Linc00152 Functions as a Competing Endogenous RNA to Confer Oxaliplatin Resistance and Holds Prognostic Values in Colon Cancer

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Accepted Article Preview: Published ahead of advance online publication Linc00152 functions as a competing endogenous RNA to confer oxaliplatin resistance and holds prognostic values in colon cancer

Ben Yue, Donglan Cai, Chenchen Liu, Changyi Fang, Dongwang Yan

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Cite this article as: Ben Yue, Donglan Cai, Chenchen Liu, Changyi Fang, Dongwang Yan, Linc00152 functions as a competing endogenous RNA to confer oxaliplatin resistance and holds prognostic values in colon cancer, Molecular Therapy accepted article preview online 16 September 2016; doi:10.1038/mt.2016.180

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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

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Received 20 May2016; accepted 07 September 2016; Accepted article preview online 16 September 2016

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Linc00152 functions as a competing endogenous RNA to confer oxaliplatin resistance and holds prognostic values in colon cancer

Ben Yue1, Donglan Cai2, Chenchen Liu1, Changyi Fang1, Dongwang Yan1*

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Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of

Medicine, Shanghai, 200080, China 2

Department of Ophthalmology, Shanghai General Hospital, Shanghai Jiao Tong University School of

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Medicine, Shanghai, 200080, China

Ben Yue and Donglan Cai contributed equally to this work.

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Correspondence should be addressed to Dongwang Yan,

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([email protected])；Tel/Fax: 86-21-63241377.

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Department of General Surgery, Shanghai General Hospital, Shanghai Jiao Tong University School of

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Medicine, 85 Wujin Road, Shanghai, 200080, China.

The work was done in Shanghai, China. Short title: Linc00152 confers chemoresistance in colon cancer

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Abstract Long non-coding RNAs (LncRNAs) act as crucial regulators in plenty of human cancers, yet their potential roles and molecular mechanisms in chemoresistance are poorly understood. Our present study showed that a novel lncRNA, Linc00152 (long intergenic non-coding RNA 152), promoted tumor progression and conferred resistance to oxaliplatin (L-OHP)-induced apoptosis in vitro and in vivo. It antagonized chemosensitivity through acting as a competing endogenous RNA (ceRNA) to modulate the expression of miR-193a-3p, and then ERBB4. Knockdown of ERBB4 in colon cancer cells decreased AKT phosphorylation, which resulted in decreased L-OHP resistance. Consistent with above findings,

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the specific AKT signaling inhibitor and activator were used, respectively, which demonstrated that

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Linc00152 contributed to L-OHP resistance at least partly through activating AKT pathway. Further studies indicated that Linc00152 was increased and appeared to be an independent prognostic factor for

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decreased survival and increased disease recurrence in stage Ⅱ and Ⅲ colon cancer patients undergoing

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L-OHP-based chemotherapy after surgery. Collectively, our findings established Linc00152 as a candidate prognostic indicator of outcome and drug responsiveness in colon cancer patients, and the

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involvement of ceRNAs mechanism in Linc00152/miR-193a-3p/ERBB4/AKT signaling axis may

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provide a novel choice in the investigation of drug resistance.

Keywords: lncRNA; Linc00152; colon cancer; oxaliplatin; chemoresistance.

Introduction As one of the most common and aggressive tumors, colon cancer remains a lethal malignancy worldwide. Despite its incidence rate decreased by approximately 3% and death rate declined by approximately 2% per year during the past decade, it is still the second leading cause of cancer-related deaths in the United States.1 Chemotherapy is a current option for stage Ⅲ and part of stage Ⅱ colon

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cancer patients after surgery. As a commonly used third-generation platinum analogue, oxaliplatin (LOHP) kills cancer cells through forming intra-strand linkage of two adjacent guanines on DNA and always used alone or combined with other drugs in the therapeutic strategies.2 However, drug resistance frequently occurs during the course of treatment. The standard-of-care first-line therapy by using L-OHP leads to only a 50% to 60% response rate.3 In fact, the exact underlying molecular mechanism responsible for L-OHP resistance remains elusive up to now. Resistance to L-OHP is a complex process and several signaling pathways, such as the nuclear factor-kappa B (NF-ΚB) pathway,4 the extracellular signal-regulated kinase

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(ERK)/Extracellular signal-regulated kinase kinase (MEK) pathway,5 and the protein kinase B

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(AKT/PKB) pathway, have been involved in this phenomenon. Accumulating evidences demonstrate that activation of the AKT signaling pathway always leads to resistance to L-OHP induced apoptosis.7

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AKT pathway is activated by phosphorylation of AKT at both Thr308 and Ser473 after recruited to plasma membrane by the upstream kinase Phosphoinositide 3-kinase (PI-3K).8 It mediates cell survival

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largely through directly phosphorylating pro-apoptotic protein BAD and causing the aberrant expression

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of anti-apoptotic protein Bcl2 family members.9, 10 Aberrant activation of AKT is a frequent event in cancers as well as chemoresistance.11 The chemotherapeutic effect of L-OHP can be enhanced by

