| Background and Objection:Gastric cancer (GC) is the second most common cause of cancer death across the world. GC is a multifactorial disease, which is closely associated with complex interactions between environmental and genetic factors.However, the detailed mechanism of the carcinogenesis remains to be further elucidated. The diagnosis of gastric cancer rely mainly on gastrduodenoscopy combined with histological biopsy. The pathological type is given priority to with adenocarcinoma. According to the infiltration depth is divided into early gastric cancer, advanced gastric cancer. The treatment of gastric cancer is based on surgery. The high mortality rate is mainly due to late presentation, since early stage of gastric cancer is either asymptomatic or presents with non-specific symptoms. In our country, only a small part of early gastric cancer can be detected in time.when they were diagnoised.,it usually turned to advanced ones. Today, surgery remains the mainstay of potentially curative treatment for gastric cancer. Nevertheless, over 50% of the patients may still develop recurrence after curative resection. The prognosis of early gastric cancer is better then advanced ones.5 years of survival rate in early gastric cancer after surgery can reach to as high as 90%, but in advanced gastric cancer, prognosis is poor. In order to improve curative effect, postoperative adjuvant treatment including radiotherapy and chemotherapy in essential. As the low sensitivity of radiotherapy, we focused more on chemotherapy. after surgery.Although in recent years, constantly upgrading of chemotherapeutic drugs and some change of treatment protocal such as preoperative neoadjuvant chemotherapy, intraoperative chemotherapy emerged, adjuvant therapy has not seen a fundamental improve, the 5-years of survival rate has not been improved significantly in advanced gastric cancers. Therefore, further explore the mechanism of gastric cancer development and explore a more effective treatment is more important. Normal gastric epithelial malignant molecular basis for the normal mucosal epithelial cell proliferation and apoptosis imbalance, and the balance is dependent on a large number of oncogene and tumor suppressor genes and cytokines, etc. Because the miRNA molecules in cancer gene, tumor-suppressor genes functions are gradually understanding, at present the miRNA is one of the hot topics in the study of tumor.miRNAs are a class of conserved endogenous small noncoding RNA, which usually contain 19-25 nucleotides. They can negatively regulate gene expression through degrading or inhibiting translation of target mRNAs, playing important regulatory functions in diverse biological processe. miRNA binding to the target genes can induce the degradation of target genes or inhibit translation.therefore, being involved in a wide range of physiological processes, such as the early embryonic development, cell differentiation, cell proliferation and apoptosis, cell metabolism, etc. Study have found that the miRNA closely associated with the occurrence of tumor development and prognosis. The miRNA can be proto-oncogenes, or can also be a tumor suppressor gene. Basically depend on their target genes in specific cells or tissues performing what kind of functions by which. Micrornas, therefore, through regulating target gene, oncogene and tumor suppressor genes expression, so as to regulate cell proliferation, differentiation and apoptosis..The mechanism of miRNA target genes form control network, the control ways to promote or inhibit the occurrence of tumor together. Abnormal expression of micrornas can be caused by many factors, such as methylation, a genetic defect, gene mutation, abnormal transcription and gene lost or abnormal amplification, etc. The miRNA target genes can be the original tumor onegene or tumor suppressor genes or an important process of cell cycle, differentiation or apoptosis gene, therefore, the abnormal expression of micrornas forward or reverse gene regulation, to directly control the proliferation, differentiation and apoptosis of tumor cells, and participate in tumor proliferation,invasion or metastasis.Previous studies have found that a series of miRNAs is dysregulated in GC and involved in carcinogenesis and cancer progression either as oncogenes or tumor suppressors. Abnormal expression of micrornas molecules through its target genes, to participate in the process of the occurrence of gastric cancer development. Over 92% of the gastric cancer patients demonstrated an upregulation of miR-21 in solid human tumors, including gastric cancerCurrently, it has been reported that miR-21 was upregulated not only in