Long Non-coding RNA H19 Participates In Metformin-mediated Regulation Of Gastric Cancer Cell Function

Objective:Gastric cancer is the most common malignancies,and its mortality lists the second place among the malignancies.Three hundred thousands of patients died of gastric cancer every year in China.Despite the diagnosis and treatment is developed,gastric cancer is diagnosed as advanced stage,whose prognosis is poor.Long non-coding RNAs?lncRNAs?are RNAs that are more than 200 nucleotides in length,which play a significant role in silencing transcription,transcription activation,chromosomal modification and intranuclear transport.Lnc RNAs regulate target genes and proteins to affect cellular function in multiple pathways and methods.Metformin is the first line oral hypoglycemic drugs for type 2 diabetes,its advantages include few side effects,low price and good hypoglycemic effect.In recent years,besides hypoglycemic effect,some researches have found metformin has an anti-tumor effect,which can lower cancer risk and improve overall survival of patients.In this present study,we found metformin could inhibit the functions of gastric cancer cells in a dose-dependent manner and time-dependent manner.By using lncRNA assay analysis,we tried to find whether metformin inhibit gastric cancer cell through regulating lncRNAs.This study provides a new orientation to understand the mechanism of metformin antitumor effect.Materials and methods:1.Cell lines purchase and cultureThe gastric cancer cell lines AGS was purchased from American Type Culture Collection?ATCC?,SGC-7901 were purchased from the Cellular Resource Center of Shanghai Institutes for Biological Sciences.All cells were incubated in a thermostat in condition of37?and 5%CO2.2.IC₅₀0 of metformin for gastric cancer cellThe AGS and SGC-7901 gastric cancer cells were cultured in certain concentration gradient of metformin for 48h.The absorbance at 450 nm was detected by microplate reader.By using GraphPad Prism 5,the IC₅₀0 curve of metformin was made and calculated.3.Migration and invasion assayAccording to the IC₅₀,AGS and SGC-7901 were treated with different concentrations of metformin?0mM,10mM,20mM,40m M?.The migration and invasion assay was used to explore the effect of metformin on the ability of migration and invasion.Both assays were repeated for at least 3 times.4.Cell proliferation assayWe used Cell Counting Kit-8 reagent to test the cell proliferation assay.After the gastric cancer cells were seeded into the 96-well plate,different concentrations of metformin were added into the groups.We detected absorbance at 450 nm at 0h,24h,48h and 72h.Growth curves were calculated according to the absorbance values.Each experiment was repeated for at least 3 times.5.The cell cycle assay and the cell apoptosis assayKeygen cell cycle kit was used to detect he cell cycle assay based on the manufacturer's protocol.First digestion of the cells and then collection of cells,70%of the pre-cold ethanol was added and then cells were stained with PI,and finally detected using flow cytometry.Keygen cell apoptosis kit was applied to detect the cell apoptosis based on the manufacturer's protocol.Annexin V-APC and PI were used as binding buffer to fix the cell and to stain the cells,and finally detected using flow cytometry.6.LncRNA microassay and screen lncRNAAfter treated with 20mM metformin for 72h,the gastric cancer cells were lysated by Trizol,and were sent to SBC to do the lnc RNA microassay.Differentiation gene expression profiling was analyzed,we screened the differentiated expressed lncRNAs according to the fold change and p-value.7.Analysis of TCGA,Oncomine and UALCANWe used TCGA to analyze the correlation of the expression of H19 between gastric cancer tissue and corresponding normal tissue,and analyze the relationship between the expression and pathological TNM stage.Oncomine database was used to explore the expression of H19 in gastric cancer tissue and adjacent normal tissue.UALCAN was used to analyze the relation between the overall survival and the expression of H19.8.Total RNA extraction,Reverse transcriptase reaction and Real-time PCR Total RNA was extracted from the gastric cancer cells which were treated with different concentration of metformin.NanoPhotometer UV/Vis spectrophotometer was used to detect the concentration and purity of total RNA.The results of A260/A280 