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inhibiting the phosphorylation and activation of AKT.12 Inversely, downregulation of AKT signaling reduces the dose of L-OHP and is considered to endure the chemotherapy.13 Some genes are known to regulate AKT pathway nowadays. For instance, ERBB4 (erb-b2 receptor tyrosine kinase 4) is acknowledged to upregulate the activation of AKT signaling.14 For non-coding transcripts, many miRNAs, including miR-203, miR-31, suppress tumor progression through deactivating AKT signaling.15, 16 Latest studies have shown that biological functions of long non-coding RNAs (lncRNAs) were involved in multiple progression of human cancers by regulating AKT pathway.17, 18 LncRNAs are typically longer than 200 nucleotides and lacking protein-coding capacity.19 They have recently been found to be linked to a series of biological processes, including not only the

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regulation of transcription and post-transcription, but also the process of genomic imprinting and chromatin modification.20, 21 Moreover, their aberrant expression have been associated with human malignancy.22 LncRNAs also affect the progression of colon cancer by regulating cell proliferation, differentiation, invasion, metastasis and so on.23, 24 Importantly, lncRNAs are considered to act as key regulators in drug resistance, and lncRNAs-associated chemoresistance is gradually becoming the focus of research.25 Recently, a novel lncRNA, Linc00152 (Long intergenic non-coding RNA 152), has been reported to exert oncogenic impacts on human cancers. It is involved in cell growth, cycle arrest, epithelial

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mesenchymal transition (EMT) and invasion,26 and could directly bind enhancer of zeste homolog 2

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(EZH2) and then repress p15 and p21 to promote tumor progression. It is noteworthy that Linc00152 promotes tumor growth through the epidermal growth factor receptor (EGFR)-mediated AKT

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pathway.28 It strongly implies that Linc00152 may regulate the AKT signaling pathway to influence the response of cancer cells to L-OHP.

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In the present study, we demonstrated a novel Linc00152/miR-193a-3p/ERBB4/AKT signaling axis

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that mediated the response of colon cancer cells to L-OHP treatment. Furthermore, Linc00152 was significantly increased and appeared to be an independent prognostic factor for survival in colon cancer

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patients undergoing L-OHP-based chemotherapy.

Results Linc00152 antagonizes L-OHP-induced apoptosis in colon cancer cells To investigate the role of Linc00152 in mediating sensitivity to chemotherapy, four colon cancer cell lines were used to determine the L-OHP effect. As a result, all the cells' viability were reduced by LOHP in a dose-dependent manner (Fig. 1a). The basal level of Linc00152 expression in SW620 and HT29 cells was higher than in SW480 and Caco2 cells (Fig. 1b), and a significant upregulation of Linc00152 levels was observed in both SW620 and HT29 cells following treatment with 10.0 µM L-

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OHP (Fig. 1c). Western blot analysis of apoptosis-related gene cleaved-PARP and cleaved-Caspase 3 showed that SW620 and HT29 cells exhibited much lower sensitivity to L-OHP-induced apoptosis than SW480 and Caco2 cells (Fig. 1d). To determine whether Linc00152 contributes to L-OHP resistance, SW620 and HT29 cells were silenced of Linc00152 effectively (Fig. 1e). CCK-8 assays showed that attenuation of Linc00152 displayed a significant inhibition of viability in response to L-OHP compared with controls in SW620 and HT29 cells not only in a dose-dependent manner but also a time-dependent manner (Figs. 1f, g).

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Linc00152 functions as a competing endogenous RNA (ceRNA) by sponging miR-193a-3p

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As non-coding transcripts, lncRNAs always exert their functions through modulating the protein expression of related target gene because of lacking of protein-coding capacity.29, 30 Recent evidences

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demonstrate that lncRNAs may participate in the ceRNAs regulatory network to affect the tumor progression. In other words, lncRNAs function as molecular sponges to regulate the expression levels of

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mRNAs by competitively binding their same miRNA responsive elements (MREs).18, 31, 32 To

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investigate whether Linc00152 has such a similar mechanism in colon cancer, we performed a prediction for miRNAs target sites via online program starbase v2.0

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(http://starbase.sysu.edu.cn/mirLncRNA.php) and found that 6 miRNAs exist relevant binding sites in Linc00152 (Supplementary Table S1). The expression levels of these miRNAs were examined in Linc00152 knockdown cells by qRT-PCR. As shown in Fig. 2a, miR-107, miR-193a-3p and miR-376c3p were significant upregulated in response to Linc00152 inhibition in both SW620 and HT29 cells. Furthermore, among all these three miRNAs, only miR-193a-3p showed a sharp decrease in colon cancer cells based L-OHP treatment (Fig. 2b). These results suggested that Linc00152 may exert an impact on deregulation of miR-193a-3p. To validate the direct binding between Linc00152 and miR-193a-3p at endogenous levels, RIP was performed and demonstrated that Linc00152 was significant enriched for miR-193a-3p compared to

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Linc00152 with mutation in miR-193a-3p binding site (Linc00152-mut) and the empty vector MS2 (Figs. 2c, d). Recently, miRNAs are known to exert the impacts of translational repression or RNA degradation on their targets in an AGO2-dependent manner. Potential miRNA targets exist in the RNAinduced silencing complex (RISC) and can be isolated via co-immunoprecipitating with AGO2, the core component of RISC.33 For further confirmation, anti-AGO2 RIP was performed and qRT-PCR analysis revealed that Linc00152 was significant enriched in miR-193a-3p transfected cells (Fig. 2e). These data supported that miR-193a-3p is a bona fide linc00152 targeting miRNA. The special association between Linc00152 and miR-193a-3p was confirmed by luciferase reporters assay. As a result, overexpression of

