malat1 rs664589 polymorphism inhibits binding to mir-194 ......biotech co. ltd., beijing, china)....

MALAT1 rs664589 polymorphism inhibits binding to miR-194-5p 1

contributing to colorectal cancer risk, growth and metastasis 2

Shenshen Wu1,2,3

, Hao Sun3, Yajie Wang

3, Xi Yang

3, Qingtao Meng

3, Hongbao Yang

4, 3

Haitao Zhu5, Weiyan Tang

6, Xiaobo Li

3, Michael Aschner

7, Rui Chen

1,2,3,8* 4

1Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital 5

Medical University, Beijing 100069, P.R. China 6

2Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, 7

Beijing 100069, P.R. China 8

3Key Laboratory of Environmental Medicine Engineering, Ministry of Education, 9

School of Public Health, Southeast University, Nanjing, China; 10

4Center for New Drug Safety Evaluation and Research, China Pharmaceutical 11

University, Nanjing, China; 12

5Department of General Surgery, Jiangsu Cancer Hospital & Jiangsu Institute of 13

Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, 14

Nanjing, China; 15

6Department of Medical Oncology, Jiangsu Cancer Hospital & Jiangsu Institute of 16

Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, 17

Nanjing, China; 18

7Department of Molecular Pharmacology, Albert Einstein College of Medicine，19

Forchheimer 209,1300 Morris Park Avenue, Bronx, NY, 10461,USA;

20

8Institute for Chemical Carcinogenesis, Guangzhou Medical University, Guangzhou 21

511436, China 22

Running title: MALAT1 polymorphism and colorectal cancer development 23

Key words: Colorectal cancer; MALAT1; polymorphism; development; survival 24

Research. on March 9, 2020. © 2019 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 16, 2019; DOI: 10.1158/0008-5472.CAN-19-0773

http://cancerres.aacrjournals.org/

*Correspondence to: Rui Chen, Department of Toxicology and Sanitary Chemistry, 1

School of Public Health, Capital Medical University, Beijing 100069, P.R. China; Tel: 2

+86 10 83911010; Fax: +86 10 83911507; Email: [email protected]. 3

Disclosure of Potential Conflicts of Interests： 4

The authors declare no conflict of interest. 5

6




Abstract 1

Metastasis associated with lung adenocarcinoma transcript-1 (MALAT1) is an 2

evolutionarily highly conserved lncRNA that contributes to colorectal cancer (CRC) 3

development. However, the exact molecular mechanisms connecting MALAT1 to 4

CRC have not been fully elucidated. Here, we performed a case-control study in 1078 5

CRC patients and 1175 healthy controls to evaluate the association between 6

potentially functional genetic variants of MALAT1 and survival outcomes in CRC 7

patients. MALAT1 rs664589 CG/GG genotypes significantly increased the associated 8

risk and decreased overall survival of CRC patients compared with the CC genotype. 9

In vitro and vivo experiments showed that the rs664589 C to G mutation facilitated 10

carcinogenesis and metastasis of CRC. Mechanistically, the microRNA miR-194-5p 11

targeted MALAT1 for degradation in the nucleus in an Ago2-dependent manner; the 12

rs664589 G allele altered the binding of MALAT1 to miR-194-5p resulting in 13

increased expression of MALAT1. CRC cells and human tissues with the rs664589 14

CG/GG genotype expressed significantly higher MALAT1 than those with the 15

rs664589 CC genotype. Multivariate cox regression analysis showed that MALAT1 16

was a poor prognostic factor of CRC. In summary, MALAT1 with the rs664589 G 17

allele demonstrates altered binding to miR-194-5p in the nucleus, leading to increased 18

