MiR-483-3p And MiR-675 Promote The Proliferation Of Gastric Cancer Based On The High Through-put Screening

MicroRNAs (miRNAs) are a class of small endogenous noncoding RNAs that participate in such biological processes as cell proliferation, differentiation, apoptosis, etc. by post-transcriptional inhibition of translation or targeting mRNA dissociation. Over 1,000 human miRNAs have been identified in the human genome, and they are expressed in a tissue-specific manner. Each miRANA is of numerous target genes, whose expression is regulated by different mechanisms including transcription factor binding, epigenetic alterations, and chromosomal abnormalities. It is found that links between miRNAs and human diseases are extremely close. Recent studies have shown that miRNAs play roles as tumor suppressors or oncogenes in gastrointestinal cancers. Therefore, regulation of miRNA expression could be a novel strategy for the chemoprevention of human gastrointestinal cancers.To further illustrate the important role of miRNA in the generation process of gastric cancer and to reveal transcriptional regulation of related miRNA in gastric mucosa cancerization and its mechanism of action on gene expression regulation, high-throughput function screening is conducted first in gastric cancer cell for miRNA related to gastric cancer proliferation based on micro-electronic transducer impedance change with proliferation phenotype as model by using of miRNA inhibitor library, and 12 anti-miRNA molecules that can suppress significantly gastric cancer proliferation are found; in which, miR-483-3p is the most significant in cancer suppression. Further research has been done for miR-483-3p and miR-675 that are tightly interlocked to No. 11 chromosome. It is found by analysis that miR-483-3p is located in the 7th introne of Insulin-like Growth Factor (IGF) 2 of the host gene, while miR-675 in the 1st exon of host H19, indicating that miR-483-3p and miR-675 may transcribe together with their respective host gene. As the imprinted genes found at the earliest, IGF2 and H19 are in immediate vicinity of No. 11 chromosome, and both of them have the important biologic function of promoting various cancer cell proliferation. Up to the present, there is not yet relevant report about miR-483-3p and miR-675 involvement in tumor formation and evolution. In that way, what is the upstream transcription regulation mechanism of miR-483-3p and miR-675? Whether both of them are involved in the new regulation mechanism of IGF2/H19 related gastric cancer promoting signal. The thesis discusses these.【Objectives】(1)Screen in high-throughput loss of function miRNA related to gastric cancer cell proliferation; (2)Study function of miR-483-3p and miR-675 involving in gastric cancer cell proliferation; (3)Discuss upstream co-transcript and downstream regulation signal pathway of miR-483-3p and miR-675; (4)Define expression and function of miR-483-3p'and miR-675's target genes in gastric cancer; (5)Probe into the regulation of miR-483-3p and miR-675 by miR-145.【Methods】(1)Using of miRNA inhibitor library and real-time cell high-throughput screening meter based on micro-electronic transducer impedance change to screen miRNA molecule related to gastric cancer cell proliferation in loss of function; (2)Constructing lentivirus vectors of miR-483-3p and miR-675; (3)Studying influence of miR-483-3p and miR-675 on tumor formation ability of gastric cancer cell by in vitro and in vivo cell proliferation assay, plating clone formation assay, soft agar colony formation assay, nude mouse tumor-bearing assay, and so on; and detecting the effect of miR-483-3p and miR-675 on cell cycle and cell DNA replication by flow cytometry and con-focal laser Edu cell nucleus proliferation experiment respectively; (4)Verifying transcriptional regulation of c-Myc on miR-483-3p and miR-675 by ChIP, EMSA and other means; (5)Screening target genes of miR-483-3p and miR-675 through united forecast of whole-genome expression profile chip and bioinformatics; (6)Structuring bi-luciferase reporter gene vector to verify negative regulation of c-Myc by miR-145 as well as target genes of miR-483-3p and miR-675 through bi-luciferase reporter gene experiment, Real time RT-PCR, Western Blot and other experiments; (7)Studying expression of miR-483-3p and miR-675's target genes in gastric cancer and their relation with gastric cancer cell proliferation through immunohistochemistry staining, MTT experiment, etc.