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miR-193a-3p reduced the luciferase activities of Linc00152 wild-type reporter vector but not the mutant

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reporter vector (Fig. 2f). Further studies showed that inhibition of Linc00152 upregulated the expression of miR-193a-3p in both SW620 and HT29 cells (Fig. 2g). In contrast, ectopically expressed Linc00152

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wild-type, but not Linc00152 mutation-type reduced the expression of miR193a-3p (Figs. 2h, i). However, we found no obvious difference in Linc00152 level after overexpression or inhibition of miR-

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193a-3p (Fig. 2j). To function as a ceRNA by playing sponge effects, the stoichiometry must match

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such that the sponge is present at sufficiently high levels to account for the endogenous levels of the miRNA. Therefore, the exact copy numbers of Linc00152 and miRNAs that have complementary base

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pairing with Linc00152 were quantified by qRT-PCR in L-OHP treated and untreated cells. We formulated standard curves using pcDNA3.1-Linc00152 and reversetranscribed miRNAs cDNA as standard templates. As a result, we found that when compared with untreated cells, L-OHP treatment induced upregulation of Linc00152 and downregulation of miR-193a-3p, however, we found no significant differences in miR-107 and miR-376c-3p levels after L-OHP treatment (Fig. 2k). All these data demonstrated that Linc00152 physically associated with miR-193a-3p and may function as a ceRNA.

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Linc00152 modulates the expression of ERBB4 through competitively binding miR-193a-3p Previous reports suggest that miR-193a-3p suppresses tumor proliferation, invasion and metastasis by directly targeting and downregulating the expression of ERBB4.34, 35 Our above findings indicated that Linc00152 function as a ceRNA by sponging miR-193a-3p. In addition, Linc00152 and ERBB4 share the same miR-193a-3p responsive element according to the prediction (Fig. 3a). Thus, we wondered whether Linc00152 could modulate miR-193a-3p and then ERBB4 in colon cancer. Interestingly, we observed that the depletion of Linc00152 decreased ERBB4 at both mRNA and protein levels, and inhibition of miR-193a-3p abrogated the decrease of ERBB4 (Figs. 3b, c). On the other hand,

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ectopic expression of Linc00152 wild-type, but not the mutant, increased ERBB4 at mRNA and protein

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levels, and restoration of miR-193a-3p relieved this increase (Figs. 3d, e).

To ascertain whether above observed effects depend on the regulation of the 3' untranslated regions

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(3'-UTR) of ERBB4, we constructed luciferase reports containing the ERBB4 3'-UTR. Luciferase plasmid or the control reporter was transfected into SW620 and HT29 cells. As a result, the depletion of

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Linc00152 decreased the luciferase activity in the pmirGLO-ERBB4 transfected cells, which was

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rescued by inhibition of miR-193a-3p. Reciprocally, overexpression of Linc00152 wild-type, but not the mutant, increased the luciferase activity in the pmirGLO-ERBB4 transfected cells, and ectopic

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expression of miR-193a-3p abolished this upregulation. Moreover, a mutation in the ERBB4 MRE (pmirGLO-ERBB4-mut) can rescue the effects of linc00152 overexpression and knockdown (Figs. 3f, g). Furthermore, Linc00152 transcript level was coexpressed with ERBB4 mRNA level in human colon cancer tissues, a positive correlation was observed between them (Fig. 3h and Supplementary Table S2). Immunohistochemical staining showed different protein expression levels of ERBB4 in colon cancer tissues (Fig. 3i). As higher ∆Ct value means lower expression, Linc00152 transcript level was also significantly correlated with ERBB4 protein level (Fig. 3j). Additionally, Linc00152 knockdown or overexpression affects the expression levels of several miR-193a-3p target genes, such as MCL1 and

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E2F1 (Supplementary Fig S1). All these findings suggested an crucial role of Linc00152 in modulating ERBB4 by competitively binding miR-193a-3p.

Linc00152 mediates drug resistance through sponging miR-193a-3p and then modulating ERBB4 As ERBB4 overexpression is associated with chemoresistance,36 to investigate whether Linc00152 confers L-OHP resistance by modulating ERBB4, we first examined the effect of ERBB4 on cell response to L-OHP. Knockdown of ERBB4 reduced cell viability (Figs. 4a, b) and increased apoptosis (Figs. 4c, d) in response to L-OHP compared with control cells. These data indicated that ERBB4 may

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protect colon cancer cells from L-OHP induced apoptosis.

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In further study, decreased cell viability was observed after knockdown of Linc00152 in both SW620 and HT29 cells treated with L-OHP, which was reversed via inhibition of miR-193a-3p (Fig.

