The Role Of Tension-responsive MicroRNAs In The Proliferation Of Hepatocellular Carcinoma Cells And Its Bioinformatics Analysis

BackgroundHepatitis-cirrhosis-liver cancer is considered to be a"trilogy"in the course of the occurrence of liver cancer.A large number of clinical data showed that most patients with liver cancer have background of liver fibrosis or cirrhosis.Median survival time decreased in liver cancer patients with liver cirrhosis background,the pressure in hepatic sinuses increased and cirrhosis promoted the progression of liver cancer.Some researchers found that the hardness of liver could be an independent risk factor for the occurrence of liver cancer.Another study showed that the higher the degree of hepatic fibrosis,the higher the intrahepatic metastasis rate of liver cancer,indicating that there is a very important relationship between hepatic fibrosis and proliferation,invasion and metastasis of liver cancer cells.In the portal hypertension,the pressure in the hepatic sinus is increased and the hepatic sinus is dilated significantly.Hepatocytes are directly or indirectly affected by the biomechanical factors such as the pressure of the hepatic sinus and the tension strain after the dilation of the hepatic sinusoids.However,the effect of tensile strain on hepatoma cells and its related MicroRNAs?miRNAs?have not been reported.As we know,miRNAs are 21-to-23-nucleotide-long,noncoding RNA molecules,and plays an important role in regulating cell proliferation,differentiation,and apoptosis.Some miRNAs are related to both tensile strain and liver cancer.So in this study,the effect of strain force on the proliferation of human hepatocellular carcinoma cell line HepG2 cells was measured by flow cytometry analysis,CCK-8 assay,BrdU assay and other methods.Agilent monochromatic marker chip was used to screen differentially expressed microRNAs,and RT-PCR was used to verify the results.Go and Pathway analysis were used to analyze genes to provide information on how the mechanical microenvironment of liver cancer affects the behavior of hepatoma cells.Methods1.Determination of optimal strain loading conditions for hepatoma cellsThere are three main parameters in the periodic strain loaded cells:the tensile strain amplitude,the tension strain frequency and the time.By fixing two of the parameters,the other parameter was changed to observe the effect of tension strain on the HepG2 cells?except these three parameters,all the other experimental conditions were the same?.?1?Tensile strain methodIn this experiment,the FX-4000T Strain Unit?Flexcell International?purchased by the Biomechanics Laboratory of Shanghai Jiao Tong University was used.The method was to synchronize HepG2 cells cultured in vitro with DMEM medium without FBS for 24 hours and then inoculate on the BioFlex amino-culture plates?Flexcell International?coated with rat tail collagen solution?Roche company?.After the cells were attached to the plate,the plate was placed on the BioFlex Culture Plate base in the incubator.First,the vacuum pump and the bridge were turned on,then the Flexercell Strain central controller was started on the connected computer,and the tension parameters were set according to the need.?2?Determination of optimal strain loading conditionsWe set up three groups of experiments:?1?Tension strain time group:fixed the tensile strain frequency to 1 Hz,and amplitude to 15%,then set the tension strain time to 2 h,6 h,12 h,24 h and 48 h respectively,a total of five experimental groups.?2?Tensional strain frequency group:fixed the tensile strain time to 24h,and amplitude to 15%,then set tensile strain frequency to 0.5 Hz and 1 Hz respectively in two experimental groups,a total of two experimental groups.?3?Tensile strain amplitude group:fixed the tensile strain time to 24h,and frequency to 1 Hz,then set tensile strain amplitude to 5%and 15%,a total of two experimental groups.All the control groups were non-tension loaded group.After the tensile strain loading was finished,the effect of different tension loading conditions on cell proliferation was evaluated by flow cytometry to determine the optimal strain loading conditions.2.Effect of strain loading on proliferation of hepatocellular carcinoma cellsThe HepG2 cells after synchronizing?24h?were also loaded on the tensile strain-treated by the Flexcell FX 4000T strain force device,and the non tension strain loading group was set as the control group.BrdU assay and CCK-8 assay were used to verify the effect of the proliferation of tensile strain-treated HepG2 cells.3.Differential miRNAs screening of tensile strain-treated HepG2 cell by miRNA microarray analysis?1?MiRNAs expression analysis using miRNA arrayTotal RNA was quantified by the NanoDrop ND-2000?Thermo Scientific?and the RNA integrity was assessed using Agilent Bioanalyzer 2100?Agilent Technologies?.The sample labeling,microarray hybridization and washing were performed based on the manufacturer's standard protocols.Feature Extraction software?version10.7.1.1,Agilent Technologies?was used to analyze array images to get raw data.Next,Genespring software?version 14.8,Agilent Technologies?was employed to finish the basic analysis with the raw data.Differentially expressed miRNAs were then identified through fold change as