Yongfu Shao

Using circular RNA as a novel type of biomarker in the screening of gastric cancer.

BACKGROUND Circular RNAs (circRNAs), a class of endogenous RNAs, have emerged as an enigmatic class of RNAs. Little is known about their value in the diagnosis of cancers. METHODS The targeted circRNA of this study was selected using two circRNA databases: CircBase (http://circbase.org/) and circ2Traits (http://gyanxet-beta.com/circdb/). Divergent primers, rather than commonly used convergent primers, for the circRNA were designed. The circRNA levels in 101 paired gastric cancer tissues and adjacent nontumorous tissues from surgical gastric cancer patients and 36 paired plasma samples from preoperative and postoperative gastric cancer patients were analyzed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The specificity of the amplified products was measured by melting curve analysis and DNA sequencing. To observe the stability of circRNA, three randomly selected samples of gastric cancer tissues were stored at room temperature, 4°C and -20°C, and then, their circRNA levels were analyzed. To verify the reproducibility of qRT-PCR, circRNA levels were detected in a set of specimens (n=15) in two independent experiments with an interval of one day. Then, the correlation of their Ct values was determined. The relationships between circRNA expression levels and clinicopathological factors of patients with gastric cancer were further analyzed by one-way analysis of variance. A receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value. RESULTS Hsa_circ_002059, a typical circular RNA, was first found to be significantly downregulated in gastric cancer tissues compared with paired adjacent nontumorous tissues (p<0.001). Its levels in plasma collected from postoperative gastric cancer patients were found significantly different from those from preoperative gastric cancer patients. The area under the ROC curve was 0.73. Importantly, we further found that lower expression levels were significantly correlated with distal metastasis (P=0.036), TNM stage (P=0.042), gender (P=0.002) and age (P=0.022). The stability of circRNAs and the reproducibility of the qRT-PCR method for detecting circRNA levels were determined. CONCLUSION These results suggested that circRNAs are highly stable in mammalian cells and that one specific circRNA, hsa_circ_002059, may be a potential novel and stable biomarker for the diagnosis of gastric carcinoma.

Long noncoding RNA associated-competing endogenous RNAs in gastric cancer

Some long noncoding RNAs (lncRNAs) play important roles in the regulation of gene expression by acting as competing endogenous RNAs (ceRNAs). However, the roles of lncRNA associated-ceRNAs in oncogenesis are not fully understood. Here, based on lncRNA microarray data of gastric cancer, bioinformatic algorithm miRcode and microRNA (miRNA) targets database TarBase, we first constructed an lncRNA-miRNA-mRNA network. Then, we confirmed it by data of six types of other cancer including head and neck squamous cell carcinoma, prostate cancer, papillary thyroid carcinoma, pituitary gonadotrope tumors, ovarian cancer, and chronic lymphocytic leukemia. The results showed a clear cancer-associated ceRNA network. Eight lncRNAs (AC009499.1, GACAT1, GACAT3, H19, LINC00152, AP000288.2, FER1L4, and RP4-620F22.3) and nine miRNAs (miR-18a-5p, miR-18b-5p, miR-19a-3p, miR-20b-5p, miR-106a-5p, miR-106b-5p, miR-31-5p, miR-139-5p, and miR-195-5p) were involved. For instance, through its miRNA response elements (MREs) to compete for miR-106a-5p, lncRNA-FER1L4 regulates the expression of PTEN, RB1, RUNX1, VEGFA, CDKN1A, E2F1, HIPK3, IL-10, and PAK7. Furthermore, cellular experimental results indicated that FER1L4-small interfering RNA (siRNA) simultaneously suppressed FER1L4 and RB1 mRNA level. These results suggest that lncRNAs harbor MREs and play important roles in post-transcriptional regulation in cancer.

Gastric juice long noncoding RNA used as a tumor marker for screening gastric cancer

Long noncoding RNAs (lncRNAs) play a crucial role in tumorigenesis. However, the value of lncRNAs in the diagnosis of gastric cancer remains unknown. To identify whether lncRNA‐AA174084 is a potential marker for the early diagnosis of gastric cancer (GC), the authors investigated its levels in tissues, blood, and gastric juices from patients with various stage of gastric tumorigenesis.

Long noncoding RNA FER1L4 suppresses cancer cell growth by acting as a competing endogenous RNA and regulating PTEN expression.

Aberrantly expressed long noncoding RNAs (lncRNAs) are associated with various cancers. However, the roles of lncRNAs in the pathogenesis of most cancers are unclear. Here, we report that the lncRNA FER1L4 (fer-1-like family member 4, pseudogene) acts as a competing endogenous RNA (ceRNA) to regulate the expression of PTEN (a well-known tumor suppressor gene) by taking up miR-106a-5p in gastric cancer. We observed that FER1L4 was downregulated in gastric cancer and that its level corresponded with that of PTEN mRNA. Both FER1L4 and PTEN mRNA were targets of miR-106a-5p. Further experiments demonstrated that FER1L4 downregulation liberates miR-106a-5p and decreases the abundances of PTEN mRNA and protein. More importantly, FER1L4 downregulation accelerated cell proliferation by promoting the G0/G1 to S phase transition. We conclude that one mechanism by which lncRNAs function in in tumorigenesis is as ceRNAs for tumor suppressor mRNAs.