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5a). Overexpression of Linc00152, but not the mutant, increased the cell viability, and ectopic expression of miR-193a-3p abolished this upregulation (Fig 5b). Consistently, depletion of Linc00152

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increased cell apoptosis in response to L-OHP, but was also abrogated through inhibition of miR-193a-

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3p (Figs. 5c, e). Reciprocally, cell apoptosis was reduced in SW620 and HT29 cells with Linc00152 overexpression. In addition, overexpression of miR-193a-3p changed the effects (Figs. 5d, f). To test

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whether Linc00152 confers L-OHP resistance by modulating miR-193a-3p and ERBB4 in vivo, a xenograft model was constructed. Results showed that tumor growth and weight decreased in Linc00152 knockdown group compared with control group, and inhibition of miR-193a-3p abrogated these effects. Reciprocally, ectopic expression of Linc00152 wild-type, but not the mutation, increased the tumor growth and weight, and overexpression of miR-193a-3p abolished this effect (Figs. 6a-e and Supplementary Table S3). Furthermore, immunochemistry analysis revealed that tumor ERBB4 expression from Linc00152 knockdown group was lower, which was rescued by inhibition of miR-193a3p. On the other hand, overexpression of Linc00152 wild-type, but not the mutant, increased the ERBB4 staining, which was also abolished by introduction of miR-193a-3p (Figs. 6f, g). These in vivo findings

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confirmed a critical role of Linc00152 in L-OHP resistance of colon cancer cells through involving in the interaction with miR-193a-3p and ERBB4, which consistent with the in vitro results.

AKT activation mediated by ERBB4 contributes to Linc00152-conferred L-OHP resistance To ascertain whether the effects we see specific to related pathway or just built upon what is always going to be a survival phenotype with ERBB4 manipulation, further study was performed. As ERBB4 could regulate AKT,37 and phosphorylation of AKT which reflects the signaling activation played roles in anti-apoptotic and chemoresistance,38 we wondered whether Linc00152 confers L-OHP resistance

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through regulating miR-193a-3p/ERBB4 and then activating AKT. We examined the activity of AKT

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signaling and found that downregulation of ERBB4 resulted in an decreased phosporylation of AKT at both Thr308 and Ser473, as also reflected by the reduction of phosphorylation of GSK3β (Fig. 7a).

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Further studies revealed that phosphorylation of Thr308, Ser473 and GSK3β were significantly decreased in cells transfected with Linc00152 shRNA, however, inhibition of miR-193a-3p abolished

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these effects (Fig. 7b). Relevant experiments also showed the effect that overexpression of Linc00152

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and Linc00152 MRE mutant has on AKT signaling (Fig. 7c). To confirm that Linc00152 regulates LOHP sensitivity at least partly through activating AKT signaling, a specific signaling inhibitor MK2206

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was used to repress the activity of AKT signaling. Consequently, MK2206 significantly decreased the phosphorylation of Thr308, Ser473 and GSK3β (Fig. 7d), and cells overexpression of Linc00152 were sensitive to L-OHP when AKT signaling pathway was blocked (Fig. 7e). IGF-1, an activator of AKT signaling was also used to evaluate the role of AKT signaling in Linc00152 mediated L-OHP resistance. Inversely, IGF-1 markedly increased the phosphorylation levels of Thr308, Ser473 and GSK3β in both SW620 and HT29 cells transfected with control or Linc00152 shRNA (Fig. 7f), and activation of AKT signaling decreased the sensitivity of cells to L-OHP (Fig. 7g). All these results demonstrated that Linc00152 has an important role in L-OHP resistance of colon cancer cells through modulating the miR193a-3p/ERBB4/AKT axis.

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Linc00152 is increased in human colon cancer tissues and is associated with poor prognosis To evaluate the clinical role of Linc00152, colon cancer tissues was used. As a result, elevated expression of Linc00152 was observed by using qRT-PCR in colon cancer tissues when compared with adjacent normal mucosa (p<0.001, Fig. 8a). To explore whether Linc00152 expression was associated with the clinicopathological characteristics, 134 stage Ⅱ and Ⅲ colon cancer patients who received LOHP-based chemotherapy after radical colectomy were divided into high group (n=98) and low group

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(n=38) in relation to the median value of Linc00152 expression.39 As shown in Supplementary Table S4, Linc00152 expression was significantly associated with pN stage (p=0.036) and recurrence

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(p=0.001) in colon cancer patients. Then, overall survival (OS) and disease-free survival (DFS) curves

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were plotted by the Kaplan-Meier analysis according to Linc00152 expression level and striking differences revealed that high Linc00152 expression level significantly correlated with a worse overall

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survival (p<0.001, Fig. 8b) and recurrence-free survival (p<0.001, Fig. 8c). In addition, univariate and multivariate analysis indicated that increased Linc00152 expression was an independent prognostic

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indicator for OS and DFS in colon cancer patients (Supplementary Table S5). These results suggested

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that Linc00152 hold significant clinical values in stage Ⅱ and Ⅲ colon cancer patients who received LOHP-based chemotherapy after radical colectomy, and determination of Linc00152 may be a useful predictor for L-OHP response. Discussion Currently, the regulatory functions of LncRNAs have been established in regulating biological processes such as cell proliferation, differentiation, cell cycle and metastasis. As a novel LncRNA, Linc00152 is increased in several cancers and exerts multiple functions to influence the tumor progression.26-28 In the present study, we report for the first time that Linc00152 promotes L-OHP resistance in colon cancer cells in vitro and in vivo. Mechanistically, Linc00152 confers resistance to L-

