microRNA-18a van M2-macrofagen remt TGFBR3 om de progressie van nasofaryngeale carcinoom en tumorgroei te bevorderen via TGF-β-signaleringsroute

Resultaten

Identificatie van M2-macrofagen

De monocyten die waren verrijkt met de hechtende methode en de monocyten die waren geïnduceerd door M-CSF werden verzameld uit perifeer bloed van gezonde donoren. Immunofluorescentiedetectie van CD68, CD206 en CD163 bevestigde dat PBMC's geïnduceerd door M-CSF in vitro veranderden in macrofagen met typische moleculaire kenmerken, die aan onze vereisten voldeden (Fig. 1a).

Identificatie van M2-macrofagen. een . CD68, CD206 en CD163 werden tot expressie gebracht op het oppervlak van macrofagen verkregen na monocytinductie in vitro. b Gepolariseerde macrofagen door IL-4 waren M2-macrofagen. c CD163 werd in hoge mate tot expressie gebracht en CD68 werd laag tot expressie gebracht in M2-macrofagen gepolariseerd door IL-4. d CCL22 werd sterk tot expressie gebracht in M2-macrofagen. e PPAR-γ werd sterk tot expressie gebracht in M2-macrofagen. v miR-18a werd sterk tot expressie gebracht in M2-macrofagen; *P <0,05; **P <0,01. Meetgegevens werden uitgedrukt als gemiddelde ± standaarddeviatie, N =3. Vergelijkingen tussen twee groepen werden geanalyseerd door t testen

De verkregen macrofagen werden gepolariseerd door IL-4 en onder een microscoop bekeken op morfologie. Het bleek dat macrofagen (M0) zonder IL-4-stimulatie divers en onregelmatig waren en een ronde, ovale of spilvorm vertoonden. Gestimuleerd door IL-4 werden M2-macrofagen groter en veranderden ze voornamelijk in een ronde vorm, wat in overeenstemming was met de morfologische kenmerken van M2-macrofagen zoals eerder beschreven [20] (Fig. 1b).

Flowcytometrie testte de oppervlakte-antigenen van de hechtende cellen gestimuleerd door 20 ng / ml IL-4 gedurende 24 uur en vond dat CD68 tot expressie werd gebracht met 21,16%, terwijl CD163 98,69% van het totale aantal cellen was (Fig. 1c), wat bevestigt de hechtende cellen zijn M2-macrofagen. RT-qPCR onthulde dat met M0-cellen daarentegen CCL22 en PPAR-γ (typische polariserende moleculen) stegen in M2-macrofagen (Fig. 1d, e), wat wijst op de succesvolle inductie van M2-macrofagen.

RT-qPCR toonde ook aan dat miR-18a-expressie toenam in M2-macrofagen in tegenstelling tot de M0-macrofagen (P ˂ 0,05) (Fig. 1f).

MiR-18a wordt sterk tot expressie gebracht en TGFBR3 wordt slecht tot expressie gebracht in NPC-cellen

Bioinformatics-website (miRanda) voorspelde de potentiële doelen van miR-18a en TGFBR3 werd beschouwd als een doelwit van miR-18a (Fig. 2a). Dubbele luciferase-reportergentest werd geïmplementeerd om te verifiëren dat miR-18a gericht was op 3'UTR van TGFBR3. TGFBR3-WT of TGFBR3-MUT werden gekloond in de pmirGLO-vector en gecotransfecteerd met miR-18a-nabootsers of NC in SUNE-1- en CNE2-cellen. miR-18a-nabootsers hadden geen invloed op de luciferase-activiteit van TGFBR3 3'UTR-MUT, maar verminderden die van TGFBR3 3'UTR-WT in SUNE-1- en CNE2-cellen, wat suggereert dat TGFBR3 een doelwitgen was dat wordt gereguleerd door miR-18a (Fig. 2b).

