Micro-ribonucleic acid143(MiR-143)inhibits oral squamous
cell carcinoma(OSCC)cell migration and invasion by downregulation of phospho-c-Met through targeting CD44v3
Ping Xu,DDS,MS,Yan Li,DDS,MS,Shuyong Yang,DDS,MS,Haiyuan Yang,DDS,MS,
Juan Tang,DDS,MS,and Mingzhe Li,DDS,MS
Objective.The aim of this study was to investigate the roles and mechanisms of Micro-ribonucleic acid143(miR-143)in oral squamous cell carcinoma cells.
Study Design.Following the detection of miR-143expression,cell proliferation,migration,and invasion assays and Western blot analysis were conducted in the oral squamous cell carcinoma(OSCC)cell lines.
Results.We found that the expression of miR-143was signi?cantly decreased in the OSCC cell lines(SCC-4,Tca-8113, CAL-27)and tumor tissues.Meanwhile,miR-143was signi?cantly correlated with the migration and invasion in OSCC cell lines.Further investigation revealed that the expression level of miR-143was opposite to that of its potential target
gene d CD44v3and was also related to phospho-c-Met activation.
Conclusions.miR-143could exert signi?cantly suppressive effects on the ability of migration and invasion in OSCC cell lines, and the mechanism of this might be related to the activity of phospho-c-met though the CD44v3/HGF signal.miR-143could thus provide new applications for the treatment of OSCC.(Oral Surg Oral Med Oral Pathol Oral Radiol2015;-:1-9)
Oral squamous cell carcinoma(OSCC)is the sixth most frequently encountered malignant neoplasm,accounting for about90%of all oral malignancies.1Despite advancements in diagnostic and therapeutic procedures, OSCC continues to have a poor prognosis and remains a lethal disease,with a mortality rate of more than50% annually.2The majority of patients with OSCC died of tumor metastasis and recurrence,which suggests that the invasion potential is strongly related to the poor prognosis.3,4Therefore,the identi?cation of new molecular targets in the invasion and metastasis of OSCC is urgently required to develop therapeutic approaches for oral cancer.5
Micro-ribonucleic acids(miRNAs)are a class of abundant,small,noncoding RNAs that negatively regulate gene expression at the posttranscriptional level by inhibiting ribosome function,decapping the5’cap structure,deadenylating the poly(A)tail,and degrading the target.6In recent years,message RNA(mRNA)have been demonstrated to play critical roles in myriad cellular function and biologic processes,including cell growth, cell signal transduction,cellular differentiation,and even apoptosis.7Overexpression of oncogenic miRNAs or underexpression of tumor suppressor miRNAs plays a critical role in tumorigenesis.8MicroRNA143(miR-143)has been previously reported to be downregulated in various types of tumors,including colorectal,9 bladder,10and non e small lung cancers.11To the best of our knowledge,miR-143has always been shown to act as a tumor suppressor and closely related to metastases of malignant tumors.Although several target genes that are regulated by miR-143have been identi?ed in recent years,the molecular mechanism of miR-143still remains unknown.
In this study,we aimed to reveal the biologic func-tion of miR-143and the mechanism of miR-143that may be involved in OSCC.Our study shows that miR-143is a negative regulator of migration and invasion in OSCC cell lines,and we further found that miR-143could inhibit the migration and invasion of OSCC cells by suppressing the expression of the phospho-c-Met signal pathway by targeting CD44v3. MATERIALS AND METHODS
Cell lines and tissue specimens
Human OSCC cell lines CAL-27and SCC-4were purchased from American Type Culture Collection (ATCC)and were properly maintained in our labora-tory.Human oral Tca-8113cell lines and human normal oral keratinocytes cell lines(hNOK)were pur-chased from the Cell Bank of Chinese Academy of
Department of Stomatology,Chengdu Military General Hospital,
NO.270,Rongdu Road,Jingniu Zone,Chengdu,610038,Sichuan, P.R.China.
Received for publication Nov15,2014;returned for revision Jan11, 2015;accepted for publication Feb20,2015.
ó2015Elsevier Inc.All rights reserved.
