| 1 Background and objectiveNephroblastoma is one of the most common malignant solid tumors in the urinary system of children,with a complex pathogenesis.It is valuable to probe the key gene targets that affect the development of nephroblastoma,which not only deepens the understand of its pathogenesis,but also provides a potentially sensitive and accurate diagnostic method.In recent years,tumor researchers have gradually shifted their interests from coding genes to non-coding genes.There are numerous non-coding RNAs and they play wide roles in the development and progression of tumors.LncRNA is a class of RNA molecules with a transcript length >= 200 nt.They do not encode proteins but regulate the expression levels of genes at various levels(such as epigenetic regulation,transcriptional regulation,and post-transcriptional regulation)in the RNA form.The mechanisms of their action are complex but can be divided into three types: epigenetic regulation,transcriptional regulation and post-transcriptional regulation.Regarding the pathogenesis of nephroblastoma influenced by LncRNA,many studies have been reported;but compared with other tumors,the known findings are still insufficient and need to be deepened.Our team has developed new peptides for nephroblastoma treatment and acquired a nephroblastoma-specific protein marker protein polypeptide M/Z6455.5Da.This study intends to study the in-vivo and in-vitro roles of polypeptide drug M/Z6455.5Da on nephroblastoma,in combination with the Microarray technology,and explore the key changed genes caused by protein polypeptide M/Z6455.5Da treatment.Thereby,this paper can be divided into the following four parts.Part I: Protein polypeptide M/Z6455.5Da treatment of nephroblastoma caused changes in the expression of LncRNA UCHL1-AS1 and mRNA UCHL1;Part II: Effects of LncRNA UCHL1-AS1 in-vitro growth of nephroblastoma and the in-vitro anti-cancer effect of protein polypeptide M/Z6455.5Da;Part III: Overexpression of LncRNA UCHL1-AS1 promotes the growth of nephroblastoma in vivo and the effect of protein polypeptide M/Z6455.5Da on in-vivo tumors;Part IV: Expression profiling of protein polypeptide M/Z6455.5Da treated nephroblastoma cell line through Microarray.Part I: Polypeptide M/Z6455.5Da treatment of nephroblastoma caused changes in the expression of LncRNA UCHL1-AS1 and mRNA UCHL1 1 Materials and methods 1.1 Experimental cells and animalsHuman nephroblastoma cell SK-NEP-1: from the ATCC cell bank;G401 cell line: from the Basic Medical Cell Center of the Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences.Nude mice: female,5 weeks old,purchased from SLAC.1.2 Cell cultureSK-NEP-1 cells were cultured in 15%FBS+Mc Coy’s 5A(Modified)and G401 cells were cultured normally in 10% FBS + DMEM medium and grew to 90% confluency before use.Add the polypeptide drug when the cell activity was normal.1.3 Polypeptide drug M/Z6455.5Da treatmentSK-NEP-1 and G401 were cultured in 6-well plates respectively at 1*106 cells per well.After adhering,the supernatant was discarded and serum-free Mc Coy’s 5A(Modified)Medium and DMEM medium were replaced.After 48 hours of polypeptide drug M/Z6455.5Da intervened,the supernatant was discarded,and the cells were extracted for RNA and protein.Cells were divided into four groups: 0 μM,10 μM,20 μM and 40 μM,according to the polypeptide drug M/Z6455.5Da concentration.1.4 Realtime PCRTotal RNA extraction was performed using the Trizol method,RNA concentration was determined,and RNA integrity was examined by agarose electrophoresis.Thereafter,Realtime PCR experiments were performed with different primers and programs.The identified genes were as follows: GAPDH,UCHL1-AS1,DLEU-1,BCYRN-1,beta-catenin,UCHL-1,Snail-1,c-Myc.1.5 Western blotAbout 100 ul of lysate was added to each cell sample to completely lyse the cells.The insoluble precipitate was removed by centrifugation for 10 minutes.The protein samples were separated by 10% SDS-PAGE.After electrophoresis,semi-dry transfer to the PVDF membrane,the