**4. Let-7 miRNAs**

The lethal-7 (let-7) family of miRNAs was first discovered in 2002 as a development regulator in *Caenorhabditis elegans* [91]. The expression of let-7 miRNAs is low in undifferentiated cells and increases gradually as the cells differentiate during development [92]. Therefore, let-7 miRNAs are also referred to as differentiation-inducing miRNAs. The let-7 mutated *C. elegans* larvae do not mature to the adult stage but keep proliferating and eventually die, earning the name "lethal-7 (let-7)" for this family of miRNAs [91]. Let-7 miRNAs are highly conserved in various animal species [93], suggesting that let-7 miRNAs regulate the same molecular pathways and biological processes in different organisms. In humans, let-7 miRNAs family comprises 12 members including let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, and miR-98 [94], which originate from eight different genomic loci [95]. Some let-7 miRNAs produced from different genomic loci at different chromosomes have the same sequence. For examples, in humans, let-7a-1, let-7a-2, and let-7a-3 have the same sequence but are encoded by loci on chromosomes 9, 11, and 12, respectively. Similarly, let-7f-1 and let-7f-2 are encoded by different genomic loci but have the same sequence [96]. Let-7 miRNAs have a common seed sequence of seven nucleotides "GAGGUAG" from nucleotide two to eight in all species, which plays an important role in

recognizing miRNA response element (MRE) in 30 -UTR of their target mRNA [96]. However, differences in non-seed flanking sequence of let-7 miRNAs affect target specificity [97,98]. Presence of similar seed sequence in all let-7 miRNAs across different species suggests that let-7 miRNAs have the same mechanism for target recognition and might have overlapping targets.

The microarray analysis data from *C. elegans* show that *let-7* miRNAs regulate the expression of thousands of genes, directly and indirectly, indicating their widespread role in biological processes [12]. *Let-7* miRNAs play profound roles in embryo development, glucose metabolism, cell pluripotency and differentiation, tumorigenesis, tissue regeneration, age of onset of puberty and menopause in humans, and organ growth [99,100]. Various studies have shown that *let-7* miRNAs induce cell differentiation and act as fundamental tumor suppressors by downregulating oncogenes [13,101–103]. At early stages of cancer development, *let-7* miRNAs are downregulated and *let-7* targeted oncofetal genes (LOG) are re-expressed [104]. Comparative bioinformatics analysis shows that *let-7* miRNAs target several oncofetal genes including high mobility group AT-hook 2 (HMGA2), insulin like growth factor 2 mRNA binding protein 1 (IMP1), IMP2, IMP3, and malignancy marker nucleosome assembly protein 1 like 1 (NAP1L1) [104]. In hematopoietic stem cells, *let-7* miRNAs inhibit transforming growth factor β (TGFβ) pathway and high mobility group AT-hook 2 (HMGA2), decide the fate of these cells, and regulate cell proliferation, self-renewal and differentiation [105,106].

*Let-7* miRNAs are synthesized following the same general mechanism for miRNA synthesis. The *let-7* loci are transcribed as pri-*let-7* miRNA, then processed into 67–80 nucleotide long pre-*let-7* miRNA by microprocessor complex [95]. Based on the mechanism of further processing, pre-*let-7* miRNAs are divided in two groups: Group I pre-*let-7* miRNAs (pre-*let-7a-2, 7c*, and *7e*) are processed in cytoplasm by direct action of Dicer, whereas group II pre-*let-7* miRNAs (all remaining *let-7s*) are mono-uridylated prior to processing by Dicer [107]. Action of Dicer produces 22 nucleotide long mature *let-7* miRNAs, called *let-7-5p*. As a part of miRNA induced silencing complex (miRISC), *let-7* miRNAs suppress a wide range of genes involved in development, cell proliferation, metabolism, and other important physiological processes [108]. There is no significant difference in expression of pri-*let-7* and pre-*let-7* miRNAs between undifferentiated and differentiated cells, however mature *let-7* miRNAs are high in differentiated cells compared to undifferentiated cells [109].

Mature *let-7* miRNA is a part of hairpin structure in pri- and pre-*let-7* miRNA. This hairpin structure contains mature *let-7* miRNA (*let-7-5p)* in the stem and a partially complimentary strand of nucleotides called *let-7-3p* miRNA, connected by a terminal loop region of different lengths called pre-element (preE) [110]. The process of generation of mature *let-7* miRNAs is more precisely regulated compared to the synthesis of other miRNAs. Different proteins regulate posttranscriptional biogenesis of mature *let-7* miRNA by binding the preE region of pri- and pre-*let-7* miRNAs [111]. One of the most prominent mechanism for regulation of *let-7* miRNAs biogenesis is through LIN28 [112].