2212-4403/$-see front matter
https://www.doczj.com/doc/a011333534.html,/10.1016/j.oooo.2015.02.486
Statement of Clinical Relevance Downregulation of Micro-ribonucleic acid143 (miR-143)was correlated with cell migration and invasion and can be a potential prognostic biomarker.
1
Vol.-No.-Month2015
Sciences(Shanghai,China)and were authenticated by
short tandem repeat analysis and polymerase chain
reaction(STR-PCR).All cell lines were cultured with
the complete DMEM-F12medium(Invitrogen)with
10%heat-inactivated fetal bovine serum(FBS,Gibco),
100units/mL of penicillin G sodium,and100m g/mL streptomycin sulfate(Sigma)in a humidi?ed atmo-
sphere containing5%CO2at37 C.We randomly ob-
tained49OSCC specimens and the corresponding
adjacent normal tissue from the Department of Stoma-
tology.This study was approved by the Institutional
Review Board and the Human Ethics Committee of the
Chengdu Military General Hospital.The histologic
diagnosis of tumors was made and agreed upon by at
least2senior pathologists at the Department of Pa-
thology,based on World Health Organization(WHO)
criteria.None of the included patients had received
chemotherapy and radiotherapy,and informed consent
was obtained from each patient before the surgery.
Tissue samples were?ash frozen and stored in liquid
nitrogen.
Quantitative real-time PCR analysis
Total RNA for quantitative real-time PCR(qRT-PCR)
analysis was extracted with the use of the74104
Rneasy Mini Kit(Qiagen)and reverse-transcribed into
cDNA with the reverse transcriptase MMLV(Takara),
according to the manufacturer’s protocol.qRT-PCR
was performed by using SYBR Green Reagents(Bio-
Rad)on the iQ5Real-Time PCR Detection System
(Bio-Rad).For analysis of miR-143expression by qRT-
PCR,reverse transcription and PCR were carried out by
using Bulge-Loop miRNA qPCR Primer Set for hsa-
miR-143(RiboBio,China)and U6snRNA(RiboBio,
China),according to the manufacturer’s instructions,
using the following parameters:95 C for20s followed
by50cycles of95 C for10s and55 C for20s.The
relative expression level of miR-143was normalized to
that of internal control U6by using2-DD Ct cycle
threshold method.
Cell transfection with miR-143
Cells were transfected with pVAX-miR-143expression
vector or blank vector pVAX(constructed plasmids
was purchased from Ribobio Life Technologies,
GuangZhou,China)by using Lipofectamine2000Re-
agent(Invitrogen,Carlsbad,CA),according to the
manufacturer’s protocol.
Cell proliferation assay
The in vitro cell viability of Tca-8113and CAL-27cells
was transfected with pVAX or pVAX-miR-143,ac-
cording to the manufacturer’s protocol.The MTT assay (Sigma)was used to determine relative cell growth
every24hours.Plates were incubated at37 C for4
hours,then the medium was replaced by0.1mL of
DMSO(Sigma),and the plates shaken at room tem-
perature for10minutes.The absorbance was measured
at490nm.
Cell migration and invasion assay
Cells were resuspended in serum-free medium after
transfection,and cell migration assays were performed
by using millicell chambers(Millipore).The chamber
was cultivated in5%CO2at37 C for24hours after the
suspension cells(200m L,5?104cells)were added to the upper place.Then,0.5%FBS medium with10%FBS
(600m L)was added to the lower chamber.Attached cells in the lower section were stained with0.1%crystal violet 24hours later.The invasion assay was similar to migration assay except that the matrigel was used in the transwell chambers(Millipore),and cells were plated in 200m L of DMEM serum-free at a number of2?105per insert in the upper chamber of the transwell in the inva-sion assay.The migration rate and invasion rate were quanti?ed by counting the migration cells in six random ?elds under a light microscope.
