Prognostic significance of microrna 200c in various types of cancer: An updated meta analysis of 34 studies

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1 MOLECULAR AND CLINICAL ONCOLOGY 4: , 2016 Prognostic significnce of microrna 200c in vrious types of cncer: An updted met nlysis of 34 studies JIA YI ZHANG 1*, YA MIN WANG 1*, LE BIN SONG 2*, CHEN CHEN 1, YI CHUN WANG 1 nd NING HONG SONG 1 1 Deprtment of Urology, The First Affilited Hospitl with Nnjing Medicl University; 2 The First Clinicl Medicl College of Nnjing Medicl University, Nnjing , P.R. Chin Received Jnury 21, 2016; Accepted Mrch 18, 2016 DOI: /mco Abstrct. Previous studies hve indicted tht mir 200c is promising cncer biomrker. However, different studies hve presented conflicting results. Therefore, the im of the present study ws to perform met nlysis of mir 200c bsed on 34 relevnt studies. The Mterils nd methods sections of ppers were crefully identified using the dtbses PubMed, Web of Science nd Embse for publictions up to December 4, Pooled hzrd rtios (HRs) nd 95% confidence intervls (95% CIs) were systemticlly clculted to investigte the ssocition between the expression of mir 200c nd cncer prognosis. The results demonstrted tht elevted expression levels of mir 200c indicted significntly worse overll survivl rtes (HR=1.37, 95% CI: 1.01, 1.85), nd high level of mir 200c ws considered n indictor of n unfvorble prognosis in ptients from Europe nd Americ (HR=1.85, 95% CI: 1.27, 2.69). Furthermore, overexpression of mir 200c ws significntly ssocited with progression of the disese in the subgroups of tissue nd blood smples (HR=0.68 nd 2.45, respectively), nd inferior overll survivl rtes for the blood subgroup were reveled (HR=2.21, 95% CI: 1.04, 4.72). In ddition, mir 200c ws of prognostic vlue in severl disese subgroups. Tken together, high expression levels of mir 200c re of significnt prognostic vlue in vrious humn mlignncies. Introduction Cncer hs become the primry cuse of mortlity in the mjority of countries nd regions worldwide, nd the incidence of cncer hs incresed substntilly in recent yers (1). In 2012, 14.1 million new cncer cses, 8.2 million cncer mortlities nd 32.6 million Correspondence to: Dr Ning Hong Song, Deprtment of Urology, The First Affilited Hospitl with Nnjing Medicl University, 300 Gungzhou Rod, Nnjing , P.R. Chin E mil: doctorurology@yeh.net * Contributed eqully Key words: cncer, mir 200c, prognosis, hzrd rtio, met nlysis individuls living with cncer (within 5 yers of dignosis) were reported worldwide. Specificlly, 57% (8 million) of new cncer cses, 65% (5.3 million) of cncer mortlities nd 48% (15.6 million) of the 5 yer prevlent cncer cses occurred in less developed regions (2). Due to the difficulty of erly dignosis nd the low survivl rte of multiple cncer types, relible biomrkers tht re ssocited with the dignosis or prognosis of cncer re urgently required. MicroRNAs re conserved smll non coding RNAs with length of ~18 25 nucleotides, which regulte the expression of trget genes nd exert vitl roles in vrious biologicl processes (3,4). They were first identified in 1993 (5). Therefter, n understnding of their roles in the cell cycle, poptosis, prolifertion nd differentition hs gretly dvnced (6,7). Furthermore, severl micrornas were identified tht function s either oncogenes or tumor suppressors, nd the expression levels of certin micrornas were ssocited with the degree of mlignncy (8 10). Due to their good stbility nd unique expression profiles in humn mlignncies, micrornas hold gret promise s conceivble biomrkers for cncer dignosis nd