| Background and Objectives:Gastric cancer (GC) is one of the most common digestive tract malignancies and causes serious damages to human health. To find the high-risk areas and population of GC for focal prevention and treatment are the important directions of country controlling strategy. Therefore,improvements of the early stage diagnosis efficiency of GC, validation and identification of diagnosis biomarkers is important to reduce the mortality of GC and become the public health research hot spot at present. The discovery of long non-coding RNA (IncRNA) in the human genome has provided a new direction in cancer study. LncRNAs, which can regulate metabolism,cell differentiation and individual development progression of life on multiple levels and related with human serious diseases. In the present study,we focus on the GC high-risk area and differentially expressed lncRNAs and mRNAs, which were identified from GC specimens and their paired adjacent non-cancerous tissues of patients in this area through microarray analysis.Subsequently, we further investigated the expression levels of significantly different IncRNAs in GC patients’ tissues specimens and analysis the distributions in different GC clinical features basis of GC patients’ information. Then, we use bioinformatics analysis to reveal the large samples RNA sequencing profiles of GC patients which were selected from the Cancer Genome Atlas (TCGA)database. Integrated analysis of high-risk area GC patients’tumor tissues microarray analysis results and TCGA database GC tissues large samples RNA sequencing bioinformatics results to find GC early diagnosis and progression candidate IncRNA biomarkers. Here, we selected IncRNA UCA1 and LOC101928316 through repeated verification to further investigate their potential roles in GC progression. Subsequently, study also deeply explores the biology functions of UCA1 and LOC101928316 which were affected the GC cell growth, proliferation, migration and invasion.Finally, study through seek and validation of UCA1 and LOC101928316 possible related signaling pathway to investigate the potential regulation mechanism in GC progression. This study will help us to elucidate the pathogenesis of GC and find the candidate biomarkers for GC screening in high-risk population provide theoretical and experimental basis.Methods:1. Analysis the China cancer registration annals to find the GC high-risk area in our country,then deeply analysis the selected GC high-risk area all malignant tumor registration data from 2003 to 2012 to clear cancer incidence and mortality situation, population distribution and cancer epidemiological characteristics in this area. In addition,summary of malignant tumor monitoring data and the literature reports of our selected high-risk area to analysis primary malignant tumors and etiology effect of local resident’s health.2. A total of 10 human advanced GC specimens and their paired adjacent non-cancerous tissue specimens were obtained for lncRNA and mRNA microarray analysis from the GC high-risk area(Wuwei, Gansu). Through bioinformatics comparing and qRT-PCR validation to reveal the GC related lncRNA and mRNA expression profile. Using Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) and IncRNA-mRNA co-expression network to analysis potential functions of primary GC related significant differently expression IncRNAs.Subsequently, apply qRT-PCR to detection the expression levels of key IncRNA in IncRNA-mRNA co-expression network in 20 early stage GC patients’ tissue specimens, in order to make clear whether the selected key IncRNAs expression levels have changed in the early stage of GC. Finally,we further investigated the expression levels of primary key lncRNA in 82 cases of GC patients ’tissues specimens and analysis the distributions in different GC clinical features basis of GC patients’ information.3. A total of 443 patients with GC were collected from the TCGA database and download GC patients tissue RNA sequence results which were included from TCGA. Bioinformatics analysis was used to description the GC related lncRNA differential expression profile of the TCGA large sample GC population. LncRNA-miRNA-mRNA co-expression network was constructed by competitive endogenous RNA (ceRNA) theory and indirect analysis potential functions of key lncRNA. Subsequently, study combined with TCGA gastric cancer patient’s information to analysis the relationship between key IncRNA expression levels and GC clinic pathological factors and overall survival.4. Compared with China GC high-risk area patients’ related IncRNA expression profile and TCGA database GC patients’ related IncRNA expression profile bioinformatics analysis results,study selected one of a common abnormal expression of IncRNA UCA1 and China GC high-risk area GC patients’ one of a uniquely abnormal expression of lncRNA LOC101928316 as target IncRNAs. Then, analysis their related potential regulation signaling pathway, GC cell lines expression levels and stability.5. RNA situ hybridization assay was performed to determine the localization