Western blot
Total cell protein was extracted in RIPA buffer(50mM Tris,1.0mM EDTA,150mM NaCl,0.1%SDS,1% Triton X-100,1%sodium deoxycholate,1mM PMSF). Isolation of mitochondrial and cytosolic protein was performed by using the Mitochondria/cytosol Frac-tionation Kit(Milipore,MIT1000).The concentrations of protein were quanti?ed by the DC protein assay kit (Bio-Rad,500-0121).Cellular lysates were resolved on sodium dodecyl sulfate polyacrylamide gel electro-phoresis(SDS-PAGE)and electrophoretically trans-ferred to polyvinylidene di?uoride membranes. Membranes were blocked with a buffer containing Tris (10mmol/L,pH7.4),NaCl(150mmol/L),Tween20 (0.1%),and bovine serum albumin(5%)and then incubated with the primary antibodies at4 C overnight. The antibodies c-Met,p-c-Met,b-actin,CD44v3(all from Cell Signal Technology,Beverly,MA).Subse-quently,the membranes were washed and treated with appropriate secondary antibodies conjugated to horse-radish peroxidase for2hours.The immunoreactivities were visualized by enhanced chemiluminescence re-agents(Millipore,WBKLS0500).b-actin was used as an internal control.
Statistical analysis
All statistical analyses were performed by using SPSS 13.0software.The data were expressed as mean?
ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 2
SD,and one-way ANOVA and an unpaired Student t test were used to determine the signi?cant differ-ences in all the results.The level of signi?cance was set at P<.05.
RESULT
miR-143is downregulated in OSCC tissues and cell lines
Real-Time qRT-PCR was used to identify the expres-sion of miR-143in human OSCC cell lines(Tca-8113, SCC-4,CAL-27)and OSCC cancer tissues.Our result suggested that the expression of miR-143was signi?-cantly decreased in human OSCC cell lines compared with human normal oral keratinocytes(hNOK) (Figure1A).Meanwhile,the expression of miR-143 was measured in49OSCC samples and adjacent normal tissues by using qRT-PCR and normalized to the control U6.As shown in Figure1B,the expression levels of miR-143were signi?cantly reduced in81.6% (40of49patients)of OSCC tumor tissues compared with corresponding adjacent normal tissues.Further investigation revealed that the expression levels of miR-143were not associated with age,gender,pT stage, TNM stage and lymphatic metastasis(Figure1C). These data showed that expression of miR-143was frequently downregulated in OSCC tissues as well as in OSCC cell lines,which indicated that the down-regulation of miR-143might be involved in OSCC carcinogenesis.
miR-143suppresses migration and invasion of OSCC cell lines but has no in?uence on proliferation in vitro
Although the expression of miR-143was signi?cantly reduced in OSCC,the functions of miR-143still remained unknown.To investigate the biologic role of miR-143in OSCC cell lines,the miR-143recombi-nant plasmid(p-miR-143)was constructed,and its ef?ciency in Tca-8113cells was con?rmed.After transfection with p-miR-143for24,48,and72hours, the expression of miR-143was notably increased in Tca-8113cells by qRT-PCR assay compared with the control vector(Figure2A).Subsequently,MTT assay showed that in Tca-8113and CAL-27cells,miR-143 did not obviously alter cell viability after transfection (Figure2B).To test the role of miR-143in the migration and invasion of OSCC cells,Tca-8113and CAL-27cells were transiently transfected with p-miR-143or a blank vector as negative control.Then the migration and invasion transwell assays were per-formed.We found that the migration and invasion abilities were signi?cantly reduced due to the over-expression of miR-143in the OSCC cell lines.The inhibition rate was70.2%and64.3%in Tca-8113cells and64%and67.3%in CAL-27cells compared with the blank control group(Figures2C and2D).All the results showed that miR-143might play an important role in migration and invasion in the OSCC cell lines.