prognosis (11,12). MicroRNA 200c (mir 200c), the most representtive member of the microrna 200 fmily, hs been widely investigted during the lst few yers. mir 200c ws reveled to exert criticl role in the regultion of epithelil to mesenchyml trnsition (EMT) nd mesenchyml to epithelil trnsition (MET) (13,14). In ddition, there hve been numerous studies demonstrting the ssocition between n berrnt expression level of mir 200c nd the prognosis of vrious humn mlignncies, including endometril cncer (8,9), gstric cncer (13,15 17), ovrin cncer (18 21), cler cell renl cell crcinom (ccrcc) (22 24), brest cncer (25 28), colorectl cncer (14,29), non smll cell lung cncer (NSCLC) (30 35), prostte cncer (36), esophgel cncer (37 39), diffuse lrge B cell lymphom (40), bldder cncer (41,42) nd pncretic cncer (43). Approximtely hlf of these studies verified the nti oncogenic function of mir 200c in certin cncer types, indicting the potentil correltion of elevted expression levels of mir 200c nd superior prognosis (13 15,18,20 22,28,29,31,35,41 43). However, other studies hve provided opposing evidence, suggesting tht mir 200c serves s n oncogene (23-27,36-39). Therefore, mir 200c is noteworthy biomrker for cncer prognosis, nd met nlysis of its precise role is required. To clrify

2 934 ZHANG et l: PROGNOSTIC VALUE OF mir-200c IN VARIOUS TYPES OF CANCER the vlue of mir 200c s prognostic biomrker, dt from studies of mir 200c in vrious cncer types were systemticlly collected nd evluted. Mterils nd methods Serch strtegy. Relevnt studies were identified by crefully serching the online dtbses PubMed, Web of Science nd Embse up to December 4th, The following combintion of keywords ws simultneously pplied for the literture serch: microrna 200c or microrn 200c or mirna 200c or mir 200c nd tumor or cncer or crcinom or neoplsm or mlignncies. In ddition, the following criteri for the study chrcteristics were used to improve the serch further: i) English lnguge publictions; ii) studies tht concentrted on ptients with mlignncies; nd iii) studies tht demonstrted the ssocition of mir 200c expression with cncer prognosis. This comprehensive online serch ws independently performed by two uthors (Ji Yi Zhng nd Y Min Wng). Inclusion nd exclusion criteri. The present met nlysis ws performed strictly following the guidelines of the Preferred Reporting Items for Systemtic Reviews nd Met Anlyses (PRISMA) sttement (44). Articles were considered eligible if they met the following criteri: i) the expression level of mir 200c hd been ssessed in tissue or peripherl blood smples from cncer ptients; ii) dichotomous ctegoriztion of expression levels of mir 200c hd been investigted ccording to cut off vlue; nd iii) n investigtion hd been mde of the ssocition of expression levels of mir 200c with survivl rtes or recurrence, together with corresponding hzrd rtio (HR) or survivl curve. If more thn one rticle hd been published on the identicl study cohort, only the most comprehensive study ws selected for the present met nlysis. In ddition, letters, review rticles nd experiments on nimls were excluded. A flow digrm of the study selection process with further detils is shown in Fig. 1. Dt extrction. All eligible studies were identified by Y Min Wng nd Le Bin Song, nd uncertin dt were ressessed by Ning Hong Song. The dt extrction included the following elements: i) the first uthor nd publiction yer; ii) chrcteristics of the studied popultion, including ptient ntionlity, number, men or medin ge, disese type nd stge, nd smple exmined; iii) study design, ssy method nd cut off definition; iv) HRs