Preliminary Study On The Role Of Mir-590-5P In Hepatocarcinoma Cells

Part I Analysis on the Expression Profiles of miR-590-5P and S100A10Objectives To analyze the expression differences of miR-590-5P and S100A10gene in hepatocarcinoma cells and normal hepatocytes.Methods Normal human hepatocytes and several hepatoma cell lines were cultured,and total RNA was extracted from the cells at logarithmic growth phase using Trizol.Specific primers for reverse transcription were used to prepare cDNA, PCR primers for thedetection of mature miR-590-5P were designed, and mature miR-590-5P content wasdetected by the quantitative fluorescence method. The total cellular protein was extractedfrom the cells at logarithmic growth phase and quantified by the BCA assay, and S100A10protein content was detected by western blot. The expression differences of miR-590-5Pand S100A10in hepatocarcinoma cells and normal liver cells were analyzed.Results Quantitative fluorescence detection results showed that the expression ofmiR-590-5P in human hepatocarcinoma cells was lower than that in normal liver cells (p<0.01) and S100A10protein content in hepatocarcinoma cells was higher than that innormal liver cells.Conclusions The results suggest that miR-590-5P expression is down-regulated inhuman hepatocarcinoma cells and S100A10is highly expressed in hepatocarcinoma cells,with a negative correlation between the both. There is a negative correlation between thetwo.Part II Analysis on the Regulatory Relation between miR-590-5P andS100A10Objectives To analyze the possible regulatory relation between miR-590-5P andS100A10expression.Methods Human genomic DNA was isolated, and the precursor sequence of miR-590-5P was amplified by PCR to construct a recombinant plasmid,pcDNA-miR-590-5P. Total human RNA was extracted, cDNA was prepared by reversetranscription, and3'UTR region of S100A10gene was amplified by PCR and cloned intopGL3-promoter vector to construct a recombinant plasmid, pS100A10A-3'UTR-PGL3.pcDNA-miR-590-5P and pS100A10A-3'UTR-PGL3were cotransfected into293cells bythe liposome method, and the dual-luciferase reporter assay system was used to detectedthe change of luciferase before and after the introduction of miRNA, with the Renillaluciferase as a reference. If miR-590-5P can inhibit the luciferase expression ofpS100A10-3'UTR-pGL3, the point mutation should be used to mutate the binding site(seed sequence) of miR-590-5P in S100A10-3'UTR, and the effect on the inhibitory roleshould be detected further.The lentiviral packaging mix and recombinantpcDNA-miR-590-5P vector were cotransfected into the lentiviral packaging cell line (293T)to produce the recombinant lentivirus, Lv-miR-590-5P. The titer was detected byproportional dilution and the optimal MOI for the infection of HepG2cells with thelentivirus was determined by preliminary experiments. HepG2cells were infected with thelentivirus at the optimal MOI and the infection efficiency was observed72hours afterinfection. Total RNA and total protein was extracted, and subjected to RT-PCR andwestern blot separately, to detect the relationship between miR-590-5P and S100A10.Results The recombinant expression vector pcDNA-miR-590-5P and therecombinant reporter vector pS100A10-3'UTR-PGL3were constructed successfully. Thetransfection of pcDNA-miR-590-5P of293cells inhibited the luciferase expression ofpS100A10-3'UTR-PGL3. In addition, pmir-590-5P did not inhibit the luciferaseexpression after the mutation of3'-UTR. The efficient expression of mir-590-5P in HepG2mediated by the lentiviral system inhibited S100A10expression.Conclusions S100A10is a target gene of miR-590-5P. Overexpression ofmiR-590-5P in HepG2cells inhibits the expression of S100A10gene.Part III Effects of MiR-590-5P on HepG2cellsObjectives To study the effects of miR-590-5P overexpression in HepG2cellsmediated by the lentiviral system on cellular proliferative activity, cell cycle, Ca2+concentration, cell invasion ability and relevant proteins.Methods HepG2cells were infected with the recombinant lentivirus Lv-miR-590-5P and the control lentivirus Lv-GFP at the best MOI, and the infectionefficiencies of the cells were determined by the fluorescence microcopy of greenfluorescent