The Mechanism Study On LncRNA-UCA1 Promotes The Migration Of Gastric Cancer Cells In Hypoxic Microenvironment

Objective:In China,the incidence and mortality of gastric cancer ranks second in all malignancies and accounts for half of the total deaths of gastric cancer in the world.Most of the advanced patients are unable to undergo surgical treatment due to invasion and metastasis of the abdominal cavity and lymph nodes.The 5-year survival rate is less than 10%.It is of great significance to deeply investigate the mechanism of gastric cancer metastasis in understanding the biological properties of gastric cancer and developing new molecular targeted drugs.Hypoxia is one of the important characteristics of solid tumors and plays an important role in promoting tumor invasion and metastasis.The hypoxic microenvironment can be divided into chronic hypoxia and acute hypoxia from its mode of occurrence.Although most previous studies of tumor-associated hypoxic microenvironment have focused on acute severe hypoxia,chronic moderate hypoxia more closely mimics the actual microenvironment of the tumor.Gastric cancer cells under hypoxic microenvironment have different degrees of therapeutic resistance to various treatment methods.Hypoxic microenvironment also promotes immune escape and distant metastasis of tumor cells,which greatly affects the therapeutic efficacy of gastric cancer patients and further worsens the prognosis of gastric cancer patients.Therefore,studies on the mechanism of action of hypoxia microenvironment for gastric cancer are of great significance for the diagnosis and treatment of gastric cancer and the understanding of the pathogenesis of the disease.Non-coding RNAs?nc RNAs?are abundant in cells and play an important role in life activities and biological processes.Among them,the mechanism of action of long-chain non-coding RNAs?lnc RNAs?in the development of tumors has become a research hotspot in recent years.Lnc RNAs can regulate the biological behavior of tumor cells through the action of molecular decoys,scaffolds,enhancer RNAs,and short peptides.There is increasing evidence that hypoxia-regulated lnc RNAs are involved in various key processes of cancer cells,including linc ROR,NEAT1,H19,UCA1,AK058003,WT1 lnc RNA,lnc RNA LET,linc-p21 and GAPLINC.However,the specific mechanisms by which these lnc RNAs promote tumor proliferation and metastasis in a chronic hypoxic microenvironment have not been elucidated.In this study,we established two novel hypoxia-tolerant gastric cancer cell lines capable of normal growth in 2% oxygen.Transcriptome sequencing detected two groups of gastric cancer cell lines and found that UCA1 was up-regulated and promoted migration in hypoxic-resistant gastric cancer cells.Current studies have shown that UCA1 is highly expressed in both plasma and diseased tissues of gastric cancer patients,suggesting that it may be a marker for the diagnosis and prognosis of gastric cancer.According to another report,HIF-1? regulates the expression of UCA1 in bladder cancer cells treated with hypoxia for a short period of time.However,in the long-term hypoxic microenvironment of gastric cancer,it is unclear how UCA1 changes and its role in the metastasis of gastric cancer cells.This study investigated the mechanism by which long-term chronic hypoxic microenvironment upregulates UCA1 promotes metastasis of gastric cancer and provides a theoretical basis for further understanding of the novel biological effects of tumorassociated hypoxia-induced lnc RNAs.Methods:1.After the treatment of MGC803 and BGC823 with the oxygen concentration in the hypoxic microenvironment down from 10% step to 2%,the survived stable passage gastric cancer cell line was named hypoxia-resistant gastric cancer cell line MGC803/Hypo,BGC-823/Hypo.2.lnc RNA high-throughput sequencing The transcriptional expression profiles of hypoxia-resistant gastric cancer cell lines MGC803/Hypo and BGC823/Hypo and their parental gastric cancer cell line lnc RNA were determined.The lnc RNAs were up-regulated in both hypoxic-resistant gastric cancer cell lines,and transcripts without Ensemble annotation information were excluded.Data-expressing transcripts and transcripts overlapping with the sense strand of the m RNA were excluded.Finally,the lnc RNA with the highest up-regulation number was selected.