The Role And Molecular Mechanism Of LncRNA KRT19P3 In Proliferation And Metastasis Of Gastric Cancer

Research purposes:Gastric cancer(GC)is one of the most common malignancies and the leading cause of cancer-related death.Gastric cancer ranks as the second most prevalent malignant carcinomas accounting for second leading cause of cancer-related deaths in china.Despite recent advances in medical treatment,patients with an advanced stage still have a poor prognosis,and the 5-year survival rate is below 20%.Recurrence and metastasis are the leading cause of death in patients with gastric cancer.Early diagnosis and treatment can reduce the mortality rate of patients with gastric cancer.However,the diagnosis and treatment rate of early gastric cancer in China is relatively low.Most of the patients have been diagnosed at the advanced stage and missed the best operation period.Even after radical surgery and chemotherapy,the recurrence and mortality rate of these patients are still high.Therefore,better understanding of the mechanisms in GC tumorigenesis and progression is essential for early diagnosis and therapy of GC.Previous studies have demonstrated that several factors are involved in the occurrence and development of GC,including activation of proto-oncogenes,inactivation of tumor suppressor genes and the aberrant expression of related signaling pathways.Long non-coding RNA(lncRNA)is a new kind of RNA molecule longer than 200nt which is non-protein coding.Lots of evidence has implicated that lncRNAs play critical roles in a variety of diseases including tumors.LncRNAs may function as oncogenes or tumor suppressors by regulating the transcription of protein-coding genes or altering the chromatin structure.LncRNAs play important roles in regulation initiation,development,proliferation,invasion and metastasis of many cancers including gastric cancer.LncRNAs can be used as molecular markers for diagnosis and therapeutic targets of cancer.Current studies have demonstrated that lncRNAs may act as important regulators in GC development and progression.Although a variety of lncRNAs with oncogenic or cancer suppressive functions have been identified in GC,the overall pathophysiological contributions of IncRNAs to GC remain unclear so far.Therefore,it is necessary to elucidate the role and molecular mechanism of IncRNAs in the occurrence and metastasis of gastric cancer which may contribute to establish a reasonable prevention and treatment program,the long-term survival rate and quality of patients' life can also be improved.LncRNAs can be used as a molecular marker and therapeutic target for the GC diagnosis and treatment.Thus,we used gene expression microarray to screen differentially expressed IncRNAs between GCs and non-tumor gastric mucosa.RT-qPCR analysis further confirmed the differential expression of selected lncRNAs in other independent samples.lncRNA KRT19P3 was chose for further study.We correlated KRT19P3 expression with clinicopathologic characteristics of gastric cancer.Kaplan-Meier analysis using the log-rank test was performed to investigate whether the expression of KRT1 9P3 was associated with GC patients' survival.We also measured the location of KRT19P3 through RT-qPCR amplified with separated cytoplasm RNA and nuclear RNA in gastric cancer cells.Using gain-and loss-of-function methods,the effects of KRT19P3 on the proliferation,apoptosis,migration an invasion of gastric cancer cells were investigated in vitro and in vivo.RNA pull-down,mass spectrometry,RNA immunoprecipitation(RIP)and Western blot were used to identify the binding protein of KRT19P3 and signaling pathway linked to KRT19P3 in GC.This study may provide a theoretical basis for KRT19P3 as a new diagnostic and prognostic marker and therapeutic target for gastric cancer.Methods:1?To identify aberrantly expressed lncRNAs in GC,we performed IncRNAs expression array analysis between ten cases of GC tissues and two cases of non-tumor gastric tissues.The differentially expressed genes were identified between the above two groups by fold changes and P value.LncRNA KRT19P3 was selected for further research.2?The expression of KRT19P3 was detected in other independent samples by RT-qPCR analysis.Moreover,we correlated KRT19P3 expression with clinicopathologic characteristics of gastric cancer.Kaplan-Meier analysis using the log-rank test was