| Background Hepatocellular carcinoma(HCC)is a common primary liver cancer in clinical practice.It has high morbidity and mortality.It is difficult to diagnose and treat early.In order to find new diagnosis and treatment methods of liver cancer,people are paying more and more attention to the discovery and function research of new carcinogenic genes in liver cancer.It has been reported that LGALS3 BP is associated with the growth and metastasis of various malignant tumors.In the early stage,we separated the total proteins in the serum of normal people and HCC patients by SDS-PAGE electrophoresis,and then identified the differentially expressed proteins by mass spectrometry.It was found that compared with normal people,the level of LGALS3 BP in the serum of HCC patients increased.However,the biological function of LGALS3 BP gene in hepatoma cells is unclear.Based on the basis of previous studies,this project further explored the differential expression of LGALS3 BP between HCC tissues and adjacent tissues or normal liver tissues,and the correlation between LGALS3 BP expression level and the prognosis of HCC patients;By the construction of a GALS3BP-RNA interference(shRNA)lentiviral vector,to study the effects of LGALS3 BP on the proliferation,apoptosis,migration and invasion of HCC cells;Through high-throughput sequencing by RNA-seq,analysis of the changes in the transcriptional genomics of HCC cells after LGALS3 BP knockdown,And,by using the bioinformatics analysis,to explore the possible mechanism of LGALS3 BP gene involved in the proliferation,apoptosis,migration and invasion of HCC cells;The expression of GO,KEGG and GSE enrichment genes were verified by PCR,and the reliability of the enrichment analysis was analyzed,which provided experimental basis for the further study of the carcinogenic mechanism of LGALS3 BP gene,and provided new diagnostic markers and new therapeutic targets for the prevention and control of clinical liver cancer.Chapter one:The differential expression of LGALS3 BP in hepatocellular carcinoma and its relationship with prognosisObjective To study the differential expression of LGALS3 BP in hepatocellular carcinoma and its relationship with prognosis.Methods1.Access to the TCGA database through UALCAN website,download the data of liver cancer tissue samples and normal liver tissue samples,and analyze the expression of LGALS3 BP gene in liver cancer tissues and normal liver tissues;2.Enter the TCGA database through UALCAN website to analyze the relationship between LGALS3 BP gene expression and pathological grade of hepatocellular carcinoma;3.The relationship between LGALS3 BP gene expression and pathological stage of HCC was analyzed by accessing to the TCGA database through UALCAN website;4.GEPIA database was used to analyze the correlation between the expression level of LGALS3 BP mRNA and overall survival(OS)in HCC tissues;5.Immunohistochemistry was used to detect the expression of LGALS3 BP in the resected liver cancer tissues and adjacent tissues.The Seville image analysis system was used to automatically read the tissue measurement area,classify the positive grade(0～3),and calculate the positive area ratio,average optical density,positive area density and H-score respectively.Results1.In the TCGA database,371 liver cancer samples and 50 normal liver tissue samples were collected.The expression of LGALS3 BP in HCC tissues was significantly higher than that in normal liver tissues(P<0.05);2.TCGA database analysis showed that the expression level of LGALS3 BP in HCC was correlated with the pathological grade of HCC(P < 0.05);3.The results of TCGA database analysis showed that the expression level of LGALS3 BP in liver cancer was correlated with the pathological stage of HCC(P < 0.05);4.GEPIA database analysis showed that the overall survival time(OS)of patients with high expression of LGALS3 BP gene was shorter than that of patients with low expression of LGALS3BP(P< 0.05),indicating that the patients with high LGALS3 BP expression had poor prognosis;5.Immunohistochemical detection showed that the expression of LGALS3 BP in HCC tissues was significantly higher than that in adjacent tissues(P< 0.05).The positive area(%),average density,area density and H-score of LGALS3 BP protein expression were 68.46 ± 7.69,0.077 ±0.006,0.053 ± 0.009115.98 ± 11.44;The adjacent tissues were 15.79 ±0.58,0.009 ± 0.004,0.001 ± 0.000 and 63.19 ± 6.82,respectively.There were significant differences between the two groups(P< 0.05).Chapter two: The biological function of LGALS3 BP gene in liver cancer cellsObjective Based on the construction of the LGALS3 BP gene short hairpin RNA(shRNA)lentiviral vector,the biological functions of LGALS3 BP gene in hepatoma cells were studied,focusing on the effects of LGALS3 BP on proliferation,cell cycle,apoptosis,migration and invasion of liver cancer cells.Methods1.Lentiviral vectors were constructed and stably infected cell lines were established.It includes the preparation of RNA interference(shRNA)lentiviral clones,lentiviral packaging and quality detection,screening for effective targets of endogenous Sh RNA,and establishment of stably infected cell lines of LGALS3BP-Sh RNA lentivirus.2.Experiment grouping and processing.Huh-7 were divided into 2groups,in which the NC group was the negative control group,which was treated with CON313 virus(NO.CON313);Group KD was treated with LV-LGALS3BP-Sh RNA virus(NO.94272-1).72 hours after virus infection in Huh-7,the expression of GFP in two groups of cells was observed by fluorescence microscope.When the cells are in good condition and the fluorescence rate is higher than 70%,collect the cells for the next detection.3.Detection of CCK-8 cell activity.The cells of NC group and KD group in logarithmic growth phase were digested,and then were resuspended and counted.Five 96 well plates(2000 cells / well)were inoculated with 100 μL cell suspension per well,and 3 multiple holes were made in each group.Add CCK solution to one 96 well plate every24 hours at the amount of 10 μ l per well.After incubation in the incubator for 2 hours,determine the absorbance at 450 nm with an enzyme reader,5 times continuously.4.Cell cycle detection.Flow cytometry was used to detect the fluorescence intensity of propidium(PI)in Huh-7 cells,and the proportion of cells in the total number of cells in each cycle(G1,s and G2 / M phases)was calculated.5.Apoptosis detection.Flow cytometry was used to calculate the apoptosis percentage of Huh-7 cells after binding with APC annexin V.6.Celigo scratch test.In the celigo scratch experiment,a special scratch tool was used to make scratches.Celigo recognized cells and took photos.Then,the migration rate of the experimental cells in XH was calculated by analyzing and processing the images after 0 h,24 h and 48 h after cell migration in the same field,and the migration ability of the Huh-7 cell was judged.7.Transwell experiment.Huh-7 cells were inoculated in Transwell chamber with a plating volume of 105.After 144 hours of culture,the cells were fixed,stained,and photographed under a microscope.The cells were counted and analyzed on the photos with magnification of 200 times to determine the movement transfer ability of Huh-7.8.Invasion assay test.The invasion chamber was coated with a layer of matrix glue on the polycarbonate membrane to imitate the extracellular matrix.Tumor cells were planted in the upper chamber.FBS or some specific chemokines were added into the lower chamber.The invasion ability of the cells was measured by counting the number of cells entering the lower chamber.Huh-7 cells were seeding into the invasion chamber with 105 plates.After 144 hours of culture,the cells were taken out,stained and photographed under the microscope,The invasive ability of Huh-7 was evaluated by cell counting analysis on the photos with magnification of 200 times.Results1.The sequencing results of positive clone transformants were consistent with LGALS3 BP sequence,the lentiviral vector of LGALS3 BP gene was constructed successfully.2.After 72 hours of lentivirus infection,the gene expression of each sample was detected by PCR,and the relative expression level 2-ΔΔCtof LGALS3 BP gene in NC group,KD1 group,KD2 group and KD3 group were1.002±0.002,0.282±0.016,0.215±0.013,and 0.150±0.003 respectively.Compared with NC group,the relative expression of LGALS3 BP gene in KD1 group,KD2 group and KD3 group decreased significantly(P<0.05).The knock-down efficiency of LGALS3 BP gene in KD1,KD2 and KD3 groups were 71.8%,78.5% and 85.0% respectively,compared with NC group.The difference was statistically significant(P<0.05).3.The OD450 values of CCK8 in 