Expression And Mechanism Analysis Of MicroRNA-873 And MicroRNA-136 Of Gastric Cancer

Gastric cancer is the fourth most common malignancy worldwide.Its morbidity and mortality are very high.According to statistics,gastric cancer accounts for about 10%of the newly diagnosed cancer,which is the second leading cause of cancer death.In recent decades,with the continuous improvement of medical level,surgery and chemotherapy technology progress,cancer prevention and control has been effectively controlled.However,as the most common malignancy,early diagnosis is still a major obstacle to successful treatment of gastric cancer.Therefore,the exploration of a new biomarker of gastric cancer can provide a potential target for early diagnosis and treatment.Previous studies have shown that noncoding RNA may be involved in the development and progression of multiple diseases,which provides a new perspective for us to explore new targets for the prevention and treatment of gastric cancer.MiRNA is a single-stranded,non-coding RNA containing 17-25 nucleotides,which was originally thought to be a noise produced during transcription.Subsequent studies have shown that miRNAs,by specifically binding to their target 3 ' UTRs,inhibit the translation of the target at post-transcriptional levels,thereby regulating a variety of physiological processes.More and more studies have shown that miRNA can regulate cell proliferation,apoptosis and differentiation,both as a proto-oncogene can also be used as tumor suppressor gene,in the tumor occurrence and development process plays an important role.Therefore,the identification of tumor-associated miRNAs and exploring the mechanism of action of miRNAs in the development of tumors has become an important issue in the study of cancer.Part ?.Expression and mechanism analysis of MicroRNA-873 of gastric cancer cell linesStudies have shown that inhibition of miR-873 expression can promote breast cancer metastasis.In contrast,overexpression of miR-873 can inhibit the invasion and spread of cancer cells,change the progress of breast cancer cell cycle,induce breast cancer cell apoptosis.In fact,miR-873 plays an important role in the development and progression of various cancers.However,the role of miR-873 in the development of gastric cancer is still not very clear.By targeting the translation of mRNAs,miRNAs can act as both oncogenes and as tumor suppressor genes.More and more studies have shown that miR-873 can inhibit tumor growth.despite this,MiR-873 inhibition of gastric cancer development mechanism is still unclear.Chemokine(CXC motif)Ligand 1(CXCL1)is a hidden growth factor produced by a signal from a G-protein coupled receptor.CXCL1 plays a role in inflammation,and the abnormal expression of the protein is closely related to the growth of certain tumors.In order to further clarify the role of miR-873 in the pathogenesis of gastric cancer,we explored the mechanism of CXCL1 in the development and progression of gastric cancer.We analyzed the effect of miR-873 on the proliferation and apoptosis of gastric cancer cells.Bioinformatics methodsmiR-873 of the targets,the preliminary draw CXCL1 may be miR-873 targets,and then verified by the dual luciferase reporter system miR-873 targets of.The miR-873 was used to knock out the targetof miR-873 by RNA interference techniqueto verifywhether miR-873 regulates the proliferation and apoptosis of gastric cancer cells by inhibiting the expression of its target.Themechanism of miR-873 on the apoptosis of gastric cancer cells wasexplored by detecting the expression of apoptosis-related proteins.Detected by immunohistochemistry CXCL1 its receptor CXCR2 expression in gastric cancer tissue,using immunohistochemical methods to identify coprecipitation CXCL1 its receptor CXCR2 affinity in gastric samples using RNA interference technology,CXCR2 rapped,gastric cancer Cell growth,proliferation,apoptosis,and migration characteristics.The main content is divided into the following two parts:Chapter ?MicroRNA-873 inhibits gastric cancer cell growth by targeting CXCL1Objective To investigate the relationship between miR-873 and gastric cancer,and to analyze the mechanism of miR-873 in the development and progression of gastric cancer.MethodsqRT-PCR was used toanalysis of 72 cases of gastric cancer cases of miR-873 expression,the patient a Kaplan-Meier survival analysis.The expression of miR-873 in MGC803,SGC7901,BGC823,HGC27 and MKN45 cells was detected by qRT-PCR.Bioinformatics predicted miR-873 targets the gastric cancer cells,dual luciferase reporter system verifies miR-873 target;using RNA interference of miR-873 targets of CXCL1 expression suppression using MTT To investigate the effect of miR-873 and its target CXCL1 on the proliferation of gastric cancer cell line HGC27.The effect of miR-873 and its target CXCL1 on the apoptosis of