-UCA1 as a research object.3.We selected the GSE72362,GSE47532 and GSE62254 gene expression profile datasets from the Gene Expression Omnibus?GEO?database to verify the expression levels of our UCA1 and mi R-7-5p in hypoxic tumor cell lines and their association with EGFR..GEO2 R network tools were used to analyze relevant data and obtain differential genes.Western blot was used to detect the expression of HIF1?,HIF2?,EGFR,p-EGFR,PKM1,PKM2,PTBP1,PAK-1,IRS1,FAK,GAPDH and Actin.5.Transwell assay for cell migration.6.Gene Chip mi RNA Array was used to screen differentially expressed mi RNAs of MGC803/Hypo and its parental cell line MGC803.7.The expression levels of UCA1, EGFR,PKM1,PKM2,and mi R-7-5p were detected by Real-time PCR.8.Double luciferase reporter assays and RNA immunoprecipitation assays identify whether UCA1 binds to mi R-7-5p.9.RNA-Pull down combined with mass spectrometry detection of UCA1-binding protein.10.The lactic acid quantification kit and the sugar ingestion kit were used to test the glycolysis of gastric cancer cells.11.Immunofluorescence detected changes in the nuclear translocation of PKM2.12.Protein chemical cross-linking assays analyze the level of changes in PKM2 tetramer and single/dimer.13.Statistical analysis: Statistical analysis was performed using SPSS 17.0 software.Each experiment was repeated 3 times and data was expressed as mean ± standard deviation?±s?.The t-test was used for the difference between groups.P<0.05 was considered statistically significant.Results:1.The migration ability of hypoxia-resistant gastric cancer cells is enhanced.Compared with the parental gastric cancer cell lines MGC-803/ and BGC-823,the migratory ability of the hypoxia-resistant gastric cancer cell lines MGC-803/Hypo and BGC-823/Hypo was enhanced.2.UCA1 promotes migration of hypoxia-resistant gastric cancer cells.Whole transcriptome sequencing screening of differentially expressed Lnc RNAs revealed that UCA1 was significantly up-regulated in both hypoxia-resistant gastric cancer cell lines,and q RT-PCR experiments also confirmed the same results.Using the GSE72362 data from the GEO online database,the results showed that UCA1 is also up-regulated in long-term hypoxic breast cancer cells.After knockdown of UCA1,the migratory capacity of both hypoxic-resistant gastric cancer cell lines MGC803/Hypo and BGC823/Hypo was significantly reduced.These results suggest that long-term chronic hypoxia-upregulated UCA1 expression may promote gastric cancer cell migration.3.There is a binding relationship between UCA1 and mi R-7-5p.MGC-803/Hypo and its parental cell MGC-803 were isolated by nuclear plasma and extracted RNA.q RT-PCR results showed that UCA1 was up-regulated in the cytoplasm and nucleus of hypoxic gastric cancer cells,but the cytoplasm was upregulated more significantly.It is suggested that UCA1 is likely to play its role by competing with endogenous RNA?ce RNA?.The mi RNA microarray detection and differential screening of MGC-803 and MGC-803/Hypo showed that compared with MGC-803,mi R-7-5p decreased most significantly in MGC-803/Hypo.Using the Mi Randa database predictions,there is a binding site between UCA1 and mi R-7-5p.UCA1 knockdown significantly up-regulated the expression of mi R-7-5p in hypoxia-resistance gastric cancer cells.Dual luciferase reporter assays and RIP experiments with Ago2 confirmed that UCA1 binds directly to mi R-7-5p.In addition,analysis using the GSE47532 dataset revealed that compared with normoxic cells,mi R-7-5p expression was decreased in hypoxic breast cancer cells for 48 hours.4.mi R-7-5p inhibits migration of hypoxia-resistant gastric cancer cells by targeting EGFR.Western blot was used to detect the expression of mi R-7-5p target genes such as EGFR,FAK,IRS-1 and PAK-1.The results showed that the expression level of EGFR in hypoxia-resistant gastric cancer cell lines was higher than that of parental gastric cancer cells..The GSE47532 dataset analysis also showed that EGFR expression levels in breast cancer cells treated with hypoxia for 48 hours were also higher than those in the normoxic group.When mi R-7-5p mimic was transfected into hypoxia-resistant gastric cancer cells,the EGFR m RNA and protein levels were decreased,the phosphorylation level of AKT was decreased,and the cell migration ability was significantly inhibited.The addition of monoclonal antibody C225 to hypoxia-resistance gastric cancer cells inhibited the migration of EGFR after the phosphorylation of EGFR,suggesting that activation of the EGFR pathway is involved in the migration