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OHP by competitively binding miR-193a-3p, upregulating ERBB4, and then inducing the activation of AKT signaling pathway. In addition, we identify the upregulation of Linc00152 in human colon cancer tissues and its expression level significantly correlates with outcome in patients undergoing L-OHPbased chemotherapy after surgery. Previous evidences suggest the existence of ceRNAs regulatory network where LncRNAs may exert functions via targeting miRNAs. For instance, lncRNA activated by TGF-β (lncRNA-ATB) promoted the invasion and induced EMT in hepatocellular carcinomas through competitively binding miR-200 family with ZEB1 and ZEB2.40 Homeobox transcript antisense intergenic RNA (HOTAIR)

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regulated HER2 expression by sponging miR-331-3p in gastric cancer.41 Our findings indicated that

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Linc00152 and ERBB4 physically associated with miR-193a-3p and may function as ceRNAs. ERBB4 is a member of the epidermal growth factor receptor (EGFR) family, its oncogenic role in the biological

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processes of human cancer has been reported recently.42 ERBB4 was also over-expressed in colon cancer and has been found to participate in the transition from colon adenoma to carcinoma following

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adenomatous polyposis coli (APC) loss.43, 44 Moreover, it has been considered to mediate acquired

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resistance to chemotherapy. Consistently, we found that inhibition of ERBB4 enhanced colon cancer cells apoptosis in response to L-OHP. In addition, Linc00152 play an important role in L-OHP

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resistance by acting as a ceRNA. It functions as a molecular sponge to modulate the expression of ERBB4 through competitively binding their same MREs on miR-193a-3p. The ceRNA network always shows a reciprocal repression between LncRNAs and miRNAs. Thus, Linc00152 can be de-pressed by depletion of miR-193a-3p, consequently, its oncogenic impacts will be magnified. Conversely, overexpreesion of miR-193a-3p overcame the influences of increased Linc00152, their functions will cancel each other out. This is why the effect of the inhibition of miR-193a-3p alone is much more dramatic than transfected with Linc00152 plus miR-193a-3p. Additionally, despite that the endogenous Linc00152 sponge is expressed at reasonable level, how significant this level is in the context of an

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entire transcriptome of potential miR-193a sites remains uncertain. Thus, it would be interesting and significant to further determine their effects. ERBB4 contributes to tumor progression, its knockdown triggers cell apoptosis and results in a decrease in AKT phosphorylation.45 As a most frequently hyperactivated signaling in plenty of malignancies, AKT pathway mediates cell survival and chemoresistance.11 Full activation of AKT requires phosphoinositide-dependent kinase 1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2) mediated phosphorylation at both Thr308 and Ser473.9, 38 Its activation also be reflected by the phosphorylation and inactivation of GSK3β.46 Inversely, AKT signaling can be negatively regulated

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through dephosphorylating AKT at Thr308 and Ser473.47 To determine whether Linc00152 contributes

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to chemoresistance through activating the AKT pathway, the specific AKT signaling inhibitor MK2206 and activator IGF-1 were used in the research, respectively.48, 49 Consistent with the speculation,

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increased cell response to L-OHP was observed when AKT signaling pathway was blocked in Linc00152-overexpressing cells. On the other hand, activation of the AKT signaling pathway decreased

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the sensitivity of cells to L-OHP induced apoptosis in colon cancer cells transfected with control or

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Linc00152 shRNA. These results demonstrated that Linc00152 contributed to L-OHP resistance at least in part through modulating the miR-193a-3p/ERBB4/AKT axis.

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In colon cancer, biomarkers associated with L-OHP resistance, for example, excision repair crosscomplementing 1 protein (ERCC1) and G-protein-coupled receptor galanin receptor 1 (GalR 1), have been reported,50, 51 revealing useful targets of L-OHP resistance are clinically important. Nevertheless, these findings have failed to translate to clinical practice and optimal prognostic biomarkers for L-OHPbased chemotherapy have not been established up to now.52 Here we found that Linc00152 was clinically significant in colon cancer patients undergoing L-OHP-based chemotherapy, and might be used as a potential biomarker for forecasting L-OHP resistance. As combination chemotheray has become the norm in colon cancer and we mostly focused on L-OHP in this study, further investigations

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that Linc00152 affects the response of colon cancer cells to other chemotherapeutic agents will enhance our understanding of LncRNAs mediated chemoresistance. Collectively, we identify the Linc00152/miR-193a-3p/ERBB4/AKT signaling axis that involves in the regulation of colon cancer cells to L-OHP treatment. To the best of our knowledge, this is the first report to suggest the significance of Linc00152 in chemoresistance via ceRNAs regulatory network. Based on the clinical evidence, Linc00152 acts as an independent prognostic factor for colon cancer patients and, as such, may hold a potential value for predicting chemoresistance. Taken together, the discovery of Linc00152 and the involvement of ceRNAs mechanism in the signaling axis may provide a

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promising option for facilitating the development of chemotherapy.

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Materials and methods Specimens

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Between July 2007 and October 2009, a total of 134 fresh colon cancer tissues and paired adjacent

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normal mucosa were collected from stage Ⅱ and Ⅲ colon cancer patients, who underwent radical

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resection and received L-OHP-based chemotherapy after surgery. The diagnosis of all specimens was

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histopathologically confirmed by a pathologist. None of the patients received any treatment before surgery. The study was approved by the Ethics Committee of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA from all tissues and cells was isolated using Trizol regent (Ambition Technology, Melbourne, Australia). For LncRNA and mRNA, RT-PCR was performed as previously described.53 The primer sequences can be found in Supplementary Table S6. For miRNAs, RT-PCT was performed

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using TaqMan miRNA assays following the manufacturer's instructions. The relative gene expression was calculated by using 2-∆∆Ct method.

