Abstract
The human small RNA miR-4443 is functionally involved in several types of cancer and in the biology of the immune system, downstream of insulin and leptin signaling. Next generation sequencing evidence and structural prediction suggest that miR-4443 is not produced via the canonical Drosha–Exportin 5–Dicer pathway of microRNA biogenesis. We tested this hypothesis by using qRT-PCR to measure miR-4443 and other microRNA levels in HCT-116 cells with Drosha, Exportin 5, and Dicer knockouts, as well as in the parental cell line. Neither of the knockouts decreased miR-4443 levels, while the levels of canonical microRNAs (miR-21 and let-7f-5p) were dramatically reduced. Previously published Ago2-RIP-Seq data suggest a limited incorporation of miR-4443 into RISC, in agreement with the functional studies. The miR-4443 locus shows conservation in primates but not in other mammals, while its seed region appears in additional microRNAs. Our results suggest that miR-4443 is a Drosha, Exportin 5, and Dicer-independent, non-canonical small RNA produced by a yet unknown biogenesis pathway.
Highlights
The human small RNA miR-4443 is functionally involved in several types of cancer and in the biology of the immune system
We previously reported that in cultured colon cancer cells, miR-4443 was upregulated by leptin and insulin in a MEK1/2-dependent manner, decreased invasion and proliferation, and directly downregulated pro-metastatic NCOA1 and TRAF4
Insulin and/or leptin resistance may neutralize this tumor-suppressive pathway, increasing the risk of developing cancer [1]. Supporting this notion, the miR-4443 locus is frequently deleted in cancers based on TCGA data; and a tumor suppressor role for miR-4443 was reported in other cancer types, i.e., osteosarcoma [2], hepatocellular carcinoma [3], ovarian cancer [4], and glioblastoma [5]
Summary
The human small RNA miR-4443 (miRbase accession MI0016786, http://www.mirbase.org/cgibin/mirna_entry.pl?acc=MI0016786) is functionally involved in several types of cancer and in the biology of the immune system. (c) Deep sequencing evidence from ~23 K reads shows only the 5′ mature miR-4443 product and no passenger strand product from pre-miR-4443. (http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0016786), with let-7f-5p and miR-21 included included for comparison. It is well known that the size and structural flexibility of the microRNA precursor terminal loop are important for both (c) Deep sequencing evidence from ~23 K reads shows only the 50 mature miR-4443 product and no passenger strand product from pre-miR-4443. The MirGeneDB database [16] (http://mirgenedb.org/) and suggest that miR-4443 is not a product of the canonical Drosha–Exportin 5–Dicer microRNA biogenesis pathway
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