| Gastric cancer(GC) has been one of the most common digestive malignant tumor and the second cause of human cancer-related deaths for several decades in China. Although gastric cancer is curable if detected early, most patients are diagnosed in the advanced stage and have poor prognosis. Thus, novel findings on prognosis or diagnosis factors, and theraupetic targets in GC would be of great clinical relevance. Apart from about 2% protein-coding genes, the vast majority of the human genome is made up of non-coding RNAs(ncRNAs). Transcription generates many long non-coding RNAs(abbreviated as ncRNA or lncRNA) that are capable of influencing diverse cellular processes like proliferation, apoptosis, or metastasis. An increasing number of studies have revealed that deregulated lncRNA expression plays a functional role in a variety of carcinomas. TINCR, an lncRNA producing a 3.7-kilobase(kb) transcript, controls human epidermal differentiation and is downregulated in human squamous cell carcinoma. Kretz et al. first confirmed that TINCR could bind to the staufen1(STAU1) protein and mediate differentiation mRNA stabilization. However, the role of TINCR in gastric cancer has not yet been reported. This study was, therefore, conducted to investigate the function of TINCR on proliferation and apoptosis in GC through cell lines, clinical tissues and xenograft tumors in vivo, and explore its underlying mechanism. Part 1: Expression level of TINCR in gastric cancer tissues and cell lines, and clinical significance of TINCR expression Objective: To detect the Expression level of TINCR in gastric cancer tissues and cell lines, investigate clinical significance of TINCR expression in prognosis or diagnosis of GC.Method: Total RNAs were extracted from 80 tumorous and adjacent normal tissues or cultured cells using Trizol reagent. RNA was reverse transcribed into cDNAs using the Prime-Script TM one step RT-PCR kit. Real-time PCR was performed in triplicate, and the relative expression of TINCR was calculated using the comparative cycle threshold(CT)(2-ΔΔCT) method with glyceraldehyde-3-phosphate dehydrogenase(GAPDH) as the endogenous control to normalize the data. Statistical analyses about the 80 patients’ clinical data were performed using SPSS 20.0 software. Results: TINCR expression was significantly higher in the tumor tissues than in the adjacent normal tissues, as investigated using qPCR assays(P < 0.01). TINCR expression was also detected in the GC cell lines, including MGC803, BGC823, MKN45 and SGC7901, and the normal gastric epithelium cell line GES1. Significantly high TINCR expression was found in SGC7901(P < 0.01), BGC823(P < 0.01), MGC803(P = 0.015), and MKN45(P = 0.031) compared to that in GES1. We used the non-tumorous tissues adjacent to the tumor tissues as a control to produce a receiver operating characteristic(ROC) curve. The cutoff value for predicting gastric cancer tissues from normal tissues was 9.05(Δ Ct value). The area under the ROC curve was 0.701(95% confidence interval [CI] = 0.619-0.782, P < 0.001; Figure 1f). The sensitivity and specificity were 0.65 and 0.71. TINCR expression levels in tumor tissues were categorized as low or high depending on whether TINCR expression was up- or down-regulated compared to the corresponding adjacent noncancerous tissue samples. Clinicopathologic factors were analyzed in the high and low TINCR expression groups. As shown in Table 1, the high TINCR group(n = 55) showed a greater depth of invasion(P = 0.005) and higher tumor stage(P = 0.002) than the lower TINCR expression group(n = 25). Kaplan–Meier analysis and log-rank test were used to evaluate the effects of TINCR expression and clinicopathological characteristics on disease-free survival(DFS). The results showed that the high-TINCR patients had higher recurrence rates(median DFS: 21 months) than the low-TINCR patients(median DFS: 29.7 months, P = 0.035). The 3-year DFS was 31.6% for high TINCR expression patients, while the low TINCR expressing patients had a 3-year DFS of 57.6%. The results of univariate analyses of the clinical variables revealed that TINCR expression was a significant prognostic indicator of DFS(hazard ratio [HR] = 2.242; 95% confidence interval [CI], 1.010-4.976; P = 0.047) in patients with GC. Further analysis in a multivariate Cox proportional hazards model showed that the TNM stage was strongly associated with