| [Objective] micro RNA(mi RNA) is a class of ~22 nt, endogenous noncoding small RNAs, which are assembled into RNA-induced silencing complex(RISC) to regulate multiple genes at posttranscriptional level. The mechanism has been well known, in which the seed sequences of mi RNA perfectly matched to target m RNA untranslted region(UTR) leading to m RNA decay; while unperfectly match results in translation suppression. However, it is reported that mi RNAs also upregulate gene expression at transcriptional and posttranscriptional levels, which depends on the specific interaction between mi RNA and target m RNA sequences, the components of mi RNP and the state of cells. In addition, the mi RNA-involved regulation netwok is complex, the accepted notion is that a single mi RNA species can regulate hundreds of targets, conversely, several mi RNAs can bind to their target m RNAs and cooperatively provide fine-tuning of a single target m RNA expression. In our previous work, we found that mi R-138 targets h TERT m RNA 3’UTR to suppress expression to inhibit cell growth in cervical cancer He La cell line, while mi R-346 target the same region of h TERT m RNA 3’UTR with mi R-138 to upregulate its expression, so mi R-138 and mi R-346 coordinately regulate h TERT to contribute to its high expression level. Based on the previous work, we aim to elucidate the detail mechanism of mi R- 346-mediated h TERT upregulation.[Methods] Firstly, qRT-PCR was used to detect the RNA levels of transfected HeLa cells treated with Dactinomycin D(Act D) to analyze the expression of h TERT m RNA and to determine whether the mi R-346 mediates its expression at transcriptional or post-transcriptional level. Next, The second structure of mi R-346/h TERT m RNA dimer was predicted by RNAhybrid algorithm to identify the motif of mi R-346, meanwhile the Target Scan 6.2 and RNAhybrid algorithm were used to predict the other target genes of mi R-346 and the second structures of them after forming dimers. Then we choosed two of the predicted target genes with and without the exposure of mi R-346 “CCGCAU” motif in their respective dimers which was similar to the motif identified in mi R-346/h TERT m RNA. EGFP reporter assay,q RT-PCR and western blot assays were performed to respectively investigate theeffects of AGO2 on mi R-346-mediated regulation of h TERT, the influences of mi R-346 with mutant motif on h TERT expression and the impacts of mi R-346 on the expression of ACVR2 B. RNA IP(RIP) using antibody against AGO2 in He La cells and q RT-PCR assays were carried out to analyze the enrichment of h TERT m RNA and mi R-346 in AGO2 complex. Secondly, MTT assay and colony formation assays were employed to detect the effects of mi R-346 on cell viability and growth. Western blot assays were used to study the influences of the mi R-138 with mi R-346“CCGCAU” motif(mi R-138/mi R-346-loop mimics) or the mi R-138 with mutant“AAAAUA” motif of mi R-346(mi R-138/mi R-346-loop mut mimics) on h TERT expression. Moreover, we altered the expression of GRSF1 or conducted combined alteration of GRSF1 and wild type/mutant mi R-346, and utilized western blot assays to detect h TERT or ACVR2 B expression levels to identify the roles of GRSF1 in mi R-346-mediated up-regulation of h TERT or ACVR2 B. RIP and RNA EMSA assays were used to indicate whether GRSF1 interacts with mi R-346 and/or h TERT m RNA.Finally, sucrose gradient sedimentation assays were used to obtain ribosome fractions from He La cells, and the q RT-PCR was performed to detect the enrichment of mi R-346 and h TERT m RNA in ribosome fractions with purpose to determine whether mi R-346 recruits h TERT m RNA to ribosome and whether this process is mediated by GRSF1.[Results] We found that mi R-346 prolonged the half life of h TERT m RNA and enhanced its stability. AGO2 knockdown did not affect the ability of mi R-346 to modulate the expression of reporter gene containing h TERT 3’UTR, and endogenous h TERT m RNA and protein. Moreover, the h TERT m RNA enrichment in AGO2 complex was positively or negatively related to mi R-138(positive control) or mi R-346 expression level, respectively. The second structure of mi R-346/h TERT m RNA dimer was predicted by RNAhybrid algorithm, showing an exposed“CCGCAU” motif by mi R-346. Mutation of mi R-346 motif sequence abolished the promoting effects on reporter expression and endogenous h TERT expression as well as cell viability and growth induced by wild type mi R-346. When replacing the mi R-138 motif with mi R-346 “CCGCAU” motif, mi R-138 with mi R-346“CCGCAU” motif promoted h TERT expression, but the promotion was abolished bymi R-138 with mi R-346 mutant motif “AAAAUA”. Furthermore, we predicted 142 target genes of mi R-346 and analyzed their second structures with mi R-346 by bioinformatics, and we chose two of them, activin A receptor, type IIB(ACVR2B)and SMAD family member 3(SMAD3) for further study, in which mi R-346/ACVR2 B m RNA had mi R-346 “CCGCAU” motif and mi R-346/SMAD3 m RNA did not. We found that wild type mi R-346 targeted ACVR2 B 3’UTR and promoted its expression, but targeted SMAD3 3’UTR and suppressed its expression.Motif mutant mi R-346 abrogated the promoting effects of wild type mi R-346 on ACVR2 B without influencing wild type mi R-346 induced regulation of SMAD3. The expression levels of h TERT and ACVR2 B correlate positively to the expression of GRSF1. GRSF1 knockdown attenuated mi R-346-induced up-regulation of h TERT and ACVR2 B, and weakened the ability of mi R-138 with mi R-346 “CCGCAU”motif to up-regulate h TERT expression. Moreover, motif mutant mi R-346 reduced GRSF1-mediated up-regulation of h TERT and ACVR2 B. mi R-346 and h TERT m RNA were enriched in GRSF1 complex. The enrichment of h TERT m RNA in GRSF1 complex was positively related to mi R-346 expression. Furthermore, GRSF1 interacted directly with mi R-346 and mi R-346/h TERT m RNA duplex. This interaction was abolished by mutation of mi R-346 motif. We further demonstrated that mi R-346 was present in the ribosome fractions and had a similar distribution pattern as h TERT m RNA. Furthermore, mi R-346 promoted the recruitment of h TERT m RNA to ribosome and facilitated the distribution curve to shift right. Mutation of the mi R-346 motif led to decreased recuitment of h TERT m RNA to ribosome and left shift of its distribution curve. What’s more, knockdown of GRSF1 resulted in a decrease in the amount of h TERT m RNA in the polyribosome fractions. Mutant motif of mi R-346 weakened GRSF-1-mediated recruitment of h TERT m RNA to polyribosomes.[Conclusion] GRSF1 mediates mi R-346 to recruit h TERT m RNA to ribosomes for translation in mi R-346 “CCGCAU” motif-depentent manner. |
PDF Full Text Request