MiR-18a wordt sterk tot expressie gebracht en TGFBR3 wordt slecht tot expressie gebracht in NPC-cellen. een miRanda voorspelde miR-18a gericht op TGFBR3. b Dubbele luciferase-reportergentest geverifieerd miR-18a gericht op TGFBR3. c miR-18a-expressie was verhoogd en TGFBR3-expressie was verlaagd in miR-18a-imitaties-getransfecteerde M2-macrofagen. d miR-18a-expressie was verlaagd en TGFBR3-expressie was verhoogd in met miR-18a-remmers getransfecteerde M2-macrofagen. e miR-18a-expressie was verhoogd en TGFBR3-expressie was verlaagd in NPC-cellijnen in vergelijking met NP96-cellen. v miR-18a-expressie was verhoogd en TGFBR3-expressie was verlaagd in miR-18a-nabootsers-getransfecteerde SUNE-1-cellen. g miR-18a-expressie was verlaagd en TGFBR3-expressie was verhoogd in met miR-18a-remmers getransfecteerde CNE2-cellen. u miR-18a-expressie was verhoogd en TGFBR3-expressie was verlaagd in SUNE-1-cellen die samen waren gekweekt met miR-18a-imitaties-getransfecteerde M2-macrofagen. ik miR-18a-expressie was verlaagd en TGFBR3-expressie was verhoogd in CNE2-cellen die samen waren gekweekt met met miR-18a-remmers getransfecteerde M2-macrofagen; *P <0,05; **P <0,01. Meetgegevens werden uitgedrukt als gemiddelde ± standaarddeviatie, N =3. Vergelijkingen tussen twee groepen werden geanalyseerd door t test. Vergelijkingen tussen meerdere groepen werden geanalyseerd door middel van one-way ANOVA, gevolgd door Tukey's post hoc test

De transfectie-efficiëntie van miR-18a-nabootsers of miR-18a-remmers in M2-macrofagen werd gemanifesteerd door bepaling van miR-18a- en TGFBR3-expressie in M2-macrofagen door RT-qPCR en Western-blot-assay. Het was duidelijk dat (Fig. 2c, d) miR-18a-overexpressie verhoogde miR-18a-expressie en verminderde TGFBR3-expressie in M2-macrofagen. Daarentegen verminderde miR-18a-remming de miR-18a-expressie en verhoogde TGFBR-expressie in M2-macrofagen. Er werd geen verschil waargenomen in de miR-18a-expressie, terwijl de TGFBR3-expressie afnam wanneer M2-macrofagen werden getransfecteerd met si-TGFBR3. Er werd geen verschil waargenomen in de miR-18a-expressie, terwijl TGFBR3-expressie groeide na OE-TGFBR3-transfectie naar M2-macrofagen.

miR-18a- en TGFBR3-expressie in CNE2-, TW03-, C666-1-, SUNE-1- en NP96-cellijnen werden getest met RT-qPCR en Western-blot-assay. In NPC-cellen nam, in vergelijking met NP96-cellen, de miR-18a-expressie toe en nam de TGFBR3-expressie af (Fig. 2e). Omdat CNE2-cellen en SUNE-1-cellen zich manifesteerden met de grootste en de kleinste verschillen in miR-18a-expressie van NP96-cellen, werden ze geselecteerd voor respectievelijk de voortgaande miRNA-downregulatie- en opregulatie-assays.

Om de effecten van miR-18a en TGFBR3 op NPC-cellen te identificeren, werden SUNE-1-cellen getransfecteerd met miR-18a-nabootsers of si-TGFBR3, terwijl CNE2-cellen met miR-18a-remmers of OE-TGFBR3. RT-qPCR en Western blot-test toonden aan dat miR-18a verhoogde miR-18a-expressie en verminderde TGFBR3-expressie in SUNE-1-cellen nabootst. Transfectie van si-TGFBR3 had geen effect op miR-18a-expressie, terwijl TGFBR3-expressie in SUNE-1-cellen verminderde. miR-18a-remmers verminderden miR-18a-expressie en verhoogde TGFBR3-expressie in CNE2-cellen. OE-TGFBR3-transfectie in CNE2-cellen had geen invloed op miR-18a-expressie maar verhoogde TGFBR3-expressie (Fig. 2f, g).