of elevted expression levels of mir 200c for cncer specific survivl (CSS), overll survivl (OS), recurrence free survivl (RFS), progression free survivl (PFS) nd disese free survivl (DFS); nd v) men or medin follow up durtion. If HRs were not directly reported in the studies, then the dt were extrcted from Kpln Meier survivl plots using Enguge Digitizer v.5.1 (license type: GPL; developed by Mrk Mitch; Ctegory: C:\Science/CAD) to clculte HRs with 95% confidence intervls (95% CIs) using methods tht re previously described (45). Furthermore, if both the univrite nd multivrite results were reported, then only the ltter ws selected, since these results were djusted for confounding fctors. All the bove mentioned dt re comprehensively shown in Tbles I nd II. Sttisticl nlysis. In the present met nlysis, HRs nd corresponding 95% CIs were combined to estimte the vlue of high expression levels of mir 200c for cncer prognosis. An individul or pooled HR of >1.0 indicted poorer prognosis in ptients with mir 200c overexpression, nd n HR of <1.0 represented n improved prognosis. Furthermore, fixed effects model using the Mntel Henszel method or rndom effects model using the DerSimonin Lird method ws pplied for the met nlysis, ccording to the heterogeneity between the pooled studies (46). Sttisticl heterogeneity ws evluted by performing the Chi squre test (ssessing the P vlue) nd by clculting the Higgins I 2 sttistic. If significnt heterogeneity ws observed (P<0.10 or I 2 >50%), the rndom effects model ws pplied; otherwise, the fixed effects model ws used. Subgroup nlyses were further performed to investigte the source of the identified heterogeneity. In ddition, sensitivity nlyses were implemented to void bises in the results due to certin low qulity studies, nd the publiction bis ws estimted using Begg's nd Egger's tests. All P vlues were two sided, nd P<0.05 ws considered to indicte sttisticlly significnt vlue. All sttisticl nlyses were conducted using Stt v.12.0 (SttCorp, College Sttion, Texs, USA), nd Microsoft Excel (v.2010, Microsoft Corportion, Redmond, Wshington, USA). Results Summry of the included studies. In totl, 987 rticles were initilly collected from primry retrievl using the dtbses PubMed, Web of Science nd Embse. Of these rticles, 33 rticles tht included 34 studies [the rticle of Mrchini et l (20) included independent studies of two different cohorts, tissue collections A nd B (20)] were ultimtely considered eligible by screening the titles, bstrcts nd full texts (Fig. 1). Of these studies of the ssocition between expression levels of mir 200c nd the survivl rte or disese recurrence in humn mlignncies, 27 were retrospective, nd seven were prospective. In totl, 3,940 ptients from Chin, Germny, Finlnd, Jpn, Spin, Austrli, South Kore, Belgium, Sweden, Polnd, USA, Itly or Denmrk were included; these ptients were dignosed with vriety of cncer types, including endometril cncer (8,9), gstric cncer (13,15 17), ovrin cncer (18 21), ccrcc (22 24), brest cncer (25 28), colorectl cncer (14,29), NSCLC (30 35), prostte cncer (36), esophgel cncer (37 39), diffuse lrge B cell lymphom (40), bldder cncer (41,42) nd pncretic cncer (43). Tissue smples were predominntly used to determine expression levels of mir 200c, lthough six studies detected expression levels of mir 200c in serum or plsm, nd one study used tissue nd blood smples (14). To ssess mirna 200c expression, quntittive rel time polymerse chin rection (RT qpcr) hd predominntly been used in 32 studies, nd in situ hybridiztion (ISH) ws performed in three studies. The chrcteristics of primry studies re systemticlly summrized in Tble I. Assocition of CSS/OS with mir 200c overexpression. In totl, 26 studies were included in the met nlysis of the ssocition between mir 200c overexpression nd CSS/OS, nd rndom effects model ws pplied due to the high