LncRNA-RMRP promotes carcinogenesis by acting as a miR-206 sponge and is used as a novel biomarker for gastric cancer

Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis. However, the mechanisms of most lncRNAs in cancers are largely unknown. Because the RNA component of mitochondrial RNA processing endoribonuclease (RMRP) is one of the dysregulated lncRNAs in gastric cancer, this study explored its molecular mechanisms in carcinogenesis. RMRP levels in 792 tissues, plasma and gastric juices from patients with various stages of gastric tumorigenesis were analyzed by quantitative reverse transcription-polymerase chain reaction. Overexpression and RNA interference were used to manipulate RMRP expression by RMRP expression vector and small interfering RNAs, respectively. Its mechanisms were evaluated by flow cytometry, real-time cell analysis, plate colony formation assays, and xenograft models. RMRP levels in tissue, plasma and gastric juices from patients with gastric cancer were significantly different from those from controls. Its levels were significantly associated with Borrmann type and metastasis. Plasma and gastric juice RMRP had higher sensitivity and specificity than commonly used markers (such as carcinoembryonic antigen and carbohydrate antigen 19–9). Knockdown of RMRP significantly inhibited cell proliferation in vitro and in vivo, whereas overexpression of RMRP promoted cell growth. Acting as a miR-206 sponge, RMRP modulated cell cycle by regulating Cyclin D2 expression. RMRP plays a crucial role in gastric cancer occurrence and can be used as a novel biomarker for gastric cancer.

lncRNA-AC130710 targeting by miR-129-5p is upregulated in gastric cancer and associates with poor prognosis

Long noncoding RNAs (lncRNAs) play an important role in cancer occurrence and development. However, there is largely unknown about lncRNAs significance in the diagnosis and treatment of gastric cancer. In our study, we focused on AC130710, one of lncRNAs. Gastric cancer tissues and adjacent tissues were gathered from 78 patients with gastric cancer. The AC130710 levels were detected by reverse transcription polymerase chain reaction (RT-PCR). Then, we further analyzed the association between AC130710 level and the clinicopathological factors of patients with gastric cancer. Finally, the molecular mechanism underling AC130710 highly expressed in gastric cancer cells was explored. The results showed that AC130710 in cancer tissues from patients with gastric cancer was significantly higher than those in adjacent noncancerous tissues (P < 0.05). Its expression level was significantly associated with tumor size (P = 0.013), tumor-node-metastasis (TNM) stages (P = 0.030), and distal metastasis (P = 0.018). AC130710 expression in MGC-803 was significantly higher than that in normal gastric mucosa cell line GES-1 (P < 0.001). Moreover, miR-129-5p may play an important role in the downregulation of AC130710 in gastric cancer cells. These results indicated that lncRNA-AC130710 may be a potential tumor marker for gastric cancer prognosis.

Non-coding RNAs and gastric cancer

Non-coding RNAs (ncRNAs) play key roles in development, proliferation, differentiation and apoptosis. Altered ncRNA expression is associated with gastric cancer occurrence, invasion, and metastasis. Moreover, aberrant expression of microRNAs (miRNAs) is significantly related to gastric cancer tumor stage, size, differentiation and metastasis. MiRNAs interrupt cellular signaling pathways, inhibit the activity of tumor suppressor genes, and affect the cell cycle in gastric cancer cells. Some miRNAs, including miR-21, miR-106a and miR-421, could be potential markers for the diagnosis of gastric cancer. Long non-coding RNAs (lncRNAs), a new research hotspot among cancer-associated ncRNAs, play important roles in epigenetic, transcriptional and post-transcriptional regulation. Several gastric cancer-associated lncRNAs, such as CCAT1, GACAT1, H19, and SUMO1P3, have been explored. In addition, Piwi-interacting RNAs, another type of small ncRNA that is recognized by gastroenterologists, are involved in gastric carcinogenesis, and piR-651/823 represents an efficient diagnostic biomarker of gastric cancer that can be detected in the blood and gastric juice. Small interfering RNAs also function in post-transcriptional regulation in gastric cancer and might be useful in gastric cancer treatment.

