| ObjectiveGastric cancer(GC) remains the fourth most prevalent types of malignant tumor and the second leading cause of global cancer-related deaths with a 5-year survival rate less than 25 %. Due to absence of desirable biomarkers for early detection, more than 80 % of GC diagnoses occur at an advanced stage of this disease. In these cases, prognosis for the majority of GC patients are hugely limited. Moreover, the unclear pathophysiologic mechanisms of GC lead to limited clinical treatment options in the same time. Therefore, reliable identification molecular characterization of GC and better understanding of the pathogenesis are the major focus of current investigation.A class of long non-coding RNA(lnc RNA), known as nucleotides more than 200 in length, have been shown to involve in multiple biological functions ranging from physiological processes to pathological processes. They are widely involved in the regulation of gene expression network in the forms of epigenetic, transcriptional and post-transcriptional levels. Besides, they are associated with the tumor invasion, metastasis and prognosis. However, the relationships between lnc RNAs and GC are still little known. So, it is important to obtain more information about the relationship between dysregulation lnc RNAs and GC. Therefore, the aim of the present study are to explore the molecular mechanisms of lnc RNAs in gastric carcinogenesis and to investigate the clinical diagnostic value when they be biomarkers for screening and predicting prognosis of GC.Methods1. Dysregulated lnc RNAs between GC tissues and the paired nontumorous tissues were screened by lnc RNA microarray. On the basis of the lnc RNA microarray results of RMRP, quantitative reverse transcription-polymerase chain reaction(q RT-PCR) was used to detect its real expression levels among GC tissues, the paired nontumorous tissues, normal gastric mucosa epithelial cell line(GES-1) and five gastric cancer cell lines(AGS, BGC-823, HGC-27, MGC-803 and SGC-7901).2. q RT-PCR was used to analyze the expression pattern of RMRP among the healthy gastric mucosa(HGM) samples, gastric ulcer(GU) tissues, erosive gastritis(EG) tissues, gastric dysplasia(GD) tissues and GC tissues.3. Cloning and sequencing were used to confirm the existence of RMRP and GAPDH m RNA in human plasma and gastric juice. Number of freeze-thaw and incubation experiments under the condition of different time and temperature confirmed the stability of body fluids RMRP and cell supernatant experiments researched on the source of RMRP in body fluid.4. Detection methods of plasma and gastric juice lnc RNAs were established, and the variation of plasma and gastric juice RMRP levels among various stages of gastric carcinogenesis were analysed. The potential relationship between clinicopathologic factors and RMRP levels in GC tissue, plasma and gastric juice of GC patients were explored. Receiver operating characteristic(ROC) curves were constructed for evaluating the diagnosis values of plasma and gastric juice RMRP.5. mi Rcode algorithm was used to predict the mi RNAs that interacted with RMRP.6. Small interfering RNA(si RNA) targeting RMRP and pc DNA3.1 expression vector of RMRP were designed and synthesized, then transfected into normal gastric mucosa epithelial cell line and gastric cancer cell lines. The expression levels of RMRP and Cyclin D2 were quantified by q RT-PCR. After overexpression or knockdown of RMRP in human gastric cell lines, cell cycle distribution, apoptosis and cell proliferation were analysed by flow cytometer, real-time cell analyzer(RTCA) and plate colony formation assay, respectively. 7. Gastric cancer xenografts in nude mice model was established to confirm the biological function of RMRP in vivo. The Variation of plasma RMRP level in nude mice was analyzed.Results1. RMRP expression levels were down-regulated in 68.2 %(90 / 132) GC tissues compared with the paired adjacent nontumorous tissues. Similar to the situation in tissues, RMRP expression levels were significantly dysregulated in five gastric cancer cell lines(AGS, BGC-823, HGC-27, MGC-803 and SGC-7901) compared with normal gastric mucosa epithelial cell line(GES-1).2. q RT-PCR results showed that RMRP expression was only significantly decreased in GD and GC tissues, but no difference in GU tissues and EG tissues. This phenomenon implies that RMRP has a very strong correlation with GC.3. Cloning and sequencing have confirmed the existence of RMRP and GAPDH m RNA in human plasma and gastric juice. GAPDH m RNA levels are stable in human plasma and gastric juice, and can be used as an ideal reference for q RT-PCR of body fluids samples.4. The results of number of freeze-thaw and incubation experiments under the condition of different time and temperature confirmed the stability of body fluids RMRP, which can meet the need of clinical routine detection. Cell supernatant experiments showed the source of RMRP in body fluid was from gastric cells.5. Compared with the healthy group, plasma RMRP levels significantly increased in the group of preoperative GC patients, but sharply declined after subtotal gastrectomy. Gastric juice RMRP levels significantly increased in GC group. Compared with serum tumor markers, plasma and gastric juice RMRP as biomarker for GC screening have a higher sensitivity and specificity. Combined use of gastric juice RMRP, CEA, and serum CEA can improve the detection rate of GC.6. RMRP levels in GC tissues were associated with several clinicopathologic factors such as Borrmann type(P=0.002), tumor invasion(P=0.037), lymphatic metastasis(P=0.014), perineural invasion(P=0.008) and tissue CEA and CA19-9 expression(P<0.001, P=0.003). Preoperative plasma RMRP levels were negatively correlated with tumor diameter(P=0.031), tumor stage(P=0.038), tumor invasion(P=0.017) and tissue CEA expression(P=0.032), whereas the individual relative changes of plasma RMRP levels 2 weeks after surgery had a significant, negative association with lymphatic metastasis(P=0.040) and tissue CEA expression(P=0.049).7. Cell and animal experiments showed knockdown of RMRP significantly inhibit cell proliferation, whereas overexpression of RMRP promote cell proliferation and tumor growth.8. Knockdown of RMRP in human gastric cells represented significant G0/G1 arrest, whereas overexpression of RMRP promoted cell cycle from G0/G1 phase to S phase. Moreover, RMRP can act as mi R-206 sponge and exert cell cycle effect through regulating the expression level of downstream target gene Cyclin D2.ConclusionsThe in vivo and in vitro mechanism researches proved that lnc RNA-RMRP plays crucial role in the occurrence and progression of GC. The large clinical sample analysis showed RMRP is a potential biomarker for screening and predicting prognosis of GC. |
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