Om de effecten van miR-18a van M2-macrofagen op NPC-cellen te onderzoeken, werden miR-18a-nabootsers- of si-TGFBR3-getransfecteerde M2-macrofagen of miR-18a-remmers- of OE-TGFBR3-getransfecteerde M2-macrofagen samen gekweekt met SUNE-1 of CNE2-cellen in de Transwell-kamer, respectievelijk. RT-qPCR en Western blot-test testten miR-18a- en TGFBR3-expressie in SUNE-1- of CNE2-cellen. SUNE-1-cellen die samen waren gekweekt met niet-getransfecteerde of miR-18a-nabootsers-getransfecteerde M2-macrofagen, vertoonden verhoogde miR-18a-expressie en gedegradeerde TGFBR3-expressie. Er werd geen verschil opgemerkt in de miR-18a-expressie en TGFBR3-expressie verminderd in SUNE-1-cellen die samen waren gekweekt met si-TGFBR3-getransfecteerde M2-macrofagen (Fig. 2h). Na co-cultuur met niet-getransfecteerde M2-macrofagen, werden CNE2-cellen gekenmerkt door verhoogde miR-18a-expressie en verlaagde TGFBR3-expressie. Echter, verminderde miR-18a-expressie en verhoogde TGFBR3-expressie gepresenteerd in CNE2-cellen die van tevoren samen waren gekweekt met miR-18a-remmers-getransfecteerde M2-macrofagen. Er werd geen verschil herkend in de miR-18a-expressie en TGFBR3-expressie nam toe in CNE2-cellen die samen waren gekweekt met OE-TGFBR3-getransfecteerde M2-macrofagen (Fig. 2i).

miR-18a van M2-macrofagen bevordert de levensvatbaarheid van NPC-cellen en het vermogen om kolonies te vormen

MTT-test en kolonievormingstest werden toegepast om de effecten van miR-18a en TGFBR3 op de levensvatbaarheid en het kolonievormende vermogen van SUNE-1-cellen en CNE2-cellen te identificeren. miR-18a-nabootsers, miR-18a-remmers, si-TGFBR3 of OE-TGFBR3 werden getransfecteerd in SUNE-1-cellen of CNE2-cellen. Er werd aangegeven dat in SUNE-1-cellen miR-18a-upregulatie of TGFBR3-downregulatie de levensvatbaarheid van de cellen en een verhoogd aantal kolonies verhoogde (Fig. 3a, e). In CNE2-cellen was de levensvatbaarheid van de cellen verminderd en het aantal kolonies werd verminderd door miR-18a-remming of TGFBR3-overexpressie (Fig. 3b, f).

miR-18a van M2-macrofagen induceert de levensvatbaarheid van NPC-cellen en kolonievormend vermogen. een miR-18a bootst of si-TGFBR3 de levensvatbaarheid van SUNE-1-cellen na. b miR-18a-remmers of OE-TGFBR3 verminderden de levensvatbaarheid van CNE2-cellen. c Co-cultuur met miR-18a bootst- of si-TGFBR3-getransfecteerde M2-macrofagen verhoogde de levensvatbaarheid van SUNE-1-cellen. d Co-cultuur met miR-18a-remmers of OE-TGFBR3-getransfecteerde M2-macrofagen verminderde de levensvatbaarheid van CNE2-cellen. e miR-18a bootst of si-TGFBR3 verhoogd kolonieaantal SUNE-1-cellen na. v miR-18a-remmers of OE-TGFBR3 verlaagden het aantal kolonies van CNE2-cellen. g Co-cultuur met miR-18a bootst- of si-TGFBR3-getransfecteerde M2-macrofagen verhoogde het aantal kolonies van SUNE-1-cellen. u Co-cultuur met miR-18a-remmers of OE-TGFBR3-getransfecteerde M2-macrofagen verminderde het aantal kolonies van CNE2-cellen; *P <0,05; **P <0,01. Meetgegevens werden uitgedrukt als gemiddelde ± standaarddeviatie, N =3. Vergelijkingen tussen meerdere groepen werden geanalyseerd door eenrichtings-ANOVA, gevolgd door Tukey's post-hoctest

Om de effecten van miR-18a van M2-macrofaag op de levensvatbaarheid en kolonievormingsvermogen van NPC-cellen te onderzoeken, werden miR-18a-nabootsers-, miR-18a-remmers-, si-TGFBR3- of OE-TGFBR3-getransfecteerde M2-macrofagen samen gekweekt met SUNE-1-cellen of CNE2-cellen in de Transwell-kamer. MTT en kolonievormingstest toonden aan dat SUNE-1-cellen die samen waren gekweekt met niet-getransfecteerde, of miR-18a-nabootsers-getransfecteerde of si-TGFBR3-getransfecteerde M2-macrofagen werden gepresenteerd met versterkte cellevensvatbaarheid en een verhoogd aantal kolonies (Fig. 3c, g) .

