colorectal cancer ( crc ) is the second leading cause of cancer related death worldwide , making it a fine and attractive area for oncologists to study ( 1 , 2 ) . in numerous cancers including crc ,
it has been documented that aberration of the growth factor signaling pathways specifically transforming growth factor- ( tgf- ) pathway play a vital role in tumor initiation , progression and metastasis ( 3 - 7 ) .
therefore , it has become a proper target for cancer therapy ( 8 - 13 ) .
it is has been accepted that a major growth factor such as tgf- , which has a wide range of effects on key physiological events , must be under extended regulation to control its expression and function .
this regulation should include mechanisms that allow a variety of effects depending on special cellular and tissue contexts ( 14 - 16 ) .
recently , a new layer of cellular mechanisms as microrna ( mirna)-mediated gene expression has been identified that is implicated in the post transcriptional negative regulation ( 17 - 20 ) .
the mirnas were well - known to interact with signaling pathway components and to involve in multiple cellular processes as well as initiation , progression and metastasis of human cancers ( 21 - 25 ) .
increasing evidences have revealed that cancer - associated mirnas can function as oncogenes or tumor suppressors ( 18 , 20 ) .
the mirnas - mediated regulation networks can strictly influence the growth factors signaling pathways ( 24 , 25 ) .
there are several studies showing relationship of tgf- with diverse micrornas ( 26 - 29 ) .
in addition , it has been shown that basic cell signaling pathways adjust the activity of the related components in mirnas biogenesis pathway to achieve a fine mirnas expression pattern ( 28 ) .
recent emerging evidence suggests manipulation of growth factors signaling by special inhibitors can alter mirnas expression in cancer cells in vivo and in vitro ( 30 - 35 ) .
several strategies based on either restoration of silenced mirnas or inhibition of overexpressed mirnas has opened a new area of research in cancer therapy including crc .
it has been proposed that restoration of normal equilibrium for cancer - related mirnas can inhibit colon tumor progression ( 36 , 37 ) . on the other hand ,
several reports imply that targeting of tgf- signaling pathway at late stages of carcinogenesis could be a helpful tool for treatment of human cancers such as crc , glioblastoma and breast cancer ( 4 , 8) .
these studies show that a series of tgri kinase inhibitors such as sd-208 could be influential in treatment of a range of cancers ( 12 ) .
our aim was to investigate the mirnas ( oncogene or tumor suppressor ) , whose expression might be altered due to inhibition of tgf- signaling pathway .
sw-48 cell line , a human colon adenocarcinoma cell line ( pasteur institute , tehran , iran ) was grown in 25 cm flask ( spl life sciences ; south korea ) containing rpmi-1640 medium ( gibco ; germany ) supplemented with 5% fetal bovine serum ( fbs ) ( gibco ; germany ) and 100 units / ml penicillin ( gibco ; germany ) .
briefly , the cells were trypsinized in exponential growth phase and were seeded in 6-well flat - bottom plates ( spl life sciences ; south korea ) at a density of 510 cells / well ( 2000 l media / well ) .
48 hr after treatment with 0.5 , 1 and 2 m concentrations of sd-208 , the cells were harvested for total rna extraction . the protocol for establishing the xenograft model of colon cancer
was approved by the committee on the ethics of animal experiments of tehran university of medical sciences ( ethical code number ; erc / s/277 ) as previously described ( 6 ) .
6-week - old female athymic c56bl/6 nude mice ( n= 8 per group ) were obtained from omid institute for advanced biomodels ( tehran , iran ) .
after cell inoculation , xenograft tumors were allowed to achieve a size of 80 mm .
then , the animals were randomly divided into two groups of 8 to receive either sd-208 ( 50 mg / kg / d ) or vehicle ( dmso - containing deionized water ) orally for three weeks .
obtained tumors after isolating from animals , were fixed in formalin or frozen for histological staining and rna extraction , respectively . to confirm colon adenocarcinoma ,
tumor tissues tissues were excised and subjected to hematoxylin and eosin ( h&e ) staining ( dako , denmark ) as previously described ( 6 ) .
