autophagy , a common mechanism in cell homeostasis and degradation , has emerged to play a critical role in crohn 's disease ( cd ) . genome - wide association studies ( gwas ) revealed that polymorphisms in autophagy - associated genes such as autophagy - related 16-like 1 ( atg16l1 ) and immunity - related gtpase family m ( irgm ) were risk loci for cd [ 1 , 2 ] . autophagy shows the inhibitory role in inflammasome activation , as indicated by the evidence that downregulation of atg16l1 leads to increased interleukin- ( il- ) 1 expression in a mouse model of cd . a cohort study suggested that atg16l1 loci variant displayed increased levels of proinflammatory cytokine il-1 and il-6 in humans . another study indicated that pro - il-1 could be specifically sequestered into autophagosomes in macrophages stimulated by toll - like receptor ligands . however , polymorphisms in nucleotide oligomerization domain 2 ( nod2 ) remain the most prominent genetic risk factor among cd - associated risk loci identified so far [ 6 , 7 ] . in recent years , several studies have linked the cd - associated nod2 mutations to autophagy appearance via the interaction of nod2 with atg16l1 [ 8 , 9 ] . besides , both cd - associated nod2 and atg16l1 mutative cd patients are defective in autophagy induction , bacterial trafficking , and antigen presentation . during bacterial invasion , nod2 and atg16l1 will interact together at bacterial entry sites , initiating the collection of the atg16-atg5-atg12 complex to the autophagosomes . however , mutant cd - associated nod2 ( l1007fsinsc ) failed to recruit atg16l1 to the plasma membrane . this evidence links nod2 and autophagy - related genes to innate immune responses against bacterial invasion , showing that the nod2-atg16l1 signaling axis disorder contributes to the deficiency of autophagy in cd . studies have highlighted that autophagy plays the critical role in maintaining intestinal homeostasis , and dysfunction of autophagy seems to be a risk factor in the onset of chronic intestinal inflammation . however , most of the autophagy - related polymorphisms were found in specific region or ethnicity . atg16l1 polymorphism can not be confirmed as one of the susceptibility loci in cd patients from asian countries [ 11 , 12 ] . the selective effects on the cell biology and specialized regulatory properties of atg16l1 or autophagy in cd patients from asia are still unclear . contactin associated protein - like 3 ( cntnap3 ) is a gene located in chromosome 9p13.1 . protein encoded by cntnap3 gene is a member of ncp family ( neuroxin - iv / cntnap / paranodin ) of cell - recognition molecules , a distinct subgroup of the neurexins which mediates neuron - glial interactions . due to our preliminary data , cntnap3 expression was found upregulated in the intestinal tissue of the patients with cd . however , it is not known how cntnap3 contributes to atg16l1 or autophagy in intestinal biology or cd pathogenesis . thus , in this paper we try to figure out if cntnap3 participates in the process of autophagy or atg16l1 pathway . a total of fifteen patients with active cd and fifteen healthy controls ( hc ) were enrolled in this study from september 2010 to february 2015 . the present research was approved by the research ethics committee of renji hospital , school of medicine , shanghai jiao tong university . written informed consents were obtained from all subjects before recruitment . patients were diagnosed based on the clinical manifestations , endoscopy , and pathology , confirmed by two gastroenterologists . tissue samples were obtained from inflamed segments of colons in patients and regions without pathological changes in hc . at the same day of colonoscopy , patients provided a fasting blood sample for measurement of serum c - reactive protein ( crp ) and erythrocyte sedimentation rate ( esr ) . hela cell line and sw620 cell line were obtained from the american type culture collection ( atcc ) and maintained in the shanghai institute of digestive disease . both hela and sw620 cells were grown in dulbecco 's modified eagle medium ( dmem ) ( gibco ) with 10% iron - supplemented calf serum ( hyclone ) , streptomycin ( 50  g / ml ) ( invitrogen ) , and penicillin ( 50  u / ml ) ( invitrogen ) at 37c with 5% co2 supplement . to downregulate the cntnap3 expression , small interfering ( si)rna oligos were purchased from genepharma . these sirna oligopairs were cntnap3-homo-2039 ( 5-cgucugggcuuuacuauautt-3 and 5-auauaguaaagcccagacgtt-3 ) and cntnap3-homo-1311 ( 5-ggaaauguguccuucucautt-3 and 5-augagaaggacacauuucctt-3 ) . the negative control pairs were 5-uucuccgaacgugucacgutt-3 and 5-acgugacacguucggagaatt-3. the cntnap3 cdna was amplified and inserted into pcdna 3.1 vector ( invitrogen ) to establish pcdna 3.1-cntnap3 for overexpression purpose . the plasmids were amplified in one shot top10f ' chemically competent cells ( invitrogen ) following the manufacturer 's guide and purified with purelink hq mini