CNE2-cellen die samen waren gekweekt met niet-getransfecteerde M2-macrofagen werden gemarkeerd met versterkte cellevensvatbaarheid en verhoogde kolonies. CNE2-cellen die samen waren gekweekt met ofwel met miR-18a-remmers getransfecteerde of OE-TGFBR-getransfecteerde M2-macrofagen vertoonden echter verminderde levensvatbaarheid van de cellen en verminderde kolonies (Fig. 3d, h).

miR-18a van M2-macrofagen bevordert NPC-celinvasie en migratiemogelijkheden

Voor een beter begrip van hoe miR-18a en TGFBR3 de migratie en invasie van NPC-cellen beïnvloedden, werden krastest en Transwell-assay geïmplementeerd. De resultaten onthulden dat SUNE-1-cellen getransfecteerd met miR-18a-nabootsers of si-TGFBR3 werden gekenmerkt door verhoogde celmigratieafstand en invasiecellen (Fig. 4a en 5a).

miR-18a van M2-macrofagen bevordert het vermogen tot NPC-celmigratie. een miR-18a bootst of si-TGFBR3 verhoogde migratie van SUNE-1-cellen na. b miR-18a-remmers of OE-TGFBR3 verminderden de migratie van CNE2-cellen. c Co-culture with miR-18a mimics- or si-TGFBR3-transfected M2 macrophages increased migration of SUNE-1 cells. d Co-culture with miR-18a inhibitors or OE-TGFBR3-transfected M2 macrophages decreased migration of CNE2 cells; *P <0.05; **P <0.01. Measurement data were expressed as mean ± standard deviation, N =3. Comparisons among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post hoc test

miR-18a from M2 macrophages promotes NPC cell invasion ability. een miR-18a mimics or si-TGFBR3 increased invasion of SUNE-1 cells. b miR-18a inhibitors or OE-TGFBR3 decreased invasion of CNE2 cells. c Co-culture with miR-18a mimics- or si-TGFBR3-transfected M2 macrophages increased invasion of SUNE-1 cells. b Co-culture with miR-18a inhibitors or OE-TGFBR3-transfected M2 macrophages decreased invasion of CNE2 cells; *P <0.05; **P <0.01. Measurement data were expressed as mean ± standard deviation, N =3. Comparisons among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post hoc test

In CNE2 cells transfected with miR-18a inhibitors or OE-TGFBR3, the reductions appeared in cell migration distance and invasion cells (Figs. 4b and 5b).

How miR-18a from M2 macrophages influenced invasion and migration abilities of NPC cells were deciphered by miR-18a mimics-, miR-18a inhibitors-, si-TGFBR3-, or OE-TGFBR3-transfected M2 macrophages co-culturing with SUNE-1 or CNE2 cells. The results highlighted that SUNE-1 cells co-cultured with untransfected, or miR-18a mimics-transfected or si-TGFBR3-transfected M2 macrophages were manifested with increased cell migration distance and invasion cells (Figs. 4c and 5c).

Both cell migration distance and invasion cells increased in CNE2 cells co-cultured with untransfected M2 macrophages. Upon co-culture with miR-18a inhibitors-transfected or OE-TGFBR3-transfected M2 macrophages, CNE2 cells were showed with reduced migration distance and invasion cells (Figs. 4d and 5d).

miR-18a from M2 Macrophages Arrests Fewer NPC Cell at G0/G1 Phase and Suppresses Apoptosis

Cell cycle distribution and apoptosis were tested by flow cytometry to stratify the effects of miR-18a and TGFBR3 on NPC cells. It was indicated that transfection of miR-18a mimics or si-TGFBR3 reduced SUNE-1 cells arrested in the G0/G1 phase, increased cells in the S and G2/M phases, and reduced cell apoptosis rate (Figs. 6a and 7a).

