| ?.BackgroundAs one of the most common malignant tumors of digestive tract in the world,gastric cancer is a serious threat to human life and health.In approximately 90% of cancer cases,invasion and metastasis are the main causes of patient death.The metastasis and recurrence of gastric cancer seriously endanger the health of patients and are associated with poor prognosis,Therefore,it is urgent to search new treatment options for gastric cancer.Although recent studies have explored a variety of new molecular targets to diagnose and treat gastric cancer,Targeted medications for gastric cancer are still lacked.Therefore,the development of drugs targeting new targets in gastric cancer has important clinical implications for improving patient outcomes.MicroRNAs(miRNAs)are small non-coding RNAs that are endogenous,And they are able to block expression of protein-coding genes via post-transcriptional regulatory mechanisms;they block the translation of mRNAs or induce their degradation by binding to the sequence(3 ? UTR)of the 3 ? untranslated region of messenger RNAs(mRNAs)of protein-coding genes.It is estimated that about 60% of human protein-coding genes are post-transcriptionally regulated by miRNAs;and in many tumors,there are also specific miRNA or miRNA family expression and function abnormalities.Recently,the function of miR-140-5p in tumors has attracted much interest from investigators.MiR-140-5p acts as a tumor suppressor in colon cancer,cholangiocarcinoma and hypopharyngeal cancer.In gastric cancer,it has been reported that the expression level of miR-140-5p is significantly increased in the plasma of patients,but others have found that the expression level of miR-140-5p is down-regulated in tissue markers and gastric cancer cell line HGC-27.More importantly,the correlation between miR-140-5p and the prognosis of gastric cancer patients as well as the role in the development of gastric cancer are unknown.It should be noted that considering that a single miRNA molecule may target and regulate a variety of protein-coding genes,what role of miR-140-5p in the development of gastric cancer needs further in-depth study.THY1 is a key regulator involved in cellular interactions between cells and the cell matrix.A previous study showed that THY1,also known as CD90 gene,high expression levels in gastric cancer and inhibited apoptosis of gastric cancer cells by regulating the expression of SPARC protein.The application of bioinformatics online prediction software as well as dual-luciferase reporter assay in that study provided evidence confirming THY1 as a direct target gene of miR-140-5p.Studies have shown that miR-140-5p can be used as a tumor suppressor in colorectal cancer,cholangiocarcinoma and hypopharyngeal cancer.In addition,down-regulation of miR-140-5p has been observed in gastric cancer,and reports have shown that it can act to inhibit gastric cell proliferation.Notch pathway is one of the important pathways involved in the regulation of cancer stem cells.Targeting the Notch pathway can inhibit tumor growth and promote apoptosis of cancer stem cells.Previous studies have revealed that activation of the Notch signaling pathway in gastric cancer cells is linked to the process of tumor formation and development.Accumulating evidence highlights the ability of THY1 to participate in regulating the Notch signaling pathway,while a lack of investigation of the relationship between miR-140-5p,THY1 and the Notch signaling pathway has been conducted.Therefore,the current study aimed to investigate the mechanism by which miR-140 affects gastric cancer cell proliferation.?.Materials and Methods1.105 pairs of freshly matched gastric cancer tissues and adjacent normal tissues were selected and analyzed analyzed the expression of expression of miR-140-5p and THY1 using real-time quantitative PCR and Western blot.2.databases such as MirDIP as well as bioinformatic prediction softeware was applied to search and predict the existence of a direct link between miR-14-5p.3.The relationship between the expression of miR-140-5p and THY1 and gastric cancer cell proliferation,migration and apoptosis was demonstrated by MTT,colony formation,scratch and transwell assays.4.The inhibition efficacy gastric cancer cell proliferation migration by MicroRNA-140-5p as well as promotion of apoptosis by THY1-mediated negative regulation of notch signaling pathway was