of previous selected IncRNA UCA1 and LOC101928316 in GC cells. Construct plasmid and lentivirus stably transfected GC cell lines, and use MTT assay, flow cytometry assay, wound healing assay and trans-well invasion assay to study target IncRNAs biological functions in GC cell growth,proliferation, migration, and invasion. Meanwhile, using western blot assay, tumor bearing nude mice assay, immunohistochemical and combination of vitro and vivo experiments to investigate the molecular mechanisms of selected target IncRNAs in regulated the GC progression and tumor development.Results:1. Chinese GC high-risk area selection, cancer incidence and mortality analysis and cancer epidemic trend studyAnalysis the cancer incidence and mortality data in Wuwei,Gansu province, a representatively area of northwestern China from 2003 to 2012, and the retrospective population based study results showed that GC incidence and mortality in Wuwei at the higher level for a long time. The GC average incidence and mortality rate were 95.56/105 and 68.65/105 in Wuwei from 2003 to 2012,which were higher than China GC average incidence and mortality 334% and 131%, respectively.Our results suggested that the epidemiological characteristics of cancers are not optimistic in Wuwei City. Gastric cancer is the major epidemic carcinomas, should be focus on prevention and control in the future.2. The identification of IncRNA expression profile in gastric cancer and its clinical significance2.1 Screening of gastric cancer related IncRNAThe IncRNA expression profile of GC patients’ tumor tissues in Wuwei suggested that there were 1046 lncRNAs (including 427 up-regulated and 619 down-regulated) were significant difference between GC tumor tissues and their paired adjacent non-cancerous tissues. The mRNA expression profile showed that there were 2996 mRNAs (including 647 up-regulated and 2349 down-regulated) were significant difference between GC tumor tissues and their paired adjacent non-cancerous tissues. LncRNA-mRNA co-expression network showed that network connections between 184 IncRNAs and 164 mRNAs. Study combined the co-expression network, mRNAs function and microarray profile differential expression to select the key lncRNAs. We found that there were 14 key IncRNAs and 21 key mRNAs were strongly associated. We selected above 14 key IncRNAs and detect their actual expression levels in 30 advanced GC samples and paired adjacent non-tumor tissue samples. Results showed that, RP5-919F19, CTD-2541M15 and UCA1 expression was significantly higher in tumor tissues than in paired adjacent non-tumor tissues(P<0.05). AP000459, LOC101928316, RP11-167N4 and LINC01071 expression was significantly lower in tumor than in adjacent non-tumor tissues (P<0.05). Expression levels of these 14 lncRNAs were approximately consistent with the microarray results.. Then, we chose significant differential expression candidate IncRNAs RP5-919F19, CTD-2541M15, UCA11 AP000459, LOC101928316,RP11-167N4, LINC01071 and MEG3 (advanced GC tissues lower expression trend, P=0.076) to further detect their actual expression levels in 20 early stage GC patient samples. Results showed that, there were 5 IncRNAs were associated with early stage GC, and the expression levels of CTD-2541M15 and UCA1 were significantly higher in 20 early stage GC patient tumor tissues than in their adjacent non-tumor tissues (P<0.05). AP000459, LINC01071 and MEG3 expression was significantly lower in tumor tissues than in adjacent non-tumor tissues (P<0.05). In addition, we also analyzed the change regularity of the significant differential IncRNAs (CTD-2541M15, UCA1,AP000459, LINC01071 and MEG3) both on 30 advanced GC and 20 early stage GC patients.Based on our results, compared with adjacent non-tumor tissues, the expression of CTD-2541M15 and UCA1 showed a significant gradual upward trend from early stage GC to advanced GC,AP000459, LINC01071 and MEG3 showed a significant gradual downward trend from early stage GC to advanced GC.2.2 Identification and functional characterization of lncRNA in gastric cancerThe relationships between the expression of the key IncRNAs RP5-919F19, CTD-2541M15,UCA1, AP000459, LOC101928316, RP11-167N4, LINC01071 and MEG3 in GC samples and associated clinicopathological characteristics were analyzed. The results of IncRNAs tissues expression detection showed that RP5-919F19, CTD-2541M15 and UCA1 were significantly higher expression in GC tumor tissues (P<0.05); and AP000459, LOC101928316, LINC01071 and MEG3 were significantly lower expression in GC tumor tissues (P<0.05). In addition, results of IncRNAs expression levels and clinical pathological parameters correlation analysis showed that RP5-919F19, LOC101928316 and MEG3 were significantly associated with tumor differentiation degree (P<0.05), RP5-919F19, UCA1 and MEG3 were significantly correlated with lymph node metastasis (P<0.05), and RP5-919F19, CTD-2541M15 and UCA1 were significantly correlated with tumor TNM stage (P<0.05). In addition, UCA1, LOC101928316 and LINC01071 were significantly correlated with tumor