cancerous tissues but also in Helicobacter pylori (H. pylori)-infected gastric mucosa. Gastric cancer is a result of multistep and long-term interactions between genetic and environmental factors which process from chronic gastritis, atrophic gastritis, intestinal metaplasia, glandular atrophy and dysplasia. The miRNA that was associated with H. pylori-induced inflammation was miR-218, overexpression of this miR abrogated nuclear factor-kappa B (NF-κB) activation. It was hypothesized that miR-21 might augment the progression of infected normal mucosa to chronic gastritis with unknown mechanisms. The signal transducer and activator of transcription 3 (STAT3) activated the induction of miR-21. Activation of NF-κB and interleukin (IL)-6 stimulated STAT3 signaling, which may explain the H. pylori-mediated upregulation of miR-21.Numerous evidence revealed that miR-21 attributed to gastric cancer through enhanced cell proliferation and inhibited apoptosis. On top of that, miR-21 also has the ability to incite cell invasion and migration. It has been reported that RECK, a tumor suppressor gene, is the target of miR-21. It involves in the process of metastasis and angiogenesis through regulating metalloproteases (MMPs). MiR-125b, miR-199a and miR-100 have been shown to be the most important progression-related miRNAs in gastric cancer and pancreatic adenocarcinoma, implicating that miRNA may have different functions depends upon the tumor site. There are some miRNAs (miR-32, miR-182 and miR-143) that are found to be associated with intestinal-type gastric cancer, this study implicated the usefulness of miRNA expression profiles may serve as diagnostic biomarkers for different subtypes of gastric cancers. Expressions of miR-143, miR-145, miR-9, miR-443, miR-31, and miR-34 have been reported to downregulate in gastric cancer. The roles of miR-143 and miR-145 on cell proliferation have also been demonstrated in other gastrointestinal cancers,In addition, miR-214 was reported to modulate hedgehog signaling, where activation of hedgehog contributes to gastric cancer. High expression of miR-214 was identified to correlate with unfavorable outcome in gastric cancer. Furthermore, miR-196b expression has been shown to be significantly higher in gastric cancer tissues than normal counterparts.Although miRNAs has always been a research hot sopt since its discovery, for the numerous types of microRNA molecules, it is still likely to exist some kind of miRNA which has not been studied or whose regulating mechanism.have not been thoroughly elucadated. Through high-throughput micrornas chip technology, the abnormal expression of micrornas molecules in gastric cancer tissue, and study its biological function and regulatory mechanism. Our purpose is to find the clinical meaningful miRNAs, which can significantly affect the biological effects of gastric cancer,or being valuable for the diagnosis of gastric cancer,.or biological target therapy and evaluating prognosis.In gastric cancer tissues, miR-34 a, miR-335 lower expression, higher expression of survivin, whether there is a connection between the two. Literature, miR-34 a by regulating the PI3K/AKT signal pathway, can inhibit the expression of survivin gene. Another study found that miR-can act directly on the transcription factors E2F2 34 a, to regulate the expression of survivin gene. So if miR-335 with the same regulatory mechanism. According to the prediction of bioinformatics website, miR-335 and survivin mRNA 3’UTR possible binding sites. Therefore, miR-34 a, miR-335 May have a common target genes, survivin. So we choose the two micrornas molecules as the research target of micrornas.Survivin, which has the gene name of baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), is an inhibitor of apoptosis protein (IAP). Survivin mainly regulates apoptosis and cell cycle control and is upregulated in almost all human tumors.Because of survivin has high specificity, high expression in tumor tissue,survivin is generally recognized as necessary for malignacy Studies have confirmed that survivin is involved in tumor cell malignant transformation and antiapoptotic physiological process. Therefore, survivin is considered to be an important target of the anti-tumor treatment. In gastric carcinoma, the expression of survivin rise significantly, and its expression is an important index of prognosis and pathology classification. A number of studies have reported survivin gene on the proliferation, invasion and metastasis of gastric cancer, or tumor cell apoptosis freshman an important regulating role in angiogenesis, but its upstream regulatory pathways were not clear, studies have confirmed