DFS. Conclusions: TINCR expression was significantly higher in the tumor tissues and gastric cancer cell lines than in the adjacent normal tissues and normal gastric epithelium cell line GES1, and TINCR may serve as a promising diagnostic and prognostic biomarker. Part 2: Detection of the upstream regulatory factors of TINCR Objective: To explore the molecular mechanism underlying the deregulated expression of TINCR in GC tissues and cell lines. Method: Bioinformatics methods were performed to predict the putative transcription factor-binding sites in TINCR promoter region. Chromatin immunoprecipitation(ChIP) assays, luciferase reporter gene assay, transfection of RNA interference, and quantitative RT-PCR, were performed to verify whether Special protein 1(SP1) regulates TINCR expression, and to detect the specific binding sites of SP1 with TINCR promoter region. Results: We performed a computational screen and found that three tandem putative SP1-binding sites at the regions-163 to-153 bp(E1),-88 to-78 bp(E2), and-16 to-6 bp(E3) in the TINCR promoter. We used ChIP assays to determine which region in the TINCR promoter mediated SP1 binding to the endogenous TINCR promoter. An obvious SP1-binding activity on the endogenous TINCR promoter was observed at the region around-0.2 kb(E1). Consistent with this finding, the-201 to +163 bp region of the TINCR promoter-driven firefly luciferase expression vector induced TINCR transcriptional activity via SP1 overexpression, as indicated by a luciferase reporter assay. To validate this finding, we deleted the E1 binding site, and repeated the reporter assay. The results showed that the deletion of the SP1-binding motif E1 significantly impaired the effect of SP1 on TINCR transcription activation, suggesting that SP1 binds to their special binding motifs to regulate TINCR transcription. We next determined whether the overexpression of TINCR is mediated by SP1. We knocked down endogenous SP1 expression in GC cells by transfection with small-interfering RNAs(siRNAs) targeting the SP1 gene. TINCR levels were signi?cantly reduced in cells transfected with siRNAs. Conclusions: These results indicate the possibility that TINCR upregulation in GC is mediated by the transcription factor SP1. Part 3: Effect of TINCR on GC Objective: To detect the role of TINCR on the proliferation, apoptosis and cell cycle of gastric cancer cells in vitro, and investigate the function of TINCR on SGC7901 xenograft tumors in vivo Methods: We used chemically synthesized siRNAs to knock down endogenous TINCR in GC cell lines, and induced ectopic overexpression of TINCR by transfecting the GC cell lines with the pcDNA3.1-TINCR expression vector. GC cells that were transfected Scrambled or Empty vector were server as controls. The Proliferation of SGC7901 and BGC823 cells were determined by MTT and colony formation assay. After transfected with siRNA-TINCR, pcDNA3.1-TINCR, Scrambled, and Empty vector, cell apoptosis, cell cycle were analyzed by flow cytometry after staining with annexin V/PI, PI/RNase. Western blot assay was performed to detect Bcl-2, Bax, and Caspase3. SGC7901 cells were injected subcutaneously into BALB/C, nu/nu mice to establish SGC7901 cells cancer xenograft models. SGC7901 cells which were infected by recombinant adenoviral vector producing shRNA, were inoculated into flanks of mice, where those infected with adenoviral vector carrying sh-Scrambled as control were inoculated into the opposite flank of the same mouse. Tumor volumes were examined once four days when the implantations were starting to grow bigger. After 28 days of treatment, mice from each group were scarified and the weight of tumor mass was measured. Results: MTT assay revealed that cells transiently transfected with siRNA had significantly inhibited growth and proliferation of GC cells. The overexpression of TINCR significantly increased the growth of both cell lines. The resulting data showed that shRNA1 and shRNA2 significantly inhibited the colony formation of GC cell lines compared to the control Scrambled RNA, while TINCR overexpression dramatically promoted the cells proliferation. The results revealed that GC cells transfected with siRNA1 or siRNA2 had an obvious cell-cycle arrest in the G0-G1 phase, and the population of cell in the S-phase was decreased. In contrast, TINCR overexpression promoted cell-cycle progression. The fraction of