Global circular RNA expression profile of human gastric cancer and its clinical significance

Circular RNAs (circRNAs) are a new class of noncoding RNAs. However, the expression profile and clinical significance of circRNAs in human gastric cancer is unclear. The global circRNA expression profile in human gastric cancer was measured by circRNA microarray. Hsa_circ_0014717, one of the most downregulated circRNAs in microarray, was selected as a targeted circRNA to explore its levels in gastric tissues and gastric juice. Freeze‐thaw experiment and incubation experiment confirmed the stability of gastric juice circRNAs. A total of 308 circRNAs, including 107 (34.74%) upregulated and 201 (65.26%) downregulated circRNAs, were found significantly aberrantly expressed in gastric cancer tissues. The top ten upregulated in gastric cancer tissues were hsa_circ_0035445, hsa_circ_0003789, hsa_circ_0063809, hsa_circ_0074362, hsa_circ_0006282, hsa_circ_0011107, hsa_circ_0084606, hsa_circ_0005556, hsa_circ_0050547, and hsa_circ_0006470, while the top ten downregulated ones were hsa_circ_0007099, hsa_circ_0001897, hsa_circ_0007707, hsa_circ_0008832, hsa_circ_0001546, hsa_circ_0002089, hsa_circ_0004680, hsa_circ_0000154, hsa_circ_0004458, and hsa_circ_0008394. The hot‐point chromosomes were chr1, chr2, chr3, chr9, and chr17. Hsa_circ_0014717 was significantly downregulated in 77.2% (74/96) gastric cancer tissues. Its levels in gastric cancer tissues were related to tumor stage (P = 0.037), distal metastasis (P = 0.048), tissue carcinoembryonic antigen (P = 0.001), and carbohydrate antigen 19‐9 expression (P = 0.021). More importantly, hsa_circ_0014717 can stably exist in human gastric juice; and its nature meets the requirements of clinical detection. Our study uncovered the circRNA expression profile in human gastric cancer. Moreover, some circRNAs can stably exist in human body fluid, and has the potential to be used as novel biomarkers for the screening of high‐risk gastric cancer patients.

Decreased expression of hsa_circ_0001895 in human gastric cancer and its clinical significances

Circular RNAs are a special class of endogenous RNAs characterized by jointing 3′ and 5′ ends together via exon or intron circularization. Recent studies found that circular RNAs are involved in the development of some human diseases. However, little is known about their roles in human gastric cancer. In this study, we chose hsa_circ_0001895 as a targeted circRNA to investigate its clinical significances in gastric cancer patients. Hsa_circ_0001895 expression levels in five gastric cancer cell lines and 257 specimens of tissues were measured by real-time quantitative reverse transcription polymerase chain reaction. Then, the potential relationship between hsa_circ_0001895 expression levels and patients’ clinicopathological factors was investigated. A receiver operating characteristic curve was constructed for evaluating the diagnostic value of hsa_circ_0001895. Hsa_circ_0001895 expression levels in five detected gastric cancer cell lines (AGS, BGC-823, HGC-27, MGC-803, and SGC-7901) were all significantly downregulated than those in normal gastric epithelial GES-1 cells. Besides, compared with healthy control tissues, it was downregulated not only in 69.8% (67/96) gastric cancer tissues but also in gastric precancerous lesions. Moreover, hsa_circ_0001895 expression levels were significantly correlated with cell differentiation, Borrmann type, and tissue carcino-embryonic antigen expression. Our results suggested that hsa_circ_0001895 may play crucial roles during gastric cancerogenesis and is a potential biomarker for clinical prognosis prediction.

Plasma circular RNA profiling of patients with gastric cancer and their droplet digital RT-PCR detection

To observe the diagnostic values of circular RNAs (circRNAs), their expression profiles between gastric cancer patients’ plasma and healthy controls were first assessed by circRNA microarray. Then, circRNA levels were measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR), respectively. A total of 343 differentially expressed circRNAs were found. The top 10 elevated circRNAs in patients were hsa_circ_0088300, hsa_circ_0075825, hsa_circ_0019172, hsa_circ_0000220, hsa_circ_0035277, hsa_circ_0000301, hsa_circ_0000189, hsa_circ_0090080, hsa_circ_0001888, and hsa_circ_0001874. The top 10 reduced circRNAs were hsa_circ_0004771, hsa_circ_0001190, hsa_circ_0061276, hsa_circ_0092337, hsa_circ_0058495, hsa_circ_0061274, hsa_circ_0075829, hsa_circ_0080845, hsa_circ_0001006, and hsa_circ_0003764. In cancer and dysplasia tissues, hsa_circ_0001017 and hsa_circ_0061276 were downregulated. Their levels were significantly associated with distal metastasis. The area under receiver operating characteristic curve in combinative use was 0.966 with 95.5% sensitivity and 95.7% specificity. Patients with low plasma hsa_circ_0001017 or hsa_circ_0061276 had a much shorter overall survival than those with high levels. Patients whose plasma hsa_circ_0001017 or hsa_circ_0061276 levels recovered to normal after operation had a longer disease-free survival. Finally, the in vitro model indicated gastric cancer cells secreting circRNAs into plasma. In conclusion, RT-ddPCR is a potent non-invasive and absolute quantification method for simultaneous detection of multiple circRNAs. Hsa_circ_0001017 and hsa_circ_0061276 are new potential biomarkers for gastric cancer.Key messageA total of 343 circRNAs are differentially expressed between gastric cancer patients’ plasma and healthy controls.Hsa_circ_0001017 and hsa_circ_0061276 are downregulated in gastric cancer tissues.The RT-ddPCR is a potent method for simultaneous detection of multiple circRNAs in plasma.Hsa_circ_0001017 and hsa_circ_0061276 are potential biomarkers for gastric cancer.