3.KD group were 0.217±0.006,0.289±0.005,0.369±0.004,0.966±0.058,0.966±0.058 on the 1st to 5th day respectively;On the 1st-5th day in NC group,they were 0.226±0.003,0.292±0.005,0.450±0.006,2.013±0.017 and 2.013±0.017,respectively.Compared with NC group,the OD450 values of KD group on the 4th and5 th day decreased significantly,and the differences were statistically significant(P<0.05).On the 1st-5th day of KD,the cell proliferation multiples(OD450/fold)were 1.000±0.029,1.335±0.025,1.702±0.021,3.045±0.028 and 4.459±0.266,respectively.In group NC,they were1.000±0.014,1.290±0.023,1.989±0.025 and 3.845±0.044、8.905±0.077,respectively.Compared with NC group,the cell proliferation ratio(OD450/fold)of KD group decreased significantly on the 4th and 5th day,and the difference was statistically significant(P<0.05).4.After 48 hours of lentivirus infection,the percentage of G1,S and G2/M cells in KD group was 37.557±1.199,45.263±1.404 and17.180±0.491,NC respectively,and the percentage of G1,S and G2/M cells(%)in NC group was 45.990±0.609,42.317±1.260,11.697±0.668,respectively.Among them,the percentage(%)of cells in G1 phase in KD group was less than that in NC group,and the difference was statistically significant(P<0.05).The percentage(%)of G2/M cells in KD group was higher than that in NC group,and the difference was statistically significant(P<0.05).The percentage(%)of S-phase cells in KD group was higher than that in NC group,and the difference had no statistically significance(P>0.05).5.After 48 hours of lentivirus infection,the percentage(%)of apoptosis of huh-7 cells in KD group and NC group were 5.827±0.192 and 1.637±0.107,respectively.The percentage(%)of early apoptosis of huh-7 cells in KD group was significantly higher than that in NC group,with statistical significance(P<0.05).6.After 24 and 48 hours of scratching,the area of cell-free scratches in KD group was significantly reduced compared with that in NC group.The migration rate(%)and multiple of KD huh-7 cells in group A were12.51±3.01 and 25.23±1.87 respectively.The migration rate(%)and multiple of NCHUH-7 cells in group A were 20.56±4.55 and 47.00±2.51,respectively.Compared with NC group,the mobility(%)and migration times of huh-7 cells in KD group at 24 and 48 hours after Celigo scratch were significantly lower than those in NC group(P<0.05).7.After inoculation of huh-7 with Transwell for 144 hours,the cell density of HUH-7 in upper chamber of KD group was significantly lower than that of NC group.In KD group,the number and migration multiple of huh-7 cells in each visual field were 24.00±0.017 and 0.56±0.017,respectively.In NC group,the number and migration multiple of huh-7cells in each visual field were 43.00±0.026 and 1.00±0.026,respectively.Compared with NC group,After 144 hours of culture in KD Transwell,the number and migration times of huh-7 cells in each visual field decreased significantly(P<0.05).8.After 144 hours of huh-7 inoculation in invasion chamber,the cell density of HUH-7 in upper chamber of KD group was significantly lower than that of NC group.In KD group,the invasion number and invasion multiple(mean standard ± deviation)of huh-7 cells in each visual field were 28.19±1.118 and 0.49±0.020 respectively.In NC group,the invasion number and invasion multiple(mean standard ± deviation)of huh-7 cells in each visual field were 56.93±1.992 and 1.00±0.035 respectively.Compared with NC group,After 144 hours of culture in KD invasion chamber,the number and invasion times of huh-7 cells in each visual field decreased significantly(P<0.05).Chapter 3: Effect of the LGALS3 BP gene on the transc-riptomics of liver cancer cells and its bioinformatics analysisObjective In chapter 2,we found that LGALS3 BP gene can promote the proliferation,inhibit apoptosis,and promote the migration and invasion of liver cancer cells,but the corresponding mechanism is unknown.In this chapter,we will study the changes of transcriptomic biological information of liver cancer cell proliferation after LGALS3 BP gene knockdown at the mRNA level,and preliminarily reveal the mechanism of LGALS3 BP gene promoting cancer.Methods1.Experiment grouping and processing.Huh-7 were divided into 2groups,in which the NC group was the negative control group,which was treated with CON313 lentivirus(NO.CON313);Group KD was treated with LV-LGALS3BP-shRNA