either sw-48 cells or tumor tissues at the mentioned time points after treatment with sd-208 , were subjected for total rna extraction using trizol reagent ( invitrogen ; germany ) according to the manufacturer s instructions . extracted total rna
four mirnas as potential onco - mirs ( mir-21 , 31 , 20a , 135b ) and four mirnas as potential suppressor - mirs ( let7-g , mir-133b , 145 , 200c ) involved in colon cancer , were selected from the sanger center mirna registry at http://www.sanger.ac.uk/software/rfam/mirna/index.shtml . microrna expression was analyzed by real - time quantitative polymerase chain reactions ( qpcr ) using the sybr green method ( parsgenom , iran ) .
after polyadenylation of total rna and cdna synthesis , mirnas were expanded by the specific primers for mature forms according to the manufacturer s instructions .
real - time pcr was performed on a bio rad cfx96 real - time pcr system .
rnu6b was used as an endogenous ( internal ) control , and the data were normalized compared to this housekeeping gene .
all reactions were performed in triplicate and the absence of contamination was verified using non - template controls .
the standard error of means was computed and analysis of variance ( anova , tukey s post tests ) completed via graphpad prism 5.0 software .
p - values less than 0.05 were considered to indicate statistically significant differences between data sets .
sw-48 cell line , a human colon adenocarcinoma cell line ( pasteur institute , tehran , iran ) was grown in 25 cm flask ( spl life sciences ; south korea ) containing rpmi-1640 medium ( gibco ; germany ) supplemented with 5% fetal bovine serum ( fbs ) ( gibco ; germany ) and 100 units / ml penicillin ( gibco ; germany ) .
briefly , the cells were trypsinized in exponential growth phase and were seeded in 6-well flat - bottom plates ( spl life sciences ; south korea ) at a density of 510 cells / well ( 2000 l media / well ) .
48 hr after treatment with 0.5 , 1 and 2 m concentrations of sd-208 , the cells were harvested for total rna extraction .
the protocol for establishing the xenograft model of colon cancer was approved by the committee on the ethics of animal experiments of tehran university of medical sciences ( ethical code number ; erc / s/277 ) as previously described ( 6 ) .
6-week - old female athymic c56bl/6 nude mice ( n= 8 per group ) were obtained from omid institute for advanced biomodels ( tehran , iran ) .
after cell inoculation , xenograft tumors were allowed to achieve a size of 80 mm .
then , the animals were randomly divided into two groups of 8 to receive either sd-208 ( 50 mg / kg / d ) or vehicle ( dmso - containing deionized water ) orally for three weeks .
obtained tumors after isolating from animals , were fixed in formalin or frozen for histological staining and rna extraction , respectively .
to confirm colon adenocarcinoma , tumor tissues tissues were excised and subjected to hematoxylin and eosin ( h&e ) staining ( dako , denmark ) as previously described ( 6 ) .
either sw-48 cells or tumor tissues at the mentioned time points after treatment with sd-208 , were subjected for total rna extraction using trizol reagent ( invitrogen ; germany ) according to the manufacturer s instructions . extracted total rna was stored at -80 c until use .
four mirnas as potential onco - mirs ( mir-21 , 31 , 20a , 135b ) and four mirnas as potential suppressor - mirs ( let7-g , mir-133b , 145 , 200c ) involved in colon cancer , were selected from the sanger center mirna registry at http://www.sanger.ac.uk/software/rfam/mirna/index.shtml .
microrna expression was analyzed by real - time quantitative polymerase chain reactions ( qpcr ) using the sybr green method ( parsgenom , iran ) .
after polyadenylation of total rna and cdna synthesis , mirnas were expanded by the specific primers for mature forms according to the manufacturer s instructions .
real - time pcr was performed on a bio rad cfx96 real - time pcr system .
rnu6b was used as an endogenous ( internal ) control , and the data were normalized compared to this housekeeping gene .
all reactions were performed in triplicate and the absence of contamination was verified using non - template controls .
data analysis was performed using the 2-cct method . the standard error of means was computed and analysis of variance ( anova , tukey s post tests ) completed via graphpad prism 5.0 software .
p - values less than 0.05 were considered to indicate statistically significant differences between data sets .