plasmid purification kit ( invitrogen ) . cntnap3 sirnas or pcdna 3.1-cntnap3 were transfected into hela cells , respectively , with lipofectamine 2000 transfection reagent ( invitrogen ) . total rna from biopsy specimens and cells was extracted using cell culture and tissue total rna extraction and preparation mini kit ( slnco ) according to the manufacturer 's instruction . the quantity and quality of rna were confirmed with a nanodrop 1000 ( nanodrop , thermo scientific - waltham ) . the primers were designed using primer premier 5.0 software and synthesized from generay biotech co. , ltd : cntnap3 ( forward 5-tcgccacccaaggaggatat-3 , reverse 5-tcaaagggaggctggagtctgt-3 ) ; atg16l1 ( forward 5-aggacagggagatgcagatga-3 , reverse 5-gattggcttcctgggcttt-3 ) ; -actin ( forward 5-gtcttcccctccatcgtg-3 , reverse 5-agggtgaggatgcctctctt-3 ) . for gene - specific reverse transcription , quantitative real - time pcrs were conducted on a stepone plus device ( applied biosystems ) with sybr premix ex taq kit ( takara ) . cell lysates were prepared using ripa buffer ( sigma ) containing protease inhibitors ( roche ) , subsequently agitated on ice for 30 minutes . pierce bca protein assay kit ( pierce ) was used to measure the protein concentration . protein electrophoresis was performed with mini - protea iii ( bio - rad ) . in 10% polyacrylamide gels ( tris / glycine ) , proteins were separated and transferred onto a polyvinylidene fluoride membrane ( bio - rad ) . antibodies were applied against cntnap3 ( rabbit polyclonal anti - cntnap3 , 1  :  1000 , sigma - aldrich ) , atg16l1 ( rabbit polyclonal anti - atg16l1 , 1  :  1000 , abcam ) , and gapdh ( rabbit polyclonal anti - gapdh ; 1  :  2500 , abcam ) . hela and sw620 cells seeded in six- or twelve - well plates were transfected with pselect - gfp - lc3 ( invitrogen ) using lipofectamine reagent ( invitrogen ) following the manufacturer 's instructions . typically , 1  10  cells / ml well in six - well plates was transfected with 0.5  g plasmids . transient transfections with cdnas were performed with lipofectamine 2000 to label autophagic vacuoles with gfp - lc3 plasmid for 24 to 72 hours . images were obtained using a fluorescent microscope ( gfp filter - ex470/40vs495 , em515/30 ; zeiss ) and gfp - lc3 punctuated dots were determined for triplicates . quantification of the average number of gfp - lc3 punctuated dots per cell was performed as previously mentioned . p < 0.05 was considered to be significant with either anova analysis or t - test . a total of fifteen cd patients and fifteen healthy controls were enrolled in the present experiment . after we included the cd patients and hcs , we found that cd patients showed a trend of lower body mass index ( bmi ) . expression of cntnap3 and atg16l1 in colonic tissue from cd patients and hcs were detected by qrt - pcr , showing that both cntnap3 ( p = 0.029 ) and atg16l1 ( p = 0.024 ) were highly expressed in cd patients compared to those in hcs ( figure 1 ) . interestingly , spearman correlation analysis indicated that atg16l1 and cntnap3 mrna expressions were significantly associated with the serum levels of crp ( r = 0.6238 for atg16l1 , p = 0.013 ) ( r = 0.5711 for cntnap3 , p = 0.026 ) ( figures 2(a ) and 2(b ) ) . cntnap3 also significantly expressed correlation with the atg16l1 transcriptomic level ( r = 0.8631 , p < 0.001 ) ( figure 2(c ) ) . unfortunately , it was found that neither cntnap3 ( p = 0.273 ) nor atg16l1 ( p = 0.231 ) was significantly correlated with esr ( figures 2(d ) and 2(e ) ) . given the evidence that cntnap3 elevated expression was closely associated with atg16l1 , we subsequently changed the expression pattern of cntnap3 by knocking down and overexpression , to determine the downstream target , namely , atg16l1 in hela and sw620 cells . seventy - two hours after transfection , the level of cntnap3 was measured by qrt - pcr ( figures 3(a ) and 3(b ) ) and validated with western blot ( figures 3(c ) and 3(d ) ) . compared with cells transfected with empty vectors , cntnap3 was significantly higher in cntnap3 overexpression cells ( hela , p = 0.001 and sw620 , p = 0.014 ) and lower in cntnap3 knockdown cells ( hela , p = 0.013 and sw620 , p = 0.002 ) . there was no significant difference between cells transfected with empty vectors and normal controls ( hela , p = 1.000 and sw620 , p = 0.794 ) . results showed that atg16l1 was lower in cntnap3 knockdown cells than that in cells transfected with empty vectors ( hela , p = 0.013 and sw620 , p = 0.005 ) ( figures 3(a ) and 3(b ) ) . on the other hand , atg16l1 was significantly higher in cells transfected with cntnap3 overexpression plasmid than that in cells transfected with empty vectors ( hela , p = 0.001 and sw620 , p = 0.052 ) ( figures 3(a ) and 3(b ) ) . there was no significant difference between cells transfected with empty vectors and normal controls ( hela , p = 0.630 and sw620 , p = 0.517 ) . similarly , western blot also indicated that