| The latest statistics show that by 2018,gastric cancer ranked fifth in the incidence of cancer worldwide,and the fatality rate ranked third,is a malignant disease that seriously threatens human health.In recent years,the number of cases of gastric cancer has decreased year by year,but the number of cases of gastric signet ring cell carcinoma(GSRCC),which accounts for 15%of gastric cancer,has increased year by year.Gastric signet ring cell carcinoma is characterized by the loss of expression of E-cadherin and the initiation of dysfunction-induced cancer,and it is highly malignant.It is usually found in the middle and late stages.It is very difficult for patients to undergo surgical resection and the cure coefficient is low.The survival rate of patients is high.Far less than non-signular cell cancer.However,studies have revealed that patients with early gastric signet ring cell carcinoma have a better survival rate than non-signosa cell death.Therefore,finding genes and their molecular mechanisms that play a critical role in the progression of gastric signet ring cell carcinoma is extremely important for improving the prognosis survival rate of patients with gastric signet ring cell carcinoma.We used the bioinformatics methods to systematically analyze the public databases The Cancer Genome Atlas(TCGA)and ACRG,and screened a series of genes that are abnormally expressed in gastric signet ring cell carcinoma and closely related to the development of gastric signet ring cell carcinoma.Among them,the expression level of Lactate dehydrogenase D(LDHD)in gastric signet ring cell carcinoma was significantly lower than that of non-signosa cell carcinoma.The Kaplan Meier-plotter(KM-plotter)database also showed that low expression levels of LDHD were significantly associated with poor prognosis in patients with signet ring cell carcinoma of the stomach.LDHD is a member of the lactate dehydrogenase family.As far as we know,its role in the development and progression of cancer has not been reported.In the patient samples collected from the cooperative hospital,we predicted the low expression of LDHD in gastric signet ring cell carcinoma compared with adjacent normal tissues at the protein level and mRNA level.Afterwards,it was found in vitro experiments that transient down-regulation of LDHD could significantly promote the invasion of gastric signet ring cell carcinoma cells and the formation of soft agar clones.Transiently overexpressing LDHD inhibited its invasion and soft agar colony formation.However,changing the expression level of LDHD has no significant effect on cell proliferation and cycle,suggesting that LDHD is not involved in the regulation of cell cycle,and is mainly involved in the invasion ability and tumor dryness of gastric signet ring cell cancer cells.To further validate our results,we constructed a cell line that stably knocked down LDHD expression using a lentiviral vector,yielding the same results as transient down-regulation of LDHD.After that,we completely knocked out the expression of LDHD using CRISPR-Cas9 technology,and constructed two knockout cell lines that did not express LDHD,and obtained the same results as transient down-regulation and stable down-regulation of LDHD.It is suggested that LDHD plays a critical role in cell invasion and soft agar colony formation.Nevertheless,the specific mechanism is still unclear.In the in vivo experiment,we used two LDHD knockout cell lines to enter the nude mice by tail vein injection.After 6 weeks,the nude mice were sacrificed to remove the lung tissue for observation.The results showed that the LDHD knockout significantly promoted the stomach ring.Lung metastasis of cancer cells.The results of HE staining also confirmed this conclusion.By immunohistochemical staining(IHC)of lung tissue of nude mice,it was observed that the cell dry markers ALDH1A1 and LGR5 were highly expressed in lung metastases formed by knockout expressing cell lines.It is therefore suggested that the down-regulation of LDLDHD HD promotes the tumor dryness of gastric signet ring cell cancer cells.Aiming at the change of matrigel invasion ability and soft agar colony formation ability of gastric signet ring cell carcinoma cells,preliminary down-regulation studies suggest that down-regulation of LDHD may up-regulate the expression of matrix metalloproteinase 28(MMP28)and activate the WNT signaling pathway.Invasive ability of gastric signet ring cell cancer cells and tumor dryness.In summary,we analyzed TCGA and ACRG database to find that LDHD is significantly down-regulated during the progression of gastric signet ring cell carcinoma and is associated with poor prognosis.In vitro and in vivo experiments have shown that LDHD can regulate the invasion ability of gastric signet ring cell carcinoma cells and tumor dryness,affecting tumor metastasis.Further studies have found that LDHD may play a biological role by regulating MMP28 and WNT pathways.The results of this study show that LDHD plays an important role in the metastasis and invasion of gastric signet ring cell carcinoma and may be a potential target for the treatment of gastric signet ring cell carcinoma. |