MALAT1 expression and enhanced CRC development. 19

20




Significance 1

Findings highlight the functional role of MALAT1 polymorphism in colorectal cancer 2

metastasis and survival as well as the underlying mechanism 3

4




Introduction: 1

Colorectal cancer (CRC) is one of the most common cancers and the fourth leading 2

cause of cancer-related death worldwide(1). In China, a total of 376,300 new cases 3

and 191,000 cancer-related deaths have been reported in 2015(2). Environmental risk 4

factors, such as smoking(3), heavy alcohol consumption(4), high consumption of red 5

and processed meat(5) contribute to the initiation and progression of CRC. Genetic 6

and epigenetic alternations intensify CRC development as well(6). 7

Long non-coding RNAs (lncRNAs), as the transcripts greater than 200 nucleotides, 8

are involved in several biological processes(7-12). Metastasis associated with lung 9

adenocarcinoma transcript-1 (MALAT1) is an evolutionarily highly conserved 10

lncRNA gene, consisting of more than 8,000 nT and maps to chromosome 11q13(13). 11

Recent studies demonstrated that MALAT1 not only promotes CRC 12

malignancy(14,15), but also affects prognosis(16). However, there is no consensus on 13

the mechanisms behind MALAT1 exotic expression. It also has been reported that 14

genetic variant in MALAT1 was associated with the risk of various tumors, such as 15

hepatocellular carcinoma (HCC)(17), breast cancer(18), and lung cancer(19). Li et al. 16

found that genetic variants in the promoter region of MALAT1 was associated with 17

colorectal cancer risk(20). However, to date, few studies have focused on the 18

association between the genetic variants in exon region of MALAT1 and risks of CRC. 19

In this study, we assessed whether the selected genetic variants in MALAT1 exon 20

region could contribute to CRC development and disclose their potential mechanisms. 21

22




Materials and methods: 1

Study subjects 2

The present study was approved by the Institutional Review Board (IRB) of Southeast 3

University (Nanjing, China), and the study was conducted in accordance with the 4

1964 Declaration of Helsinki. All the participants signed written informed consent 5

form before recruitment. 6

Briefly, a total of 1,078 CRC patients and 1,175 healthy controls were enrolled for 7

genotyping in this study. All patients were newly diagnosed with histologically 8

confirmed CRC and consecutively recruited from the Jiangsu province (China) 9

between January 2007 and October 2011. Besides, CRC patients who had history of 10

metastasis or received preoperative chemo/radiotherapy were excluded. The 11

pathological stage of CRC was assessed based on the 6th edition of American Joint 12

Committee on Cancer (AJCC) cancer staging manual. Cancer-free controls 13

genetically unrelated to the cases were randomly selected from individuals who were 14

seeking routine physical examination in the same district. 15

In addition, a total of 96 pairs of CRC tissues and the corresponding adjacent 16

noncancerous tissues, which were used to evaluate MALAT1 levels in patients with 17

different rs664589 genotypes, were obtained to from patients undergoing surgery in 18

the Jiangsu Tumor Hospital between 2014 and 2015. 19

MALAT1 SNP selection and prediction of secondary structure 20

The location of the MALAT1 was determined in chromosome 11, position 21




65263738-65276556 (data were obtained from the LNCipedia). Two SNPs were 1

selected from the LncRNASNP database according to the following criteria: (a) minor 2

allele frequency (MAF) > 0.05 in global population; (b) SNPs in MALAT1 gene were 3

predicted to locate in the miRNA binding sites using LncRNASNP database; (c) SNPs 4

may modulate the secondary structure of MALAT1 calculated by RNAfold. 5

Genotyping 6

The genomic DNA of controls was extracted from peripheral blood with the 7

RelaxGene Blood DNA System (Tiangen Biotech Co. Ltd., Beijing, China). Due to 8

lack of peripheral blood in CRC cases, paraffin-embedded sections were used to 9

isolate the genomic DNA by EZNA Tissue DNA Kit (Omega Bio-Tek Inc., Norcross, 10

GA, USA). The genotypes of selected polymorphisms were determined by TaqMan 11

allelic discrimination method using the Quant Studio 6 Flex System (Applied 12

Biosystems, Foster city, CA, USA). Approximately 10% of the random samples were 13

selected for validation of the genotyping, with a 100% concordance rate in the results. 14