blots were blocked with Blocking Buffer for 2 hours and then washed 3 times with TBST for 10 minutes.Appropriately diluted primary antibodies were added and incubate overnight at 4C.The membrane was washed 5 times with TBST for 10 minutes each time.Appropriately diluted secondary antibodies were added and incubated for 2 hours at room temperature.The membrane was washed 5 times with TBST for 10 minutes each time.Chemiluminescence detection was performed using an ECL chemiluminescence kit,and the abundance of each blot was analyzed.The detected proteins were: GAPDH,beta-catenin,UCHL-1,Snail-1,c-Myc.1.6 data analysisGraphpad prism 6 software was used for data analysis and mapping.One-way ANOVA analysis of variance was used for comparison between groups at one time point.The specific statistical difference was recorded as p < 0.05.2 Results: 2.1 Protein polypeptide M/Z6455.5Da down-regulates lncRNA UCHL1-AS1 and mRNA UCHL1 expressionRealtime PCR results showed that lncRNA UCHL1-AS1 and mRNA UCHL1 expression levels were down-regulated dose-dependently with protein polypeptide M/Z6455.5Da(20 μM and 40 μM Group vs Control group p<0.01)in SK-NEP-1 cells treated with polypeptide drug M/Z6455.5Da for 48 hours.In addition,the expression levels of two LncRNAs DLEU1 and BCYRN1 were also significantly down-regulated(p < 0.01 or 0.05).At mRNA levels,UCHL1 and c-Myc were also down-regulated in a dose-dependent manner(p < 0.01 or 0.05).In G401 cells,the results were consistent with SK-NEP-1cells.LncRNA UCHL1-AS1 and mRNA UCHL1 expression levels were down-regulated with protein polypeptide M/Z6455.5Da treatment(20 μM and 40 μM vs Control p<0.05).DLEU1 levels were significantly down-regulated(20 μM vs 40 μM vs.Control group p<0.05),while BCYRN1 levels were significantly up-regulated at 20 μM,as opposed to SK-NEP-1 cell experiments.At mRNA expression levels,c-Myc was also down-regulated in a dose-dependent manner(20 μM vs Control group p<0.05;40 μM vs vs Control group p<0.01).2.2 Protein polypeptide M/Z6455.5Da down-regulates UCHL1 protein expression levelWestern blot showed that after 48 hours of treatment with protein polypeptide M/Z6455.5Da,UCHL1,CDK-1 and c-Myc showed consistent changes with Realtime PCR results in SK-NEP-1 and G401 cells,that were dose-dependently down-regulated by protein polypeptide M/Z6455.5Da.Part II: Effects of LncRNA UCHL1-AS1 in-vitro growth of nephroblastoma and the in-vitro anti-cancer effect of polypeptide M/Z6455.5Da 1 Materials and methods 1.1 LncRNA UCHL1-AS1 overexpression vector and lentiviral packagingThe UCHL1-AS1 plasmid was constructed by using the p SGLV plasmid vector,and the LncRNA UCHL1-AS1 sequence was inserted.The vector plasmid,the packaging plasmid and the VSVG plasmid were transferred into 293 T cells,and it was verified by GFP fluorescence.The virus supernatant was purified and concentrated,and the virus titer was identified by fluorescence method.1.2 Lentivirus transfection of SK-NEP-1 cellsThe SK-NEP-1 stably transferred cells were cultured using the following medium: 84% Mc Coy’s 5A(Modified)Medium + 15% FBS + 1% Penicillin/Streptomycin.A volume of 0.5-1 ml of the virus solution was added to each 10-cm dish.After 48 hours,Puromycin was added to kill the cells which were not transfected with the virus.Cells were treated by Puromycin twice in 24 hours,and the fluorescence was checked.The expression of GFP was observed under a microscope.When the positive cells reached 90% or more,the cells were expanded and the expression of GFP was observed again.The identified stable strains were used for cell function experiments.Subsequently,the overexpression of UCHL1-AS1 gene in stable transgenic cells was detected by QPCR.1.3 Cell proliferation activityThe cell proliferation activity was measured by the CCK8 method,and the amount of formazan produced by the CCK-8 reagent reflected the number of viable cells.The light absorption value was measured at a wavelength of 450 nm by a microplate reader.Cell proliferation activity levels