Cell culture Human colon mucosal epithelial cell line NCM460 and four colon cancer cell lines SW480, Caco2, SW620 and HT29 were purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (IBCB, Shanghai, China). All cells were cultured in DMEM medium (Gibco BRL, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Invitrogen, Camarillo, CA,

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USA). Cells were maintained in a humidified atmosphere at 37 ℃ with 5% CO2.

Plasmid construction and Luciferase reporter assay

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The cDNA encoding Linc00152 was PCR-amplified and subcloned into pcDNA3.1 vector. The

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shRNAs specifically targeting Linc00152 or ERBB4 were synthesized by GenPharma Co. (Shanghai, China). Hsa-miR-193a-3p mimic/negative control mimic and inhibitor/negative control inhibitor were

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purchased from Genechem, (Shanghai, China). Linc00152 constructs with mutations at the putative

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miR-193a-3p response elements using a QuikChange Site-Directed Mutagenesis kit (Stratagene) and named Linc00152-mut. Transfections were performed using the Lipofectamine 2000 kit (Invitrogen, Carlsbad, CA, USA) following the manufactuer's instructions. 48-72 hours after transfenction, cells were harvested for qRT-PCR and western blot analyses, or collected for other following assays. miRNA mimics and negative control were introduced into cells at a final concentration of 100nM/ml. PmirGLO, pmirGLO-Linc00152, or pmirGLO-Linc00152-mut was transfected with miR-193a-3p mimics or negative control, and pmirGLO, pmirGLO-ERBB4 or pmirGLO-ERBB4-mut was transfected into different cell clones using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The relative luciferase activity was normalized to Renilla luciferase activity 48 h after transfection. A dual-luciferase

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reporter gene assay system was used in this assay (Promega, Madison, WI, USA). Primer sequences for plasmid construction were listed in Supplementary Table S6.

Cell proliferation assay and apoptosis assay Cell proliferation assays were assessed with Cell Counting Kit-8 (Byotime, Haimen, China) following the manufacturer's protocol and detected at 24, 48 and 72 h. 100 µl/well CCK-8 solution was added and the mixture was incubated at room temperature for 2 h. The cell proliferation curves were plotted using the 460 nm absorbance (A460) at each time point. Experiments were independently

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repeated in triplicate.

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For the flow cytometric analysis, cells were collected and analyzed. Annexin V-APC/PI Apoptosis Kit (BD, CA, USA) was used following the manufacturer's instructions.

Western blot analysis

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Whole cell lysates were prepared at 100 ℃ for 5 min. Equivalent amounts of protein were

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separated by SDS-PAGE at 80 V for 2.5 h and transfected to PVDF membranes for 2 h. The membranes

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were washed using 1% TBST for 30 min after incubation with specific antibodies targeting ERBB4, cleaved PARP, cleaved Caspase 3, AKT, pAKT(T308), pAKT(S473), GSK3β, pGSK3β or GAPDH (1:1000, Cell Signaling Technology, USA) at 4 ℃ overnight, then incubated with secondary antibodies (1:5000, BioTNT, China) for 2 h and washed by 1% TBST again, finally, detected by chemliuminescence system (Amersham Biosciences, Piscataway, NJ, USA).

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RNA immunoprecipitation (RIP) Cells were co-transfected with pMS2-GFP (Addgene) and pcDNA3.1-MS2 or pcDNA3.1-MS2Linc00152 or pcDNA3.1-MS2-Linc00152-mut. After 48 h, experiments were carried out using a GFP antibody (Abcam, Cambridge, MA) and the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA) according to the manufacturer's instructions. qRT-PCR was performed to analyze miR-193a-3p in the precipitates after the antibody was recovered. For anti-AGO2 RIP, cells were transfected with miR-193a-3p or miRNA negative control. RIP experiments were performed using an AGO2 antibody (Millipore) as described above, and Linc00152 or

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Linc00152-mut was detected by qRT- PCR after 48 h.

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Immunohistochemical analysis

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Immunostaining was performed using a GT Vision III Kit (Genetech, Shanghai, China) following the manufacturer's instructions. ERBB4 expression was analyzed using human colon cancer tissues. The

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staining intensity was scored as 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong). The staining area

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was scored as 0 (<10% positive staining), 1 (10%–25% positive staining), 2 (25%–50% positive staining), 3 (50%–75% positive staining) and 4 (>75% positive staining). The final staining scores were

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determined by the formula: overall scores = percentage score × intensity score. The overall score of ≤3 was defined as negative, of >3 - ≤6 as weak positive, and of >6 as strong positive.54

Nude mice xenograft models HT29 cells (1.0×106) stably expressing sh-NC or sh-Linc00152 or miR-193a-3p inhibitor or shLinc00152+miR-193a-3p inhibitor were subcutaneously injected into the frank of 4-week-old nude mice (10 mice per group). When the tumor volumes reached 0.2 cm3, every group was randomized for 2 subgroups: inject or not inject L-OHP which was dissolved in PBS and 5 mg/kg/day was injected intraperitoneal twice a week. All the mice were killed after 30 days and tumors were subjected to

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immunohistochemical analysis of ERBB4. All animal experiments were performed in accordance with the Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine Animal Care and Use guidelines. Immunohistochemitry was performed on formalin-fixed paraffin-embedded tumor tissue using a primary antibody against ERBB4 (1:200, Abcam) following ImmunoCruz goat ABC Staining System protocol (Santa Cruz, CA, USA).