miR-18a from M2 macrophages arrests fewer NPC cell at G0/G1 phase. een miR-18a mimics or si-TGFBR3 decreased SUNE-1 cells in the G0/G1 phase, and increased SUNE-1 cells in the S and G2/M phases. b miR-18a inhibitors or OE-TGFBR3 increased CNE2 cells in the G0/G1 phase, and decreased CNE2 cells in the S and G2/M phases. c Co-culture with miR-18a mimics- or si-TGFBR3-transfected M2 macrophages decreased SUNE-1 cells in the G0/G1 phase, and increased SUNE-1 cells in the S and G2/M phases. d Co-culture with miR-18a inhibitors or OE-TGFBR3-transfected M2 macrophages increased CNE2 cells in the G0/G1 phase, and decreased CNE2 cells in the S and G2/M phases; *P <0.05; **P <0.01. Measurement data were expressed as mean ± standard deviation, N =3. Comparisons among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post hoc test

miR-18a from M2 macrophages inhibits NPC cell apoptosis. een miR-18a mimics or si-TGFBR3 decreased apoptosis of SUNE-1 cells. b miR-18a inhibitors or OE-TGFBR3 increased apoptosis of CNE2 cells. c Co-culture with miR-18a mimics- or si-TGFBR3-transfected M2 macrophages decreased apoptosis of SUNE-1 cells. d Co-culture with miR-18a inhibitors or OE-TGFBR3-transfected M2 macrophages increased apoptosis of CNE2 cells; *P <0.05; **P <0.01. Measurement data were expressed as mean ± standard deviation, N =3. Comparisons among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post hoc test

Upon transfection with miR-18a inhibitors or OE-TGFBR3, CNE2 cells in the G0/G1 phase trended toward an elevation while those in the S and G2/M phases toward a reduction, and cell apoptosis rate raised (Figs. 6b and 7b).

With the purpose to decode the mechanism of miR-18a from M2 macrophages in NPC cell cycle distribution and apoptosis, M2 macrophages transfected with miR-18a mimics, miR-18a inhibitors, si-TGFBR3, or OE-TGFBR3 were co-cultured with SUNE-1 cells or CNE2 cells in the Transwell chamber. Co-cultured with untransfected, or miR-18a mimics-transfected or si-TGFBR3-transfected M2 macrophages, reduced SUNE-1 cells were displayed in the G0/G1 phase and increased cells in the S and G2/M phases, and SUNE-1 cell apoptosis rate decreased (Figs. 6c and 7c).

Co-cultured with untransfected M2 macrophages, CNE2 cells in the G0/G1 phase reduced, cells in the S and G2/M phases increased, and apoptosis rate declined. On the contrary, co-cultured with miR-18a inhibitors-transfected or OE-TGFBR3-transfected M2 macrophages, CNE2 cells in the G0/G1 phase elevated while those in the S and G2/M phases decreased, and apoptosis rate elevated (Figs. 6d and 7d).

miR-18a from M2 Macrophages Reduces p-Smad1/t-Smad1 and Elevates p-Smad3/t-Smad3 in NPC Cells

Western blot assay detected TGF signaling pathway-related proteins in NPC cells to further explain the effects of miR-18a and TGFBR3 on TGF signaling pathway.

It was explained that transfection of miR-18a mimics or si-TGFBR3 reduced p-Smad1/t-Smad1 and elevated p-Smad3/t-Smad3 in SUNE-1 cells (Fig. 8a).

miR-18a from M2 macrophages decreases p-Smad1/t-Smad1 and increases p-Smad3/t-Smad3 in NPC cells. een miR-18a mimics or si-TGFBR3 in SUNE-1 cells decreased p-Smad1/t-Smad1 and elevated p-Smad3/t-Smad3. b miR-18a inhibitors or OE-TGFBR3 in CNE2 cells increased p-Smad1/t-Smad1 and decreased p-Smad3/t-Smad3. c Co-culture with miR-18a mimics- or si-TGFBR3-transfected M2 macrophages decreased p-Smad1/t-Smad1 and elevated p-Smad3/t-Smad3. d Co-culture with miR-18a inhibitors or OE-TGFBR3-transfected M2 macrophages increased p-Smad1/t-Smad1 and decreased p-Smad3/t-Smad3; *P <0.05; **P <0.01. Measurement data were expressed as mean ± standard deviation, N =3. Comparisons among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post hoc test