Construction of tissue microarray (TMA) and in situ hybridization (ISH) 15

The CRC TMAs were constructed by the National Engineering Center for Biochip 16

(Shanghai, China). Duplicate 1.0 mm diameter cores of tissue from each sample were 17

punched from paraffin-embedded tumor blocks and corresponding non-tumor CRC 18

tissues. As a tissue control, the biopsies of normal CRC epithelium tissues were then 19

inserted in the four corners and the center of each slide. 20

MALAT1 expression levels in CRC cells, fresh paraffin-embedded tissues, and TMA 21




were analyzed with the RNAscope 2.0 HD-Brown Manual Assay (Advanced Cell 1

Diagnostics, Newark, CA, USA) according to manufacturer’s instructions. In brief, 2

samples were incubated with MALAT1 probes (NR_002819.2, nt 6820-7930) at 40 °C 3

for 2 h. For the fresh paraffin-embedded tissues, the samples were rehydrated for 15 4

min, and then digested with protease for 30 min before incubation with the probe. 5

After hybridization, a series of single amplification procedures were performed. 6

MALAT1 expression levels were eventually visualized with DAB 7

(3,3-Diaminobenzidine) staining. 8

Assessment of staining of MALAT1 9

The staining score of MALAT1 were calculated by two pathologists who were blinded 10

to the results of genotype according to a semi quantitative immunoreactivity score 11

(IRS) as previously reported(21). Briefly, the score was assessed based on the 12

intensity of staining and percentage of gram-positive cells. The receiver operating 13

characteristic (ROC) curve was used to obtain the optimum cutoff value of MALAT1 14

IRS. In addition, the area under the curve (AUC) at different cutoff values of 15

MALAT1 IRS for survival time from 1 to 9 years were calculated, respectively. In 16

presence of these conditions, samples with IRS 0-3 and IRS 4-12 were defined as low- 17

and high-expression level for MALAT1, respectively. 18

Cell culture and transfection 19

Human CRC cells (SW480 and SW620) were purchased from the Shanghai Institute 20

of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) in 21




2014, and cultured at Dulbecco's modified Eagle medium (DMEM) (HyClone, Logan, 1

UT, USA) at 37 °C in 5% CO2. The culture medium was supplemented with 10% fetal 2

bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), penicillin (100 U ml-1), 3

and streptomycin (100 μg ml-1) (HyClone, Logan, UT, USA). The cells were 4

periodically tested and validated to be free of mycoplasma. All the cell lines were 5

certified by short tandem repeat (STR) analysis and used within 6 months. In addition, 6

all the cell lines were cultured within 25 passages for all experiments. 7

The miR-194-5P mimics and miR-194-5P inhibitors were synthesized by RiboBio 8

(Guangzhou, China). All transfections for small RNAs were conducted with 9

Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The cells were 10

harvested 48 h after transfection as well. 11

Construction of CRC cells with indicated genotypes 12

CRC cells with indicated genotypes were generated by CRISPR/Cas9 strategy (Fig. 13

s1). Briefly, CRISPR/Cas9 expression plasmid and ssDNA donor were co-transfected 14

into the CRC cells using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, 15

USA). After 24 h, the culture medium was replaced with 2 ml of complete medium 16

containing 0.6 μg/ml Puromycin (VWR International Pty Ltd., Brisbane, Australia), 17

and the cells were grown for additional 2 weeks. Positive allele-specific colonies were 18

obtained by limiting dilution and were ultimately verified by DNA sequencing. For 19

the negative control group, cells were co-transfected with CRISPR/Cas9 expression 20

plasmid and the same wild type ssDNA donor carrying with C allele (Fig. s1). 21




Analysis of cell colony formation, invasion, and migration ability 1

For colony formation assays, 500 CRC cells were seeded in 10 cm culture dishes and 2

allowed to grow in complete medium for 10 d. For the cell invasion assays, 1×105

3

cells were plated into the Transwell insert coated with Matrigel (CytoSelect 24-Well 4

Cell Invasion Assay Kit; Cell Biolabs Inc., San Diego, CA, USA), and cultured with 5

complete medium for 48 h. The migration assays were performed with a similar 6

procedure without Matrigel coating on the filters. All the cells were ultimately fixed 7

with ethanol, stained with crystal violet, and counted. 8

Flank tumor growth in vivo 9

All the mouse experiments were performed in accordance with the approval of the 10