were determined in different groups at 0,24,48 and 72 hours.1.4 Clone formation abilityThe in vitro clone formation ability was evaluated by the soft agar method,and the numbers of clones formed after 7 days of cell culture in different groups were compared.1.5 Apoptosis detectionApoptosis levels were measured by flow cytometry,stained with Annexin-V/PI double stain,and the percentage of total apoptotic cells in early and late stages was analyzed by flow cytometry.1.6 Cell cycle detectionThe fluorescence intensity was measured by flow cytometry,and various phases of the cell cycle were reflected.About 3 million cells were counted and analyzed the proportion of cells in each cell cycle,especially the percentage of cells in G0-G1 phase.1.7 Cell invasion ability testCell invasion was assessed using a Trans-well chamber.The upper and lower chambers are separated by a polycarbonate membrane.SK-NEP-1 cells were seeded in the upper chamber,and the number of cells invading to the lower layer was observed to reflect the invasive ability of the cells.The cells were cultured for 48 h,stained with 0.1% crystal violet for 20 min,washed 3 times with PBS,and the number of cells in the lower layer was observed.1.8 Experimental groupingFollowing groups were divided:SK-NEP-1: wild type SK-NEP-1 cells;SK-NEP-1 + M/Z6455.5Da: wild type SK-NEP-1 cells added with polypeptide drug M/Z6455.5Da(40 μM);SK-NEP-1-NC: empty vector stably transformed SK-NEP-1 cells;SK-NEP-1-NC+M/Z6455.5Da: empty vector stably transformed SK-NEP-1 cells,cells or animals treated with polypeptide drug M/Z6455.5Da(40 μM);SK-NEP-1-UCHL1-AS1:SK-NEP-1 cells transfected with UCHL1-AS1 overexpressing virus;SK-NEP-1-UCHL1-AS1+M/Z6455.5D: SK-NEP-1 cells transfected with UCHL1-AS1 overexpressing virus,cells or animals treated with polypeptide drug M/Z6455.5Da(40 μM).1.9 Data AnalysisFor molecular expression,cell and animal experiments,was used for data analysis and mapping.Two-way ANOVA analysis of variance was used for comparison between groups at multiple time points.One-way ANOVA analysis of variance was used for comparison between groups at one time point.The LSD method was used to compare the two pairs,and the specific statistical difference was recorded as p < 0.05.2 Results 2.1 LncRNA UCHL1-AS1 promotes proliferation of nephroblastoma in vitroThe cell proliferation activity of SK-NEP-1 nephroblastoma was measured by CCK8 method for 72 hours.The results showed that overexpression of UCHL1-AS1 significantly increased cell proliferation activity compared with the empty vector control(SK-NEP-1-NC)(48 and 72 hours,SK-NEP-1-UCHL1-AS1 vs SK-NEP-1-NC,p < 0.01);and for each stable cell,M/Z6455.5Da significantly inhibited cell viability at time points(48 and 72 hours,SK-NEP-1-UCHL1-AS1+ M/Z6455.5Da vs SK-NEP1-UCHL1-AS1,p < 0.01;SK-NEP-1-NC+M/Z6455.5Da vs SK-NEP1-NC,p < 0.01;SK-NEP-1+M/Z6455.5Da vs SK-NEP1,p < 0.01).However,under the protein polypeptide M/Z6455.5Da exposure,the overexpressed UCHL1-AS1 group still had a highly markedly enhanced cell viability compared to the SK-NEP-1-NC group(72 hours: SK-NEP-1-UCHL1-AS1)+M/Z6455.5Da vs SK-NEP-1-NC+M/ Z6455.5Da,p < 0.01).These results suggest that overexpression of UCHL1-AS1 promotes proliferation of nephroblastoma in vitro.2.2 LncRNA UCHL1-AS1 promotes in vitro clonal formation of nephroblastomaSoft agar experiments showed that overexpression of UCHL1-AS1 significantly increased the clone number compared to the empty vector control(SK-NEP-1-NC),whereas for each group of virus transfer,the number of clone formation was slightly reduced by protein polypeptide M/Z6455.5Da treatment.The present results suggest that high expression of UCHL1-AS1 has a promoting effect on the clonal formation of nephroblastoma.2.3 LncRNA UCHL1-AS1 inhibits the apoptosis level of nephroblastoma in vitroThe in vitro apoptosis level of SK-NEP-1 cells was detected by flow cytometry.The results showed that overexpression of UCHL1-AS1 significantly attenuated the level of apoptosis in vitro compared