HGC27 cells was detected by flow cytometry.Using WB detected by miR-873 mimic or miR-873 after inhibitor treatment HGC27 cells of Caspase3 and p53 expression levels.Results miR-873 was down-regulated in gastric cancer and gastric cancer cell lines.Clinical data show that the low expression of miR-873 is also associated with poor prognostic features of gastric cancer(GC),including serum alpha alpha-fetoprotein(AFP)levels,tumor size and TNM staging.Bioinformatics analysis showed that the 3'UTR region of CXCL1 mRNA contained the binding site of miR-873,and the double luciferase reporter system confirmed that CXCL1 was a direct target of miR-873.MTT assay showed that miR-873 could be inhibited HGC27 cells proliferated and flow cytometry showed that miR-873 could induce HGC27 cell apoptosis.CXCL1 gene silencing counteracts the effect of miR-873 deletion on GC cells to reduce cancer cell proliferation and increase its apoptosis.The results of WB showed that miR-873 could increase the expression of caspase3 and p53 invitro.Conclusion Our results suggest that miR-873 can act as a tumor suppressor and regulate the proliferation and apoptosis of gastric cancer cells by direct action of CXCL1.This process may be mediated by the expression of p53 and Caspase3.Chapter?CXCL1 promotes the development and progression of gastric cancer by mediating with its receptor CXCR2Objective To explore the expression pattern and mechanism of CXCL1 and its receptor CXCR2 in the development and progression of gastric cancer.Methods qRT-PCR was used to analysis of 72 cases of gastric cancer cases in CXCR2 the expression by immunohistochemistry assay CXCL1 its receptor CXCR2 expression in gastric cancer tissues;co-immunoprecipitation method identified CXCL1 its receptor CXCR2 gastric samples in affinity;vitro gastric cancer cell HGC27,use of RNA interference technology,gastric cancer cells HGC27 of CXCR2 gene was knocked out.The effect of CXCR2 on the proliferation of HGC27 cells was detected by MTT assay.The effect of CXCR2 on the apoptosis of HGC27 cells was detected by flow cytometry.The effect of CXCR2 knockout on the expression of Caspase3 and p53 in HGC27 cells was detected by WB method.Results CXCR2 was significantly up-regulated in gastric cancer tissues.Immunohistochemical test results showed that,compared with normal tissue,gastric cancer tissue samples The expression of CXCL1 and its receptor CXCR2 were significantly up-regulated.The results of immunoprecipitation assay showed that CXCL1 had a significant interaction with CXCR2.MTT assay showed that the proliferation of HGC27 cells was inhibited by CXCR2 knockoutcytometry.Analysis showed that CXCR2 knockout significantly increase HGC27 apoptosis.The results of WB showed that the expression of caspase3 and p53 was also increased after CXCR2 knockout.Conclusion Our results suggest that CXCL1 is involved in the proliferation,apoptosis and migration of gastric cancer by specifically binding to its receptor CXCR2.Part ?.Expression and mechanism analysis of MicroRNA-136 of gastric cancer cell linesObjective To investigate the relationship between miR-136 and gastric cancer,and to analyze the mechanism of miR-136 in the developent and progression of gastric cancer.Methods qRT-PCR was used toanalysis of 26cases of gastric cancer cases of miR-136expression.The expression of miRl36 in AGS,BGC-823,MGC-803,SGC-7901cells was detected by qRT-PCR.Bioinformatics predicted miR-136 targets the gastric cancer cells,dual luciferase reporter system verifies miR-136target.miR-136 inhibitor was introduced into MGC-803 and SGC-7901 cells and the transfection efficiency was assessed by using RT-qPCR.The proliferation of transfected cells was determined using CCK8 method.Transwell cell invasion assay was used to investigate the effect of miR-136 underexpression on gastric cancer cell invasion.we detected AKT and p-AKT expression in MGC-803 and SGC-7901 cells after transfection with miR-136 inhibitor or NC inhibitor.Results We found that the expression of miR-136 was aberrantly upregulated in both gastric cancer tissues and cell lines.Suppression of miR-136 inhibited cell proliferation and invasion in gastric cancer.Besides,phosphatase and tensin homolog deleted on chromosome ten(PTEN)was identified as a direct target gene of miR-136 in gastric cancer.PTEN was lowly expressed in gastric cancer tissues and negative correlated with miR-136 expression.Furthermore,PTEN overexpression mimics the effects of miR-136 knockdown on gastric cancer cells.Moreover,miR-136 underexpression reduced p-AKT expression,but not affect AKT expression in gastric cancer cells.Conclusion Our data suggested that miR-136 acts as an oncogene in gastric cancer by regulation of PTEN/AKT/p-AKT signaling pathway,and it may potentially serve as a novel therapeutic target for the treatment of gastric cancer.