with PNO1 gene interference were significantly decreased using Western Blotting The protein expression level of PNO1 in shPNO1 group was significantly lower than that in shCtrl group.2.3 The cell growth and proliferation was significantly inhibited in the PNO1 interfering 5637 and T24 human bladder cancer cells(1)Detection of cell growth by Celigo cell counting The number of cells in the shPNO1 group began to decline gradually from the third day of observation of self-scanning plate,while the number of cells in the shCtrl group continued to increase,indicating that the proliferation of bladder cancer cells in the shPNO1 group was significantly inhibited.(2)Cell activity test The results showed that the absorption rate and the change of absorption rate at 490 nm of bladder cancer cells in the control group were significantly higher than those in shPNO1 group,indicating that the prolif-eration activity of bladder cancer cells in shPNO1 group was significantly inhibited,compared with the control group.(3)Cell cloning formation detection The number of clones formed and the average number of clones in shPNO1 group were significantly lower than those in shCtrl group.This indicated that in 5637 and T24 human bladder cancer cells,the cloning ability of cancer cell in shPNO1 group was significantly inhibited,suggesting that knockdown of PNO1 gene could inhibit the proliferation of bladder cancer cells.2.4 The apoptosis rates of 5637 and T24 human bladder cancer cells with PNO1 gene interference were significantly increasedThe results showed that the apoptosis rate of shPNO1 group was significantly higher than that of shCtrl group.This indicated that in 5637 and T24 human bladder cancer cells,the apoptosis number in shPNO1 group was increased,suggesting that PNO1 gene could inhibit the apoptosis of bladder cancer cells.2.5 The downstream genes of PNO1 gene were initially screened by the gene chip combined with IPA analysisBy comparing the gene chip detection results of these two groups,genes with significant differences in gene expression between the two groups were preliminarily screened out.Compared with the shCtrl group,the up-regulated gene number in the shPNO1 group was 675,and the down-regulated gene number was 868.Then,the results of gene chip analysis were input into the IPA data analysis software,and the downstream genes regulated by PNO1 gene-CD44,PTGS2,CCND1,CDK1,F3,CXCL8 and FOSL1 were preliminarily screened.2.6 The downstream genes of PNO1 gene were verified by Western blottingWestern Blotting was used to detect the protein products expressed by CD44,PTGS2,CCND1,CDK1,F3,CXCL8 and FOSL1 genes.The results showed that,except for F3 protein,the expressions of CD44,CCND1,FOSL1,PTGS2,CDK1 and CXCL8 were significantly down-regulated in shPNO1 group compared with shCtrl group.This suggests that PNO1 plays a role in T24 bladder cancer cells by regulating CD44,PTGS2,CCND1,CDK1,CXCL8 and FOSL1 genes,while F3 is excluded.2.7 Downstream regulatory pathways of PNO1 genesTen signal pathways with enriched differently expressed genes were preliminarily screened,including the mTOR signal pathway,ERKS signal pathway and other classical signal pathways.Then antibody microarray was performed to compare the changes of critical signal molecules in the signal pathway between shCtrl and shPNO1 group of T24 human bladder cancer cell lines.The results showed that the expression of multiple critical signal molecules were down-regulated in the shPNO1 group,and the expression levels of mTOR and p70 S6 kinase were significantly down-regulated,with statistical significance.This suggests that PNO1 gene may affect bladder cancer cells by regulating the mTOR signaling pathway.2.8 The mRNA expression of mTOR gene was decreased after PNO1 gene interferenceThe mRNA expression of mTOR gene in shPNO1 group were significantly lower than those in shCtrl group.3.1 PNO1 gene interference can inhibit the growth of transplanted tumor Results showed that the difference in tumor volume between the two groups increased with the extension of time.The volume and weight of transplanted tumor of shPNO1 group were significantly smaller than that of shCtrl group on the day of death.This indicates that inhibiting the expression of PNO1 gene can reduce the proliferation rate of bladder cancer cells in vivo,and inhibit the tumorigenic ability at the same time.3.2 In vivo imaging showed that the growth of transplanted tumor was inhibited after PNO1 gene interferenceThe total fluorescence expression in shPNO1 group was significantly lower than that in shCtrl group,suggesting that interfering with PNO1 gene could inhibit the proliferation of bladder cancer cells and the growth of transplanted tumors in vivo.3.3 The expression of Ki67 protein in transplanted tumor was significantly down-regulated after PNO1 gene interference The expression of Ki67 in shPNO1 group was significantly lower than that in shCtrl group.This suggests that reducing PNO1 gene expression may inhibit the proliferation rate and tumorigenic ability of bladder cancer cells in vivo.3.4 The mRNA expression of Bcl-2 was significantly down-regulated while Bax mRNA expression was up-regulated after the PNO1 interference The mRNA expression of Bcl-2 in shPNO1 group was significantly lower than that in shCtrl group,while the expression of Bax in shPNO1 group was significantly higher than that in shCtrl group.This suggests that reducing PNO1 gene expression can induce the apoptosis of bladder cancer cells in vivo.3.5 The mRNA expression of mTOR gene was decreased in transplanted tumor after PNO1 gene interferenceThe mRNA expression of mTOR gene in shPNO1 group were significantly lower than those in shCtrl group in transplanted tumor.This suggests that PNO1 gene can promote the proliferation of bladder cancer cells in vivo via mTOR signaling pathway.Conclusion1.The expression of PNO1 gene was increased in human both bladder cancer clinical specimens and cell lines.Meanwhile,its expression correlated with the grade and stage of the tumor.The higher the pathological grade and clinical stage,the higher the expression of PNO1 protein.2.The growth status,cell vitality and cloning ability of bladder cancer cells were significantly inhibited after PNO1 gene interference,and the number of apoptotic cell was significantly increased,indicating that PNO1 gene can promote cell proliferation and inhibit cell apoptosis in bladder cancer.Further mechanism studies showed that PNO1 gene influences the growth and proliferation of bladder cancer cells by regulating CD44,PTGS2,CCND1,CDK1,CXCL8,FOSL1 genes,and mTOR signaling pathway is involved.3.The growth of bladder cancer xenografts and amount of living fluorescence expression in nude mice were significantly suppressed after PNO1 gene interference in vivo.Meanwhile,the expression level of Ki67,Bcl-2 and mTOR gene decreased obviously,while the expression of Bax was significantly increased,which suggests that PNO1 gene interference could effectively inhibit cell proliferation and promote cell apoptosis of bladder cancer xenograft in vivo via mTOR signaling pathway. |
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