miR-143targets CD44v3and downregulates its expression in OSCC cells
We proved that the expression of miR-143was signi?-cantly reduced in the OSCC cell lines(Tca-8113,SCC-4, CAL-27)and that upregulated miR-143could signi?-cantly inhibit cell migration and invasion in the OSCC cell lines.In addition,Ma Qing-ping et al.also found that the upregulated miR-143could signi?cantly inhibit cell migration and invasion in non e small cell lung cancer by targeting CD44v3.11Thus,to test whether miR-143 could regulate CD44v3in the OSCC cell lines,we examined the potential interaction between miR-143and CD44v3by using Western blot and qPCR analyses.We found that the expression of CD44v3in Tca-8113and CAL-27cells were signi?cantly reduced in p-miR-143 groups at the mRNA and protein levels compared with the blank control group(Figures3A and3B).These data demonstrated that miR-143could inhibit CD44v3 expression.To determine the in?uence of CD44v3on migration and invasion in OSCC cells,CD44v3was knocked down by a speci?c si-CD44v3(GenePharma, Shanghai,China),and through Western blot assay, signi?cantly lower expression of CD44v3was detected in Tca-8113and CAL-27cells after transfection with si-CD44v3(Figure3C).Moreover,compared with Tca-8113and CAL-27transfected with si-NC,the invasion capability of the groups transfected with si-CD44v3was inhibited by87.2%and85.1%,respectively.The migration capability of Tca-8113and CAL-27trans-fected with si-CD44v3groups was inhibited by89.3% and87%,respectively(Figure3D).All these results revealed that CD44v3might be a crucial target of miR-143in the OSCC cell lines and that the mecha-nism of the miR-143e mediated inhibition of the inva-sion and migration in OSCC might be targeting CD44v3. miR-143suppresses the expression of c-Met by targeting CD44v3
c-Met is a proto-oncogene that encodes for the re-ceptor tyrosine kinase and is usually upregulated in a wide variety of tumors to promote tumor growth and metastasis.c-Met was also known as the hepatocyte growth factor(HGF)receptor and associated with a poor prognosis and tumor metastasis.12,13It had been demonstrated that CD44v3could promote HGF-induced phosphorylation of c-Met,whereas HGF itself was able to induce phosphorylation of c-Met moderately.14Moreover,both CD44v3and c-Met are
OOOO ORIGINAL ARTICLE Volume-,Number-Xu et al.
upregulated in OSCC.15-18Our previous studies have shown that the overexpression of miR-143is closely related to invasion and migration ability in the OSCC cell lines.To investigate whether c-Met was associ-ated with the role of miR-143e mediated inhibition of invasion and migration in OSCC cells,the expression level of phosphorylation c-Met was identi ?ed by
using Western blot.As shown in Figure 4A,after transfection with miR-143,the expression level of phospho-c-Met was signi ?cantly decreased in Tca-8113.Furthermore,we found that the expression of phospho-c-Met was obviously downregulated when CD44v3was interfered with by si-CD44v3,which indicated that CD44v3could regulate
phospho-c-Met
Fig.1.miR-143is downregulated in OSCC tissues and cell lines.A ,The expression levels of miR-143in hNOK cells and 3OSCC cell lines (SCC-4,Tca-8113,and CAL-27)were measured by quantitative real-time polymerase chain reaction (qRT-PCR).The values represent the mean ?standard deviation of triplicates (*P <.05).B ,Forty-nine pairs of OSCC tissues and adjacent nonneoplastic oral tissues from patients with OSCC were assessed by qRT-PCR analysis (*P <.05).The values were standardized to the U6expression level.C ,Relationship between clinicopathologic factors and miR-143expression levels.miR-143,micro-ribonucleic acid 143;OSCC,oral squamous cell carcinoma;qRT-PCR,real-time quantitative polymerase chain reaction;hNOK,human normal oral keratinocytes;AN,adjacent nonneoplastic oral tissues.
ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 4
in Tca-8113cells.To go a step further and validate the relationship among miR-143,CD44v3,and p-c-Met,HGF was used as an inducer of phospho-c-Met.We found that HGF could improve the cell ability of invasion and migration in Tca-8113by inducing the expression of phospho-c-Met.However,in the p-miR-143group,the addition of HGF could not improve the ability of cell invasion and
migration
Fig.2.miR-143suppresses the migration and invasion of OSCC cell lines but has no in ?uence on proliferation in vitro.A ,qRT-PCR assay was used to determine the expression level of p-miR-143recombinant after 24,48,72hours transfection in Tca-8113(*P <.05).B ,MTT assays were performed to detect the viability of OSCC cells transfected with p-miR-143,pVAX and blank control.C ,Transwell assays were performed to detect the for cell migration and invasion activity of OSCC cells transfected with p-miR-143,pVAX,and blank control.D ,Relative migration and invaded cells transfected with p-miR-143,pVAX,and blank control.miR-143,micro-ribonucleic acid 143;OSCC,oral squamous cell carcinoma;qRT-PCR,real-time quantitative polymerase chain reaction;p,pVAX empty vector;p-miR-143,pVAX-miR-143recombinant plasmid.