【Results】1.miR-483-3p and miR-675 can significantly promote gastric cancer cell proliferation.miRNA inhibitor library was used to perform microelectronic transducer impedance change based real-time cell high-throughput afunction screening on the basis of proliferation phenotype of gastric cancer cell line SGC7901 and 12 anti-miRNA molecules that significantly inhibit the gastric cancer cell proliferation were screened. In which, miR-483-3p is the most obvious in cancer suppression. Analysis found that miR-483-3p and miR-675 are two miRNA molecules close on No. 11 chromosome, and there are not yet relevant reports on their involvement in tumor formation and evolvement. Study was carried out in depth for miR-483-3p and miR-675 involvement in gastric cancer cell proliferation. Real-time fluorescence quantitative PCR detection revealed that expression levels of miR-483-3p and miR-675 in gastric cancer cell line SGC7901, AGS and MKN45 were significantly higher than in immortalized cell line GES-1 of normal gastric mucosa; in 12 cases of fresh gastric cancer tissues, expression levels of them in 8 cases were significantly higher than in corresponding paracancerous tissues. By building lentiviral carrier of miR-483-3p and miR-675 to transfect stably SGC7901 and MKN45 cells and to carry out function experiments in vitro and vivo, it is found that up-regulated miR-483-3p and miR-675 expression could significantly improve capacities of plating clone in vitro, soft agar colony formation and nude mouse tumor-bearing in gastric cancer cells; transiently transfecting miR-483-3p and miR-675 inhibitors could obviously inhibit tumor formation ability of gastric cancer cells in vitro and in vivo. Results of flow cell cycle test showed that miR-483-3p and miR-675 can promote the progress of gastric cancer cell in phase G1, and the laser con-focal results showed that both of them could significantly promote DNA replication of gastric cancer cell. These results suggested that miR-483-3p and miR-675 could promote proliferation of gastric cancer cell.2. miR-483-3p and miR-675 regulate signal channel of gastric cancer with IGF2BP1, BRCA2 and PITX1 as target genes respectively.Target genes of miR-483-3p and miR-675 were further screened by combined prediction of gene expression profile and target gene. Transiently transfecting the whole-genome expression profile of miR-483-3p in SGC7901, 1,812 genes were down-regulated; By combining TargetScan and miRanda, 402 target genes of miR-483-3p were predicted, and there were 13 genes in the intersection of above-mentioned results, in which, Breast Cancer Susceptibility Gene (BRCA) 2 was down-regulated by 2.17 times as the only tumor suppressor gene. Similarly, transiently transfecting the whole-genome expression profile of miR-675 in SGC7901, 490 genes were down-regulated; By combining TargetScan and miRanda, 208 target genes of miR-675 were predicted, and there were 25 genes in the intersection of above-mentioned results, in which, Pituitrin Homeobox (PITX) 1 was down-regulated by 2.54 times as the only tumor suppressor gene. Real-time fluorescence quantitative PCR and Western blot confirmed that miR-483-3p and miR-675 could respectively down-regulate mRNA and protein levels of BRCA2 and PITX1; In contrast, miR-483-3p and miR-675 inhibitors could respectively up-regulate mRNA and protein levels of BRCA2 and PITX1. Bi-luciferase reporter gene experiments confirmed that miR-483-3p and miR-675 separately could combine directly 3'UTR of BRCA2 and PITX1. In addition, combined prediction of TargetScan, miRanda and Pictar found that translation inhibitory factor's Insulin-like Growth Factor IGF2 Binding Protein (IGF2BP) 1 was a potential target gene of miR-483-3p; real-time fluorescence quantitative PCR and Western blot confirmed that miR-483-3p could down-regulate protein level of IGF2BP1, but would not affect its mRNA level. Bi-luciferase reporter gene experiments confirmed that miR-483-3p could bind directly 3'UTR of IGF2BP1.3. IGF2BP1, BRCA2 and PITX1 play the role of tumor suppressors in gastric cancer.In order to further clarify the role of tumor suppressors of miR-483-3p'and miR-675's target genes in gastric cancer, further study was conducted. RT-PCR and Western blot results showed