to assess the potential toxicity effects of sd-208 , the expression levels of mirnas was examined by real time rt - pcr ( table 1 ) .
then the appropriate numbers of sw-48 cells were injected into 8 mice to develop tumors .
following sd-208 treatment period , we could not observe any changes in animal behavior , body weight or lifespan compared to controls .
these data suggest that sd-208 lacks toxic effects on animals ( data not shown ) .
mirnas differentially expressed in sw-48 cells after 48 hr treatment with sd-208 colon adenocarcinoma confirmation the h&e staining confirmed marked cellularity with significant hyperchromatism and pleomorphism ( figure 1 ) .
the pattern of adenocarcinoma was alike to the human origin ( figure 1 a , b ) .
nude mice bearing developed sw-48 tumors were divided into two groups : sd-208 treatment ( a ) and vehicle ( b ) .
hematoxylin and eosin staining of tumor tissues confirmed colon adenocarcinoma in both treated ( a ) and non - treated ( b ) tumor - bearing mice .
the staining demonstrates marked cellularity with profound hyperchromatism and pleomorphism ( arrows ) and low differentiated tumor cells similar to human colon cancer expression pattern of selected onco / suppressor mirnas in colon adenocarcinoma sw-48 cell line real - time pcr analysis detected differentially expression of all selected mirnas in sw-48 cell line ( table 2 ) . among the studied mirnas , mirna-135b and let7-g expressed at the highest and lowest levels , respectively ( figure 2 ) .
mirnas differentially expressed in sw-48-derived tumors after treatment with sd-208 electrophoresis of mirnas genes pattern expressed in sw-48 cell line .
as shown , all selected onco / suppressor mirnas express differentially in sw-48 cell line alteration of the mirnas expression resulted by sd-208 treatment evaluation of mirnas expression by q - pcr showed that the expression of mir-135b significantly down - regulated after treatment by sd-208 ( p=0.006 , figure 3a ) . in the tumors treated with sd-208 ,
mir-135b expression also was down - regulated significantly ( p=0.018 ) , compared to control tumors ( figure 3b ) .
our results showed that all the treated tumors express a lower number of mir-135b , but not other mirnas , compared to the control tumors .
however , there was no change in cell proliferation or tumor size ( data not shown ) .
the expression of mir-135b significantly was down - regulated either in cell culture ( a ) or developed tumors ( b ) in sd-208 groups compared to controls prediction of the target genes of mir-135b using bioinformatics analysis in order to determine the biological function of the down - regulated mirna , mir-135b , we further predicted the putative downstream targets of this mirna .
we focused our attention on mir-135b because it was the only mirna affected by sd-208 either in vitro or in vivo . in silico analysis using targetscan ( http://www.targetscan.org/ ) showed that mir-135b potentially targets transcripts encoding known tumor - suppressor factors , such as apc ( adenomatosis polyposis coli ) , foxo1 ( forkhead transcription box1 ) , runx1 ( runt - related transcription factor 1 ) and esrra ( estrogen - related receptor alpha ) .
to assess the potential toxicity effects of sd-208 , the expression levels of mirnas was examined by real time rt - pcr ( table 1 ) .
then the appropriate numbers of sw-48 cells were injected into 8 mice to develop tumors .
following sd-208 treatment period , we could not observe any changes in animal behavior , body weight or lifespan compared to controls .
these data suggest that sd-208 lacks toxic effects on animals ( data not shown ) .
mirnas differentially expressed in sw-48 cells after 48 hr treatment with sd-208 colon adenocarcinoma confirmation the h&e staining confirmed marked cellularity with significant hyperchromatism and pleomorphism ( figure 1 ) .
the pattern of adenocarcinoma was alike to the human origin ( figure 1 a , b ) .
nude mice bearing developed sw-48 tumors were divided into two groups : sd-208 treatment ( a ) and vehicle ( b ) .
hematoxylin and eosin staining of tumor tissues confirmed colon adenocarcinoma in both treated ( a ) and non - treated ( b ) tumor - bearing mice .
