The Role And Potential Mechanisms Of LncRNA-AK058003in The Metastasis And Invasion Of Gastric Cancer Under Hypoxia

Gastric cancer (GC) is the second most common cause of cancer-related deaths in ourcountry, the main causes of deaths are distant metastasis. In recent years, the research islimited to the the tumor cells and always ignored the important roles of tumormicroenvironment in the invasion and metastasis. As we know, hypoxia is a feature ofmost solid tumors and is associated with malignant progression, invasion, angiogenesis,and increased risk of metastasis. More and more studies have attempted to characterize thetumor response to hypoxia and to identify predictive markers of disease. Among the manyfactors regulated by hypoxia, the transcriptional factor of hypoxia-induced factor (HIF),plays a central role in the ability of tumor cells to sense and respond to hypoxia. HIFinvolved in many aspects of cancer, including cell invasion and metastasis, by regulating anumber of downstream protein-coding genes. As a result, investigation ofhypoxia-induced signaling pathways and seek the effective molecular target will help todevelop new tools for the treatment of hypoxic tumors.LncRNAs, which are broadly defined as transcribed RNA molecules greater than200nt in length and lacking an open reading frame, have attracted much attention in recent years. They can regulate gene expression from various angels（Epigenetic Regulation、Transcriptional Regulation and Post-transcriptional Regulation）. It has benn a populartopic in tumor study. There have been many studies confirmed that the expression of somelncRNAs are increased in hypoxic tumors. So to deep exploration the roles and molecularmechanisms of lncRNAs in hypoxic tumors will help us to comprehensive understand theprocess and regulating mechanisms of hypoxia induced invasion and metastasis in GC.ObjectiveTo analyze the expression profile variation of long non-coding RNAs(lncRNAs) ingastric cancer cell lines under normoxia and hypoxia through high throughput lncRNAmicroarrays; To verify the expression of the key molecules and explore the relationship ofits expression between carcinoma and pericarcinous tissues; To further investigate its rolesand mechanisms in the hypoxia-induced invasion and metastasis in GC.Methods1. High throughput lncRNA microarrays(12x135K LncRNA Expression Microarray：lncRNA from NCBI RefSeq, UCSC, RNAdb, lncRNAs from literatures and UCRs andreferences) are used to compare the lncRNA expression profile difference betweennormoxia-induced and hypoxia-induced GC cell lines (SGC7901,MKN45and MKN28).2. Screen hypoxia-induced invasion and metastasis related lncRNAs through strategiessuch as change fold, neighboring genes analysis and characteristics (length) analysis.3. The different expression of the key molecular between hypoxia-induced GC andnormoxia-induced GC were valitaed using RT-PCR; The expression levels of the keymolecular in20GC tissues and corresponding non-tumor mucosa were detected byRT–PCR.4. SiRNA-mediated antisense lncRNA-AK058003gene transfer technique was employedto downregulate AK058003expression in human GC cell lines SGC7901and MKN45.Transwell migration and invasion assays, would-healing assays between normoxia andhypoxia and tail vein injection metastasis assays were employed for investigating thehypoxia-induced metastatic properties of each cell subline.5. Bioinformatics analysis were performed to identify the target gene of AK058003. The expression of the target gene was verified by RT-PCR and western blots.6. The SNCG expression was further confirmed by immunohistochemistry in GC cancertissues and adjacent tissues.7. SNCG was silenced in GC cells with antisense oligonucleotide or SNCG wasoverexpressed in GC cells with a SNCG expression vector respectively and confirmed theeffects using western blots. The effects of SNCG expression on cell invasion andmetastasis were determined by transwell migration and invasion assays under normoxia.8. To further ascertain whether SNCG is one of AK058003target genes,we transfected theSNCG expression vector or a negative control into cells that stably knocked downAK058003ectopically. Then transwell migration and invasion assays were performedunder normoxia.9. To further determine whether SNCG is involved in AK058003-induced GC cellmetastasis and invasion under hypoxia, we first detected the expression level of SNCGunder hypoxic conditions in the cells that stably down-expression of AK058003and thenegtive control by western blots. Then transwell migration and invasion assays wereperformed under hypoxia.10. The cells of SGC7901-LV-siAK and MKN45-LV-siAK were transiently transfectedwith the SNCG over-expression vector, after confirmed the co-transfection successfully.Transwell migration and invasion assays were performed to explore the role of SNCG inthe AK058003-mediated GC metastasis and invasion under hypoxia.Results1. Compare with the gastric cancer cell lines under normoxia, and254lncRNAs whichhave more than1.5times variation and significant diference(P<0.05) by statistical analysisare regarded as lncRNAs with differential expression, accounting for3.6%of alllncRNAs：while84increase more than1.5times and5increase more than3times；2. After screening hypoxia-induced invasion and metastasis related lncRNAs throughsome strategies, we found that lncRNA-AK058003may play the key role in thehypoxia-induced GC metastasis and invasion.3. RT-PCR showed