values between 1.9 and 2.0 indicate high RNA purity.The Takara reverse transcription kit was used as reverse transcriptase reaction and real-time quantitative PCR was performed using LightCycler?480-Real-time PCR instrument.The data were analyzed by 2^-??CT??CT method.9.Construction of vectors and stable cell linesWe send the sequence of the sequence of H19 searched by NCBI to Shanghai Gene Pharma Co.,Ltd to synthesize the target gene and construct it as low expression vector.AGS and SGC-7901 were transfected to kock-down H19,and we used puromycin to construct the stable cell lines.The real-time PCR was conducted to detec the transfection efficiency.After repeated passage,stable expressed cells were constructed successfully when the expression of fluorescence and the transfection efficiency continuously elevated.10.Animal experiments1×10⁶ shNC or sh H19 cells were injected into the tail veil of four-week-old female BALB/c nude mice divided into four groups.Two weeks after the injections,one group that was injected with shNC cells and one group that was injected with shH19 cells were treated with metformin by intraperitoneal injection daily for two weeks.The other groups were treated with saline solution.Six weeks after gastric cancer cells injections,18-fluorodeoxyglucose Positron Emission Tomography?18F-FDG PET?scans were performed using a PET scanner?Metis 1800,Madic Technology Co,Ltd?.For semi-quantitative analysis,the maximum standardized uptake value?SUVmax?was measured and calculated.After PET scan,the whole lung tissues were isolated from the mice and tissue sections were stained with hematoxylin and eosin?HE?,the metastatic cancer nests were observed at 10×10 magnifications using an inverted microscope?Leica DMI300B?Results:1.Metformin can suppress the migration,invasion and proliferation of gastric cancer cellThe results of migration and invasion showed that metformin could inhibit the migration and invasion of gastric cancer cell,which was concentration-dependent,with the increased concentration of metformin,the inhibited effect was gradually getting stronger.The results of cell proliferation assay showed that the proliferation ability of gastric cancer cell were suppressed by metformin,the inhibited effect was concentration-dependent and time-dependent.2.The results of cell cycle experiment and cell apoptosis detection experimentThe results of cell cycle assy showed that metformin could arrest the gastric cancer cell in G₁ phase.In cell apoptosis assay,metformin could inhibit early and late apoptosis,the rate of apoptotic cells was time and dose dependent,with the time and concentration increased,the proportion of apoptotic cell increased significantly.3.The results of lncRNA microarray and differentiated expression of lncRNAThe results of lncRNA microarray demonstrated that 693 lncRNAs were differentially expressed?407 up-regulated and 286 down-regulated?.We observed that the fold change range for down-regulated lncRNAs was much more than for up-regulated lncRNAs.We used real-time PCR to verify that H19 was definitely down-regulated following metformin treatment,accordant with the results of lncRNA microarray.4.Metformin inhibits the invasion ability through downregulating H19After H19 was knocked down in AGS and SGC7901 cell lines,the invasion ability of AGS and SGC-7901 were surpressed.In knocking down H19 cell,metformin could not inhibit further invasion ability.The results showed that metformin could inhibit the invasion ability of gastric cancer cell through downregulating H19.5.The results of animal experiments showed the metastases of lung were decreased after metformin treatment in nude miceKnock-down of H19 could also decrease the metastasis of lung.Metformin could not further inhibit the metastasis of lung after H19 knock down.The results showed that metformin could inhibit the metastatic ability of gastric cancer cell in vivo.Conclusion:1.Metformin could inhibit the proliferation of gastric cancer cell,the cell cycle,induce apoptosis and surpress the invasion ability.2.H19 is overexpressed in gastric cancer tissue,and it is correlated with advanced T stage and TNM stage.3.Metformin decreased the expression of H19.4.Metformin inhibited the invasion ability of gastric cancer cell through down-regulaitng H19.5.Metformin inhibited the pulmonary metastasis of gastric cancer cell in vivo down-regulaitng H19.