The Expression Of MicroRNA-125a In Human Gastric Cancer And Its Clinical Significance

Background and ObjectiveGastric cancer ranks fourth in incidence (after lung, breast and colorectal) and second in mortality (after lung cancer) among all cancers worldwide, with an estimated 990,000 new cases and 738,000 deaths registered in 2008.Gastric cancer is a multifactorial disease. Despite the advances in current therapeutic approaches of gastric cancer, the development of adjuvant chemotherapy, which improve the clinical therapeutic effect in some way. On the contrary, the prognosis of gastric cancer still remains poor in those advanced cases with lymph node metastasis. Therefore, some reporters believed that some genes seem to contribute to the malignant potential of gastric cancer, but it remains extremely important that the identification of the precise factors which predict the prognosis and recurrence of gastric cancer.MicroRNAs (miRNA) are a type of short, non-coding RNAs that suppress the expression of protein-coding genes by partial complementary binding, especially to the 3’untranslated regions (UTRs), of messenger RNAs (mRNAs). miRNA in the nucleus and cytoplasm is transcribed as an long and the initial fragment (pri-miRNA), and then through the cut by inner RNA endonuclease Ⅲ-Drosha and a series of complex processes, it generates miRNA precursor with 70 nt length (pre-miRNAs). Finally, pre-miRNAs are transported to the cytoplasm and cut by Dicer enzyme to become a mature miRNA. So far, it has been discovered hundreds of miRNAs. In the expression profile of all human genes, about 30% of miRNAs are involved in the regulation of a variety of protein-coding genes. Alterations in miRNA expressions are involved in the initiation, progression and metastasis of human cancer, and it is believed that miRNAs function as tumor suppressors and oncogenes in cancer development.More and more evidence indicates that miR-125 family has abnormal expression in tumor tissues. miR-125 family consists of miR-125a, miR-125b-1 and miR-125b-2. miR-125a is located on chromosome 19q13, and it includes 2 mature molecules, ie.miR-125a-3p and miR-125a-5p, which are from 3’end and the 5’end of miR-125a precursor, respectively. A number of studies have shown that miR-125a is an important tumor suppressor gene, and reduced miR-125a expression has been detected in many types of human cancer, including breast, lung and ovarian cancer, as well as glioblastoma. Nishida et al reported that a reduced expression of miR-125a-5p is associated with enhanced potential to develop gastric cancer. Furthermore, Hashiguchi et al reported that miR-125a-3p, commonly ignored by investigators, has almost the same function in gastric cancer as miR-125a-5p. There are also reports that germline mutations in the miR-125a coding region can reduce miR-125a expression and are associated with human breast cancer. These findings strongly suggest that miR-125a-5p and-3p variants can act as tumor suppressors and reduce miR-125a expression, thereby serving as genetic markers for gastric cancer diagnosis and treatment.Is there a mutation in the coding region of miR-125a in gastric cancer? Does this mutation lead to changes of miR-125a expression? What is the underlying mechanism? To answer these questions, in the present study, we first examined the expression level of miR-125a-5p and-3p in gastric cancer tissue and adjacent healthy gastric tissue. And then we genotyped the miR-125a coding region in gastric cancer patients and healthy controls. We found a germline mutation in the pri-miR-125a coding region that was associated with gastric cancer and the reduction of miR-125a expression, suggesting that miR-125a is likely to function as a tumor suppressor gene in human gastric cancer.Methods1.Collected gastric specimens:A total of 75 pairs of histopathologically confirmed gastric cancer and adjacent non-cancer tissue samples were obtained from patients in Central Hospital of Zibo. Control samples from a total of 287 healthy Han-Chinese individuals were also collected at the Central Hospital of Zibo.2.Cell line culture:Human gastric adenoma cell lines (MGC-803 and BGC-823) were purchased from the Cell Bank of Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cells were routinely cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37℃ in a humidified atmosphere with 5% CO2.3.Reverse transcription-quantitative polymerase chain reaction (RT-qPCR):The expression level of miR-125a-5p and -3p was assessed by TaqMan miRNA real-time (quantitative) RT-PCR. Total RNA was extracted from tissues and cells using TRIzol according to the manufacturer’s instructions. Single-stranded cDNA was synthesized using the TaqMan MicroRNA Reverse Transcription kit and then usingthe fluorescence quantitative kit instructions of TAKARA company fluorescence quantitative PCR detection.4.DNA collection and genotyping:DNA from tumor and adjacent healthy tissues of the gastric cancer cohort was isolated using the TIANamp Genomic DNA kit. DNA from blood samples was extracted using the TIANamp Blood DNA kit. DNA samples were amplified using standard PCR protocols. The PCR products were sequenced in the forward direction on an ABI 3730x1 sequencing platform. The sequencing results were analyzed using the open-source software DNAMAN and Chromas Lite. The PCR primers used for miR-125a sequencing were:5’-TGT GTC TCT TTC ACA GTG GAT C-3’and 5’-CCA TCG TGT GGG TCTCAAG-3’.5. Secondary structure prediction:The secondary structure of the 217-bp pri-miR-125a sequence, which includes the mutation site, was predicted using the RNAfold web server.6. miR-125a expression vectors:To construct miR-125a expression vectors,1,016-nt fragments corresponding to pri-miR-125a and its flanking regions (previously confirmed to bear the two alleles) were amplified from cDNA and cloned into the pcDNA3.1 vector (Invitrogen Life Technologies). The sequences of the two resulting vectors were confirmed by direct sequencing, with the only difference being in the mutation site. The primers used were:miR125a-F/XhoI,5’-CCG CTC GAG GGT AGG AGG TTG TAT AGT TGA GGA GG-3’and miR-125a-R/XbaI,5’-GCT CTA GAC CTC TGG GCC TCT CCT GC-31.7.Cell proliferation assay (CCK8):MGC-803 and BGC-823 cells were seeded into 96-well plates at low density (5×103) in Dulbecco’s modified Eagle’s medium (DMEM) and were allowed to attach overnight. The cells were then transfected with different haplotype miR-125a expression vectors, with the empty pcDNA3.1 vector used as the control. Ten microliters CCK-8 were added into each well 48 h following transfection, and the cells were incubated for an additional 3 h. Following their absorbance was recorded at 450 nm on a 96-well plate reader.8.Dual luciferase assay. The full-length region (618 bp) of the 3’UTR of the erythroblastic leukemia viral oncogene homolog 2 (ERBB2) gene was cloned downstream of the coding region of the firefly luciferase gene within the pmirGLO Dual-Luciferase miRNA Target vector to generate the luciferase reporter vector. For luciferase reporter assays, MGC-803 and BGC-823 cells were seeded into 48-well plates. The miR-125a expression and the luciferase reporter vectors were co-transfected.The cells were harvested and assayed with the Dual-Luciferase Assay system. Each assay was performed in triplicate, in three independent experiments. The results were expressed as relative luciferase (LUC) activity (firefly LUC/RenillaLUC).9.Statistical analysis:Data were analyzed using the SPSS statistical package version 16. Continuous variables normally distributed represented as mean±standard deviation. Comparisons between two independent groups were performed with the Student’s t-test. Expression data for miR-125a were compared using the rank sum test. P<0.05 was considered to indicate a statistically significant difference.Results1. Decreased miR-125a-5p and-3p in gastric cancer tissues. In order to explore the role of miR-125a in gastric carcinogenesis, the expression patterns of miR-125a were analyzed in 75 pairs of human gastric cancer and adjacent healthy gastric tissues using RT-qPCR. Each sample consisted of pooled RNAs from cancer tissues of three patients (25 samples for 75 pairs). The levels of miR-125a-5p and -3p were significantly decreased in 92 (23/25) and 80%(20/25 samples) of gastric cancer tissues, respectively.2.A germline mutation was detected in the pri-miR-125a coding region. Since nucleotide variants can alter miRNA expression and are associated with many types of human diseases, we genotyped the coding region of pri-miR-125a by sequencing the DNA extracted from gastric cancer tissues. We found five patients carrying the minor G allele, which existed in the +43 relative to the mature miR-125a-5p and+29 relative to pre-miR-125a. Furthermore, we sequenced genomic DNAs isolated from adjacent healthy gastric tissues and found the