apoptotic cells was significantly increased among the siRNA1- and siRNA2-treated cells as compared with the Scrambled RNA-treated cells. Furthermore, western blot analysis indicated that BCL-2 expression and cleaved caspase-3 levels were altered in the siRNA treated GC cells. Ad-shRNA significantly inhibited the tumorigenicity in vivo, as tumor weight and size were obviously decreased compared with the controls. Moreover, we detected stronger Ki-67 expression in tumors derived from Scrambled RNA than those derived from TINCR-shRNA Conclusions: These results indicated that TINCR may drive GC development. Part 4: Detection and validation of the downstream target genes of TINCR Objective: To probe and verify the TINCR-associated pathway and the downstream target genes. Methods: We performed RNA transcriptome sequencing from control or TINCR-depleted SGC7901 cells using two independent siRNAs(siRNA1 & siRNA2) against TINCR. SGC7901 cells were treated with a control(Scrambled) or TINCR-specific siRNAs for 48 h. Bioinformatic analysis was conducted for the altered gene data, including cluster analysis, Gene Ontology(GO) analysis, and pathway analysis dependent on the Kyoto Encyclopedia of Genes and Genomes(KEGG). The qRT-PCR assay was performed to further confirm the changed genes.Western blot assay was conducted to detect CDKN1A/P21, CDKN2B/P15, KLF2 protein expression. Immunohistochemical(IHC) analysis was used to quantify protein expression of CDKN1A/P21,CDKN2B/P15,KLF2 in xenograft tumor tissues.Results: Analyses of the RNA transcriptome sequencing data from triplicate samples revealed that a common set of 326 mRNAs showed ≥1.5-fold increased abundance and 304 reduced the ≤1.5-fold abundance TINCR-depleted cells. To further study the involved pathways activated by TINCR, we analyzed these genes using data collected from the Kyoto Encyclopedia of Genes and Genomes(KEGG) and Gene Ontology(GO) databases. Cell cycle and apoptosis were both involved in the affected biological process in TINCR-depleted cells. Using qRT-PCR, we validated the changes in the cellular levels of a large number of mRNAs involved in cell-cycle progression and apoptosis. Based on our RNA sequencing data, KLF2 mRNA was the most upregulated transcript among the transcripts that were commonly regulated upon TINCR downregulation. CDKN1A/P21 and CDKN2B/P15 were also upregulated after TINCR depletion. Knockdown of TINCR by specific siRNAs substantially increased their expression, whereas ectopic expression of TINCR reduced their mRNA levels. Consistently, western blot analysis revealed that knockdown/overexpression of TINCR increased /decreased respectively their protein level in GC cells. Knockdown of TINCR dramatically increased the levels of KLF2, CDKN1A/P21, and CDKN2B/P15 in the xenograft tumors. Conclusion: Cell cycle and apoptosis were both involved in the affected biological process in TINCR-depleted cells. TINCR may contribute to the malignant phenotype by inhibiting KLF2, CDKN1A/P21, and CDKN2B/P15.Part 5: Mechanisms of TINCR regulating GC proliferation and apoptosis Method: Isolation of cytoplasmic and nuclear RNA was used to detect the location of TINCR in GC cells. The qRT-PCR assay was performed to detect the KLF2 mRNA level upon STAU1 and UPF1 depletion. RNA immunoprecipitation assay, RNA pulldown assay was used to verify whether STAU1 protein interate with TINCR and KLF2 mRNA. pcDNA3.1-STAU1-FLAG expression vector, pLUC-KLF2 3’-UTR vector, Rluc-ARF1 SBS vector, and phCMV-MUP vector were contructed. SGC7901 cells were transfected with the above test plasmids, the two latter of these served as a positive and a negative control, respectively, for STAU1-FLAG binding. Immunoprecipitation assay was conducted to detect whether STAU1 protein binds to KLF2 3’-UTR. KLF2 mRNA half-life was detected upon TINCR deleption or overexpression. Results: TINCR was mainly present in the cytoplasm of GC cells. The mRNA level of KLF2 increased upon STAU1-depleted and UPF1-depleted GC cells. The RIP assays with STAU1 antibody, showed a significant enrichment of KLF2 and TINCR by STAU1 antibody compared with IgG control, indicating STAU1 is associated with KLF2 and TINCR mRNA. The RNA pull-down assay revealed that TINCR interacted with KLF2 mRNA, STAU1 protein, and UPF1 protein. Compared to control siRNA, siRNA-TINCR reduced by ~1.5-fold the co-IP of STAU1 with KLF2 mRNA and the depletion