with the empty vector control(p < 0.05).Protein polypeptide M/Z6455.5Da administration significantly increased the level of apoptosis(M/Z6455.5Da vs Vehicle,p < 0.05).These results suggest that high expression of UCHL1-AS1 can reduce the level of apoptosis in nephroblastoma in vitro,while protein polypeptide M/Z6455.5Da can play a therapeutic role by promoting apoptosis.2.4 LncRNA UCHL1-AS1 reduces the proportion of G0-1 cell cycleSimilar to the above results,overexpression of UCHL1-AS1 significantly reduced the G0-1 cell cycle ratio compared to SK-NEP-1-NC(SK-NEP-1-UCHL1-AS1 vs SK-NEP-1-NC,p < 0.01),in SK-NEP-1 cells and SK-NEP-1-NC cells,protein polypeptide M/Z6455.5Da treatment significantly increased the G0-1 cell cycle ratio(p<0.05).Under the exposure of protein polypeptide M/Z6455.5Da,the proportion of G0-1 cell cycle in UCHL1-AS1 overexpression group was still significantly lower than that in the control group(SK-NEP-1-UCHL1-AS1+M/ Z6455.5Da vs SK-NEP-1-NC+M/Z6455.5Da,p < 0.01).These results suggest that high expression of UCHL1-AS1 can reduce the cell cycle arrest level of nephroblastoma in vitro,and protein polypeptide M/Z6455.5Da can play a therapeutic role by promoting cycle arrest.2.5 LncRNA UCHL1-AS1 promotes in vitro invasion of nephroblastomaThe invasion ability of SK-NEP-1 cells was evaluated by48-hour Trans-well chamber test.The results showed that under protein polypeptide M/Z6455.5Da exposure,the invasive ability of each group was lower than that of the control;while LncRNA UCHL1-AS1 was overexpressed.The number of invasive cells was significantly higher than that of the empty vector control,and this trend was consistent with protein polypeptide M/Z6455.5Da exposure under protein polypeptide M/Z6455.5Da exposure.The above results suggest that the high expression of UCHL1-AS1 can promote the invasion of nephroblastoma in vitro,and protein polypeptide M/Z6455.5Da can inhibit the invasion of nephroblastoma in vitro,thereby exerting the against anti-nephroblastoma effect in vitro.Part III: Overexpression of UCHL1-AS1 promotes the growth of nephroblastoma in vivo and the effect of polypeptide M/Z6455.5Da on in-vivo tumors 1 Materials and methods 1.1 Nephroblastoma tumor-bearing nude mouse modelFive-week-old female BALB/c nude mice were kept in an SPF-class animal laboratory.The breeding environment was 12 hours/12 hours day and night,23 °C.All inoculation operations were performed in a sterile environment.The above groups of SK-NEP-1 cells were resuspended in Mc Coy’s 5A medium and BD Matrigel Matrigel(volume ratio 1:1),and the final inoculation concentration was 1 × 10 7 / 0.2 ml.After disinfection at the left forearm armpit,cells were injected into the left forelimb axilla with a 0.5 m L syringe to form a subcutaneous hillock.Continuous observation was performed after inoculation to record the feeding,mental state and daily activity.When the diameter of the nephroblastoma reached 0.5 cm,the tumor-bearing nude mouse model was considered successfully established.When the volume increased to about 200 mm3,each cell line was divided into two groups.One group was intraperitoneally injected with polypeptide drug M/Z6455.5Da(4 mg/kg),and the other group was injected with an equal volume of normal saline.The tumor volume was measured every two days for one week and the luciferase activity was measured on the eighth day.The method of measuring volume is to record the long diameter(L)and short diameter(S)of the transplanted tumor with a vernier caliper.The tumor volume was calculated by the following formula: V = 0.5 * L * S * S 1.2 In vivo luciferase imagingSince the lentiviral plasmid vector has a luciferase reporter gene,in vivo imaging can be performed by injecting a substrate to visually evaluate the tumor-bearing level of a living animal.Animals in 4 groups with virus transfection were injected with Luciferin(100 mg/kg)intraperitoneally,before M/Z6455.5Da administration(day 0)and 4 hours before sacrifice(day 8).1.3Tumor and kidney samplingAll