Statistical analysis

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Data were analyzed using the SPSS 19.0 software package (SPSS, Chicago, IL, USA). Between-

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group differences were analyzed using Student's t-test and one way analysis of variance (ANOVA). The associations between categorical variables and outcomes were determined using the Fisher's exact test.

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Spearman correlation coefficient was used to assess the expression correlation assay. Overall survival (OS) and disease-free survival (DFS) were estimated by the Kaplan-Meier method with log-rank test,

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and Cox regression was utilized to analyze the univariate and multivariate hazards models. All statistical

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analyses were defined as significant for p-values <0.05.

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Conflict of interest: None.

Acknowledgments: This work was supported by the Natural Science Foundation of Shanghai (16ZR1427700) and Doctoral Innovation Fund Projects from Shanghai Jiao Tong University School of Medicine (BXJ201630).

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Figure legends Fig. 1 Colon cancer cells display different response to L-OHP treatment and Linc00152 antagonize

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L-OHP-induced apoptosis. (a) Different colon cancer cells were treated with increasing concentrations

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of L-OHP for 72 h and exhibit different sensitivity to L-OHP. (b) The expression of cleaved PARP and cleaved Caspase 3 were higher in SW480 and Caco2 than in SW620 and HT29 after 10 µM L-OHP

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treatment for 72h. (c) Higher Linc00152 levels in colon cancer lines compared with normal colon mucosal epithelial cell line NCM460. (d) qRT-PCR analyses indicated that Linc00152 was increased in

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SW620 and HT29 cells treated with 10 µM L-OHP for 72 h. (e) Linc00152 was knocked down

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effectively in both SW620 and HT29 cells. (f) CCK-8 assays showed decreased cell viability in Linc00152 knockdown cells compared with the control cells following L-OHP treatment in a dose-

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dependent manner. (g) Linc00152 knockdown cells displayed a significant reduction in proliferation in a time-dependent manner following 10 µM L-OHP treatment. *p<0.05, **p<0.01; *p or #p or +p<0.05, **p or ##p or ++p<0.01 (sh-NC vs sh-Linc00152-1 or sh-Linc00152-2 or sh-Linc00152-3, respectively).

Fig. 2 Linc00152 functions as a competing endogenous RNA (ceRNA) by sponging miR-193a-3p. (a) miR-107, miR-193a-3p and miR-376c-3p were upregulated significantly in response to Linc00152 inhibition. (b) Attenuation of miR-193a-3p was observed in both SW620 and HT29 cells following 10 µM L-OHP treatment for 72 h. (c) Putative Linc00152 base pairing with miR-193a-3p as identified by

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prediction program. Mutation was generated on the Linc00152 RNA sequence for the seed region of miR-193a-3p. (d) RIP followed by qRT-PCR to examine miR-193a-3p endogenously associated with Linc00152. (e) Anti-AGO2 RIP was performed followed by qRT-PCR to examine Linc00152 associated with AGO2 in SW620 and HT29 cells transiently overexpressing miR-193a-3p. (f) SW620 and HT29 cells were cotransfected with luciferase reporters containing nothing, wild-type Linc00152 or mutant Linc00152, and miR-193a-3p mimics, the luciferase activity was measured using a dual-luciferase reporter gene assay system after transfection 48 h. (g) Upregulation of miR-193a-3p by inhibition of Linc00152. (h, i) pcDNA3.1-Linc00152 transfection was validated and mutant-type Linc00152 clone

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exert limited suppression on miR-193a-3p compared with wild-type Linc00152. (j) Expression of

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Linc00152 in SW620 and HT29 cells transfected with miR-193a-3p mimic, inhibitor and control. (k) The exact copy numbers of Linc00152 and miR-193a-3p in SW620 and HT29 cells treated or untreated

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with L-OHP were quantified with qRT-PCR. *p<0.05, **p<0.01.

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Fig. 3 Linc00152 modulates the expression of ERBB4 through competitively binding miR-193a-3p. (a) Linc00152 and ERBB4 share the same miRNA responsive element in their sequences. (b, c) The

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mRNA or protein levels of ERBB4 in stable SW620 and HT29 cell clones transfected with sh-NC, shLinc00152, sh-linc00152+miR-193a-3p inhibitor and miR-193a-3p inhibitor. (d, e) The mRNA or protein levels of ERBB4 in stable SW620 and HT29 cell clones transfected with pcDNA-3,1, pcDNA3.1-Linc00152, pcDNA3.1-Linc00152-mut, pcDNA3.1-Linc00152+miR-193a-3p mimics and miR-193a-3p mimics. (f, g) Luciferase activity in SW620 and HT29 cells transfected with luciferase reporters containing ERBB4 3'-UTR or nothing. (h) Positive correlation between Linc00152 transcript level and ERBB4 mRNA level in 134 colon cancer tissues. (i) Immunohistochemical staining with specific anti-ERBB4 antibody was done in human colon cancer tissues. (j) Linc00152 transcript level was also significantly correlated with ERBB4 protein level. **p<0.01.