of STAU1 significantly reduced the interaction of TINCR with KLF2 mRNA, corroborating that STAU1 is required for the association between TINCR and KLF2 mRNA. The KLF2 mRNA half-life was signi?cantly increased upon downregulation of STAU1 or TINCR, while it was decreased after TINCR overexpression. Conclusion: TINCR recruits STAU1 to the 3’UTR of KLF2 mRNA, degrading KLF2 through the UPF1-dependent mRNA decay mechanism. Part 6: Effect of KLF2 on GC and exploration of the molecular mechanism Method: We used chemically synthesized siRNAs to knock down endogenous KLF2 in GC cell lines, and induced ectopic overexpression of TINCR by transfecting the GC cell lines with the FLAG-KLF2 expression vector. GC cells that were transfected Scrambled or Empty vector were server as controls. The Proliferation of SGC7901 and BGC823 cells were determined by MTT and colony formation assay. After transfected with FLAG-KLF2 expression vector and Empty vector, cell apoptosis were analyzed by flow cytometry after staining with annexin V/PI. Western blot assay was performed to detect CDKN1A/P21 and CDKN2B/P15. SGC7901 cells which were stable infected by FLAG-KLF2 expression vector were inoculated into flanks of BALB/C, nu/nu mice, where those infected with Empty vector as control were inoculated into the opposite flank of the same mouse. Tumor volumes were examined once two days when the implantations were starting to grow bigger. After 16 days of treatment, mice from each group were scarified and the weight of tumor mass was measured. ChIP assay was conducted to detect whether KLF2 regulates the transcription of CDKN1A/P21 and CDKN2B/P15. qRT-PCR and Immunohistochemistry were used to detect the expression of KLF2 in gastric cancer tissues and cell lines. SPSS 20.0 was used to analyze the correlation between TINCR and KLF2, and to detect the clinical significance of KLF2. A rescue experiment was carried out to investigate whether KLF2 was involved in the TINCR-depleted induced cell proliferation inhibition and apoptosis with SGC7901 cells. Results: MTT and colony formation assay revealed that the overexpression of KLF2 significantly inhibited the growth of both cell lines, while transiently transfected with siRNA had significantly promoted proliferation of GC cells. The fraction of apoptotic cells was significantly increased among the FLAG-KLF2 expression vector-treated cells as compared with the Empty vector-treated cells. Ectopic overexpression of KLF2 significantly inhibited the tumorigenicity in vivo, as tumor weight and size were obviously decreased compared with the controls. Western blot and qRT-PCR analysis indicated that CDKN1A/P21 and CDKN2B/P15 expression were upregulation or downregulation upon KLF2 overexpression or depletion. KLF2 regulated transcription of CDKN1A/P21 and CDKN2B/P15 through binding the promoter regions. KLF2 expression is downregulated in cancer tissues and cell lines and is inversely correlated with TINCR expression, and low KLF2 expression was associated with GC progression. MTT assay and Flow cytometric analysis indicated that the cotransfection could partially rescue the TINCR silencing-mediated suppression of SGC7901 cell proliferation and cell apoptosis induction. Moreover, we found that cotransfection of siTINCR and siKLF2 could rescue the increased expression of CDKN2B/P15 and CDKN1A/P21 proteins induced by TINCR depletion Conclusions: KLF2 inhibited proliferation and induced apoptosis through regulated the transcription of CDKN2B/P15 and CDKN1A/P21, and KLF2 was involved in the TINCR-depleted induced cell proliferation inhibition and apoptosis increase in GC In Conclusion: TINCR was upregulated in tissues and cell lines, and was regulated by SP1. TINCR is a diagnostic marker and high TINCR expression is associated with poor prognosis in GC patients. TINCR may drive GC development through influencing KLF2, CDKN1A/P21, and CDKN2B/P15 expression. The pathway via which TINCR regulates cell cycle and cells apoptosis has been depicted below. The nuclear transcription factor SP1 induces TINCR overexpression. TINCR recruits STAU1 to the 3’UTR of KLF2 mRNA, degrading KLF2 through the UPF1-dependent mRNA decay mechanism. Subsequently, the reduced KLF2 abundance contributes to decreased CDKN2B/P15 and CDKN1A/P21 transcripts, promoting cell-cycle progression and tumorigenicity. |
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