3 MOLECULAR AND CLINICAL ONCOLOGY 4: , Figure 1. Flow digrm with detils of the study selection process. level of heterogeneity (P<0.001, I 2 =86.2%). Three studies were excluded, since they enrolled cncer ptients of only stge I (20,42). The pooled vlue of HRs from individul studies ws 1.37 (95% CI: 1.01, 1.85), long with P vlue of (Fig. 2A). Therefore, this result indicted significnt correltion of CSS/OS with high expression levels of mir 200c. Furthermore, subgroup nlyses were performed on specific study chrcteristics, including region, disese type nd smple detection. All the pooled HRs with 95% CIs of the subgroups re shown in Fig. 2B nd C. First, in the subgroup nlysis of ptients from Europe nd Americ, the pooled outcome demonstrted tht high expression level of mir 200c ws significntly ssocited with worse OS (HR=1.85, 95% CI: 1.27, 2.69). Secondly, the subgroups of esophgel cncer nd endometril cncer exhibited n identicl ssocition, with HR vlues of 1.68 nd 2.18, respectively. However, the colorectl cncer subgroup demonstrted the opposite result (HR=0.54, 95% CI: 0.32, 0.90). Thirdly, the result for the blood smple subgroup significntly reveled tht mir 200c overexpression ws ssocited with worse OS, with n HR vlue of 2.21 (95% CI: ). No significnt results were identified in the other subgroups. Assocition of disese progress with mir 200c overexpression. A totl of 16 studies were included in the present met nlysis of the ssocition of mir 200c overexpression nd RFS/PFS/DFS; the rndom effects model ws used due to the high level of heterogeneity (P<0.001, I 2 =87.1%). Two studies from the literture were excluded, since they enrolled cncer ptients t only stge I (20). The pooled HR from individul studies ws 1.03, long with P vlue of 0.880, indicting lck of sttisticl significnce (Fig. 3A). Therefore, subgroup nlyses were performed to reduce the confounding influence of the pprent heterogeneity (Fig. 3B). First, high expression levels of mir 200c were shown to correlte significntly with improved disese progression in ptients with NSCLC (HR=0.48, 95% CI: 0.34, 0.68; fixed effects model: P=0.235, I 2 =29.2% for the heterogeneity test). Secondly, the pooled HR of the tissue subgroup ws 0.68 (95% CI: 0.48, 0.96, rndom effects model; P<0.001, I 2 =82.3% for the heterogeneity test), suggesting n ssocition between mir 200c overexpression nd fvorble ptient prognosis. However, the blood subgroup reveled significnt correltion of expression levels of mir 200c with unfvorble ptient prognosis (HR=2.45, 95% CI: 1.85, 3.26; fixed effects model: P=0.722, I 2 =0.0% for heterogeneity test). Sensitivity nlysis. In the studies of CSS/OS nd RFS/PFS/DFS, our sensitivity nlyses did not revel ny ltertions in the results due to the inclusion of ny individul study (Fig. 4A nd B), indicting tht no single study significntly influenced the pooled HRs nd 95% CIs. Publiction bis. Egger's test nd Begg's funnel plot were used for the nlysis of publiction bis. The funnel plots of the CSS/OS nd RFS/PFS/DFS nlyses were lmost symmetricl, nd ll P vlues from the Egger's test were >0.05 (Fig. 4C nd D). Therefore, no significnt publiction bis ws observed in the present met nlysis. Discussion The EMT is well estblished mechnism tht includes intercellulr contct disruption nd enhnced cell motility (47). Additionlly, burgeoning body