atg16l1 protein expression was significantly associated with the expression of cntnap3 manipulations . atg16l1 protein expression significantly decreased or increased 72 hours after cntnap3 was interfered by sirna and cntnap3 overexpressed , respectively ( figures 3(c ) and 3(d ) ) . to determine whether autophagy can be regulated by cntnap3 , hela and sw620 cells were cotransfected with lc3-gfp plasmid and subsequently observed using fluorescent microscope 72 hours after cntnap3 modulation . it was shown that autophagy vacuoles significantly increased ( hela , p = 0.161 and sw620 , p = 0.036 ) ( figures 4(a ) ( iii ) and 4(b ) ( iii ) ) and decreased ( hela , p = 0.003 and sw620 , p = 0.004 ) ( figures 4(a ) ( iv ) and 4(b ) ( iv ) ) after cntnap3 overexpression and sirna interference compared with cells transfected with empty vectors ( figures 4(a ) ( ii ) and 4(b ) ( ii ) ) . no significant difference was found between empty vector group and normal control ( hela , p = 0.066 and sw620 , p = 0.166 ) . in hela cells , average number of gfp - lc3 punctuated dots per cell of cntnap3 overexpression group was 5.434  0.2658 . it was 1.507  0.08273 in cntnap3 knockdown group and 3.714  0.3373 in empty vector group . in sw620 cells , the average number of gfp - lc3 punctuated dots per cell of cntnap3 overexpression group was 5.365  0.3412 . it was 1.989  0.05458 in cntnap3 knockdown group and 3.899  0.3226 in empty vector group ( figures 4(a ) and 4(b ) ) . atg16l1 is a classic gene that is associated with the development of crohn 's disease . in early research , hampe et al . identified that atg16l1 variant was associated with susceptibility to crohn 's disease . and then a nonsynonymous snp was identified to show association of a threonine - to - alanine substitution ( t300a ) in atg16l1 . with atg16l1 ( t300a ) , the innate immune cells can not be triggered by the specific microbial structures to form the autophagosomes . although atg16l1 polymorphisms can lead to subsequent bacterial persistence in the gut mucosa , 25% of caucasian populations with the risk allele do not have severe gut inflammation . on the other hand , many patients with crohn 's disease do not have the risk allele atg16l1 ( t300a ) . in italian , brazilian , moroccan , japanese , and korean   populations , atg16l1 ( t300a ) although atg16l1 ( t300a ) was found in asian populations , the relationship of the variants and asian crohn 's disease is still to be confirmed . in some other autoimmune diseases , atg16l1 expression in colonic mucosal samples from cd patients was found to be increased , and mrna expression of atg16l1 was correlated with crp of patients . this phenomenon showed that atg16l1 played an important role in the course of the disease , and the relationship between atg16l1 and cntnap3 showed that cntnap3 might be of importance in autophagy process of crohn 's disease . as newly reported in gut related disease , cntnap3 is a distinct subgroup of the neurexins . it was first found in nervous system , the function of cntnap3 remaining undiscovered . in our study , we found cntnap3 was highly expressed in colonic samples from crohn 's disease patients , and it might increase the autophagy process in cell models . as most reports have confirmed , variants of atg16l1 gene were related to autophagy deficiency . atg16l1 was found to work together with atg12 and atg5 in the formation of autophagosomes , of which the process was dependent on the correct localization of atg16l1 to sites of lc3-lipidation . in our research . with the increase of cntnap3 and atg16l1 , autophagosomes increased in hela and sw620 cells subsequently . bacterial immunity was found disabled in autophagy deficiency related to mutations of atg16l1 or nod2 [ 26 , 27 ] . in our research , autophagosomes increased without the stimulation of bacteria . necroptosis , a programmed cell death which is believed to link with the pathogenesis of ibd , shows the features of necrosis , including mitochondrial swelling and extensive vacuole formation . atg16l1 was well correlated with the crp values in patients indicating that atg16l1 might be a biomarker for the severity of the disease .
autophagy is a common physiological process in cell homeostasis and regulation . 
 autophagy - related gene mutations and autophagy disorders are important in crohn 's disease ( cd ) . 
 the nucleotide oligomerization domain 2autophagy genes autophagy 16-like 1 ( nod2atg16l1 ) signaling axis disorder contributes to the dysfunction of autophagy . 
 this paper is focused on the relationship between contactin associated protein - like 3 ( cntnap3 ) and atg16l1 expression in crohn 's disease . 
 the results indicated that the expression of atg16l1 is higher in some cd patients compared to normal controls . 
 atg16l1 was well correlated with the c - reactive protein ( crp ) in some cd patients . in vitro study 
 revealed that cntnap3 could upregulate the expression of atg16l1 and increase autophagy vacuoles .