that lncRNA-AK058003was frequently up-regulated under hypoxic GC cell lines relative to expression under normoxia. Expression of AK058003reach amaximum at16hr in SGC7901cell lines,24hr in MKN45cell lines and48hr in MKN28cell lines, respectively.The AK058003was also detected in20pairs of GC and corresponding noncancerousgastric samples using RT-PCR.The results showed that the expression of AK058003wassignificantly up-regulated in GC when compared to the noncancerous gastric samples.The overall expression level of AK058003increased in15GC samples (75%), wasunchanged in3samples and was down-regulated in2samples, indicating that up-regulation of AK058003is a frequent event in GC.4. Functional studies of AK058003revealed that down-expression of AK058003resultedin an reduction in migration and invasion.This suggests that AK058003involved in GCmetastasis and invasion.To further explore whether AK058003mediated the hypoxia-induced GC metastasisand invasion. Transwell migration and invasion assys revealed that hypoxia can promotethe GC cells metastasis and invasion, but when we silenced AK058003with an antisenseoligonucleotide inhibitor in the GC cells, the ability of GC cells metastasis and invasiondecreased compared with the negative control. Consistent with the above data, thewounding assays also showed the same tendency.To further investigate the promotion of in vivo tumor metastasis by AK058003, weimplanted SGC7901-LV-siAK cells that were stably knocked down AK058003or controlcells into nude mice through the tail vein injection. The average liver metastasis nodulenumbers were apparent in mice that injected with SGC7901-LV-control cells. In contrast,fewer metastasis nodules were detected in mice injected with SGC7901-LV-siAK cells.The results showed that liver metastasis tumor nodules were approximately56in averageinjected with SGC7901-LV-control cells; in contrast, only approximately6metastasistumor nodules were found in mice injected with SGC7901-LV-siAK cells. All these resultsdemonstrated that AK058003invovled in the hypoxia-induced metastasis and invasion.5. Bioinformatics predicted that SNCG (Breast cancer metastasis associated genes) may bea target for AK058003, SNCG is in the downstream of AK058003. To further test the hypothesis, we analyzed the expression of AK058003and SNCG in GC cell lines.Weobserved that SNCG mRNA and protein levels were decreased when AK058003wasknocked down in GC cells.6. The expression of SNCG in the GC tissue was significantly higher than in para cancertissues. Next, we analyzed the correlations between SNCG expression andclinicopathological parameters of GC patients. SNCG expression was correlated closelywith the depth of tumor invasion, TNM stage, lymph node metastasis, but not withpatients’ age, gender and differentiation.7. To explore whether SNCG mediated GC metastasis and invasion. Transwell migrationand invasion assys revealed that when we silenced SNCG with an antisense RNAexpression vector in the GC cells, the ability of GC cells metastasis and invasiondecreased compared with the negative control the GC cells. On the contrary,overexpressed SNCG can promote the GC cells metastasis and invasion. These suggeststhat SNCG mediated the GC metastasis and invasion.8. If SNCG is indeed the functional target of AK058003in GC, then restoration of SNCGin AK058003downregulated GC cells should abrogate the effects of AK058003. To testthis hypothesis, we introduced the SNCG expression vector into GC cells loss ofAK058003expression in GC cells. After the restoration of SNCG, the migration andinvasion of SGC7901and MKN45cells inhibitied by si-AK058003were significantlyincreased. These results indicated that SNCG is a functional target of AK058003in GCcells.9. Western blots showed that the expression of SNCG in SGC7901-LV-NC andMKN45-LV-NC cells was increased under hypoxia when compared to its expression undernormoxia. When we use SGC7901-LV-siAK and MKN45-Lvsi-AK,the cells thatAK058003was inhibited, the increase in SNCG induced by hypoxia was abrogated.Theseindicated that the elevation in SNCG was mediated by the up-regulation of AK058003under hypoxic conditions.The transwell migration and invasion assays which were performed under hypoxiashowed that hypoxia can promote the GC cells metastasis and invasion, but when we silenced SNCG with an antisense RNA expression vector in the GC cells, the ability ofGC cells metastasis and invasion decreased compared with the negative control.10. To further determine the function of SNCG in AK058003–induced GC metastasisunder hypoxia, the SNCG expression vector and negative control were cotransfected intoSGC7901-LV-siAK and MKN45-Lvsi-AK cells. In transwell experiments, the impairmentof siRNA-mediated AK058003on the hypoxia-induced GC cell migration and invasionwas partially relieved by SNCG, but not by the negative control.Conclusions1. Based on high-throughput screening of lncRNAs microarrays, we reported a group ofhypoxic gastric cancer related lncRNAs for the first time.2. LncRNA-AK058003is one of the most key moleculars invovled in hypoxia inducedGC metastasis and invasion.3. SNCG is the functional target of AK058003in GC. It overexpressed in metastatic GCtissue. Furthermore, SNCG can promoted hypoxia induced GC metastasis and invasion.4. The siRNA-mediated AK058003on the hypoxia-induced GC cell migration andinvasion can partially relieved by overexpressing SNCG, but not by the negative control.