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obviously,probably because miR-143suppressed the expression of phospho-c-Met (Figures 4B and 4C).
DISCUSSION
In this study,we clearly identi ?ed that miR-143was downregulated in primary OSCCs and the OSCC cell lines Tca-8113and CAL-27.Moreover,we char-acterized that miR-143could suppress the expression of phospho-c-Met by targeting CD44v3to attenuate the ability of invasion and migration in OSCC cells.
To our knowledge,miR-143expression is signi ?-cantly decreased in several human malignancies,including lung cancer,19prostate cancer,20colorectal cancer,9and so on.Consistent with previous studies,we found that the expression levels of miR-143was signi ?cantly downregulated in the majority of OSCC tissues and several OSCC cell lines compared with noncancerous counterparts.It was the ?rst time that it was con ?rmed that miR-143was downregulated in oral cancer.We also found that the expression levels
of
Fig.3.miR-143targets CD44v3and downregulates its expression in OSCC cell lines.A ,qRT-PCR assay was used to determine the expression level of CD44v3after 48h of transfection in Tca-8113and CAL-27(*P <.05).B ,The expression level of CD44v3protein transfected with p-miR-143or pVAX was determined by Western blot assay,and then a small interfering RNA (siRNA)targeting CD44v3was used to block the CD44v3protein expression.C ,Transwell assays were performed to detect the migration and invasion activity of Tca-8113and CAL-27cells transfected with p-miR-143,si-CD44v3,and negative control (NC).D ,Relative migration and invasion cells transfected with p-miR-143,si-CD44v3,and NC (*P <.05).miR-143,micro-ribonucleic acid 143;OSCC,oral squamous cell carcinoma;qRT-PCR,real-time quantitative polymerase chain reaction;p,pVAX empty vector;p-miR-143,pVAX-miR-143recombinant plasmid.
ORAL AND MAXILLOFACIAL PATHOLOGY OOOO 6
miR-143were not associated with age,gender,pT stage,TNM stage,and lymphatic metastasis.These ?ndings suggest that miR-143may be a tumor sup-pressor gene in OSCC.
miRNAs,which were frequently altered in various human cancers,could also act either as tumorigenic or anti-tumorigenic molecules in tumorigenesis.8According to previous studies,miR-143might func-tion as a tumor suppressor in human cancers,which was consistent with our primal hypothesis.For example,Zhang et al.found that miR-143decreased the prolif-eration and migration of prostate cancer cells through suppression of KRAS and enhanced cell sensitivity to docetaxel.20Osaki et al.also showed that miR-143could regulate human osteosarcoma cell metastasis by regulating matrix metalloprotease-13expression.21To verify our hypothesis,we validated the functional roles of miR-143in 2different OSCC cell lines d Tca-8113and CAL-27.Cell viability assay demonstrated that the overexpression of miR-143could not inhibit OSCC cell growth,which was also observed recently in non e small cell lung cancer cell lines by Ma-Q.11However,numerous studies have shown that miR-143could certainly in ?uence the tumor cell growth.22Thus,our results suggest that the roles of miR-143might be varied in different types of cancers.Never-theless,overexpression of miR-143in OSCC cells could dramatically suppress the ability of cell migration and invasion in vitro,which was similar to recent observa-tions in osteosarcoma 21and bladder carcinoma.23Taken together,miR-143was demonstrated,for the ?rst time,to exert its inhibitory effect on cell migration and in-vasion in the OSCC cell
lines.
Fig.4.miR-143suppress the expression of c-Met by target-ing CD44v3.A ,Western blot assay was used to determine the expression level of CD44v3,c-Met (total c-MET),and phospho-c-Met after 48hours of transfection in Tca-8113.B ,HGF was used as an inducer of phospho-c-Met and the expression level of CD44v3,c-Met (total c-MET)and phospho-c-Met were detected after 48hours of transfection with p-miR-143and pVAX,with or without HGF (10nM).C ,Transwell assays were performed to detect the migration and invasion activity of Tca-8113transfected with p-miR-143or pVAX with or without HGF (10nM).miR-143,micro-ribonucleic acid 143;pVAX empty vector;p-miR-143,pVAX-miR-143recombinant plasmid;HGF,hepatocyte growth
factor.