lentivirus(NO.94272-1)inserted into the LV-LGALS3BP-shRNA fragment.72 hours after virus infection in Huh-7,the expression of GFP in two groups of cells was observed by fluorescence microscope.2.Library construction and quality inspection.Total RNA was extracted from the sample,then mRNA with poly A tail was enriched by Oligo(d T)magnetic beads,and the RNA was random Ly broken into fragments,and then cDNA fragments were synthesized fromRNA fragments by random primers and reverse transcriptase.Then,the end of the cDNA fragment was repaired and connected to the sequencing adapter,and the cDNA of about 250～300 bp was screened by AMPure XP beads,and the PCR product was purified by AMPure XP beads again.Finally,the library is obtained.3.Sequencing on the computer.After the library is qualified,different libraries are pooled according to the requirements of effective concentration and target download data volume,and then Illumina sequencing is carried out,and 150 bp paired-end readings are generated.4.Analysis of sequencing data.Focus on differential gene expression analysis,differential gene enrichment analysis(GO,KEGG,GSEA).The GSEA enrichment analysis only performed on the KEGG isogenic datasets。5.Real-time PCR detected the mRNA expression of differential genes related to IL-17 signaling pathway in Huh-7 cells,and verified the results of KEGG and GSEA enrichment analysis.Results1.A total of 1711 differentially expressed genes were screened from the two groups,including 1337 differentially expressed genes up-regulated and 374 differentially expressed genes down-regulated.2.The enrichment analysis of GO pathway found that all the different gene sets were enriched in 6566 different GO functional categories,the up-regulated differential gene sets were enriched in 5,902 different GO functional categories and the down-regulated differential gene sets were enriched in 122 different GO functional categories.With padj less than 0.05 as the threshold of significant enrichment,All differential gene sets were significantly enriched in 170 GO functional categories,the up-regulated differential gene sets were significantly enriched in 164 GO functional categories,and the down-regulated differential gene sets were significantly enriched in 83 GO functional categories.3.The enrichment analysis of KEGG pathway found that all the differential gene concentrations were enriched in 292 different KEGG pathways,the up-regulated differential gene aggregation are enriched in269 different KEGG pathways and the down-regulated differential gene sets are enriched in 198 different KEGG pathways.With padj<0.05 as the threshold of significant enrichment,all differential gene sets were significantly enriched in the following KEGG pathways:KEGGID were hsa04974、hsa04512、hsa04978、hsa04360、hsa04080、hsa04060、hsa04657、hsa03008.4.GSEA enrichment analysis showed that the enrichment of CXCL5、CXCL8、SRSF1、 IKBKE、 CXCL3、CXCL2、CXCL10 and FOSL1 genes in IL-17 signal pathway in KD group decreased significantly,which was consistent with KEGG enrichment analysis.5.After 72 hours of lentivirus infection,Real-time PCR was GSEA to detect the mRNA expression of different genes related to IL-17 signaling pathway in Huh-7 cells.Except CXCL10,the expressions of CXCL5、CXCL8、SRSF1、IKBKE、 CXCL3、CXCL2 and FOSL1 in KD group samples were significantly reduced,and the difference was statistically significant(P<0.05).The results were the same as those of KE.Conclusions1.The expression level of LGALS3 BP in liver cancer tissues is significantly higher than that in adjacent/normal tissues,and patients with high LGALS3 BP expression have poor prognosis;2.LGALS3 BP gene can promote the proliferation,migration and invasion of liver cancer cells and inhibit the apoptosis of liver cancer cells;3.After knocking down the LGALS3 BP gene,the transcriptome of liver cancer cells changed significantly;there were 1711 differential genes between the NC group and the KD group,with 1337 up-regulated genes and 374 down-regulated genes.Among them,IL-17 signaling pathway gene set related molecules CXCL5,CXCL8,SRSF1,IKBKE,CXCL3,CXCL2,FOSL1 were significantly down-regulated,indicating that the LGALS3 BP gene may affect the biological function of liver cancer cells through the IL-17 signaling pathway. |
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