tumors were harvested on the 8th day after injection of M/Z6455.5Da or normal saline.The animals were sacrificed by dislocation.The tumors were removed in one minute and fixed in 4% paraformaldehyde.The kidneys were removed and placed in paraformaldehyde.1.4 HE staining of tumor sectionsThe fixed tumor tissue was dehydrated by gradient ethanol,decolorized by xylene,embedded in paraffin,sliced in a paraffin sectioner into 5 μm sections,and subjected to conventional HE staining.Specific steps include: dewaxing,dehydration,hematoxylin staining,water washing,0.25% hydrochloric acid alcohol color separation,washing,eosin staining,dehydration,xylene treatment,and neutral gum sealing.Tumor tissue was observed under a microscope.1.5 Masson staining of kidney tissuesTo test whether the expression regulation of UCHL1-AS1 and the intraperitoneal administration of M/Z6455.5Da affect renal fibrosis in nude mice,the kidneys were collected,fixed,dehydrated and sectioned,and subjected to Masson staining.Specific steps include: dewaxing,dehydration,Regaud’s hematoxylin,water washing,1% hydrochloric acid alcohol color separation,acid fuchsin staining,washing,1% phosphotungstic acid treatment,aniline blue treatment,1% acetic acid rinse,dehydration,Toluene treatment,and neutral gum sealing,etc.The kidney tissue was observed under a microscope.1.6 Experimental groupingFollowing groups were divided:SK-NEP-1: wild type SK-NEP-1 cells;SK-NEP-1 + M/Z6455.5Da: wild type SK-NEP-1 cells added with polypeptide drug M/Z6455.5Da(40 μM);SK-NEP-1-NC: empty vector stably transformed SK-NEP-1 cells;SK-NEP-1-NC+M/Z6455.5Da: empty vector stably transformed SK-NEP-1 cells,cells or animals treated with polypeptide drug M/Z6455.5Da(40 μM);SK-NEP-1-UCHL1-AS1: SK-NEP-1 cells transfected with UCHL1-AS1 overexpressing virus;SK-NEP-1-UCHL1-AS1+M/Z6455.5D: SK-NEP-1 cells transfected with UCHL1-AS1 overexpressing virus,cells or animals treated with polypeptide drug M/Z6455.5Da(40 μM).1.7 Data AnalysisFor all animal experiments,Graphpad prism 6 software was used for data analysis and mapping.Two-way ANOVA analysis of variance was used for comparison between groups at multiple time points.One-way ANOVA analysis of variance was used for comparison between groups at one time point.The LSD method was used to compare the two pairs,and the specific statistical difference was recorded as p < 0.05.2 Results 2.1 LncRNA UCHL1-AS1 promotes the growth of nephroblastoma in vivoThe tumor volume was measured by subcutaneous inoculation and intraperitoneal injection of protein polypeptide M/Z6455.5Da.The results showed that overexpression of LncRNA UCHL1-AS1 significantly increased the tumor growth rate.From day 4,the volume of UCHL1-AS1 overexpression was significantly higher than that of the group.In both cells,protein polypeptide M/Z6455.5Da treatment significantly reduced tumor volume(day 4-8: SK-NEP-1-UCHL1-AS1+M/Z6455.5Da vs SK-NEP-1-UCHL1-AS1 p < 0.01,SK-NEP-1-NC+ M/Z6455.5Da vs SK-NEP-1-NC p < 0.01,SK-NEP-1+M/Z6455.5Da vs SK-NEP-1 p < 0.01).In addition,in the SK-NEP-1-UCHL1-AS1 and SK-NEP-1-NC groups,Luciferin injection was performed on days 0 and 8,and the fluorescence intensity signal was recorded by in vivo imaging to observe the subcutaneous tumor burden,and the results were similar to the above findings.These results suggest that LncRNA UCHL1-AS1 can promote the growth of nephroblastoma in vivo;Protein polypeptide M/Z6455.5Da can play a role in fighting nephroblastoma in vivo.2.2 HE staining observationHE staining showed that protein polypeptide M/Z6455.5Da can cause necrosis and cavities in multiple tumors,while LncRNA UCHL1-AS1 overexpressing cells have less necrosis.Even under systemic administration of protein polypeptide M/Z6455.5Da,the necrosis and void levels were relatively low.2.3 Kidney Masson staining observation resultsThe level of renal fibrosis was observed by Masson staining,and no significant difference was observed between groups.Part IV: Expression profiling of polypeptide M/Z6455.5Da