that survivin expression levels in gastric cancer is regulated by the miRNA, such as miR-34 a, etcmiR-335 and miR-34a are two miRNAs usually downregulated in GC. Both of them are considered as tumor suppressor in GC. miR-335 can suppress metastasis of GC cells by targeting Bcl-w and specificity protein 1 (SP1), while miR-34a can inhibit the growth, invasion, and metastasis of gastric cancer by targeting platelet-derived growth factor receptor (PDGFR) and hepatocyte growth factor receptor (MET). However, miRNAs usually have multiple targets and play a role in complex regulative networks. Exact regulative roles of the dysregulatedOverexpression of survivin was considered as an important diagnostic and prognostic marker for gastric cancer. However, how the dysregulation is related to aberrant expression of miRNAs is not quite clear. In the current study, we studied the association between miR-335/-34a expression and overall survival of GC patients and explored the regulative role of miR-335/-34a over survivin expression and GC cell growth and invasion.Methods and materials1.8 patients with primary gastric cancer were recruited from the People’s Hospital of Yibin, China, in 2010.1-2010.2, This study protocol was approved by the Ethics Review Board of the hospital. The patients had not received previous chemotherapy or radiotherapy. The gastric cancer tissues and adjacent normal tissues were obtained in gastrectomy. Informed consent was obtained from the patients before the surgical resection. Cancer staging was performed according to the International Union against Cancer’s (UIAC) tumor-node-metastasis (TNM) system by two pathologists without authorship to this study. Briefly, total miRNAs in the tumor and adjacent normal tissues and in the cell samples were extracted using the miRVana miRNA Isolation Kit (Ambion, USA) according to manufacturer’s instruction. Then, the samples were sent to Shanghai Biotechnology Corporation for array hybridization on an Agilent Human miRNA array (v.12.0). Fluorescein-labeled (Cy3 or Cy5) miRNAs were used for hybridization on Affymetrix miRNA Chip. Background subtraction and normalization were performed. The miRNAs with at least threefold difference between tumor and adjacent tissues were selected for further analysis (p<0.05).2. The putative binding sites between miR-335 and BIRC5 were predicted using Targetscan 6.2. Wild-type (WT) or mutant (MUT) sequence of BIRC5 3’UTR containing the predicted miR-335 binding site were chemically synthesized and inserted between the SacⅠ and SalⅠ sites in the downstream of the firefly luciferase in pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega). The insertion was verified by sequencing. The recombinant vectors are named as pmirGLO-BIRC5-WT and pmirGLO-BIRC5-MUT, respectively. HEK 293 T and SGC-7901 cells were co-transfected with 200 ng pmirGLO-BIRC5-WT or pmirGLO-BIRC5-MUT plasmids and 100 nM miR-335 mimics. Twenty-four hours after transfection, both firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega) in a Promega GloMax 20/20 luminometer. The firefly luciferase activity was normalized to the renilla luciferase activity.3. Further study miR-34 a, miR-335 for its target genes of survivin expression regulation function. In gastric cancer cell line SGC-7901 in the transient transfection miR-34 a, miR-335 mimics the way overexpression of miR-34 a, miR-335, through transfection miR-34 a, miR-335 antibody way cut miR-34 a, miR-335. Using the qRT-PCR technology, western blot experiments survivin gene was detected in changes in the expression of mRNA and protein levels.4. in gastric cancer cell line SGC-7301, respectively, using the CCK 8, flow cytometry, transwell experimental study miR-34 a, miR-335 in gastric cancer cell proliferation, apoptosis, invasion ability. Clear miR-biological effects after 34 a, miR-335, on the basis of the experiment, further explore its regulatory mechanism. Common transfection miR-34 a or miR-335 with pcDNA3.1-survivin (through genetic modification of no.3’UTR survivin), detect the biological effects of the change again. Due to this survivin expression 3’UTR, does not exist and the miRNA site. The test results from the reverse side confirmed miR-34 a/335 a role needs to combine with survivn gene specificity.5.50 patients with primary gastric cancer were recruited from the People’s Hospital of Yibin, China, in 2010.1-2011.1, using the qRT-PCR technique to detect expression level of miR-34a, miR-335, divided them into relatively high expression and the relatively low expression group according to the median value.in order to observe the difference between the two groups on clinical pathologic feature