well as P value calculated using t-test.The threshold set for up-and down-regulated genes was a fold change>=2.0 and a P value<=0.05.?2?Verification of the differential miRNAsThe total RNAs of HepG2 cells in each group were extracted by Trizol method.The concentration and purity of RNA were detected by UV detector at 260 nm and 280 nm,respectively.The cDNA was synthesized by reverse transcription kit?Thermo Fisher?and used as template for real-time fluorescence quantitative PCR amplification.U6 was used as internal reference in the experiment.The expression of mRNA was analyzed by comparing Ct(2^-??Ct).The results showed that the differential miRNAs was accurate or not.4.Bioinformatic analysis of tensile strain regulating liver cancer cell proliferationTarget genes of differentially expressed miRNAs were the intersection predicted with the3databases:Targetscan?http://www.targetscan.org/?,microRNAorg?http://www.microrna.org?,PITA?https://genie.weizmann.ac.il/?.Two hundred and twenty-four target genes were selected from the intersection of 3 databases,of which SMAD7 and SP1 are worthy of attention.GO and pathway analysis were performed on predicted mRNA targets of the miRNAs using DAVID Bioinformatics resources and the KEGG database,respectively.Specific biological process categories and pathways were enriched.Finally,the unsupervised hierarchical clustering of differential miRNA was carried out,and the expression pattern of differential miRNA in different samples was demonstrated by thermal map,and the possible biological process affecting the function of hepatoma cells was explored.Results1.Determination of optimal strain loading conditions for hepatoma cellsThe method of using type I rat tail collagen coated with BioFlex six-hole plate to make HepG2 cells stably adhere to the plate was found.The following experimental conditions were as follows:tensile strain amplitude 15 Hz,frequency 1 Hz and time 24 h.2.Effect of strain loading on proliferation of hepatocellular carcinoma cellsThe results of BrdU assay and CCK-8 assay showed that compared with control group,the tensile strain amplitude of 15%and the frequency of 1 Hz for 24 h significantly promoted the DNA synthesis of the cells,P<0.001.3.Differentially expressed miRNAs in response to tensile strain?1?Screening of differential miRNAsTo investigate the effects of tensile strain on the miRNA profile of HepG2 cells,tensile strain was applied to the activated HepG2 cells,and RNA was extracted and subjected to microarray analysis.A total of 2557 miRNA were detected,and we identified 7significantly Differentially expressed miRNAs with a threshold of Fold Change>=2.0 and P-value<0.05,including5up-regulations?hsa-miR-296-5p,hsa-miR-6752-5p,hsa-miR-6794-5p,hsa-miR-6889-5p,hsa-miR-7845-5p?and2down-regulations?hsa-miR-4428,hsa-miR-503-5p?in tensile strain-treated group compared with control group.?2?Validation of miRNAs microarrayThe qRT-PCR was performed to validate the microarray results,and the changes of 7miRNAs detected by qRT-PCR are consistent with those of miRNA microarray.4.Bioinformatic analysis of tensile strain regulating liver cancer cell proliferationPotential target genes for those 7 differentially expressed miRNAs were searched by 3bioinformatics:miRNAorg?PITA and TargetScan,predicted 2306,1277 and 16205 target genes,respectively.In order to remove the false results,we took the intersection of 3databases for 224 target genes.Among them,SMAD7 and SP1 are worthy of attention.we use GO analysis and Pathway analysis to understand the biological characteristics of these target genes.Through GO analysis,the target genes are concentrated in cyclin-dependent protein kinase holoenzyme complex,neuromuscular junction,microtubule cytoskeleton,protein kinase binding,protein binding,transcription factor activity,RNA polymerase II distal enhancer,positive regulation of transcription,DNA-templated,negative regulation of cell migration,positive regulation of skeletal muscle tissue development,patterning of blood vessels.Through KEGG pathway analysis,we identified 52 significant KEGG pathways,including diseases?Breast cancer,Melanoma,Prostate cancer,Endocrine resistance,Bladder cancer?and signaling pathway?TGF-?signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,FoxO signaling pathway,Ras signaling pathway?.Conclusion1.Using type I rat tail collagen coated with BioFlex six-hole plate can make HepG2cells grow stably,and strain loading can promote the proliferation of human hepatoma cell line HepG2 cells.Especially under the condition that the pulling amplitude is 15,the pulling frequency is 1 Hz and the time is 24 hours,the promotion effect is the most remarkable.2.The expression of miRNAs in HepG2 cells was significantly changed after loading under the optimal conditions.Seven differentially differentiated miRNAs and 224 target genes were screened.Among them,SMAD7 and SP1 are worthy of further exploring the related functions and mechanisms.3.Go and KEGG analysis showed that the biological functions of the target genes were mainly related to various cancers,including TGF-?mitogen-PI3K-Akt and other signaling pathways closely related to various cancers.The possible mechanism to be verified is the regulation of SMAD7 gene and TGF-?signaling pathway by hsa-miR-503-5p.