Transfection with miR-18a inhibitors or OE-TGFBR3 led to declined p-Smad3/t-Smad3 and increased p-Smad1/t-Smad1 in CNE2 cells (Fig. 8b).

miR-18a from M2 macrophages influencing TGF signaling pathway in NPC cells was determined by Western blot assay through testing TGF signaling pathway-related proteins in SUNE-1 cells and CNE2 cells which had co-cultured with miR-18a mimics-, miR-18a inhibitors-, si-TGFBR3-, or OE-TGFBR3-transfected M2 macrophages in the Transwell chamber.

SUNE-1 cells co-cultured with untransfected, miR-18a mimics-transfected, or si-TGFBR3-transfected M2 macrophages were manifested with reduced p-Smad1/t-Smad1 and incremental p-Smad3/t-Smad3 (Fig. 8c).

Co-cultured with untransfected M2 macrophages, CNE2 cells trended toward declined p-Smad1/t-Smad1 and elevated p-Smad3/t-Smad3. In an opposite way, CNE2 cells were demonstrated with increased p-Smad1/t-Smad1 and decreased p-Smad3/t-Smad3 when co-cultured with miR-18a inhibitors-transfected or OE-TGFBR3-transfected M2 macrophages (Fig. 8d).

miR-18a from M2 Macrophages Induces Tumor Growth in Nude Mice with NPC

Tumor xenografts were conducted on nude mice to further elucidate the impacts of miR-18a and TGFBR3 on tumor growth of NPC.

It was indicated that injected with miR-18a mimics-transfected or si-TGFBR3-transfected SUNE-1 cells, mice were manifested with enlarged tumor volume and heavier tumor weight (Fig. 9a).

miR-18a from M2 macrophages promotes tumor growth in nude mice with NPC. een miR-18a mimics or si-TGFBR3 in SUNE-1 cells increased tumor volume and weight. b miR-18a inhibitors or OE-TGFBR3 in CNE2 cells decreased tumor volume and weight. c Co-culture with miR-18a mimics- or si-TGFBR3-transfected M2 macrophages increased tumor volume and weight. d Co-culture with miR-18a inhibitors or OE-TGFBR3-transfected M2 macrophages decreased tumor volume and weight; *P <0.05; **P <0.01. Measurement data were expressed as mean ± standard deviation, three nude mice in each group. Comparisons among multiple groups were analyzed by one-way ANOVA, followed by Tukey’s post hoc test

Reduced tumor volume and weight were presented in mice with injection of miR-18a inhibitors-transfected or OE-TGFBR3-transfected CNE2 cells (Fig. 9b).

Tumor growth was observed in mice which had injected with miR-18a mimics-, miR-18a inhibitors-, si-TGFBR3-, or OE-TGFBR3-transfected M2 macrophages to illustrate the mechanism of miR-18a from M2 macrophages in NPC.

After co-culture with untransfected, miR-18a mimics-transfected or si-TGFBR3-transfected M2 macrophages, SUNE-1 cells were injected into mice and mice were observed with larger tumor volume and heavier tumor weight (Fig. 9c).

CNE2 cells were co-cultured with M2 macrophages and injected into mice with the results suggesting growing tumor volume and weight. Both tumor volume and weight were inclined to reduce when mice were injected with CNE2 cells which had co-cultured with miR-18a inhibitors-transfected or OE-TGFBR3-transfected M2 macrophages (Fig. 9d).

Discussion

NPC refers to a polygenic disease threatened by a wide range of factors [21]. MiRNAs are previously implied to participate in the pathogenesis of NPC via regulation of their target genes which are indicators of cellular processes and pathways [22]. Concretely, miR-18a advances NPC progression by miRNA biogenesis impairing [6]. Given that this study goes forward to decipher the combined interactions of miR-18a from M2 macrophages and TGFBR3 in NPC with the conclusion elucidating that miR-18a from M2 macrophages stimulates NPC progression via TGFBR3 inhibition (Fig. 10).

