Mir-18b Regulates The Invasion Of Gastric Cancer Cells By Inhibiting HPSE Expression

【Background】Gastric cancer is one of the most common malignant tumors, with lower mortality thanlung cancer and liver cancer in China. The gastric cancer patients were increased bymillions each year in the world, of which41%occurred in China. There were about35%people died of gastric cancer each year in china. Gastric cancer invading the serosa, lymphnode metastasis and hematogenous metastasis was the main reason of failure of radicalsurgery, postoperative recurrence and death. Tumor cell must first breaks through the tissuebarrier constituted by the basement membrane (basilar membrane BM) and extracellularmatrix (Extracellular Matrix ECM) when accrues invasion and metastasis. Recent studieshave found that heparanase (HPSE) is the only beta-D-glucuronide enzyme in the humanbody to degrade of the barrier-sulfate heparan proteoglycan (heparan sulfateproteoglycan,HSPG). The HPSE cracks heparan sulfate glycosaminoglycan chains (HS-GAG) todegrade of HSPG. And it can also promote tumor invasion and metastasis collaboratedmany factors in many ways directly and indirectly. Thus HPSE becomes a hot topic tostudy the mechanism of tumor metastasis and countermeasures recently.MicroRNA (miRNA) are a size of about18-25nt non-coding single-stranded RNAmolecules, which is mainly involved in post-transcriptional gene regulation, promotestarget mRNA degradation or inhibits protein translation through incompletecomplementary pairing with the target gene3’-untranslated region (3’-UTR). Functionalstudies shows that miRNA involved in a variety of physiological and pathological cellproliferation, differentiation, apoptosis, tumorigenesis and metastasis by up-regulating ordown-regulating the expression of cell to effect the cell development and disease processes.We use the interaction between miRNA and cancer-related proteins to play a biologicalfunction to promote or inhibit tumor formation.AimsIn this study, we detected HPSE relative expression levels in gastric cancer tissues andtried to find the targeted endogenous miRNA by bioinformatics screening methods. Andthen we identified the miRNA expression in gastric carcinoma in hope of investigating thespecific correlation between abnormal expression of miRNA in the process of metastasis ofgastric cancer and the HPSE up-regulation. This study was to explore the molecular mechanisms of gastric cancer development, tumorigenesis and metastasis and to determinethe metastasis targets, and also to provide a reference and a breakthrough to seek newintervention strategies of gastric cancer.MethodsReal-time quantitative PCR method was used to detect the expression of HPSE ingastric carcinoma. We used bioinformatics methods to filter out potential miRNA targetedHPSE based on the combination of MiRNA and HPSE3’-untranslated region (3’-UTR)sequence, and then we detect the expression of miRNA in gastric carcinoma by Real-timequantitative PCR. The dual luciferase reporter gene and western blot method were used todetermine the relationship between the miRNA and HPSE. Gene transfection was used toeffect the expression of targeting miRNA in gastric cancer cells. The cells vitality wasidentified by MTT method and flow cytometry to determine apoptosis, cell-penetratingexperimental identification of cell invasion.Result s1. HPSE expression in stomach cancer.The tissues RNA sample extracted was measured by1%agarose gel electrophoresis,the electrophoresis strip of28S,18S and5S small molecular RNA take clear. RT-PCRshows that he HPSE gene express more higher in paraneoplastic tissues than in normaltissues, which is significant higher than in cancer tissue(P<0.01).2. The relationship between HPSE gene expression level and the clinical pathologiccharacteristic of stomach cancer.According to the expression level of heparinase mRNA in stomach cancer tissues andparaneoplastic tissues, Analyses the relationship between heparinase mRNA and clinicalpathologic characteristic of stomach cancer (SPSS10.0was used in this experiment). Theheparinase mRNA high expression has no relationship to sufferer’s sex and has significantrelationship to tumor diameter size, T stages, the lymph transfer and the clinical stages.3. The relationship between miRNA and HPSE targeting.MiR-18b, miR-137, miR-299-3p and miR-502may act in HPSE gene according tobioinformatics prediction; above all, miR-18b and miR-137have two Combining Sites inHPSE gene each. RT-PCR shows that miR-18s, miR-137, miR-299-3p and miR-502express at low level in stomach cancer tissue than in normal stomach tissue (50%,50%,45%and69%respectively compared to each in normal stomach tissue).4. miR-18b control HPSE expression Dual-luciferase reporter gene assay shows that miR-18can specific inhibit luciferaseactivity in3’UTR area which contain HPSE gene. Western blot shows miR-18b overexpression in stomach cancer cell can inhibit the expression of endogenous HPSE proteinbut do not affect the mRNA level.5. miR-18b control stomach cancer cell invasivenessCell growth activity detection shows that miR-18b do not affect the reproductiveactivity of stomach cancer cell. Flow cytometry shows that miR-18b do not affect theapoptosis of stomach cancer cell. Cell trans-membrane experiment shows that stomachcancer cell over-expressing miR-18b has low trans-membrane than normal stomach cancercell (72.5%lower than control group).Conclusion1. HPSE express higher in stomach cancer tissue than normal stomach tissue. Afteranalysis the relationship between HPSE expression and clinical pathologic characteristic,we can confirm that it is relative to the stomach caner growth, infiltrate and metastasis2. The result of bioinformatics prediction, dual-luciferase reporter gene assay andwestern blot shows that miR-18b can significant inhibit the translational level after HPSEtranscription, HPSE is a target gene of miR-18b.3. miR-18b can significant inhibit the stomach cancer cell invasiveness and is veryimportant in the stomach cancer evolution process.4. It is a new research direction to ensure how miR-18b controls HPSE expression.Does some deep study in miRNA regulatory network will be meaningful to the Cause,mechanism of stomach cancer and search for new gene therapy.