performed to investigate whether the expression of KRT19P3 was associated with GC patients' survival.3?In order to elucidate the role of KRT19P3 in GC progression,we overexpressed KRT19P3 using pcDNA3.1(+)plasmid vector or down-regulated KRT19P3 with siRNA in SGC7901 and MKN45 cells.The overexpression and knockdown efficiency were confirmed 48h after transfection by qRT-PCR.To explore the role of KRT19P3 on cell proliferation,CCK-8 and EdU assay were performed.The apoptotic rate of gastric cancer cells was detected using Annexin V-FITC/PI double staining flow cytometry.4?we also assessed the roles of KRT19P3 on GC cell migration and invasion capabilities by cell migration and invasion assay.5?In vivo tumorigenic and metastasis assay.To analyze the effects of KRT19P3 on GC cells in vivo,xenograft nude mouse model experiments were performed.SGC7901 cells were transfected with lentivirus vector LV5-KRT19P3 or LV5-NC,and then cells were injected into the right dorsal flanks or the lateral tail vein of 4-week-old male Nu/Nu mice(Vital River,Beijing,China).Six weeks after tumor inoculation,the animals were sacrificed,the tumor nodules and lungs were extracted and stained by Hematoxylin and Eosin(HE)for assessment.6?Molecular mechanism of KRT19P3 in gastric cancer.KRT19P3 and its antisense RNA were in vitro transcribed and biotin-labeled.The RNA pull-down assay was used to explore the KRT19P3 potential binding protein.The associated proteins were resolved by SDS-PAGE and detected using a standard western blotting technique or by mass spectrometry identification.Western blot and RNA Immunoprecipitation(RIP)technologies were performed to verified the target protein of KRT19P3.Signaling pathway linked to KRT19P3 was identified by rescue experiments,Western blot and luciferase assay.7?Statistical analysis.The significance of the differences was confirmed using the Student s t-test between two groups or with one-way ANOVA for three groups.Chi-squared test was used to analyze the association between KRT19P3 expression and clinicopathological parameters.Survival curves were plotted using the Kaplan-Meier method and the differences in overall or disease-free survival were assessed with the log-rank test.P-value<0.05 was considered statistically significant.Results:1?lncRNA KRT19P3 expression in gastric cancer.579 down-regulated genes and 1075 up-regulated genes were detected in gastric cancer tissues by lncRNAs expression array analysis.lncRNA KRT19P3 was found to be downregulated more than 5-fold(absolute fold change=8.58)in GC compared with adjacent non-tumor tissues.KRT19P3 is located in chromosome 4.To further validate these results,we detected KRT19P3 expression in 84 cases of gastric cancers and 29 cases of non-tumor gastric mucosa by RT-qPCR.KRT19P3 expression was significantly decreased in gastric cancer tissues,and KRT19P3 level was even lower in primary GC tissues with lymph node metastasis(LNM)compared with those without LNM.Lower expression of KRT19P3 was found to be associated with lager tumor size(?5 cm),advanced TNM stage,Lauren's classification and positive lymph node metastasis.Patients with lower expression of KRT19P3 had worse overall survival(OS)and disease-free survival(DFS)compared with these patients with higher KRT19P3 expression.We found that most of the KRT19P3 RNA is located in the nucleus of the GC cells.2?The effects of KRT19P3 on the biological behaviors of gastric cancer cells.we overexpressed KRT19P3 using pcDNA3.1(+)plasmid vector or down-regulated KRT19P3 with siRNA in SGC7901 and MKN45 cells.The overexpression and knockdown efficiency were confirmed 48h after transfection by RT-qPCR.CCK-8 assays results suggested that overexpression of KRT19P3 reduced proliferation of SGC7901 and MKN45 cells,whereas KRT19P3 knockdown significantly enhanced cell growth.Furthermore,EdU assay confirmed upregulation of KRT19P3 reduced EdU staining.Consistently,inhibition of KRT19P3 expression increased EdU staining.Additionally,it was found that overexpression of KRT19P3 induced apoptosis rate compared with the negative control in SGC7901 and MKN45 cells,whereas KRT19P3 knockdown decreased the apoptosis rate of GC cells.SGC7901 and MKN45 cells displayed significantly decreased migration and invasion capability after transfection with KRT19P3 overexpression plasmid,compared with that of the cells transfected with