MiR-198 Inhibits Cell Proliferation And Migration By Down-regulation Of TLR4 In Gastric Cancer

Backgrounds and ObjectivesGastric cancer(GC)is one of the most common human malignant tumors.Especially in China,GC is the second leading cause of cancer-related morbidity and mortality.Hence,prevention and control of GC remain challenging and are a research priority.The mechanism of malignancy and development of gastric cancer(GC)remains unclear.Hence,the development and molecular mechanisms of GC invasion and metastasis must be comprehensively studied to provide theoretical basis and guidance for early diagnosis,molecular classification,treatment strategy selection and prognosis determination in GC.Changes in microRNA(miRNA)expression are related to tumors,including GC.Aberrantly expressed miRNA molecules in GC are involved in the proliferation,apoptosis and migration of cancer cells and may change the sensitivity of radiotherapy and chemotherapy by regulating different tumor-related target genes.Results of previous research and the present study indicate that miR-198 expression is downregulated in tumor tissues of patients with GC and low miR-198 expression is correlated with early metastasis and survival of patients with GC.However,the role of miR-198 in GC development and prognosis and its underlying molecular mechanism remain unclear.Therefore,in-depth study of miR-198 and its role in the function of target genes may elucidate the molecular mechanism of GC development and provide a potential therapeutic target.This study includes the following three parts:the first part is to investigate effects of miR-198 overexpression on the biological characteristics of GC cells and the xenografts in nude mice;the second part is screening and identification of miR-198 target gene and verification of its targeting;the third part is to knockout TLR4 gene by CRISPR/Cas9 technique in gastric cancer cells and to verify the the effect of TLR4 KO on cell proliferation,apoptosis and invasion of GC cells.Part One Effects of miR-198 overexpression on the biologicalcharacteristics of GC cells and the xenografts in nude miceMethods:1.miR-198 expression in the GC cell line SGC-7901 was detected by real-time quantitative PCR(qPCR).2.The miR-198 lentiviral expression vector was constructed and transfected to GC cells.SGC-7901 GC cells with stable miR-198 expression were selected and identified.3.The effects of miR-198 on the proliferation,apoptosis and migration of GC cells were studied by MTT,clone formation assay,flow cytometry,cell scratch and transwell assay.4.The effect of miR-198 overexpression on the transplanted tumor was observed by subcutaneous injection of stable miR-198-overexpressing GC cell line into nude mice.Results:1.qPCR results show that the miR-198 expression significantly decreased in GC cells.2.The miR-198 expression in GC cells significantly increased compared with that in the lentiviral vector control group(NC group)after transfection of the lentiviral overexpressing vector miR-198.GC cell lines with stable miR-198 expression were successfully established.3.Experiments on cell growth characteristics show that miR-198 overexpression significantly inhibited the proliferation,migration and invasion of GC SGC-7901 cells and promoted the apoptosis of tumor cells.In the SGC-7901 cells,the OD450 nm value of NC group for 72h and 96h were 1.813±0.057 and 3.107±0.089,respectively,and the OD450 nm value of NC group for 72h and 96h were 1.159±0.048 and 1.710±0.061(P<0.05)by MTT assay.The numbers of clones in the SGC-7901 control group and the NC groups were 232±21 and 201 ± 246,respectively,and in miR-198 overexpression group was 139±11(P<0.05).The number of invasion cell in the NC group was 212± 14 and in miR-198 overexpression group was 161 ± 17(P<0.05).4.The growth rate and tumor size(854.01±58.20mm3)of SGC-7901 cells transfected with miR-198 overexpressing lentiviral vector were significantly lower than those of SGC-7901 cells(2589.28±119.60mm3)and SGC-7901 cells transfected with the the lentiviral vector.The difference was statistically significant(P<0.05).Summary:1.The expression level of miR-198 in gastric cancer cells significantly