of evidence hs clerly demonstrted the involvement of EMT in the invsion nd migrtion of tumor cells (14,32,43). Intrcellulr nd extrcellulr fctors re known to be cpble of promoting or inhibiting EMT progression. In prticulr, the mir 200 fmily hs been proposed to suppress EMT by directly trgeting the trnscriptionl repressors of E cdherin, zinc finger E box binding homeobox 1 (ZEB1) nd zinc finger E box binding homeobox 2 (ZEB2), thus inducing E cdherin upregultion (48). Conversely, inhibition of the mir 200 fmily would induce mesenchyml like spindle morphology, which promotes cncer metstsis. As the most representtive microrna mong the mir 200 fmily, mir 200c fulfills importnt roles in EMT inhibition nd in MET promotion. For instnce, Mrchini et l (20) demonstrted tht severl downstrem trgets of mir 200c, including vsculr endothelil growth fctor A (VEGFA)

4 936 ZHANG et l: PROGNOSTIC VALUE OF mir-200c IN VARIOUS TYPES OF CANCER Tble I. The predominnt chrcteristics of the included studies. Ptient's Ptient's Men or Study Mlignnt Disese Detected Men or medin Authors, yer ntionlity number medin ge design disese stge smple follow up time Refs. Antolín et l, 2015 Spin R Brest cncer I IV Blood OS /235.3 PFS weeks (27) Butz et l, 2015 Cnd 425 NM R ccrcc I IV Tissue NM (24) Song et l, 2015 Chin R Brest cncer I IV Tissue NM (28) Zho et l, 2015 Chin P NSCLC IIB IIIB Tissue NM (35) Zhou et l, 2015 Chin R Gstric cncer IIB IV Tissue NM (13) Go et l, 2015 Chin 93 NM R Ovrin cncer I IV Blood NM (18) Vergho et l, 2014 Germny R ccrcc I IV Tissue 45.6 months (22) Tuomril et l, 2014 Finlnd P Brest cncer I IV Tissue 9.7 yers (25) Toiym et l, 2014 Jpn 156 T /182 S 68.0 R Colorectl cncer I IV Both 20 months (14) Tejero et l, 2014 Spin R NSCLC I III Tissue 43 months (30) Song et l, 2014 Chin R Gstric cncer I IV Tissue 35 months (17) Lin et l, 2014 Austrli P Prostte cncer III IV Blood 12 months (36) Li et l, 2014 Chin R NSCLC IIIB, IV Tissue 16.7 months (31) Kim et l, 2014 South Kore R NSCLC I IV Tissue 31 months (32) Diz et l, 2014 Spin R Colorectl cncer I III Tissue 113 months (29) Co et l, 2014 Chin R Ovrin cncer I IV Tissue 36.8 months (19) Berghmns et l, 2013 Belgium P NSCLC NM Tissue NM (33) Yu et l, 2013 Chin R Esophgel cncer III, IV Blood 20 months (37) Wotschofsky et l, 2013 Germny 111 >60.0 R ccrcc NM Tissue NM (23) Tng et l, 2013 Chin R Gstric cncer I IV Tissue NM (15) Tnk et l, 2013 Jpn R Esophgel cncer II IV Blood NM (38) Berglund et l, 2013 Sweden 61 >60.0 R DLBCL I IV Tissue NM (40) Mdhvn et l, 2012 Germny 193 NM R Brest cncer III IV Blood NM (26) Vlldres Ayerbes et l, 2012 Spin P Gstric cncer I IV Blood 24 months (16) Torres et l, 2013 Polnd P Endometril cncer I IV Tissue NM (9) Liu et l, 2012 Chin P NSCLC I IV Tissue NM (34) Kryvz et l, 2012 USA 34 NM R Endometril cncer I IV Tissue NM (8) Wszolek et l, 2011 USA 57 >60.0 R Bldder cncer NM Tissue 92 months (41) Mrchini et l, 2011 Itly R Ovrin cncer I Tissue 110 months (20) Mrchini et l, 2011 Itly R Ovrin cncer I Tissue 108 months (20) Hmno et l, 2011 Jpn 98 >60.0 R Esophgel cncer I IV Tissue 28.8 months (39) Wiklund et l, 2011 Denmrk 100 NM R Bldder cncer I Tissue NM (42) Yu et l, 2010 Jpn R Pncretic cncer I IV Tissue NM (43) Leskelä et l, 2010 Spin R Ovrin cncer I IV Tissue NM (21) The study design is described s prospective (P) or retrospective (R) T, tissue smple of ptients ws ssyed; S, serum smple of ptients ws ssyed; ccrcc, cler cell renl cell crcinom; NSCLC, non smll cell lung cncer; DLBCL, diffuse lrge B cell lymphom; NM, not mentioned; OS, overll survivl; PFS, progression free survivl.

