The Fuction And Mechmism Of LncRNA-uc003uxs In The Invasion And Metastasis Of Gastric Cancer Under Hypoxia

BackgroundWith the advances in diagnosis and treatment technology, five-year survival rate ofpatients with early gastric cancer(GC) have improved significantly. However,the prognosisof gastric cancer patients with lymph node metastasis and distant metastasis is still noimprovement.Currently,the metastasis in gastric cancer patients have an impact on theefficacy and obtain long-term survival. Initially the study of tumor invasion and metastasisis often limited to changes in the internal tumor cells while ignoring the interactionbetween tumor cells and tumor microenvironment. As we all know, hypoxia is animportant development process microenvironment of solid tumor..It can contribute to theincidence of tumor’s malignant phenotypes. Therefore,the role of tumor hypoxia inmetastasis is more and more attention from scholars. Hypoxia could induces thetranscriptional factor of hypoxia-induced factor (HIF) and other factors, activation ofmany genes involved in tumor invasion and metastasis downstream,which Resulting intreatment failure. Therefore, further researching hypoxia-induced signaling pathway andlooking for the molecular target which can effectively reverse the cancermetastasis,thatcould provide adequate theoretical basis to prevent metastasis of gastric cancer. lncRNAs are a class of non-coding RNAs.The length is greater than200nt. They arethe current high-profile cancer research hotspot and can regulate gene expression atmultiple levels.Existing literature confirms that lncRNAs play an important role in tumorinvasion and metastasis. Therefore, further research the function and mechanism oflncRNAs in hypoxic tumors will help us clarify the hypoxia-induced gastric cancers’invasion and metastasis processes and molecular mechanisms.Objective1.To find the different expression of lncRNAs under hypoxia through high throughput Microarrays;2.To validate the expression of the key lncRNAs,and explore the relationship of theexpression between carcinoma and pericarcin ous tissues;3.To futher research the function and machnism of the key lncRNAs in invasion andmetastasis in GC under hypoxia.Methods1. Three gastric cancer cell lines(SGC7901,MKN28and MKN45) were incubated innormoxia and hypoxia after24h.To compare the lncRNA expression profile differencebetween the three paired gastric cancer cells by high-throughput microarray.2. Through the strategies such as neighboring genes analysis, change fold and length orother conditions characterized to screen the key molecules in invasion and metastasis ofgastric cancer cells induced by hypoxia.3. Using real-time quantitative PCR(RT-PCR) to detection the different expression levelsof lncRNA-uc003uxs under normoxia and hypoxia conditions of GC. Further testing itsexpression levels in40cases of gastric cancer tissues (relative to corresponding non-tumortissues)4. Construction of siRNA lentivirus,stable downregulate uc003uxs expression in humanGC cell lines SGC7901and MKN28.Transwell assays,wound-healing assays and veininjection metastasis assays were used for researching the hypoxia induced metastasis inhuman GC cells.5.Through bioinformatics analysis to identified the target gene of uc003uxs.The targetgene’s expression was verified by Western Blot and real-time quantitative PCR. 6.The expression of SERPINE1was further notarized by immunohistochemistry in GCtissues and adjacent tissues.7.Using antisense oligonucleotide to silenced SERPINE1or a SERPINE1expressionvector to overexpressed it respectively and verified the effect through Western Blot.Theeffect of SERPINE1expression on cell metastasis and invasion were determined bytranswell assays under normoxia.8.To further notarize whether SERPINE1is the target gene of uc003uxs,we transfected theSERPINE1expression vector or a control into cells which stably knocked downSERPINE1.Then transwell assays were performed under normoxia.9.To further verify whether SERPINE1is involved in uc003uxs-induced GC cell invasionand metastasis under hypoxia. So we detected the expression of SERPINE1under hypoxiathat stably down expression of uc003uxs and control by Western Blot.10.The cells of MKN28-LV-siuc and SGC7901-LV-siuc were