RNA interference: new mechanistic and biochemical insights with application in oral cancer therapy

Figures & data

Figure 1 The common features of ncRNAs.

Notes: The ncRNAs are transcribed from the DNA and through multiple processing steps are exported into the cytoplasm, in a manner that bears a higher or lower degree of similarity with the processing of mRNA. In the cytoplasm, the main function of the ncRNAs is to repress the translation of mRNAs into proteins.

Abbreviations: circRNA, circular RNA; lncRNA, long noncoding RNAs; miRNA, microRNAs; mRNA, messenger RNA; ncRNAs, noncoding RNAs; piRNA, piwi-interacting RNA; siRNA, small interfering RNA.

Figure 2 siRNA therapeutic implication.

Notes: (A) Some relevant examples of nanosystems used for siRNA application, comprising a great variety of viral/nonviral delivery systems, some of which are used in oral cancer: lipid nanoparticles, nanorods, peptides, dendrimers, PEG nanoparticles, or adeno-associated viruses with application in oral cancer. (B) Schematic view of siRNA mechanism. siRNAs can have an endogenous origin, being transcribed from the genome and processed by Dicer or it can be delivered by artificial nanosystems. Both the exogenous and the endogenous siRNAs are loaded in the RISC complex, with the help of which only the guide strand is kept for further interaction with its corresponding mRNA.

Abbreviations: dsRNA, double-stranded RNA; mRNA, messenger RNA; OSCC, oral squamous cell carcinoma; PEG, polyethylene glycol; RISC, RNA-induced silencing complex; siRNA, small interfering RNA.

Figure 3 An efficient therapeutic siRNA design has to consider: 1) the siRNA sequence design; 2) modification made to the sequence as to escape the blood nuclease; 3) an efficient nanoparticle size range essential for efficient siRNA delivery; and 4) the off-target effects exogenous siRNA.

Abbreviations: 2′-FANA, 2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid; miRNA, microRNAs; PKR, protein kinase R; RIG-I, retinoic acid-inducible gene I; RISC, RNA-induced silencing complex; siRNA, small interfering RNA; TLR, Toll-like receptor.

Table 1 Studies using siRNA-targeted delivery as treatment strategies in oral cancer

Targeted gene	Expression	Delivery system	Cell line	Animal model	Methods	Effect on	Reference
CIP2A	Up	599 peptide (viroporins) + Lipofectamine	CAL 27 and SCC-25	–	Agarose gel shift assay, fluorescence microscopy, RT-PCR, Western blot, CellTiter kit for cell proliferation, CytoSelect 24-well cell invasion assay kit, anchorage-independent growth assay	Regulate anchorage- independent growth and inhibition of invasion	^Citation42
Cip2A	Up	EGFR-binding peptide and endosome-disruptive peptide	CAL 27 and SCC-15	Nude mice	Cell proliferation assay, hydrodynamic diameter and zeta potential measurement, BIO-RAD ChemiDoc XRS system	Prevent invasion and tumor growth	^Citation28
CxcR4	Up	Lentiviral vectors	SCC-9, SCC-4, and Tca8113 cell lines	Nude mice	RT-PCR, Western blot, propidium iodide staining for cell cycle, Annexin V for apoptosis, CCK8, IHC, and microarray	Regulate cell cycle and proliferation	^Citation67
VEGF	Up	Folic acid-decorated polyamidoamine dendrimer G4	HN-12	Female athymic nude mice	ELISA, qRT-PCR, tumor size, H&E staining	Inhibit angiogenesis	^Citation68
MDR1 and MDR2	Up	Adeno-associated viral vector	KB	No	RT-PCR, Western blot, flow cytometry, MTT assay	Regulate MDR	^Citation51
MDR1	Up	Polymer polyethylenimine	Doxorubicin	KBV cells, BALB/c nude mice	Cytotoxicity assays in vitro, drug loading rate and release assays, agarose gel retardation assay, qRT-PCR, apoptosis by flow cytometry	Regulate MDR	^Citation69

Abbreviations: CCK8, cell counting kit 8; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; MDR, multidrug resistance; KBV, oral squamous cells; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; siRNA, small interfering RNA.

Figure 4 The main pathway targeted using siRNA delivery systems in oral cancer.

Notes: Examples of siRNA therapeutics alter multiple hallmarks applied for oral cancer. Cell growth and proliferation are impaired by siRNA for Pa28α, Sox4, or Angptl4. Local inflammation is reduced by IL-8. Invasion and metastasis are decreased by siRNA for Sox4, Ang2, Angptl4, Wnt, Ctsb, Slug, Ck14, Has2, Versican, Frmd4A, Cxcr4, and Cip2A. Angiogenesis is inhibited by siRNA targeting VEGF, Angptl4, and Ang2. MDR is inhibited by siRNA for Mdr1 and Mdr2. Phagocytosis is stimulated by siRNA for Cd47. Tumor growth is reduced in the presence of siRNA for Cip2A and Frmd4A. The heat-shock response is decreased by siRNA for Bag3.

Abbreviations: IL-8, interleukin-8; MDR, multidrug resistance; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.

