LncRNA HOXA11-AS Promotes Proliferation,Migration And Invasion Of Gastric Cancer Cells By Regulating MiR-148a/WNT1/β-catenin

Research background Gastric cancer is one of the most common malignant tumors in the world,and it is also an important factor in cancer death in China.Its morbidity and mortality rank among the top in all tumors.At present,although traditional treatments such as surgical treatment,chemotherapy and radiation therapy have prolonged the survival time of patients to some extent,due to the invasion and metastasis of tumor cells,the prognosis of most patients is not satisfactory,and the survival rate is low.The limitations of treatment and the low early diagnosis rate make the diagnosis and treatment of gastric cancer in China very serious.Therefore,how to effectively reduce the malignant proliferation and metastasis of tumor cells is of great significance for the treatment of gastric cancer.Long noncoding RNA(lncRNA)is a kind of RNA that does not encode a protein with a length of more than 200 nt.It can specifically bind microRNAs(miRNAs)through a sponge adsorption mechanism to reduce the negative regulation of miRNA on target genes.It plays an important role in regulating biological processes such as cell proliferation,migration and invasion,and participates in the physiological and pathological processes of various tumor diseases including gastric cancer.Homeobox-containing A11 antisense RNA(HOXA11-AS)is a lncRNA closely related to tumor development,which is highly expressed in various tumor cells and plays a carcinogenic role.miR-148 a is a member of the miR-148/152 family located on the7p15.2 chromosome.It affects the development of various tumors by the role ofoncogenes or tumor suppressor genes.WNT1 is a member of the WNT gene family and is also an important component of the classical WNT/β-catenin signaling pathway.It activates intracellular scattered proteins by binding to transmembrane receptors,and increases intracellular β-catenin accumulation.The nucleus activates the expression of related genes,which in turn affects cell function.The development of gastric cancer is a complex process of multiple genes,multiple stages and multiple mechanisms.The exact mechanism is still unclear;although the role of HOXA11-AS,miR-148 a and WNT1/β-catenin signaling pathways in gastric cancer has been reported.However,the regulatory mechanism of the gene network is very large and complex.Whether there is a link between the three,it is not known whether the gastric cancer process can be promoted through related effects.We observed the expression of HOXA11-AS in gastric cancer tissues and analyzed the relationship between HOXA11-AS expression and clinicopathological features.Starbase bioinformatics software predicted that there were targeted binding sites between HOXA11-AS 3’UTR and miR-148 a,and Target Scan Human software predicted that WNT1 3’UTR contained nucleotide sequences complementary to miR-148 a.Therefore,it is speculated that HOXA11-AS may promote the progression of gastric cancer by targeting the adsorption of miR-148 a and then activating its target gene WNT1-mediated WNT1/β-catenin signaling pathway.In order to verify the above conjecture,we conducted experiments on gastric cancer SGC-7901 and MGC-803 cells.The experiment is divided into three parts:Part one Mechanism of lncRNA HOXA11-AS Regulating Gastric Cancer Progression by Regulating miR-148 a Objective: To investigate the effect of HOXA11-AS targeting miR-148 a on proliferation,migration and invasion of gastric cancer cells.Method: The expressions of HOXA11-AS in 50 gastric cancer tissues and SGC-7901,MGC-803 cells were detected by RT-PCR,and the relationships between HOXA11-AS expression and clinicopathological features,prognosis were analyzed.HOXA11-AS overexpressed gastric cancer SGC-7901 cell lines and HOXA11-AS silenced MGC-803 cell lines were constructed.The effects of HOXA11-AS on the proliferation,cloning,migration and invasion of gastric cancer cells were detected by CCK-8,cloning and Transwell assay.The targeting relationship between HOXA11-AS and microRNA-148 a was predicted by using Starbase bioinformatics software,and were analyzed by double luciferase reporter gene experiment and RIP experiment.Effect of HOXA11-AS on miR-148 a expression and the expression of microRNA-148 a in gastric cancer tissues and cells were measured by real-time fluorescence quantitative PCR.MiR-148 a upregulated SGC-7901 cells and miR-148 a downregulated MGC-803 cells were constructed.The effects of miR-148 a on proliferation,cloning,migration and invasion of gastric cancer cells which HOXA11-AS overexpressed or silenced were examed by CCK-8,cloning and Transwell assay.The lentivirus-mediated HOXA11-AS gene was transfected into gastric cancer SGC-7901 cells,and the axillary fossa was subcutaneously inoculated to construct a nude mouse xenograft model of gastric cancer SGC-7901 cells.The interference of HOXA11-AS expression on the growth of gastric cancer xenografts and miR-in nude mice was observed.The effect of 148 a expression.Result: HOXA11-AS is highly expressed in gastric cancer tissues and cells.The expression of HOXA11-AS was not correlated with age and sex(P>0.05),but with tumor size,lymph node metastasis and TNM stage(P<0.05).The survival rate of gastric cancer patients with low expression of HOXA11-AS was significantly higher than that of patients with high HOXA11-AS expression.Overexpression of HOXA11-AS can promote the proliferation,clonal