transfected with theSERPINE1over expression vector then verified the co-transfection successfully.Transwell assays were accomplished to research the role of SERPINE1in the uc003uxsmediated GC invasion and metastasis under hypoxia.Results1.A high-throughput microarray analysis comparing lncRNA found that the GCcells(SGC7901,MKN28and MKN45)which incubated under normoxic conditionscompared with hypoxia induced cells. There were5significantly molecules increased inhypoxia induced GC cells.2.Through the screening by some strategies, we found that the lncRNA-uc003uxs mayplay the key role in hypoxia induced GC invasion and metastasis.3.RT-PCR showed that lncRNA–uc003uxs was significantly increased under hypoxia GCcells relative to the expression under normoxia. Expressio n of uc003uxs reach a peak at24hr in MKN28cell line,48hr in MKN45cell line and24hr in SGC7901cellline,respectively. Further RT-PCR showed that the expression of uc003uxs was upregulated in GC compared to the noncancerous gastric samples.4.Under normoxic conditions,knock down uc003uxs,the invasion and migration capacityof MKN28and SGC7901decreased significantly. More Transwell assays demonstratedthat hypoxia would increase the ability of invasion and migration in MKN28andSGC7901under hypoxia. 5.Bioinformatics analyzed that SERPINE1may be the target of uc003uxs.SERPINE1issituated in the downstream of uc003uxs.To further confirm the hypothesis, we pay closeattention to the expression of uc003uxs and SERPINE1in GC cells. We found thatSERPINE1mRNA and protein levels all decreased when uc003uxs was knocked-down inGC cells.6.The expression of SERPINE1in GC tissues was much higher than in adjacent cancertissues. Then we compared the correlations between SERPINE1expression and clinicalparameters of GC patients. We found that SERPINE1expression closely associated withTNM stage, lymph node metastasis and the depth of cancer invasion,but not withpatients’gender, age and degree of differentiation.7.To explore whether SEERPINE1mediated gastric cancer invasion and metastasis.Transwell assays resulted that when we silenced SERPINE1with an antisense RNAexpression vector in gastric cancer cells,the invasion and migration ability of GC cellsdecreased compared with control in the GC cells. Otherwise, overexpressed SERPINE1could promote the GC cells invasion and migration.8.We transfected the SERPINE1expression vector into gastric cancer cells which loss ofuc003uxs expression in gastric cancer cells.After the recovery of SERPINE1,the abilityof invasion and migration in MKN28and SGC7901cells that inhibited by si-uc003uxswere significantly increased. These results demonstrated that SERPINE1is the functionaltarget of uc003uxs in the GC cells.9.Western Blot displayed that the expression of SERPINE1in MKN28-LV-NC andSGC7901-LV-NC cells was significantly increased under hypoxia compared to itsexpression under normoxia. When we use cells which uc003uxs were inhibited, theincrease in SERPINE1induced by hypoxia was disappeared. These illustrated that theelevation in SERPINE1was mediated by the up regulation of uc003uxs under hypoxia.The transwell assays that completed under hypoxia displayed that hypoxia couldpromote gastric cancer cells invasion and migration. But when we silenced the expressionof SERPINE1in GC cells, the ability of invasion and migration decreased compared withcontrol.10.To further explore the function of SERPINE1in uc003uxs-induced gastric cancermetastasis under hypoxia, the SERPINE1expression vector and control were transfectedinto MKN28-LV-siuc and SGC7901-LV-siuc cells. In transwell assays, the weakening ofsiRNA mediated uc003uxs on the hypoxia induced GC cell invasion and migration was partly recovered by SERPINE1but not by the control.Conclusions1.A high-throughput microarray analysis comparing lncRNA found a gro up of lncRNAsin GC cells which were related to the hypoxia.2.LncRNA-uc003uxs is the most important molecule which involved in hypoxia inducedGC cell invasion and migration.3.SERPINE1is the functional target of uc003uxs in GC.SERPINE1also can promotehypoxia induced GC invasion and migration.4.The siRNA mediated uc003uxs on the hypoxia induced GC cell invasion and migrationcan partly recovered by overexpressing SERPINE1but not by the control.