the staining demonstrates marked cellularity with profound hyperchromatism and pleomorphism ( arrows ) and low differentiated tumor cells similar to human colon cancer expression pattern of selected onco / suppressor mirnas in colon adenocarcinoma sw-48 cell line real - time pcr analysis detected differentially expression of all selected mirnas in sw-48 cell line ( table 2 ) . among the studied mirnas , mirna-135b and let7-g expressed at the highest and lowest levels , respectively ( figure 2 ) .
mirnas differentially expressed in sw-48-derived tumors after treatment with sd-208 electrophoresis of mirnas genes pattern expressed in sw-48 cell line .
as shown , all selected onco / suppressor mirnas express differentially in sw-48 cell line alteration of the mirnas expression resulted by sd-208 treatment evaluation of mirnas expression by q - pcr showed that the expression of mir-135b significantly down - regulated after treatment by sd-208 ( p=0.006 , figure 3a ) . in the tumors treated with sd-208 ,
mir-135b expression also was down - regulated significantly ( p=0.018 ) , compared to control tumors ( figure 3b ) .
our results showed that all the treated tumors express a lower number of mir-135b , but not other mirnas , compared to the control tumors .
however , there was no change in cell proliferation or tumor size ( data not shown ) .
the expression of mir-135b significantly was down - regulated either in cell culture ( a ) or developed tumors ( b ) in sd-208 groups compared to controls prediction of the target genes of mir-135b using bioinformatics analysis in order to determine the biological function of the down - regulated mirna , mir-135b , we further predicted the putative downstream targets of this mirna .
we focused our attention on mir-135b because it was the only mirna affected by sd-208 either in vitro or in vivo . in silico analysis using targetscan ( http://www.targetscan.org/ ) showed that mir-135b potentially targets transcripts encoding known tumor - suppressor factors , such as apc ( adenomatosis polyposis coli ) , foxo1 ( forkhead transcription box1 ) , runx1 ( runt - related transcription factor 1 ) and esrra ( estrogen - related receptor alpha ) .
in this study , we evaluated the effect of a tgf- receptor kinase inhibitor , sd-208 , on some potential onco / suppressor - mirnas expression in crc .
sd-208 is a tgf- signaling pathway inhibitor that could exert anticancer effects on several tumor cells by reduction of growth rate or modification of other cell functions ( 6 , 9 , 12 ) .
some investigators revealed sd-208 reduces tumor growth and metastasis in different cancers ( 9 , 12 ) , whereas , others showed this agent regulates the growth of tumors without changes in proliferation , apoptosis or angiogenesis and can not reduce proliferation of cells ( 8) .
another study reported that sd-093 ( as an sd-208 analog ) failed to alter morphology and growth rate of pancreatic carcinoma cells ( 38 ) .
also it has been reported that sd-208 has no effect on the growth of primary and metastatic r3 t mammary tumors in athymic nude mice ( 9 ) .
these findings indicate that anti - cancer effect of sd-208 may be not due to suppression of cell growth and proliferation ( 36 ) . since efficacy of this inhibitor is a controversial issue in cancer treatment , we hypothesized the unknown mechanism(s ) including alteration of cancer - related mirnas might be involved .
several chemopreventive agents have been shown to modulate the expression of numerous mirnas in cancer cells that lead to sensitization of cancer cells to chemotherapeutic agents ( 39 , 40 ) , suggesting the potential of mirnas as targets for anti - cancer drugs ( 18 , 20 ) .
hence , evaluation of the possible effects of chemotherapeutic drugs on the expression profile of mirnas may have an important outcome for cancer therapy strategies ( 21 , 33 ) .
although a number of studies have shown that chemotherapy drugs alter mirnas expression in many cancer cells , there is no report on the effect of tgf- receptor kinase inhibitors on mirnas in human crc .
we selected some mirnas that have already been confirmed to function as onco - mir ( mir-21 , mir-31 , mir-20a , and mir-135b ) or suppressor mir ( mir-133b , mir-145 , mir-200 and let7-g ) in crc ( 36 - 39 ) .
the present study focused on the alteration of these mirnas in colon cancer treated by sd-208 .
expression analysis of mirnas by a q - pcr revealed that expression of mir-135b significantly down - regulated after in vitro treatment with sd-208 ( p=0.006 ) . in the tumor tissues treated with sd-208 , mir-135b expression significantly
the results showed that all the treated tumors significantly expressed a lower number of mir-135b , but not other oncogenic - mirnas , compared to controls ( p=0.018 ) .