treated nephroblastoma cell line through Microarray 1 Materials and methods 1.1 Expression profiling MicroarrayThe experiment of the expression profiling Microarray was performed by Shanghai Bohao Biotechnology Co.,Ltd.,and the lncRNA probe was detected by SBC human lncRNA V6 array.1.2 Experimental groupingIn the gene expression profiling analysis,two groups were divided:SK-NEP-1: Wild type SK-NEP-1 cells;SK-NEP-1 + M/Z6455.5Da: Wild type SK-NEP-1 cells treated with polypeptide drug M/Z6455.5Da(40 μM).1.3 Data AnalysisFor expression profiling,data analysis included: normalization,box plot analysis,differential gene screening,normalized data scatter plotting,GO enrichment analysis of differential mRNA,KEGG pathway enrichment analysis of differential mRNA,LncRNA target gene prediction.2 Results 2.1 Microarray analysis of differential genesAfter differential screening gene(DEG)quasi-screening,the following differential genes were obtained: 212 mRNA differential genes,of which 69 up-regulated genes and 80 down-regulated genes;for LncRNAs,there were 666 differential genes,among which 317 were up-regulated and 349 down-regulated.UCHL1-AS1 was also verified,and its expression level decreased to 0.0285.2.2 GO and KEGG enrichment analysisThe enrichment of biological processes was found by GO enrichment analysis of differential genes,especially the enrichment of neutral amino acid transport,organic acid transmembrane transport and L-amino acid transmembrane transporter activity.The KEGG pathway enrichment analysis showed that Taste transduction and Melanoma had the highest enrichments.2.3 LncRNA target predictionAmong above differences lncRNA,1323 genes were cis targets and 18098 were trans targets.Then,it is predicted that the cis target and the differential gene of the same direction of mRNA share a common target,and the target of the common target of the trans target and the mRNA reverse change differential gene is predicted.The obtained cis co-occurred 32 targets(rejected duplicate values),and the obtained Trans co-occurrence targets 32(excluding duplicate values).Among them,there are three targets in the above two types(Trans target and differential mRNA common gene and cis target and differential mRNA common gene): CPPED1,LCTL and EREG.It can be considered that up-regulated CPPED1 and down-regulated LCTL and EREG are the three most important targets for the effect of protein polypeptide M/Z6455.5Da treatment on nephroblastoma in vitro.Conclusion1.UCHL1(protein and mRNA)and lncRNA UCHL1-AS1 expression levels were dose-dependently downregulated by M/Z6455.5Da treatment in nephroblastoma cell lines.Meanwhile,Protein polypeptide M/Z6455.5Da significantly down-regulated the in-vitro expression levels of DLEU1,CDK-1 and c-Myc.2.lncRNA UCHL1-AS1 promotes in vitro proliferation,clonal formation and invasiveness of nephroblastoma cell lines,inhibits apoptosis and cell cycle arrest.3.Overexpression of lncRNA UCHL1-AS1 promots in vivo growth of nephroblastoma but had no significant effect on renal tissue structure.4.A large number of differential genes,including 395 differential mRNAs and 666 differential lncRNAs,were detected by expression profiling of protein polypeptide M/Z6455.5Da in vitro.Among them,lncRNA UCHL1-AS1 is also one of the genes which are significantly down-regulated by protein polypeptide M/Z6455.5Da treatment.Protein polypeptide M/Z6455.5Da treatment can cause GO enrichment represented by neutral amino acid transport,organic acid transmembrane transport,L-amino acid transmembrane transporter activity,etc.Protein polypeptide M/Z6455.5Da causes KEGG pathway enrichment represented by Taste transduction,Melanoma,and VEGF signaling pathway.Differential mRNAs caused by Protein polypeptide M/Z6455.5Da included CPPED1,LCTL and EREG,which were highly consistent with the target gene of LncRNAs and may play important roles in the process of Protein polypeptide M/Z6455.5Da regulation of nephroblastoma. |
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