Schematic representation of macrophage-derived exosomal miR-18a in NPC and the involvement of TGFBR3-mediated TGF-β signaling pathway

At the start of this study, macrophages are stimulated by IL-4 to differentiate to M2 macrophages which are found to enrich the expression of miR-18a. As we know, during macrophage polarization, miRNA’s expression was altered [23]. In addition, M2 polarization enriches genes which are involved in the cell cycle and metabolic processes, and the M2 phenotype is conducive to tumor growth and angiogenesis in neoplastic tissues [24]. Based on the M2 macrophages-enriched miR-18a, a series of experiments were successfully conducted.

Initially, our study has uncovered that miR-18a is highly expressed while TGFBR3 is poorly expressed in NPC cells. Drawn from a previous study, it is concluded that miR-18a is overexpressed in NPC tissues with its association with lymph node metastasis and clinical stage [5]. Besides that, miR-17-92 cluster members including miR-18a are documented to be overexpressed in NPC tissues [25]. Furthermore, upregulated miR-18a is reported to demonstrate in NPC tissues which is connected with tumor node metastasis stage and tumor size [4]. Experimentally, except for the downregulated TGFBR3 in tongue squamous cell carcinoma [26], there has been another study depicting reduced TGFBR3 in clear-cell renal cell carcinomas accompanied by unwanted prognosis [27]. Anyhow, the results in this study are consistent with these study findings to some extent.

In order to explore the roles of miR-18a and TGFBR3 in NPC cell progression, we have conducted a series of assays with the results indicating that upregulated miR-18a or downregulated TGFBR3 triggers NPC cell progression while miR-18a repression or TGFBR3 elevation has the opposite effects on NPC cells. Widely, suppression of miR-18a is evidenced to hamper cell progression in malignancies including ovarian cancer, colitis-associated colorectal cancer, and hepatocellular carcinoma [28,29,30]. Narrowly, an existed study has pronounced that upregulated miR-18a promotes NPC cell progression via mediation of DICER1 [6]. In addition, it is noticed that overexpressed miR-18a in NPC is believed to connect with NPC metastasis and repressed miR-18a partially contributes to better prognosis of NPC patients [31]. Lately, it is surveyed that downregulation of miR-18a is capable of discouraging NPC proliferation, invasion, and migration [4]. Additionally, a decrease in TGFBR3 expression is regarded to link with laryngeal squamous cell carcinoma (LSCC) invasion and miR-223/TGFBR3 axis regulation takes part in LSCC progression inhibition [32]. TGFBR3 elevation is documented to restrict NPC cell viability, induce apoptosis, and activate pro-apoptosis signaling pathways [14]. Previously, a study has indicated that upregulation of TGFBR3 promotes apoptosis and cells arrested in the G2/M phase, resulting in impaired cell viability and migration in salivary gland adenoid cystic carcinoma [33]. Intriguingly, it is formerly described that induction of TGFBR3 contributes to disrupt intrahepatic cholangiocarcinoma progression [34].

Despite the protective role of lowly expressed miR-18a and overexpressed TGFBR3 in NPC cell in vitro, we have performed tumor xenografts in nude mince in vivo for further verification with the results explaining that miR-18a knockdown or TGFBR3 elevation restrains tumor growth in nude mice. As demonstrated in a prior study, miR-18a-injected nude mice show with enhanced tumor growth [5] and conversely, the miR-18a antagomir-injected nude mice are displayed with suppressed tumor growth in NPC [4]. In the light of the TGFBR3 reduction in tumor growth, it is suggested that poorly expressed TGFBR3 provokes tumor formation in clear-cell renal cell carcinoma [27]. In the opposite way, an increase in TGFBR3 is recognized to hinder tumor growth in lung cancer with the presence of long non-coding RNA ADAM metallopeptidase with thrombospondin type 1 motif, 9 antisense RNA 2 elevation, and miR-223-3p suppression [35]. This study has also predicted and verified that TGFBR3 is a target gene of miR-18a. But, more studies still need to be conducted for further verification.

Conclusion

Generally speaking, this study elaborates the concrete mechanisms that miR-18a from M2 macrophages inhibits TGFBR3 expression to exacerbate the progression of NPC via TGF-β signaling pathway, the results of which is abrogated by miR-18a knockdown or TGFBR3 elevation. This study updates the therapeutic target for NPC. However, a large cohort researches are still in need for in-depth explorations.

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