Fig. 5.Graphic illustration for the interaction among miR-143,CD44v3,and phospho-c-Met.CD44v3could promote HGF-induced phosphorylation of c-Met,while HGF itself was able to induce phosphorylation of c-Met moder-ately.The transfection of miR-143was signi ?cantly reduced the expression level of CD44v3and downregulated phospho-c-Met.miR-143,micro-ribonucleic acid 143;HGF,hepato-cyte growth factor.
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The mechanism of miR-143in cell migration and invasion in OSCC still remains unknown.Until now, various target genes,including CD44v3,11MACC1,9 MMP-13,24K-Ras,25and BCL-226have been demonstrated to be regulated by miR-143.These target genes were closely correlated with human tumor progression.CD44v3was a variant of the hyaluronate receptor CD44(v1-v10),which was localized at the cellular plasma membrane and was involved in several cell functions.27Previous studies have shown that the high expression CD44v3is associated with cell migration and invasion in breast cancer,28colon cancer,29prostate cancer,30and ovarian cancer.31In our study,we found that in both mRNA and protein levels,the expression of CD44v3was signi?cantly downregulated after transfection with miR-143in Tca-8118and CAL-27.Besides,“we con?rmed that miRNA-143directly targets CD44v3in oral cancer.”Which was consistent with the previous studies. Moreover,we found that when CD44v3was interfered with by si-CD44v3,the ability of invasion and migration was also reduced in Tca-8113cells.Taken together,our results demonstrated that miR-143could regulate CD44v3and affect the ability of invasion and migration in OSCC cells.
It is well known that CD44v3can promote HGF-induced phosphorylation of c-Met14and that abnormal c-Met activation is generally correlated with poor prognosis and metastasis in various human cancers,including oral cancer.32The binding of CD44v3and HGF can promote HGF-induced phos-phorylation of c-Met,which is more ef?cient than single HGF signal.33Because our study showed that the expression of miR-143could downregulate CD44v3in OSCC cell lines,the relationship between miR-143and phospho-c-Met was identi?ed by Western blot.As for the result,we found that the phospho-c-Met was both obviously reduced when transfected with miR-143or si-CD44v3.To further investigate the regulatory mechanism of miR-143,HGF was used to induce the expression of phospho-c-Met in Tca-8118.34We found that HGF could improve the ability of invasion and migration in the p-Tca-8113group by inducing phospho-c-Met.However,in the p-miR-143-Tca-8113 group,adding HGF could not improve invasion and migration abilities,obviously because miR-143sup-pressed the expression of phospho-c-Met.Thus,our results demonstrated that miR-143could suppress the expression of phospho-c-Met by targeting CD44v3to attenuate the ability of invasion and migration in OSCC cells(Figure5).
CONCLUSIONS
Our study indicated that miR-143is signi?cantly downregulated in OSCC and that the overexpression of miR-143could diminish the ability of migration and
invasion in the OSCC cell lines,whereas it had no
signi?cant in?uence on proliferation,as shown for the ?rst time by our study.Furthermore,our study was also the?rst to show that the downregulation of CD44v3
attenuated tumor migration in OSCC cells,which
might be closely related to the activity of c-met though
the CD44v3/HGF signal.Thus,our results demon-
strated that miR-143could suppress the expression of
phospho-c-Met by targeting CD44v3to attenuate the
ability of invasion and migration in OSCC cells.
Finally,this study indicated the potential application of
miR-143in therapy for oral squamous cell carcinoma. The authors are grateful to all study participants.
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Reprint requests:
Mingzhe Li,DDS,MS
Department of Stomatology
Chengdu Military General Hospital
NO.270,Rongdu Road
Jingniu Zone
Chengdu,610038
Sichuan
P.R.China
biolimingzhe@https://www.doczj.com/doc/a011333534.html,
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