| Background:Gastric cancer is the most frequent cancer-related cause of death second only to lung cancer.It’s mortality rate is the highest among all cancers in China and represents25%of gastric cancer mortality worldwide. The overall5-year survival rate in China is poor at40%as most gastric cancer is diagnosed at stage III or IV and has the high rate of lymph node metastasis (50%-75%). Therefore, it is of great clinical value to further elucidate the molecular mechanisms in gastric cancer and to develope reliable earlier diagnostic markers and novel therapeutic strategies. MicroRNA (miRNA) are a family of21to25nucleotide, it plays a important role in cancer or anti-cancer effect by adjusting mRNA translation and degradation. Differential expression of miRNAs in tumor tissues reflected the closely relationship between miRNA and the initiation and development of tumor. miRNA involved in the process of gene regulation is very complex that target genes of miRNA involved in the regulation of tumor differentiation, proliferation, apoptosis, invasion and migration.So as a new starting point, the study of miRNA would open a new way for studying the molecular mechanism of proliferation, invasion and metastasis and targeted therapy of gastric cancer. Purpose:miR-125b can function as oncogenes or tumor suppressors in many human cancers. However, the clinical significance and accurate molecular mechanism of miR-125b in gastric cancer has never been sufficiently investigated. In this study,we aimed to elucidate the functions,molecular regulated pathway and clinical application of miR-125b in gastric cancer by different approach so as to find the molecular mark of early diagnosis and targeting therapy for gastric cancer.Methods:Total RNA was extracted from tissues and cells using Trizol and miRNAeasy mini kit according to the manufacturer’s instructions,cDNA synthesis was carried out with the miScript Reverse Transcription Kit,the specific reverse primer for miR-125b and U6were provided by Qiagen,the resulting cDNA was amplified with the QuantiTect SYBR Green PCR Master Mix using ABI7500FAST realtime PCR;To determine the effect of miR-125b on cell proliferation, clone formation,migration and invasion assays of HGC-27and MKN-28,5.0x10cells were seeded in6-well culture plates and were maintained at37℃under an atmosphere of5%CO for24h.5.0x10cells were transfected with either2μl(50pmol/μl) miR-125b inhibitor or Negative Control using Lipofectamine2000according to the manufacturer’s instructions in6-well plates;MTT assay was performed at24h,48h and72h post-transfection,migration assays was performed at48h post-transfection,invasion assays was performed at72h post-transfection; The expression of miR-125b, PPP1CA and phosphorylated RB were detected by quantitative real-time PCR and/or Western blotting24h after transfection; To construct two luciferase reporter plasmids containing the wild-type3’UTR of PPP1CAmRNA and mutant-type3’UTR of PPP1CAmRNA so as to confirm the interaction of miR-125b with predicted targeted PPP1CA mRNA;In situ hybridization was performed in300human gastric cancer tissues according to manufacturer’s instructions of sensitivity-enhanced in situ hybridization kits.Results:The expression of miR-125b in50pairs of matched gastric tumour tissues were much higher in gastric tumours (0.37±0.23) than in non-tumour tissues (0.19± 0.13^P<0.01), while in gastric tumour group, lymphatic metastasis group (n=32)(0.47±0.23) were higher than non-lymphatic metastasis group (n=18)(0.25±0.12,P<0.05);In addition, miR-125b was significantly up-regulated in AGS, MKN-28, BGC-823, HGC-27, SGC-7901compared to than non-malignant gastric epithelial cell line GES-1except MKN-45;miR-125b inhibitor significantly inhibited proliferation and clone formation of HGC-27and MKN-28cells,the24h,48h,72h relative proliferation and cell clone formation of HGC-27inhibited rate were59%,55%,52%and48%(P<0.01), respectively, the24h,48h,72h relative proliferation and cell clone formation of MKN-28inhibited rate were62%,59%,56%and49%(P<0.01), respectively;Gastric cancer cells transfected with miR-125b inhibitor displayed significantly lower transmembrane migration capacity compared with those transfected with control,the migration activity of miR-125b inhibitor-infected HGC-27and MKN-28was specifically reduced by approximately52%and49%, respectively,while the invasion activity was substantially reduced by approximately48%and51%, respectively;miR-125b significantly decreased the relative luciferase activity of wild-type3’UTR of PPP1CA mRNA through luciferase test(P<0.01),but no significance to mutant-type3’UTR of PPP1CA mRNA; The mRNA and protein levels of PPP1CA in miR125b inhibitor transfected HGC-27and MKN-28cells were much higher than control-transfected group, while the Rb protein phosphorylation level in miR125b inhibitor infected HGC-27and MKN-28cells were much lower than control group;High levels of miR-125b expression were detected in174(58%) tumors, and low miR-125b expression levels were detected in126(42%) tumors. Higher expression levels of miR-125b correlated with size of tumor, depth of invasion, lymph node metastasis, histological grade,distant metastasis, and TNM stage (P<0.05), but was not associated with age, sex, location of gastric tumor (P>0.05). In stages I, II, and III, the5-year survival rate of gastric cancer patients with high levels of miR-125b expression was significantly lower than that in gastric cancer patients with low levels of expression (P<0.05),but in stage IV, the expression of miR-125b did not correlate with the5-year survival rate (P=0.85). By multivariate Cox regression analysis revealed that lymph node metastases(P=0.001), distant metastases (P=0.001), TNM stage (P=0.003), invasion depth (P=0.001), and expression of miR-125b (P=0.001) were significant difference in gastric cancer survival. However, no significant differences were observed regarding size of tumor(P=0.999), histological grade (P=0.710), regional lymph nodes (P=0.170).Conclusions:1. miR-125b is up-regulated in gastric tumour tissues and cell lines.2.miR-125b promoted gastric cancer cell proliferation, clone-formation,migration and invasion in vitro, may be by directly repressing the PPP1CA-RB signaling pathway in gastric cancer cells.3. Higher expression levels of miR-125b correlated with size of tumor, depth of invasion, lymph node metastasis, distant metastasis, TNM stage and the5-year survival rate of gastric cancer patients.4. miR-125b can be a mark of independent prognosis in gastric cancer. |
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