empty plasmid.In addition,downregulation of KRT19P3 could significantly enhance the migration and invasion abilities of GC cells.Tumors in the LV-KRT19P3 group were non-invasive or well-encapsulated.However,tumors in the negative control group invaded locally into fibrous capsules in subcutaneous implanted nude mouse models.The lungs in the LV-KRT19P3 group had less metastatic GC foci than the negative control group.Moreover,the metastatic foci in the LV-KRT19P3 group had smaller volumes than those in the control group.These results showed that KRT19P3 played an important role in regulating proliferation,invasion and metastasis of GC cells.3?Study on the molecular mechanism of abnormal expression of KRT19P3 in gastric cancer.We found several bands pulled down by vitro-transcribed biotinylated KRT19P3 sense transcript using silver staining,mass spectrometry identification indicated that COPS7A(CSN7A)was potential KRT19P3-interacting protein.We further verified such interaction independently by western blot analysis and RNA immunoprecipitation(RIP)experiment.We found that overexpression of KRT19P3 effectively induced the COPS7A protein level in SGC7901 and MKN45 cells.Western blot analysis showed that overexpression of KRT19P3 enhanced COPS7A,protein stability in GC cells.We found that the tumor suppressive effect of KRT19P3 could be attenuated through overexpression of COPS7A into the GC cells.CCK-8 assay and transwell migration and invasion assay results had indicated a significant rescue effect of COPS7A on proliferation,migration and invasion of GC cells with KRT19P3 inhibition.These data demonstrated that KRT19P3 suppressed GC cell proliferation and metastasis partly through regulation of COPS7A expression.To understand the molecular basis of the COPS7A's tumor-suppressing effects,we analyzed the putative interaction between COPS7A and I?B?.We found that I?B?expression was increased with COPS7A plasmid transfection in GC cells and COPS7A regulated the degradation of the I?B? protein via the ubiquitin-proteasome pathway.COPS7A suppressed the degradation of I?B? through deubiquitination.Results of luciferase reporter assay showed that the NF-KB-dependent reporter activity was significantly decreased in COPS7A overexpression group compared with that of the negative control.Western blot analysis showed that p65 phosphorylation was decreased in GC cells with COPS7A plasmid transfection,indicating reduced degradation of I?B?and inactivation of NF-?B signaling pathway following COPS7A overexpression.we investigated the NF-?B luciferase activity in KRT19P3 vector or siRNA transfection GC cells.We found that KRT19P3 overexpression dramatically decreased NF-?B reporter activity compared with that of the negative control.whereas knockdown of KRT19P3 generated an opposite effect and the upregulation of NF-?B reporter activity was reversed when COPS7A plasmid was simultaneous co-transfected.Western blot analysis showed that IKBa was decreased and p65 phosphorylation was increased and then NF-?B signaling activity was activated in KRT19P3 downregulated cells,and this result could be reversed by COPS7A overexpression.These results demonstrated that KRT19P3 was involved in the regulation of the NF-?B pathway by modulating COPS7A.Conclusions and significance:1?lncRNA KRT19P3 was found to be downregulated in GC by lncRNAs expression array analysis and RT-qPCR further confirmed the array results in other independent samples.Lower expression of KRT19P3 was correlated with lager tumor size(?5 cm),advanced TNM stage,Lauren's classification,positive lymph node metastasis and poor prognosis of GC patients.2?KRT19P3 exerted tumor suppressive function in GC.KRT19P3 inhibited the proliferation,invasion and metastasis ability of gastric cancer cells in vitro and in vivo.KRT19P3 also induced the apoptosis ability of GC cells significantly.3?KRT19P3 could bind to COPS7A in the GC cells,KRT19P3 regulated COPS7A expression by enhancing its protein stability in GC cells.KRT19P3 exerted tumor-suppressive effects by binding with COPS7A directly.KRT19P3 may act as a tumor-suppressor by interacting with COPS7A directly and then regulating NF-?B signaling pathway.These findings demonstrate that KRT19P3 is an important molecular marker in GC tumorigenesis and has the potential to be a novel promising candidate for the prognosis and therapy for GC.