Abstract
Oral squamous cell carcinoma (OSCC) is among the leading causes of cancer-associated deaths worldwide. Family members in miR-371/372/373 miRNA cluster, which is localized at human chromosome 19q13.4, are co-expressed in both human stem cells and malignancies. The individual miRNA in this cluster are also involved in modulating the pathogenesis of malignancies as either oncogenes or suppressors. The 19q13 region is frequently gained in head and neck cancers. High expression of miR-372 and miR-373 are survival predictors for OSCC. However, the role of the miR-371/372/373 cluster in oral carcinogenesis remains to be fully investigated. We use the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system to establish OSCC cell subclones that had the miR-371/372/373 cluster deleted. In addition, further subclones were established that had the promoter of this cluster deleted. Concordant silencing in SAS cells of miR-371/372/373 decreased oncogenic potential, increased cisplatin sensitivity, activated p53, and upregulated the expression of Bad and DKK1. We also employed the CRISPR/dCas9 synergistic activation mediator system, which allowed robust transcriptional activation of the whole miR-371/372/373 cistron. Upregulation of endogenous miR-371/372/372 expression increased both oncogenicity and drug resistance. These were accompanied by a slight activation of AKT, β-catenin, and Src. This study identifies the oncogenic role of the miR-371/372/373 cluster in OSCC. Using CRISPR based strategy can be a powerful paradigm that will provide mechanistic insights into miRNA cluster functionality, which will also likely help the development of targeting options for malignancies.
Highlights
Oral squamous cell carcinoma (OSCC) is one of the prevalent head and neck cancers worldwide [1,2]. miRNAs are non-coding RNAs that play various important roles in regulating -targeted transcription events during pathogenesis [1,2,3,4]
Multiple members of the chromosome 19q13 miRNA cluster (C19MC), which consists of more than 40 miRNAs, have been identified as being involved in modulating a wide variety of biological activities; these include self-renewal, apoptosis, and oncogenesis [6,7,8]. miR-371, miR-372, and miR-373, all to be found in the miR-371/372/373 cluster, which is around 1 Kbp in length, is close to the telomere of chromosome 19
Upon the binding of Wingless-related integration site (Wnt) to receptors, the de-phosphorylated cytosolic β-catenin translocates into nucleus as active β-catenin, which transactivates T-cell factor/lymphoid enhancer factor (Tcf/Lef) elements to turn on the expression of Wnt targets [10]
Summary
Oral squamous cell carcinoma (OSCC) is one of the prevalent head and neck cancers worldwide [1,2]. miRNAs are non-coding RNAs that play various important roles in regulating -targeted transcription events during pathogenesis [1,2,3,4]. Wnt signaling has been shown to direct upregulation of miR-371/372/373 cluster members by transactivating Tcf/Lef elements in the promoter. The members of the miR-371/372/373 cluster have been shown to target a number of important genes such as LATS, p62, and ZBTB1A, which drive oncogenic progression in OSCC [15,16,17]. MiR-371/372/373 members are co-upregulated in OSCC, but the various effects of miR-371/372/373 cistron during the regulation of target genes still remain elusive. The concordant silencing of the members of the miR-371/372/373 cluster by deletion of either the whole cistron or the promoter using a CRISPR/Cas editing strategy was carried out in this study [3,17]. Soefqtuheencc6in0gsuobfctlhoenec6c0onsfiurbmcleodnethceonlofisrsmoefd18th40e blopsssooffD1N84A0 tbhpast oinfcDluNdAes tthhaet minicRl-u3d7e1s/37th2e/37m3iRcl-u3s7t1e/r37(F2i/g3u73rec1luBs, tRetr).(FUigsiunrge t1hBe, DRNt).AUosfinpg49thseubDcNloAneo, af mp4p9lifisucabtciloonneu,sianmgpthlieficFa3t/iRo2n pursiimngerthpeaiFr3g/Ren2eprraitmederapnaaimr gpelniceornattehdaatnwaams ~pl1i.c4oKnbthsastmwaallse~r1t.h4aKnbtshasmt oaflltehrethpaanretnhtaatl ocfeltlhse, wpahrielentianl pcaelrlasl,lewl hthileeFi3n/Rp3aprarlilmelerthpeaiFr3d/Rid nportimyieerldpaanir admidplincootn y(Fieilgduraen1Cam, Mpliidcodnle)(.FiSgeuqrueen1Cci,ngMoidf dthleis). aSmeqpuliecnocnins gcoonffirtmhiesdatmheplloicsosnosf 1c3o7n6fibrmpseodf DthNe Alotshsatoifnc1l3u7d6esbtphse omfiRD-3N7A1/3t7h2a/3t73incclluusdteers ptrhoemmotieRrr3e7g1io/3n72(F/3ig7u3recl1uCs,teRrt)p. rOotmheorteerxarmegpiloens o(fFtihgeuprese1rCie,s Rsut)b.clOontheesr, inexclaumdpinlgesp5o,fpt2h9e, apndsepr4i4e,swseurbecfloounneds, tionchluavdeinhgepte5r,opz2y9g,oaunsddpe4le4t,iownesreanfdouinddenttoifhyainvge phreotemrooztoyrgoduesledtieolnetisounbsclaonndesidbeyntciefyllinsgorptirnogmwotaosr udnesleutcicoensssfuublcwlointhesthbeysececlllosnoersti(nngowt sahsouwnns)u.ccessful with these clones (not shown)
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