same genotypes as the ones from gastric cancer tissues.The frequency of the minor G allele was examined in the population of 287 healthy individuals (control group) collected in the same region. We found no individual carrying the G allele in this population, while this allele was also not found in the 1000 Genomes database, suggesting that the T allele is a mutation. Furthermore, since no individual in the control group from the same area carried the G allele, the presence of the G allele among gastric cancer patients is unlikely to be due to a founder effect. Together, these results suggest that a germline mutation in pri-miR-125a is associated with gastric cancer tumorigenesis.3.The G mutation can enhance the predicted stability of pri-miR-125a and reduce miR-125a expression. To explore the function of the mutation, we first compared the predicted secondary structure of pri-miR-125a molecules bearing or not the minor allele. The minor allele G causes an apparent change in loop size (from 8 nucleotides loop to 6 nucleotides loop and from 10 paired nucleotides stem to 12 paired nucleotides stem) and a reduction of the predicted △G from-74.30 to-77.08 kcal/mol. Using RT-qPCR and two different expression vectors carrying the alternative miR-125a-5p alleles, we quantified the expression level of the mature miR-125a-5p in the two gastric cancer cell lines MGC-803 and BGC-823. The presence of the mutation was associated with a ～40% reduction in mature miR-125a-5p expression, in agreement with RT-qPCR analyses on gastric cancer tissue samples.4. Altered miR-125a expression attenuates the inhibitory effect of miR-125a on ERBB2 expression and gastric cancer ell proliferation. The ERBB2 gene, a confirmed target of miR-125a-5p, is a member of the epidermal growth factor receptor (EGFR/ERBB) family. The protein is commonly overexpressed in numerous types of cancer cells and has been suggested to promote cell proliferation. We used the ERBB23’ UTR reporter system to study the effect of the identified mutation on ERBB2 gene expression. As shown in Fig.3A, the reduction in ERBB2expression caused by transfection with the miR-125a vector was significantly attenuated in cells bearing the G compared to those bearing the A allele. To explore the in vivo expression pattern of ERBB2, we measured theERBB2mRNA level by using RT-qPCR in gastric cancer and adjacent healthy gastric tissues. The ERBB2expression level was significantly higher in cancer tissues compared to healthy controls, which had reduced miR-125a expression.5. Based on the predicted function of ERBB2. the reduction of miR-125a expression is expected to promote cell proliferation. Therefore, a proliferation assay was carried out to explore potential differences in antitumor activity caused by different miR-125a genotypes in gastric cancer cells. MGC-803 and BGC-823 cells were transfected with different pri-miR-125a expression vectors, and. as expected, the proliferation of MGC-803 cells was significantly reduced (P=0.0008 and 0.0067 compared to the control) in pri-miR-125a genotypic backgrounds (bearing the A and the mutant G allele). while the A to G mutation attenuated this effect by almost 20%(P=0.023 compared to the A allelic background). In BGC-823 cells, proliferation was also reduced in the pri-miR-125a genotype bearing the A allele (P=0.0075). but this effect was not significantly attenuated by the G mutation.Conclusions1. Decreased miR-125a-5p and-3p in gastric cancer tissues.2. A germ line mutation was detected in the pri-miR-125a coding region.3. The G mutation can enhance the predicted stability of pri-miR-125a and reduce miR-125a expression.4. Altered miR-125a expression attenuates the inhibitory effect of miR-125a on ERBB2 expression and gastric cancer ell proliferation.5. The over-expression of miR-125a is expected to inhibit cell proliferation. miR-125a is a cancer suppressor gene.The significance of studyThere are significant differences in the expression of miR-125a between gastric cancer tissues and normal tissues. The expression of miR-125a may play an important role in the development of gastric cancer, which could provide an effective basis for early diagnosis and prognosis of gastric cancer patients. However, its specific regulatory mechanisms and how it promotes the development of gastric cancer need further studies. The study on the target genes of miR-125a and its regulatory pathways has important clinical value.