size (P<0.05). Results emphasis suggest that the IncRNAs RP5-919F19, LOC101928316, CTD-2541M15, UCA1 and MEG3 were closely related with the invasion and metastasis of GC, and worth for further study as novel candidate biomarkers.3. Gastric cancer related IncRNA expression profile identification in TCGA databaseBioinformatics analyzed the GC patient’s tissue samples’ RNA sequencing data of TCGA database we found 25 key lncRNAs, 22 key miRNAs, and 489 key mRNAs were closely related with GC. Based on these GC related key genes, study constructed the IncRNA-miRNA-mRNA ceRNA network; and further analyzed the clinical related informations found there were 14 GC specific lncRNAs were significantly different in comparison of clinical features. Results showed that ATP8B5P, FOXD2-AS1, UCA1, GUCY1B2, RHPN1-AS, TSPEAR-AS2, LOC100128164 and SLC26A4-AS1 were linked to tumor grade (P<0.05), PVT1, H19 and PART1 were linked to TNM stage (P<0.05), LOC553137, HOTAIR and TINCR were linked to lymphatic metastasis (P<0.05).Furthermore, we also found there were 8 lncRNAs were significantly associated with GC patients’overall survival (log-rank p<0.05). Among the 8 significant IncRNAs, IncRNA RPLPOP2,FOXD2-AS1 and H19 were negatively associated with overall survival (P<0.05), IncRNA TINCR,SLC26A4-AS1, SMIM10L2A, SMIM10L2B and SNORD116-4 were positively correlated with overall survival (P<0.05).4. Target IncRNAs selection, cell expression levels detection and stability analysisCompared with China GC high-risk area GC patients’ related IncRNA expression profile,lncRNA-mRNA network and TCGA database GC patients’ related IncRNA expression profile,ceRNA network, study selected one of a common abnormal expression of IncRNA-UCA1 and China GC high-risk area GC patients’ one of a uniquely abnormal expression of lncRNA-LOC 101928316 as target lncRNAs. Bioinformatics analysis the UCA1 and LOC101928316 potentially related mRNAs functions and selected PI3K-Akt-mTOR signaling pathway to further study the regulating relationship. UCA1 and LOC101928316 cell lines expression levels detection results showed that UCA1 with a higher expression in GC cell lines,which compared with GES-1, the BGC-823, SGC-901 and MGC-803 were upregulated 30.81,19.37 and 7.31 times, respectively; LOC101928316 with a lower expression in GC cell lines,which compared with GES-1, the MKN-28, SGC-7901 and BGC-823 were downregulated 20.81, 12.23 and 10.27 times, respectively. Gastric cancer cell lines expression levels of UCA1 and LOC101928316 were approximately consistent with the microarray results, GC patients’ tissues validation results and TCGA analysis results. In addition, UCA1 and LOC101928316 expression levels are relatively stable in total RNA of GC cells under the condition of less repeated freezing and thawing, which can meet the testing requirements of transcriptomics.5. Study of the effect of UCA1 on biological behavior of gastric cancer and its mechanismRNA situ hybridization assay results suggested that the localization of UCA1 in BGC-823 cell was existed in the cytoplasm. MTT assay revealed that BGC-823 cell growth were significantly impaired in lenti-UCA1-siRNA compared with negative control (P < 0.01); And the lentivirus-UCA1-Overexpression group, gastric cells growth appears increasing trend compared with negative control (P < 0.05). BGC-823 cell apoptosis was significantly increased after lentivirus-UCA1-siRNA transfected compared with negative control (P<0.05). Wound healing assay results showed that compared with negative control migration ability was appearing decreasing tendency in BGC-823 cell when lenti-UCA1-siRNA stable infection (P<0.05); and migration ability was increased in BGC-823 cell when stable infected with Lenti-UCA1-Overexpression (P<0.01). Meanwhile, trans-well invasion assay also suggested that UCA1 lenti-UCA1-siRNA stable infection can lowered the number of BGC-823 invading cells (P<0.05). Western blot results revealed that Lenti-UCAl-siRNA stable infected can remarkably inhibited the expression level of AKT3, p-AKT, p-mTOR, S6K and increase the expression level of EIF4E in BGC-823 cells, which compared with blank and negative control, respectively (P<0.05);Meanwhile, Lenti-UCA1-Overexpression can remarkably increase the expression level of AKT3,p-AKT, p-mTOR, S6K and inhibited the expression level of EIF4E in BGC-823 cell, which compared with blank and negative control, respectively (P<0.05). Tumor bearing nude mice assay results showed that the average tumor weight at the end of the experiment was markedly reduce in the UCA1-siRNA group (0.46 ±0.12g),which compared to the blank group (0.84 ±0.17g) and the empty vector group (0.88 ± 0.35g) (P < 0.05). Immunohistochemical results suggested that lenti-UCA1-siRNA stable transfected BGC-823 cells can significantly inhibit the p-AKT and p-mTOR proteins expression levels which compared with BGC-823 blank and negative control groups.6. Study of the effect of LOC101928316 on biological behavior of gastric cancer and its mechanismRNA situ hybridization assay results suggested that the