The Identification Of Differentially Expressed Mirnas In Tumor Samples And Function Study Of Mir-200b

miRNAs are ~22nt small non-coding RNAs which regulate gene expression post-transcriptionally. miRNA is usually transcribed by RNA polymerase II as primary miRNA (pri-miRNA) transcript, which is sequentially cleaved by Drosha/Pasha, generating a~70 nt stem loop pre-miRNA. The pre-miRNA is then exported to the cytoplasm by Exportin-5 and processed by Dicer to liberate mature miRNA. miRNA then associates with the Argonaute proteins and forms the RNA-induced silencing complex (RISC) for binding to the 3' UTR of targeted mRNAs. This results in the decay of target mRNAs or the inhibition of translation.miRNAs play important role in tumor research: firstly, the expression of miRNAs are different in diverse tumors, research on miRNA expression profile will contribute to the classification of tumors. Compared to the traditional mRNA profile, miRNA profile is more exact and could directly represent to genes expression levels. The miRNA profile could also be used as biomarker in diagnosis and prognosis. Secondly, miRNAs are involved in many important process, such as proliferation, apoptosis, differentiation, metablism and tumor metastasis. To inhibit the oncogene-like miRNAs or to overexpress the anti-tumor miRNAs will be a novel method on tumor therapy. Thirdly, tumor generation is a complicated process including many genes, the research on miRNA will conduce the exploration of molecular machenism in tumor generation and development.In our research, we identified the differentially expressed miRNA in bladder cancer, endometrial cancer, squamous cell carcinoma of lung and adenocarcinoma of lung, investigated the function of mir-200b, and established a new strategy to identify miRNA functional target genes. This paper could be divided into three parts:①miRNA expression profile in tumor samples;②function study on mir-200b;③a new strategy in miRNA functional target identification.In the partⅠ, we deteced the expression of miRNA in four kinds of tumors and paracancer tissues and found dozens of differentially expressed miRNAs each, including many tumor metastasis related miRNAs. Among these miRNAs, the let-7 family, mir-21, mir-29, mir-125, mir-15 and mir-16 are differentially expressed in all four tumors.In the partⅡ, we investigated the function of mir-200b. In our previous study, we demonstrated that the mir-200 family is overexpressed in endometrial adenocarcinomas, and that mir-200b showed the most significant change. Other groups have demonstrated that the mir-200 family is abnormally expressed in tumors of many other cancers, such as hepatocellular, ovarian and gastric cancers. To investigate the function of mir-200b further, we transfected a mir-200b mimic into different cancer cell lines. HeLa cells transfected with the mir-200b mimic showed a high percentage of S-phase entry. Luciferase assays indicated that overexpression of mir-200b reduced the luciferase activity from the reporter vector containing the wild-type RND3 3′UTR, but it had less effect on activity from the first deletion construct and no effect on activity from the second. Thses results suggest that mir-200b regulates RND3 expression by targeting both putative target sites 1 and 2. Using quantitative (q) PCR and western blotting, we demonstrated that mir-200b could directly reduce the expression of RND3 in HeLa cells. By comparing the data with that generated using an siRNA against RND3, we demonstrated that mir-200b could regulate the downstream protein CCND1 and promote S-phase entry by targeting RND3. T24 cells transfected with the mir-200b mimic changed in morphous, and were inhibited in migration ability. The mir-200b mimic, siRNA against WASF-3 or the negative control RNA duplex was transfected into T24 cells, qRT-PCR and western blotting were used to detect the expression levels of WASF-3 mRNA and protein, respectively, the migration of T24 cells was detected by Transwell assay. The mRNA and protein levels of WASF-3 were reduced in cells transfected by the mir-200b mimic and siRNA compared with the negative control, the migration was also inhibited in these cells.In the partⅢ, we established a new strategy in miRNA functional target identification.①Identification of miRNA binding sequences with the help of Hfq. Hfq has two RNA binding site, we demonstrated that Hfq could bind with let-7a and poly(A) in the two different sites respectively by EMSA. Then, His-Hfq, the 3'UTR library, let-7a/NC and poly(A) were incubated to find the let-7a binding sequences, RT-PCR result demonstrated that there are un-specific binding between 3'UTR library and Hfq, further research will try to optimize the binding condition of Hfq and 3'UTR library.②Identification of functional miRNA targets by cytoplasmic/nucleic ratio of mRNA levels in cultural cells. In our previous study, we found that the ratios of mRNA levels in cytoplam and nucleus (C/N ratios) were closely related to the corresponding miRNA targets, we hypothesized that a rapid strategy may be established to identify miRNA target genes by combination of bioinformatic prediction with C/N ratios of potential miRNA target genes. To verify our hypothesis, hsa-mir-93 was chosen as an example to identify its target genes in HeLa and HepG2 cells, individually. We first used bioinformatic software to predict the potential target genes of mir-93. Among all the predicted potential target genes, seven genes including E2F1, RB1, EGR2, MXD1, RBL2, RBL1 and P21 were chosen randomly. Then we used mRNA microarray to investigate the C/N ratios of above genes both in HeLa and HepG2 cells, the results were identified by qRT-PCR. Based on the C/N ratios results, we considered that RBL1 may be the functional target of mir-93 in HeLa cells, but E2F1 is not; E2F1 may be the functional target of mir-93 in HepG2 cells, but RBL1 is not; EGR2, P21 and RBL2 may all be the functional targets of mir-93 both in HeLa and HepG2 cells. We also speculated that RB1 and MXD1 may not be the functional targets of mir-93 both in HeLa and HepG2 cells. To testify our inference, Western blot assay was performed to directly detect these genes expression after transfection of mir-93 and all the Western blotting results were exactly in accord with our speculation. To further investigate the accuracy of this strategy, we identified novel functional targets of miR-200b in HeLa cells by similar approach. We chose the mRNAs with higher C/N ratios ( TFAP2A, SFRS1 and CDYL ) and lower C/N ratios ( MSN, WASF3 and ZEB1 ) to testify whether these genes were the functional targets of miR-200b in HeLa cells, Western blot results were also exactly consistent with our speculation.In summary, we deteced the expression levels of miRNA in four kinds of tumors and paracancer tissues and found dozens of differentially expressed miRNAs each. We investigated a novel role for mir-200b in cell cycle progression and identified a novel mir-200b target, RND3. We provide the concept of"functional targets of miRNAs"for the first time, and established a strategy to identify miRNA functional target genes by combination of bioinformatic prediction with C/N ratios of mRNAs.