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Fig. 4 ERBB4 promotes the L-OHP resistance in colon cancers. (a) The protein levels of ERBB4 in SW620 and HT29 cells were suppressed effectively by specific shRNA. (b-d) ERBB4 knockdown and control cells were treated with increasing concentrations of L-OHP for 72 h, CCK-8 assays, western blot analysis of cleaved PARP and cleaved Caspase 3, and flow cytometry analysis showed that inhibition of ERBB4 expression conferred resistance to L-OHP induced apoptosis. *p or #p<0.05, **p or ##p<0.01 (sh-NC vs sh-ERBB4-1 or sh-ERBB4-2, respectively).

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Fig. 5 Linc00152 mediates drug resistance through sponging miR-193a-3p and then modulating

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ERBB4 in vitro. (a) Decreased cell viability was observed after knockdown of Linc00152 in cells treated with L-OHP, which was rescued through inhibition of miR-193a-3p. (b) Increased cell viability

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was observed after overexpression of Linc00152 in cells treated with L-OHP, which was abolished by

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overexpression of miR-193a-3p. (c, e) Depletion of Linc00152 increased cell apoptosis in response to LOHP, but was also abrogated through inhibition of miR-193a-3p. (d, f) Cell apoptosis were reduced in

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SW620 and HT29 cells with Linc00152 overexpression, in addition, overexpression of miR-193a-3p changed the effects. *p or #p<0.05, **p or ##p<0.01 (sh-NC vs sh-Linc00152 or inh miR-193a-3p, respectively).

Fig. 6 Linc00152 mediates drug resistance through sponging miR-193a-3p in vivo. (a-e) Xenograft model showed that tumor growth and weight decreased in Linc00152 knockdown group compared with control group, and inhibition of miR-193a-3p abrogated these effects. Reciprocally, ectopic expression of Linc00152 wild-type, but not the mutation, increased the tumor growth and weight, and

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overexpression of miR-193a-3p abolished this effect. (f, g) Immunochemistry analysis revealed that tumor ERBB4 expression from Linc00152 knockdown group was lower compared with the control group, and inhibition of miR-193a-3p abrogated this decrease. On the other hand, overexpression of Linc00152 wild-type, but not the mutant, increased the ERBB4 staining, which was also abolished by introduction of miR-193a-3p.

Fig. 7 AKT activation mediated by ERBB4 contributes to Linc00152-conferred L-OHP resistance. (a) Knockdown of ERBB4 resulted in the downregulation of AKT phosphorylation and GSK3β

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phosphorylation. (b) Knockdown of Linc00152 resulted in the downregulation of AKT phosphorylation

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and GSK3β phosphorylation, and inhibition of miR-193a-3p abolished this effect. (c) The effect that overexpression of Linc00152 and Linc00152 MRE mutant has on AKT signaling (d) AKT

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phosphorylation and GSK3β phosphorylation were decreased by MK2206 in Linc00152-overexpressing cells. (e) Increased cell response to L-OHP was observed when AKT signaling pathway was blocked in

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Linc00152-overexpressing cells. *p or #p or +p<0.05, **p or ##p or ++p<0.01, (pcDNA3.1-Linc00152

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vs pcDNA3.1 or pcDNA3.1 plus MK2206 or pcDNA3.1-Linc00152 plus MK2206, respectively). (f) AKT phosphorylation and GSK3β phosphorylation were increased by IGF-1 in Linc00152 knockdown

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cells. (g) Activation of the AKT signaling pathway decreased the sensitivity of cells to L-OHP induced apoptosis in colon cancer cells transfected with control or Linc00152 shRNA. *p or #p or +p<0.05, **p or ##p or ++p<0.01, (sh-Linc00152 vs sh-NC or sh-NC plus IGF-1 or sh-Linc00152 plus IGF-1, respectively).

Fig. 8 Aberrant expression of Linc00152 in colon cancer samples and its association with patients' prognosis. (a) Expression levels of Linc00152 in 134 colon cancer tissues and paired adjacent normal mucosa. (b, c) Kaplan-Meier analyses of the correlations between Linc00152 expression level and

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overall survival (OS) or disease-free survival (DFS) of 134 colon cancer patients received oxaliplatinbased chemotherapy after surgery. The OS and DFS of the Linc00152 high group (n=96) was significantly shorter than that of the low expression group (n=38).

Supplementary Fig S1. Linc00152 knockdown or overexpression affects the expression levels of several miR-193a-3p target genes. (a) The expression levels of E2F1 and MCL1 in stable SW620 and HT29 cell clones transfected with sh-NC, and sh-Linc00152. (b) The expression levels of E2F1 and MCL1 in stable SW620 and HT29 cell clones transfected with pcDNA3.1, pcDNA3.1-Linc00152-mut

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and pcDNA3.1-Linc00152.

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Supplementary Table S1. miRNAs that have putative binding site with Linc00152.

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Supplementary Table S2. The expression levels of Linc00152 and ERBB4 (∆Ct) in 134 pairs of colon cancer tissues.

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Supplementary Table S3. Tumor volume (mm3) of mice in different treatment groups.

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Supplementary Table S4. Association between clinicopathologic features and Linc00152 expression.

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Supplementary Table S5. Univariate and multivariate analysis of overall survival and disease-free survival after surgery.

Supplementary Table S6. Detailed sequence (5'-3') information.

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