Table 2 Relevant studies of siRNA as a silencing mechanism in OSCC using commercial delivery systems used to furnish new mechanistic insights

Targeted gene	Expression	Delivery system	Type of cell line/animal model	Methods	Effect on	Reference
Sox4	Up	Lipofectamine 3000	UM1 cell line	MTT assay, cell counting kit, matrigel invasion assay	Reduce proliferation and invasion	^Citation78
Pa28α	Up	Lipofectamine™ 2000	CAL 27 and HSC-3 cell line BALB/c nude mice	Western blot, MTT assay, plate clonogenic assay, Edu assay, TUNEL assay, single scratch wound assay, tumor size measurement, immunohistochemical analysis of PCNA and CD34	Cell growth and proliferation	^Citation93
Cd47	Up	Lipofectamine RNAiMAX	HSC-3 cell line cocultured with CD68+ and CD163+ macrophage	Flow cytometry CFSE+ CD33+- engulfed cells	Anti-phagocytic	^Citation94
IL-8	Up	Lipofectamine RNAiMAX	SCC-4, SCC-9, SCC-25 cell lines	qRT-PCR, Western blot, immunohistochemical staining, MTT assay, clonogenic assay, Lipofectamine 2000 siRNA transfection	Reduce proliferation and colony formation	^Citation81
Ck14	Up	Lipofectamine 2000	3D cultures from fresh tissues of patients with salivary adenoid cystic carcinomas	Collective invasion in an ex vivo 3D culture assay, Western blot, micrographs of the border	Reduce proliferation rate and metastatic capacity	^Citation84
Cxcr4	Up	Lipofectamine 2000	TSCC cell line	Wound healing assay, transwell assay, Western blot, Annexin V-FITC apoptosis detection kit, histopathological analysis, immunohistochemistry analysis	Regulate EMT and prevent metastasis	^Citation83
Frmd4A	Up	Lipofectamine RNAiMAX	CAL 27 cell line	Western blot, propidium iodide staining and flow cytometry, cell counting kit, wound healing assay, matrigel invasion assay	Reduce tumor progression and metastasis	^Citation75
Has2	Up	Lipofectamine 2000	Primary fibroblasts from 48 patients – NF and CAF, the CAL 27 cell line	Western blot, immunofluorescence, wound healing and matrigel invasion assay, bromodeoxyuridine assay, RayBio antibody array	Regulate EMT and reduce invasion	^Citation85
Versican	Up	Lipofectamine 2000	SCC-15 cell line	Western blot, transwell assay, matrigel invasion assay	Reduce migration, invasion, and tumor progression	^Citation95
Angptl4	Up in late- stages	Lipofectamine 3000	TSCC cells	Immunohistochemistry staining, qRT-PCR, Western blot, transwell plate invasion, colony-forming assay, cell counting kit	Reduce cell growth, angiogenesis, and metastasis	^Citation89
Ctsb	Up	Not specified	OC2 and CAL 27 cell lines	Western blot, transwell migration assays	Prevent metastasis	^Citation96
MMP-10 and Slug	TGFβ-induced expression	Lipofectamine RNAiMAX	HSC-4 cell line	qRT-PCR, Western blot, Boyden chamber assay	Regulate TGFβ-induced invasion	^Citation87
Ang2	Up	FuGENE HD transfection reagent	TCA8113 cell line, BALB/c nude mice	Western blot, MTS assay, fluorescence channel analysis, flow cytometry, transwell chamber, ELISA, tumor volume measurement, TUNEL assay, immunohistochemical and quantification analysis of CD31 protein	Regulate EMT, prevent angiogenesis, invasion, and metastasis Inhibit apoptosis	^Citation90

Abbreviations: ELISA, enzyme-linked immunosorbent assay; EMT, epithelial to mesenchymal transition; MTS, 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; siRNA, small interfering RNA; TGFβ, transforming growth factor β; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Table 3 Studies using siRNA in combination with other therapies in oral cancer

Targeted gene	Expression	Delivery system	Combination	Animal model, cell line	Methods	Target mechanism	Reference
Hsp70	Up	Gold nanorods	Plasmonic photothermal therapy	HSC (oral)	XTT assay, PI staining, Annexin- V-FITC, immunofluorescence, immunoblot	Inhibition of proliferation and activation of apoptosis	^Citation98
WNT3	Up	Nanoparticles of PEG, Ce6, and PEI	Photodynamic therapy	KB	Singlet oxygen generation measurement, particle size determination with TEM, MTT, Annexin V/PI staining, qRT-PCR, Western blot	Regulate EMT and prevent invasion	^Citation97, ^Citation100
VEGF	Up	Calcium phosphate lipid nanoparticles	Photodynamic therapy	SCC-4 and SAS cell lines, C57BL/6 mice	Western blot, tumor measurement, hematoxylin and eosin, IHC staining for Ki-67, cleaved caspase-3, α-SMA, CD31, VEGF, TUNEL, liver and kidney function assessment via blood measurement of ASP, ALT, and urea nitrogen	Inhibit angiogenesis	^Citation101
Hif1a	Up	Calcium phosphate lipid nanoparticles	Photodynamic therapy	SCC-4 and SAS cell lines, female nude mice	Western blot, Texas Red-DNA oligos, MTT, qRT-PCR, IHC, TUNEL assay, DAB detection kit, liver toxicity with ALT, AST, total bilirubin, kidney toxicity with creatinine, blood urea nitrogen, blood total albumin, protein, globulin, LDH, creatine kinase	Inhibit angiogenesis via hypoxic-related pathways	^Citation102
Bag3	Up	Gold nanorods	Photodynamic therapy	CAL 27 cell line, BALB/c nude mice	TEM, MTT, RT-PCR, Western blot, fluorescence microscopy, flow cytometry, IHC, TUNEL assay	Implicated in heat- shock response	^Citation55

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; EMT, epithelial to mesenchymal transition; FITC, fluorescein isothiocyanate; LDH, lactate dehydrogenase; PEG, polyethylene glycol; PEI, polyethylenimine; PI, propidium iodine; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; siRNA, small interfering RNA; TEM, transmission electron microscope; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VEGF, vascular endothelial growth factor; α-SMA, α-smooth muscle actin; XTT, 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide.

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