of hypoxia-resistant gastric cancer cells.These results suggest that mi R-7-5p inhibits the migration of hypoxia-resistant gastric cancer cells by targeting EGFR.5.UCA1 promotes HRGC cell metastasis through the mi R-7-5p/EGFR axis.Knockdown of UCA1 in hypoxic gastric cancer cells reduced the expression of EGFR m RNA and protein.Mi R-7-5p inhibitor significantly reversed the cell migration and EGFR expression induced by knockdown of UCA1,suggesting that UCA1 forms ce RNA with mi R-7-5p,which relieves the inhibitory effect of mi R-7-5p on EGFR.Promote the migration of hypoxia-resistant gastric cancer cells.In addition,analysis of the GSE62254 dataset containing 300 microarrays expressing gastric cancer patients revealed a positive correlation between m RNA expression of UCA1 and EGFR?NM₀05228?.6.UCA1 enhances the glycolytic ability of hypoxia-resistant gastric cancer cells.Lactic acid assay results showed that compared with the parental gastric cancer cells,the level of lactic acid production in hypoxia-resistant gastric cancer cells was significantly increased;after knocking down UCA1,the level of lactic acid metabolism in hypoxia-resistant gastric cancer cells decreased,suggesting that UCA1 promotes hypoxia-resistant gastric cancer cells.The level of glycolysis increased.7.UCA1 promotes the expression of PKM2 and binds to PKM2 to promote the incorporation of its monomer into the nucleus.In vitro amplification of the lysates containing desthiobiotin-UCA1 and MGC803/Hypo cells was incubated with RNA-Pull down technology combined with mass spectrometry to identify proteins that bind to UCA1.Both mass spectrometry and RIP experiments demonstrated that PKM2 can bind to UCA1.Protein chemical cross-link detection showed that PKM2 expression was up-regulated in hypoxia-resistant gastric cancer cells compared with parental gastric cancer cells,and PKM2 expression in hypoxia-resistant gastric cancer cells was down-regulated after knocking down UCA1.At the same time,the intracellular localization of PKM2 was detected by immunofluorescence and protein nuclear plasma separation.Compared with parental gastric cancer cells,PKM2 was increased in hypoxic gastric cancer cells.Knockdown of UCA1 reduced the nuclear PKM2 in hypoxic gastric cancer cells.These results suggest that UCA1 promotes the expression of PKM2 and binds to PKM2 to promote the incorporation of its monomer into the nucleus.8.UCA1 promotes PKM2 expression through binding to PTBP1.The mass spectrometric analysis of UCA1 binding protein involved the alternative splicing nuclear heterogeneous ribonucleoprotein PTBP1,and RIP experiments demonstrated that UCA1 indeed binds to PTBP1.Westen blot and RT-PCR methods showed that the proportion of PKM1/PKM2 in hypoxic gastric cancer cell lines was lower than that of their parental cell lines.After Knockdown of UCA1,the proportion of PKM1/PKM2 was increased.These results suggest that UCA1 promotes PKM2 expression through binding to PTBP1.9.PKM2 monomer polymerization reverses the effect of UCA1 on cell migration.After silencing UCA1 in hypoxia-resistance gastric cancer cells,transwell experiments showed that the migration ability was down-regulated.After the PKM2 inhibitor alanine inhibited the tetramerization and enzyme activity of PKM2,the migratory ability of hypoxia-resistant gastric cancer cells partially recovered.When increasing the concentration of DASA-58 in the medium,DASA-58 can promote the formation of PKM2 tetramer and reduce the nuclear translocation of PKM2,thereby reversing the enhanced migration ability of hypoxia-resistant gastric cancer cells caused by overexpression of UCA1.These results suggest that UCA1 affects the configuration and expression of PKM2,and promotes the nuclear translocation of monomeric PKM2,thereby promoting the migration of hypoxia-resistant gastric cancer cells.Conclusion: 1.The expression of lnc RNA-UCA1 in hypoxia-resistant gastric cancer cells was up-regulated under chronic hypoxia microenvironment.2.In hypoxia-resistant gastric cancer cells,UCA1 adsorbs mi R-7-5p and inhibits its function.As a result,UCA1 up-regulates EGFR expression and promotes cell metastasis.3.UCA1 combined with PTBP1 regulates PKM alternative splicing and promotes PKM2 expression.4.UCA1 promotes the formation of PKM2 tetramer and enhances glycolysis of hypoxic gastric cancer cells.5,UCA1 promote PKM2 monomer into the nuclear enhanced hypoxia-resistant gastric cancer cell metastasis.