localization of LOC101928316 in SGC-7901 cell was existed in the cytoplasm. MTT assay revealed that SGC-7901 cell growth were significantly impaired in lenti-LOC 101928316-Overexpression group, which compared with negative control (P<0.01). Wound healing assay results showed that compared with negative control migration ability was appearing decreasing tendency in SGC-7901 cell when lenti-LOC 101928316-Overexpression stable infection (P<0.05). Meanwhile, trans-well invasion assay also suggested that lenti-LOC 101928316-Overexpression stable infection can lowered the number of SGC-7901 invading cells (P < 0.05). Western blot results revealed that lenti-LOC 101928316-Overexpression stable infected can remarkably inhibited the expression level of PI3K, p-AKT, mTOR, p-mTOR and BCL-XL in SGC-7901 cells, which compared with blank and negative control, respectively (P<0.05); Meanwhile, lenti-LOC101928316-siRNA stable infected can remarkably increase the expression level of AKT3, mTOR, p-mTOR, BCL-XL and inhibited the expression level of PTEN in SGC-7901 cells, which compared with blank and negative control, respectively (P<0.05). Tumor bearing nude mice assay results showed that the average tumor weight at the end of the experiment was markedly reduce in the lenti-LOC101928316-Overexpression group (0.55±0.27g),which compared to the blank group(0.94±0.22g) and the empty vector group (1.16±0.54g) (P<0.05). Immunohistochemical results suggested that lenti-LOC101928316-Overexpression stable transfected SGC-7901 cells can significantly inhibit the p-AKT and p-mTOR proteins expression levels which compared with SGC-7901 blank and negative control groups.Conclusion1. The GC average incidence and mortality rate were 95.56/105 and 68.65/105 in Wuwei from 2003 to 2012, which were higher than China GC average incidence and mortality 334% and 131%,respectively. Results suggested that GC is the major epidemic carcinomas in Wuwei, should be focus on prevention and control in the future.2. Identification the lncRNA expression profile of GC in China high-risk area GC population,using bioinformatics analysis, qRT-PCR large samples validation and clinical pathologic factors related analysis we found compared with adjacent non-tumor tissues, the expression of CTD-2541M15 and UCA1 showed a significant gradual upward trend from early stage GC to advanced GC,AP000459, LINC01071 and MEG3 showed a significant gradual downward trend from early stage GC to advanced GC. In addition, lncRNAs: RP5-919F19, CTD-2541M15, UCA1,LOC101928316 and MEG3 were closely related with the invasion and metastasis of GC, and worth for further study as novel candidate biomarkers.3. Based on the analysis of the TCGA database GC patients’ tissues RNA sequencing,study constructed the lncRNA-miRNA-mRNA ceRNA network; and found there were 14 GC specific lncRNAs: TP8B5P, FOXD2-AS1, UCA1, GUCY1B2, RHPN1-AS, TSPEAR-AS2, LOC100128164,SLC26A4-AS1, PVT1, H19, PART1, LOC553137, HOTAIR and TINCR, which were significantly different in comparison of clinical features. In addition, there were 8 GC specific lncRNAs:RPLPOP2, FOXD2-AS1, H19, TINCR, SLC26A4-AS1, SMIM10L2A, SMIM10L2B and SNORD116-4, which were significantly associated with GC patients’ overall survival. These results suggested that above IncRNAs as key GC related genes in TCGA database are worth to deeply investigate.4. Bioinformatics analysis the UCA1 and LOC 101928316 potentially related genes functions and selected PI3K-Akt-mTOR signaling pathway to further study the regulating relationship.Gastric cancer cell lines expression levels of UCA1 and LOC 101928316 were approximately consistent with the microarray results, GC patients’ tissues validation results and TCGA analysis results. In addition, UCA1 and LOC101928316 expression levels are relatively stable in total RNA of GC cells under the condition of less repeated freezing and thawing, which can meet the testing requirements of transcriptomics.5. Study found that UCA1 expression was significantly upregulated in gastric cancer tissues and cell lines and upregulated UCA1 was correlated with gastric cancer TNM stage and lymph-node metastases. UCA1 silencing can significantly inhibited gastric cancer BGC-823 cell proliferation, migration, and invasion, increased its apoptosis and inhibited the tumorigenicity of GC cells in nude mice. Taken together, UCA1 may function as oncogene and play an important role in GC tumorigenesis.6. Study found that LOC 101928316 expression was significantly downregulated in gastric cancer tissues and cell lines and downregulated LOC101928316 was correlated with gastric cancer tumor differentiation degree. LOC101928316-Overexpression can significantly inhibit gastric cancer SGC-7901 cell proliferation, migration and invasion. Vivo experiment also revealed that LOC101928316-Overexpression can inhibit the tumorigenicity of GC cells in tumor-burdened experimental nude mice. Taken together, LOC101928316 may function as anti-oncogene and also play an important role in GC tumorigenesis. |
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