that expressions of IGF2BP1, BRCA2 and PITX1 in gastric cancer cells SGC7901 and MKN45 were significantly lower than in immortalized normal gastric mucosa cell GES-1. Immunohistochemistry results showed that expression of IGF2BP1, BRCA2 and PITX1 in 76 cases of gastric cancer tissues was significantly lower than in paracancerous tissues, and the lower the differentiation of gastric cancer tissue, the lower its expression level is (p <0.01). Using their specific siRNA to transfect GES-1 cells could significantly promote cell proliferation and cycle progression (p <0.05).4. miR-483-3p and miR-675 are jointly under direct transcriptional regulation of c-Myc.Some researches have shown that as host gene of miR-483-3p and miR-675, IGF2 and H19 may be activated by c-Myc transcription, prompting that miR-483-3p and miR-675 may also be regulated by direct transcription of c-Myc. Combining transcription factor database and using its prediction software, it was also found that c-Myc was a potential upstream transcription factor of miR-483-3p and miR-675. Chromatin Immuno-Precipitation (ChIP) experiment and gel Electrophoretic Mobility Shift Assays (EMSA) further confirmed that c-Myc could directly combine the 8th and 4th transcription binding sites in the upstream promoter region of miR-483-3p and miR-675 respectively; increasing c-Myc expression in SGC7901 and MKN45 could markedly up-regulate miR-483-3p and miR-675 expression, while inhibiting c-Myc expression in SGC7901 and MKN45 could markedly down-regulate miR-483-3p and miR-675 expression in SGC7901 and MKN45.5. miR-145 performs indirect regulation on miR-483-3p and miR-675 through negative regulation on c-Myc.Some researches have shown that miR-145 activated by p53 could perform negative regulation with c-Myc as its target gene; in addition, it was found that c-Myc could directly transcribe and activate miR-483-3p and miR-675, therefore, it can be inferred that miR-145 can conduct negative regulation on miR-483-3p and miR-675 expression by c-Myc, and miR-145 could inhibit in vitro plating clone formation capability of gastric cancer cell SGC7901 through retardation in phase G1; real-time RT-PCR and Western blot confirmed that miR-145 in gastric cancer could down-regulate protein level of c-Myc and miR-145 inhibitors were able to up-regulate protein level of c-Myc. Bi-luciferase reporter gene experiments confirmed that miR-145 could directly combine 3'UTR of c-Myc. Further study found that miR-145 in gastric cancer tissue was of negative correlation respectively to expression level of miR-483-3p and miR-675; transfecting mimic of miR-145 in SGC7901 and MKN45 could significantly inhibit expression levels of miR-483-3p and miR-675, while transfecting miR-145 inhibitor in GES-1 could markedly up-regulate expression level of miR-483-3p and miR-675; if treating GES-1 cell with miR-145 inhibitor and specific small-molecule inhibitor 10058F4 of c-Myc, up-regulation function of miR-145 on expression level of miR-483-3p and miR-675 will be reversed.6. miR483-3p/miR675-miR145 positive feedback loop is involved in c-Myc mediated gastric cancer occurrence promotion.BRCA2 and PITX1 is the target gene of miR-483-3p and miR-675 respectively; some researches have shown that BRCA2 may serve as a cancer suppressor together with p53, while PITX1 is able to play the same role by directly transcribing and activating p53, which can directly transcribe and activate miR-145. It was found that down-regulating PITX1/BRCA2 expression could significantly inhibit the protein level of p53/miR-145 and expression level of miR-145 at the same time. Thus, in the tumorigenesis of gastric cancer, there formed a new mechanism of c-Myc gastric cancer regulation mediated by positive feedback loop of miR4 83-3p/miR675-miR145.【Conclusion】miR-483-3p and miR-675 are novel miRNAs related to gastric cancer proliferation promotion, whose upstream is directly transcribed and regulated by c-Myc, while the downstream performs negative regulation on IGF2BP1, BRCA2 and PITX1 respectively before regulating miR-145 by P53 dependently or independently; and miR-145 regulates miR-483-3p and miR-675 indirectly through negative regulation on c-Myc. Therefore, in the tumorigenesis of gastric cancer, there forms a new mechanism of c-Myc gastric cancer regulation mediated by positive feedback loop of miR4 83-3p/miR675-miR145.