validated.?.Results1.Low expression of miR-140-5p and high expression of THY1 in GC tissuesIn light of our speculation on whether miR-140-5p has the possibility to affect GC by regulating THY1,attention and focus shift in the experiment to identify the expression of miR-140-5p and THY1 in GC tissues and tissues.Based on the results of RT-qPCR and Western blot analysis,differences in miR-140-5p and THY1 expression were observed between GC tissues and the tissues.A correlation study between miR-140-5p and THY1 expression in GC tissue samples was performed,and the results showed that miR-140-5p was negatively correlated with THT1.These results provide conclusive evidence that the expression of miR-140-5p is lower in GC tissues compared to that in tissues,in contrast to the high expression of THY1.2.The expression of miR-140-5p and THY1 correlates with the progression of GCBased on the analysis of the relationship between miR-140-5p and THY1 expression as well as clinicopathological features in GC,Clinicopathological features of all GC patients were analyzed and organized accordingly.According to the median levels of miR-140-5p and THY1,GC patients were divided into low expression group and high expression group.The results showed that there was no correlation between miR-140-5p expression with gender or age,but a significant correlation with differentiation(p = 0.026),lymph node metastasis(p = 0.002),and clinical stage.(Fig.P = 0.033)and permeability(p = 0.018)GC patients.Similarly,there was no correlation between THY1 expression and gender and age in GC patients,while there was a significant correlation for differentiation(p < 0.001),lymph node metastasis(p < 0.001),clinical stage(p = 0.003)and degree of invasion(p = 0.001).3.MiR-140-5p can regulate the expression of THY1Microrna.Org's online bioinformatics software predicts THY1 as a downstream target gene of miR-140-5p.Subsequently,the results of dual-luciferase reporter assay showed that miR-140-5p could down-regulate the expression of THY1 by binding to the THY1 mRNA 3 ? UTR.4.MiR-140-5p overexpression inhibits THY1 expression and Notch signaling pathwayThe experimental part identifies the mechanism by which miR-140-5p affects the Notch signaling pathway through THY1.THY1 and Notch signaling pathway related NICD,HES1 and HES5 were inhibited or overexpressed at the mRNA and protein levels by miR-140-5p overexpression and knockdown in gastric cancer SGC-7901 cell line.This result confirmed that miR-140-5p negatively regulates THY1 expression and Notch signaling pathway in gastric cancer SGC-7901 cell line.5.MiR-140-5p inhibits the proliferation,migration and invasion of GC cellsMTS assay confirmed that the addition of miR-140-5p mimic to gastric cancer SGC-7901 cell line significantly inhibited the proliferation of cells,while the addition of miR-140-5p inhibitor promoted the proliferation of cells.Cell scratch assay confirmed that the addition of miR-140-5p mimic to gastric cancer SGC-7901 cell line significantly inhibited the migration of cells,while the addition of miR-140-5p inhibitor promoted the migration of cells.Transwell assay confirmed that the addition of miR-140-5p mimic to the gastric cancer SGC-7901 cell line significantly inhibited the invasion of gastric cancer cells,while the addition of miR-140-5p inhibitor promoted the invasion of gastric cancer cells.The above experiments confirmed that miR-140-5p could inhibit the proliferation,migration and invasion of GC cells.6.miR-140-5p promotes apoptosis of gastric cancer cellsCell flow assay showed that miR-140-5p mimic promoted the apoptosis of gastric cancer cells,while the addition of miR-140-5p inhibitor inhibited the apoptosis of gastric cancer cells.Western blotting showed that miR-140-5p promoted apoptosis by up-regulating the expression of Bax and caspase-3 and PARP1 and down-regulating Bcl-2 and Notch1?.Conclusion1.miR-140-5p was down-regulated in gastric cancer tissues compared with normal tissues by tissue sample detection combined with bioinformatics in gastric cancer patients.2.miR-140-5p negatively regulates the proliferation,migration and invasion of gastric cancer cells,and promotes the apoptosis of gastric cancer cells.3.miR-140-5p mainly plays a biological role in inhibiting the development of gastric cancer by negatively regulating THY1 and Notch signaling pathways. |
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