interestingly , the mir-135b has been documented as a tumor promoting factor and to play a role in migration and metastasis in different cancers as well as crc ( 37 - 39 ) . in order to address the question of how can the possible molecular mechanisms of the mir-135b on crc cell signaling pathways be defined
, we performed an in - silico study . in silico analysis using targetscan ( http://www.targetscan.org/ ) showed that mir-135b potentially targets key tumor - suppressor genes involved in crc : apc and foxo1 .
apc and foxo1 genes have also been validated as targets of mir-135b using luciferase reporter assay ( 41 ) , hence , we would like to discuss these genes whose functions could potentially affect the cell signaling in crc .
apc gene acts a tumor suppressor the inactivation of which is the key initiating event in colorectal carcinogenesis ( 42 ) .
recently , one study demonstrated that up - regulation of mir-135b in crc is associated with low apc mrna levels ( 42 ) . in the present study
, we advocate a novel molecular mechanism : a kinase inhibitor molecule can reduce mir-135b expression , by which apc activation could be mediated .
moreover , it has been reported that mir-135b affects foxo1 as an endogenous target and suppresses protein expression .
several lines of evidence indicate that foxo transcription factors might play an important role in tumor development ( 41 , 43 ) . on the other hand , foxo1 alteration by mir-135b can affect sensitivity to chemotherapeutic drugs , such that tumor cells overexpressing mir-135b were more resistant to specific anticancer drugs .
these results suggest the possibility that mir-135b may confer chemoresistance to tumor cells through foxo1 modulation ( 41 ) .
therefore , it is rational to hypothesize that the properties of anti - cancer drugs may be related to their alteration of mirna profiles ( 43 , 44 ) .
for example , suppression of mir-21 has been shown to sensitize mcf-7 cells to topotecan ( 44 ) . as well , 5-fu was reported to be able to modify the expression of several mirnas in human colon cancer cells ( 44 ) .
overall , we primarily predicted that combination of sd-208 and one anticancer drug such as 5-fu , may show stronger inhibition of colon tumor cell growth by modification of onco - mirs .
additionally , as a novel approach in crc therapy , pre - treatment by sd-208 could improve chemosensitivity in resistant cancerous cells . however , the mechanism by which these agents alter mirna expression may be dependent on genomic context .
the receptor kinase inhibitor sd-208 may partially inhibit colon tumorigenesis as well as chemoresistance by alteration of mirnas .
hence , the tgri kinase inhibitor - based treatment may possibly uphold chemosensitivity in resistant colon cancer cells .
 objective(s):transforming growth factor- ( tgf- ) is involved in colorectal cancer ( crc ) . 
 the sd-208 acts as an anti - cancer agent in different malignancies via tgf- signaling . this work aims to show the effect of manipulation of tgf- signaling on some mirnas implicated in crc.materials and methods : we investigated the effects of sd-208 on sw-48 , a colon adenocarcinoma cell line . the cell line was treated with 0.5 , 1 and 2 m concentrations of sd-208 . 
 then , the xenograft model of colon cancer was established by subcutaneous inoculation of sw-48 cell line into the nude mice . 
 the animals were treated with sd-208 for three weeks . 
 a quantitative real - time pcr was carried out for expression level analysis of selected oncogenic ( mir-21 , 31 , 20a and 135b ) and suppressor - mirnas ( let7-g , mir-133b , 145 and 200c ) . 
 data were analyzed using the 2-ct method through student s t - test via the graphpad prism software.results:our results revealed that sd-208 could significantly down - regulate the expression of one key onco - mirna , mir-135b , in either sw-48 colon cells ( p=0.006 ) or tumors orthotopically implanted in nude mice ( p=0.018 ) . 
 our in silico study also predicted that sd-208 could modulate the expression of potential downstream tumor suppressor targets of the mir135b.conclusion:our data provide novel evidence that anticancer effects of sd-208 ( and likely other tgf- inhibitors ) may be owing to their ability to regulate mirnas expression . 