formation,migration and invasion of SGC-7901 cells,whereas inhibit the proliferation,clonal formation,migration and invasion of MGC-803 cells.Dual luciferase reporter gene test and RIP test confirmed that miR-148 a was the target gene of HOXA11-AS.Real-time fluorescence quantitative PCR detection confirmed that HOXA11-AS could negatively regulate the expression of miR-148 a,and the expression of miR-148 a was decreased in gastric cancer tissues and cells.Upregulation of the expression of miR-148 a can reverse the effect of HOXA11-AS overexpression on SGC-7901 cell proliferation,clonal formation,migration and invasion;as well as down-regulation of its expression can reverse the inhibition of HOXA11-AS silencing on proliferation,clonal formation,migration and invasion of gastric cancer cells.At the same time,after the expression of HOXA11-AS was successfully down-regulated,the growth of xenografted gastric cancer in nude mice was significantly inhibited,and the expression of miR-148 a was significantly increased.Conclusion: HOXA11-AS is highly expressed in gastric cancer and can promote the proliferation,migration and invasion of gastric cancer cells by targeting microRNAs-148 a.It is expected to become a new target for diagnosis and treatment of gastric cancer.Part 2 : Mechanism Research of miR-148 a Regulating Gastric Cancer Progression Through Regulation of WNT1/ β-catenin signaling pathway Objective: To investigate the effects of miR-148 a on proliferation,migration and invasion of gastric cancer cells by regulating WNT1/β-catenin signaling pathway.Methods: The targeting relationship between microRNA148 a and WNT1 was predicted by Target Scan Human software;and be analysed by double luciferase reporter gene assay and RIP assay.The expression of WNT1 in 50 gastric cancer tissues was detected by immunohistochemistry and RT-PCR.The expression of WNT1 mRNA in gastric cancer SGC-7901 and MGC-803 cells was detected by RT-PCR.The SGC-7901 cell lines overexpressing miR-148 a and MGC-803 cell lines silencing miR-148 a wereconstructed,and the effects of miR-148 a on the expression of WNT1/ β-catenin signaling pathway related proteins WNT1 and β-catenin were detected by Western blot.The SGC-7901 cell line overexpressed by WNT1 and MGC-803 cell line silenced by WNT1 were further constructed,and the effects of miR-148 a targeting WNT1 on proliferation,clone formation,migration and invasion of gastric cancer cells was detected by CCK-8 assay,clone formation assay and Transwell chamber assay.Results:A complementary nucleotide sequence with miR-148 a was contained in WNT13’UTR.The double luciferase reporter gene and RIP assay confirmed that WNT1 was a potential target gene of miR-148 a.WNT1 was highly expressed in gastric cancer tissues and cells,WNT1 and β-catenin proteins were significantly decreased in SGC-7901 cells overexpressed by miR-148 a,while WNT1 and β-catenin proteins were significantly increased in MGC-803 cells silenced by miR-148 a.The inhibitory effect of overexpression of microRNAs on proliferation,clone formation,migration and invasion of SGC-701 cells was reversed by WNT1 upregulation,while the promoting effect of microRNAs silencing on proliferation,clone formation,migration and invasion of MGC-803 cells could be significantly reversed by WNT1 down-regulation.Conclusion: WNT1 is a potential target gene of miR-148 a,which is highly expressed in gastric cancer tissues and cells.MiR-148 a can inhibit the proliferation,migration and invasion of gastric cancer cells by targeting WNT1 and regulating WNT1/β-catenin signaling pathway.Part3: Mechanism Research of HOXA11-AS Mediated miR-148 a Expression Regulating WNT1/β-catenin Signaling Pathway Objective:To investigate the effects of HOXA11-AS mediated miR-148 a on the WNT1/β-catenin signaling pathway.Methods:According to the expression levels of HOXA11-AS,miR-148 a and WNT1 ingastric cancer tissues were detected by RT-PCR,Pearson correlation was used to analyze the correlation among them.After constructing SGC-7901 cell lines overexpressed by HOXA11-AS and miR-148 a and MGC-803 cell lines silenced by HOXA11-AS and miR-148 a,the effects of co-expression and silencing of HOXA11-AS and miR-148 a on WNT1/β-catenin signaling pathway related proteins WNT1 and β-catenin were detected by Western blot.Results: There was a significant negative correlation between the expression of HOXA11-AS and miR-148 a.WNT1 mRNA was negatively correlated with the expression of miR-148 a,while HOXA11-AS and WNT1 mRNA were positively correlated.The expression of WNT1 and β-catenin proteins in SGC-7901 cells could be upregulated and the activation of WNT1/ β-catenin signaling pathway could be induced by HOXA11-AS overexpression,and the activation of WNT1/β-catenin signaling pathway induced by HOXA11-AS overexpression could be reversed by miR-148 a upregulation.The expression of WNT1 and β-catenin protein in MGC-803 cells could be downregulated and the activation of WNT1/β-catenin signaling pathway could be inhibited by HOXA11-AS silencing,and the inhibition of HOXA11-AS silencing on the activation of WNT1/ β-catenin signaling pathway could be reversed by miR-148 a downregulation.Conclusion:HOXA11-AS is negatively correlated with the expression of miR-148 a,and positively correlated with the expression of WNT1,while the expression of WNT1 and miR-148 a is negatively correlated.HOXA11-AS can induce the activation of WNT1/ β-catenin signaling pathway through mediating miR-148 a expression.