protein (GFP)72hours after infection.The cells infected with virus wereprepared into cell suspension by trypsinization, seeded into96-well plates and culturedunder normal conditions for24,48or72hours.10μl CCK-8reagent was added into eachwell, and the cells were incubated at37℃for another4hours. The cell proliferative abilitywas determined by cell counting, which was measured by the absorbance at450nm (A450)in a microplate reader.72hours after the infection, the viable cells were counted by trypanblue staining. The cells were seeded into6-well plates and cultured under normalconditions for24hours. The cells at exponential phase were collected and fixed in75%ethanol for24hours, treated with RNase, and stained with PI. The cell cycle was detectedwith flow cytometry.72hours after infection, the cell suspension was prepared bytrypsinization and subjected to Transwell assay to detect the change of cell invasion ability.The cells suspension was prepared with chemokine-free medium (containing5%BSA toadjust the osmotic pressure) at a concentration of1.0×106/ml.250μl of cell suspensionwas added into each insert, and500μl complete medium with10%FBS was added intoeach receiver well. The culture was covered, and incubated in an incubator at37°C and5%CO2for48hours. The cell suspension on the insert was aspirated, and the membranewas cut off with a scalpel and treated for fluorescence photography; at the same time, thefluorescence staining analysis was performed to count the cells on membrane.Afterinfection, the viable cells were counted by trypan blue staining. The cells were seeded to6-well plates and cultured under normal conditions for24hours Ca2+concentrationdetection was conducted on the cells at exponential phase. Fluo3-AM was solved inDMSO to prepare the stock solution of Fluo3-AM, which was diluted with dPBS to makethe working solution. The medium was removed and the cells were washed with cellbuffers three times, and added with Fluo-3-AM working solution and incubated at37°Cfor30min. The Fluo3-AM working solution was removed and the cells were washed withcell buffers three times to remove residual Fluo3-AM working solution thoroughly. Thecells were then incubated at37°C for20-30min, and then detected at an excitationwavelength of480-500nm and an emission wavelength of525-530nm.The cells infectedwith virus were prepared into the cell suspension by trypsinization, seeded into6-wellplates and cultured under normal conditions for24hours. The cells at exponential phase were collected for the detection of relevant protein levels. Total cellular protein wasextracted with the cellular protein extraction kit and quantified with the BCA method.Western blot was used to detect the levels of relevant proteins in Wnt pathway (WNT5a,E-cadherin, Caspase3, cMyc, CyclinD1and MMP7) and the phosphorylation level ofβ-catenin. The proteins were isolated by SDS-PAGE, and transferred to PVDF membranesat a constant current of400mA. The membranes were blocked with TBST containing5%nonfat milk for2hours at normal temperature, and incubated with the primary antibody at4°C overnight, and then reacted with the secondary antibody for1hour. The bands weredetected by chemiluminescence and imaged with X-ray films. The optical density analysissoftware was used to analyze the gray values of target bands on the scanned films.Results The lentiviral system used in HepG2cells resulted in a transductionefficiency of100%. The overexpression of miR-590-5P in HepG2inhibited the cellularproliferative activity, caused cell cycle arrest, and suppressed the invasion ability of tumorcells effectively. Moreover, the overexpression of miR-590-5P reduced the expression ofWNT5a, cMyc, CyclinD1, and MMP7, and increased the expression of E-cadherin andCaspase3. Meanwhile, miR-590-5P expression induced the phosphorylation of β-catenin,so as to speed up its degradation.Conclusions The overexpression of miR-590-5P in human hepatocarcinoma cellscan inhibit the activation of Wnt pathway and proliferative activity of tumor cells, causecell-cycle G1arrest, reduce intracellular Ca2+content, and inhibit the invasion ability oftumor cells. The overexpression of miR-590-5P in HepG2cell line effectively inhibits itstumor activity.