Taken together, this study demonstrates that miR-34a may be required for the anti-cancer effects of DADS, both in human breast cancer cell lines and in xenograft models. Furthermore, SRC may be a critical target of miR-34a. DADS may therefore suppress proliferation and invasion in human breast cancer cells by up-regulating miR-34a and consequently reducing SRC/Ras/ ERK signaling, indicating that miR-34a may serve as a potential gene therapy target for enhancing the antitumor effects of DADS in breast cancer therapy. MicroRNAs, a class of small non-coding RNA, are widely studied as crucial regulatory molecules able to modulate broad regulatory networks at the post-transcriptional levels. miRNA is generated from primary miRNA and precursor miRNA in animals, with the premiRNA presenting a stable stem-loop structure. Both of the two arm products, miR5p and miR3p, have been reported to form mature and functional miRNAs, with the loop sequences connecting miR5p and miR3p in the stem-loop structure typically degraded during miRNA biogenesis. These transitory intermediates are rarely examined due to an unseen direct role in the miRNA regulation process. Instead, most studies have focused on the functional and regulatory roles of miRNAs during miRNA-mRNA recognition as it relates to expression or translational repression. However, indeed, both pri-miRNA and pre-miRNA may contribute to the regulatory process. Specifically, the loop nucleotides may tune and alter miRNA activity, controlling the processing precision during the miRNA maturation process. Recently, the potential impact of hairpin loops has been examined, with short hairpin RNA loops possibly influencing effectivity and alternative loop conformations in miRNAs potentially effecting expression. The loop sequence may also affect Dicer recognition and possibly specificity, thus affect miRNA cleavage during the maturation process. As mentioned above, the loop sequence in premiRNAs has an important role in regulating the activities and specificities of related molecules that can facilitate Clofentezine Drosha and Dicer, with a mutation in the loop possibly affecting miRNA processing. Furthermore, miRNAs have been a key focal point because a miRNA locus can yield multiple sequences have diverse 59 and/or 39 ends and expression levels. These miRNA variants, or physiological miRNA isoforms, may be mainly generated by imprecise Drosha and Dicer cleavage during pre-miRNA processing. Additionally, the loop sequences may also impact pre-miRNA processing by affecting Drosha and/or Dicer directly or indirectly. Even though premiRNA loop sequences have been shown to directly or indirectly impact Drosha and Dicer during miRNA processing, few studies have performed an evolutionary analysis of the loop sequences, especially one focused on homology and clustered miRNAs across Atractylenolide-III different animal species. Herein, we attempt to perform an evolutionary analysis of the loop sequences in let-7 and locate related miRNAs across different animal species. The let-7 gene family has been widely detected in metazoans, with its associated miRNAs thought to have coexpanded with the HOX gene clusters. A series of homologous miRNAs can be found in a miRNA gene family, including multicopy pre-miRNAs from specific animal species. Simultaneously, some members can be located in a cluster with other related miRNAs, with miRNAs within a close proximity on a chromosome being co-transcribed as a polycistronic transcript. Therefore, a classical miRNA gene family and related miRNAs are typically selected to track and reveal the evolutionary patterns of the ever present yet ignored loop sequences. Thus, this study will examine the roles of loops during miRNA maturation and processing.