reduced.2.miR-198 overexpression inhibited the proliferation,migration and invasion of GC SGC-7901 cells and promoted cell apoptosis.Overexpression of miR-198 can effectively inhibit xenograft tumor growth in nude mice.Part Two Screening and identification of miR-198 target gene andverification of its targetingMethods:1.Differentially expressed cytokines in GC tissues were screened by high-throughput protein chip proteomics.2.Luciferase reporter assay was further performed to confirm that TLR4 was the target gene of miR-198.3.In order to identify the target genes of miR-198,qPCR and Western blot were performed to determine the mRNA and protein expressions of the candidate gene in the GC SGC-7901 cells transfected with miR-198.Results:1.Differentially expressed cytokines were detected through protein chip and bioinformatics analysis.Results show the presence of 105 differentially expressed proteins in GC tissues relative to that in adjacent tissues(P<0.05).Based on the results of KEGG enrichment analysis,105 proteins are involved in several important biological functions and signaling pathways.The results of high-throughput protein microarray were analysed by three databases(Target Scan,Pictar,Diana).Bioinformatics prediction results show that among the 105 differentially expressed proteins,six cytokines could be candidates for miR-198 target genes and include TLR4,growth differentiation factor(GDF)1,GDF 5,GDF11,human Fas-related cell death domain protein and epidermal growth factor.2.Luciferase reporter assay results show that miR-198 overexpression significantly inhibited the fluorescence intensity of the wild-type TLR4 in GC SGC7901 cells but did not affect the fluorescence intensity of the TLR4 mutant.3.The expression level of the TLR4 gene in GC SGC-7901 cells is significantly higher than that in SGC-7901 cells transfected with miR-198.The TLR4 expression gene significantly decreased in miR-198-overexpressing SGC-7901 cells.Western blot analysis shows that miR-198 overexpression inhibited TLR4 expression.Summary:1.Expression of six cytokines(TLR4,GDF1,5,11,FADD,EGF)wereup-regulated in GC tissues.2.TLR4 could be a target gene of miR-198 in GC cells.Part Three Effect of knockout of TLR4 gene on the cell biologicalcharacteristics of GC cellsMethods:1.sgRNA targeting the second exon of DNA for the human TLR4 gene was designed and cloned into the px330 vector.The recombinant plasmid was sequenced,transformed into GC SGC-7901 cells and identified by restriction endonuclease digestion.The monoclonal cell clones were screened,and monoclonal cell lines were obtained.2.Western blot analysis was conducted to determine the effect of TLR4 knockout on cell lines.3.MTT assay was used to detect the proliferation of knockout cell lines.4.The effect of TLR4 knockout on the apoptosis of GC cell was detected by flow cytometry.5.The effect of TLR4 knockout on cell invasion was analyzed by the transwell assays.Results1.Monoclonal TLR gene-knocked out GC cells were screened and obtained by CRISPR/Cas9 gene editing technique.2.Western blot results showed that the TLR4 protein significantly decreased in TLR4 gene knockout monoclonal cell lines.This finding indicates the successful construction of the TLR4 gene knockout stable cell line.3.MTT assay was conducted to confirm the effect of TLR4 knockout on the growth and proliferation of GC cells.Compared with that of normal GC cells,the growth of GC cells after TLR4 knockout was slower and cell proliferation significantly decreased.4.TLR4 gene knockout could obviously promote the apoptosis of GC cells decrease GC cells invasion.Summary:1.Knockout of TLR4 gene can promote the apoptosis of GC cells and inhibit the proliferation and invasion of GC cells.Conclusions1.The expression level of miR-198 in gastric cancer cells significantly decreased.Overexpression of miR-198 inhibites the proliferation,migration and invasion of GC SGC-7901 cells and promoted cell apoptosis.2.Overexpression of miR-198 can effectively inhibit xenograft tumor growth in nude mice.3.TLR4 gene is a target gene of miR-198 in GC cells.4.miR-198 negatively regulates TLR4 gene and inhibits proliferation and invasion of GCr cells.