5 MOLECULAR AND CLINICAL ONCOLOGY 4: , Tble II. HRs of included studies. HRs OS/ RFS/PFS/ Authors, yer Min ssy of mir200c Cut off Resource of HR CSS DFS Refs. Antolín et l, 2015 RT qpcr Men Reported (27) Butz et l, 2015 RT qpcr Men Reported (24) Song et l, 2015 ISH Men (28) Zho et l, 2015 RT qpcr Medin Reported 0.35 (35) Zhou et l, 2015 RT qpcr Medin 0.49 (13) Go et l, 2015 RT qpcr Men Reported 0.32 (18) Vergho et l, 2014 RT qpcr 2.73 Reported 0.95 (22) Tuomril et l, 2014 RT qpcr Medin (25) Toiym et l, 2014 RT qpcr Medin Reported 0.56 T /2.67 S 4.51 S (14) Tejero et l, 2014 RT qpcr Men 1.95 (30) Song et l, 2014 RT qpcr Lowest qurtile Reported (17) Lin et l, 2014 RT qpcr Medin Reported 2.30 (36) Li et l, 2014 RT qpcr Reported (31) Kim et l, 2014 RT qpcr Men Reported 3.67 (32) Diz et l, 2014 RT qpcr NM (29) Co et l, 2014 RT qpcr 3.84 Reported (19) Berghmns et l, 2013 RT qpcr Medin Reported 1.51 (33) Yu et l, 2013 RT qpcr Medin Reported 1.67 (37) Wotschofsky et l, 2013 RT qpcr Medin Reported 1.40 (23) Tng et l, 2013 RT qpcr/ish Men Reported (15) Tnk et l, 2013 RT qpcr Medin Reported 2.79 (38) Berglund et l, 2013 RT qpcr Men 2.68 (40) Mdhvn et l, 2012 RT qpcr Lower qurtile (26) Vlldres Ayerbes et l, 2012 RT qpcr 62.4 Reported (16) Torres et l, 2013 RT qpcr Medin Reported 2.72 (9) Liu et l, 2012 RT qpcr 2.00 Reported 6.02 (34) Kryvz et l, 2012 RT qpcr (8) Wszolek et l, 2011 RT qpcr Men 0.09 (41) Mrchini et l, 2011 RT qpcr Medin Reported (20) Mrchini et l, 2011 RT qpcr Medin Reported (20) Hmno et l, 2011 RT qpcr Medin 1.71 (39) Wiklund et l, 2011 ISH NM 0.52 (42) Yu et l, 2010 RT qpcr 0.64 Reported 0.45 (43) Leskelä et l, 2010 RT qpcr Medin Reported 0.85 (21) The study design is described s prospective (P) or retrospective (R). Dt extrcted from the survivl curve. In the OS/CSS nd RFS/PFS/DFS columns, ʻTʼ denotes the HR of mir 200c overexpression in tumor tissue, nd ʻSʼ denotes the HR of mir 200c overexpression in serum smple; HR, hzrd rtio; OS, overll survivl; CSS, cncer specific survivl; RFS, relpse free survivl; PFS, progression free survivl; DFS, disese free survivl; RT qpcr, quntittive reverse trnscription polymerse chin rection; ISH, in situ hybridiztion. nd tubulin, bet 3 clss III (TUBB3), were significntly upregulted in ptients with ovrin cncer who relpsed (20). Leskelä et l (21) suggested n inverse correltion between mir 200c nd TUBB3 expression in dvnced ovrin cncer. Furthermore, mrked inverse correltion of the expression levels of mir 200c nd the mrna levels of VEGFA ws demonstrted in two independent cohorts of ccrcc nd norml tissues (49). Thus, mir 200c hs been considered tumor suppressor. However, potentil oncogenic role of mir 200c in humn mlignncies hs lso been reported. Tuomril et l (25) demonstrted tht progesterone receptor (PR) negtive cses with locl or distnt recurrence hd higher expression levels of mir 200c compred with those without recurrence, suggesting tht high expression level of mir 200c is n independent fctor for predicting poor survivl rtes in PR negtive brest cncer. Tejero et l (30) reported tht high expression levels of mir 200c were ssocited with shorter OS of ptients with NSCLC due to MET nd ngiogenesis (30). In ddition, Hmno et l (39) indicted tht the mir 200c induced chemoresistnce of esophgel cncer ws medited by the Akt pthwy, showing tht mir 200c overexpression ws significntly correlted with shortened OS (39). In the present met nlysis, significnt ssocition of the expression of mir 200c with outcome ws observed for pooled CSS/OS (Fig. 2A). However, there ws heterogeneity

6 938 ZHANG et l: PROGNOSTIC VALUE OF mir-200c IN VARIOUS TYPES OF CANCER A B C Figure 2. Forest plots of the combined nlyses of the ssocition of CSS/OS nd expression levels of mir 200c. (A) Forest plots of the pooled nlysis of CSS/OS. Squres nd horizontl lines correspond to study specific HRs nd 95% CIs, respectively. The re of the squres correltes with the weight, nd the dimonds represent the pooled HRs nd 95% CIs. (B) Forest plots of the pooled nlysis of CSS/OS in different disese type subgroups. (C) Forest plots of the pooled nlysis of CSS/OS in blood smple subgroup. CSS, cncer specific survivl; OS, overll survivl; HR, hzrd rtio; CI, confidence intervl.