such as sex, age, histological type, clinical pathologic stage, lymph node metastasis are undifferentiated. After regular follow-up for 60 months, Kaplan Meier-survival curve were used to estimate overall survival of two groups..Statistical analysisAll statistical analysis was carried out using SPSS 18.0 software (IBM Corporation). Paired Wilcoxon test was performed to compare miR-335 and miR-34a expression between tumor samples and adjacent normal tissues.Patients were divided into high/low miR-335 and high/low miR-34a expression groups based on the median miRNA expression. Overall survival curves were estimated using the Kaplan-Meier method, and the difference between the curves was calculated using the log-rank (Mantel-Cox) test. The association between miR-335 or miR-34a expression and clinicopathologicalcharacteristics was assessed using Pearson’s χ2 test. Group comparison was performed using unpaired t test. A two-sided P value of <0.05 was regarded as statistically significant.Results:1.We used a miRNA microarray chip to measure miRNA expression in eight gastric tumor tissues and corresponding adjacent normal tissues. When analyzing the miRNAs with the highest level of changes, six upregulated and six downregulated miRNAs were significantly associated with GC. This data suggested that miRNAs are frequently dysregulated in GC, and we hypothesized that these miRNAs may play certain roles in regulating GC. Since the functions of some of the miRNAs were reported in previous studies and our recent work try to explore the role of BIRC5-related miRNAs in gastric cancer, we therefore focused on miR-335 and miR-34a2. Through prediction in online databases, we observed that miR-335 has a putative binding site in the 3’UTR of BIRC5 Therefore, we designed and reconstructed two dual luciferase reporters carrying the wide type and mutant binding sequence, respectively. In both HEK 293 T and SGC-7901 cells, miR-335 could only significantly weaken the relative luciferase activity of the reporter carrying wide-type sequences, but not the mutant construct.In fact, previous studies reported that miR-34a can directly target E2F3, a transcription factor promoting transcription of BIRC5 gene. Therefore, it is quite possible that miR-34a and miR-335 can modulate survivin expression at transcriptional and translational level, respectively.3. We firstly measured miR-335 and miR-34a expression in GES-1, AGS, BGC-823, MGC-803, and SGC-7901 cells.qRT-PCR analysis showed that MGC-803 and AGS cell line had the lowest and highest miR-335 expression, respectively, while SGC-7901 and AGS cells had the lowest and highest miR-34a expression, respectively. SGC-7901 cells were transfected with miR-335 or miR-34a mimics for overexpression, while AGS cells were transfected with anti-miR-335 or anti-miR-34a for knockdown In SGC-7901 cells, miR-335 or miR-34a overexpression substantially decreased survivin expression, while knockdown of miR-335 or miR-34asignificantly increased survivin expression in AGS cells at both mRNA and protein level. These results suggest that both miR-335 and miR-34a are negatively correlated to survivin expression.4. miR-335 and miR-34a overexpression could dramatically reduce cell viability of SGC-7901 cells. But,this effect was abrogated by expression of BIRC5 gene without 3’UTR. miR-335 and miR-34a overexpression could also significantly enhance cell apoptosis, and reduce cell invasion capability. But, these effects were all abrogated by expression of BIRC5 without 3’UTRm These results indicated thatmiR-335 andmiR-34a can simultaneouslymodulate growth and invasion of GC cells.5. We assessed the association of miR-335 and miR-34a expression with clinicopathological features of the GC patients recruited. The results showed that low miR-335 or low miR-34a expression were associated with higher clinical stage and lymph node metastasis Follow-up with patients showed that low miR-335 or miR-34a expression is associated with poor overall survival. Patients with combined low miR-335 and miR-34a expression had even poorer overall survival.Conclusion1. miRNA microarray chip shows that miRNAs are frequently dysregulated in GC.2. miR-335 directly targets 3’UTR of BIRC5 gene and regulates its translation.3. miR-335 and miR-34a may directly and indirectly modulate survivin expression,respectively. These twomiRNAs can regulate GC cell growth,apoptosis, and invasion at least partly through a combined regulation over survivin.4. low miR-335 or miR-34a expression is associated with higher clinical stage and lymph node metastasis and may predict poor overall survival of GC. |
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