7 MOLECULAR AND CLINICAL ONCOLOGY 4: , A B Figure 3. Forest plots of the pooled nlysis of (A) the ssocition of RFS/PFS/DFS nd mir 200c expression in different smple subgroups, nd (B) RFS/PFS/DFS in the in the non smll cell lung cncer subgroup. RFS, recurrence free survivl; PFS, progression free survivl; DFS, disese free survivl; HR, hzrd rtio; CI, confidence intervl. A B C D Figure 4. Sensitivity nlyses nd Begg's funnel plots. (A) Sensitivity nlysis of the effect of individul studies on the CSS/OS results. (B) Sensitivity nlysis of the effect of individul studies on the RFS/PFS/DFS results. (C) Begg's funnel plots to test for the publiction bis in the overll nlysis of CSS/OS. Ech point represents seprte study. (D) Begg's funnel plots to test for publiction bis in the overll nlysis of RFS/PFS/DFS. RFS, recurrence free survivl; PFS, progression free survivl; DFS, disese free survivl; HR, hzrd rtio; CI, confidence intervl.

8 940 ZHANG et l: PROGNOSTIC VALUE OF mir-200c IN VARIOUS TYPES OF CANCER in both groups of outcomes (P<0.001, I 2 =86.2% for CSS/OS; P<0.001, I 2 =87.1% for RFS/PFS/DFS). Heterogeneity my be cused by the different chrcteristics of the ptients, including rce, disese type nd clinicl stge, s well s the selected cut off vlue of the expression level of mir 200c. To minimize the influence of these confounding fctors, subgroup nlyses tht focused on region, disese type nd smple detection were performed. On the bsis of the subgroup nlyses of the included studies, heterogeneity in severl subgroups ws gretly reduced, nd vluble results were obtined (Fig. 2B nd C). These results indicte tht mir 200c my be used s prognostic biomrker; however, severl detils require further refinement. First, different cut off vlues were used in the studies of mir 200c. The mjority of the vilble studies estblished medin or men expression cut off vlue, lthough certin studies used lower qurtile vlue. Furthermore, severl studies used ternry method, seprting the expression levels of mir 200c into high, intermedite nd low ctegories (50). Due to the lck of stndrdized mir 200c expression dt, evluting its prognostic role in mlignncies would produce inccurte results. Secondly, whether mir 200c functions s n independent tumor biomrker or, more likely, s component of predictive microrna signture, hs yet to be firmly estblished. For instnce, Yeh et l (51) demonstrted tht the downregultion of mir 141 nd mir 200c serves s n independent predictor of DFS in heptocellulr crcinom. In the multivrite nlysis performed by Blnco Clvo et l (52), the combintion of high levels of growth differentition fctor 15, mtrix metlloproteinse 7 nd mir 200c ws considered n independent predictor of mortlity in gstric cncer. In ddition, using liner combintion of the microrna cycle threshold vlues nd Cox regression coefficients s weights, Berghmns et l (33) reveled tht certin microrna signture (mir 200c, mir 124, mir 29c nd mir 424) is of prognostic vlue for OS in ptients with NSCLC (33). Thirdly, lthough significnt results were identified for severl subgroups bsed on the subgroup nlyses, the results for other subgroups were not decisive. Empiriclly, prognostic fctor is considered decisive when the HR is >2.0 or < 0.5 (53). Finlly, severl HRs were extrcted from the survivl curves, which unvoidbly resulted in severl slight sttisticl errors. In conclusion, we hve demonstrted tht high expression levels of mir 200c re of significnt prognostic vlue in vrious humn mlignncies. In ddition, expression levels of mir 200c in tumor tissue nd blood smples were considered to serve s relible predictive biomrker for disese progression in cncer ptients. 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