Institutional Animal Care & Use Committee of Southeast University. The lentiviral 11

plasmids encoding firefly luciferase were constructed and lentiviruses were generated 12

as reported previously(22). In brief, the culture medium was replaced with complete 13

medium containing 7.5 μg/ml Blasticidin S after infection with lentivirus for 12 h. 14

Then, the medium was replaced every 2 days until all the control cells died. Positive 15

cells were maintained in 1 μg/ml Basticidin S for 2 consecutive weeks. Of note, cells 16

less than fifteen passage after the initial drug selection were implanted in the flank site 17

of 6-week-old female BALB/c nude mice. After 14 d, tumor, liver and lung tissues 18

were harvested and the biochemical luciferase activities were determined by a 19

luminometer (Sirius, Berthold Detection Systems GmbH, Germany). 20

RNA pull-down assay 21




The RNA pull-down assay was performed as described previously(23). For this 1

purpose, the 1000-bp MALAT1 fragments flanking the rs664589C or G allele were 2

amplified and cloned into pcDNA3.1 vector. Biotin-labeled RNA was generated by an 3

in vitro transcription reaction with the Biotin RNA Labeling Mix (Roche Diagnostics 4

GmbH, Mannheim, Germany) and T7 RNA polymerase (Roche Diagnostics GmbH, 5

Mannheim, Germany), and then treated with RNase-free DNase I (Takara, Kusatsu, 6

Japan). After purified with the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA, USA), 7

the biotin-labeled RNA (2 μg) was then incubated with cells extracted from CRC cells 8

at room temperature for 1 h. Washed streptavidin agarose beads were then added to 9

each binding reaction and incubated at room temperature for another 1 h. The bound 10

RNAs were detected by quantitative reverse transcription polymerase chain reaction 11

(RT-qPCR) analysis. 12

Dual Luciferase Reporter Assay 13

Reporter plasmids containing 160-bp MALAT1 exon region fragments flanking the 14

rs664589C or G allele were synthesized based on the reporter vector psiCHECK-2 15

(Promega, Madison, WI, USA). Briefly, 800 ng reported plasmids along with 16

miR-194-5p mimics or inhibitors were co-transfected into cells using Lipofectamine 17

2000 reagent (Invitrogen, Carlsbad, CA, USA). After 24 h, the cells were collected 18

and Renilla-Firefly Luciferase activities were detected using the Dual-Luciferase 19

Reporter Assay System (Promega, Madison, WI, USA). 20

RNA immunoprecipitation 21




RNA immunoprecipitation assay was performed using EZ-Magna RIP RNA-Binding 1

Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). Nuclei was 2

isolated from colorectal cancer cells using Nuclei EZ Prep Kit (Sigma-Aldrich, St. 3

Louis, MO, USA), and then incubated with magnetic beads conjugated with human 4

anti-Ago2 antibody (Millipore, Billerica, MA, USA), or negative control normal igG 5

at 4 °C. After 6 h, the beads were washed with wash buffer and incubated with 6

Proteinase K to remove proteins. Besides, MALAT1 and miR-194-5p were finally 7

measured by RT-qPCR. 8

RNA extraction and RT–qPCR analysis 9

Total RNAs were extracted from the cell lines with the TRIzol reagent (Invitrogen, 10

Carlsbad, CA, USA). Furthermore, cDNA synthesis was performed with 1 μg total 11

RNA according to the manufacturer’s protocol (Takara, Kusatsu, Japan). 12

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal controls 13

in SYBR®

Green Realtime PCR Master Mix-Plus kit (Toyobo, Osaka, Japan). The 14

relative RNA levels of MALAT1 were determined with the Quant Studio 6 Flex 15

system (Applied Biosystems, Foster city, CA, USA). 16

We designed specific primers for MALAT1 (Forward: 17

5′-GTGATGCGAGTTGTTCTCCG-3′ ； Reverse: 18

5′-CTGGCTGCCTCAATGCCTAC-3′) and GAPDH (Forward: 19

5′-CCATCAATGACCCCTTCATTGACC-3′; Reverse: 20

5′-GAAGGCCATGCCAGTGAGCTTCC-3′). 21




Statistical analysis 1

The chi-square goodness-of-fit test was applied to calculate the Hardy–Weinberg 2

equilibrium (HWE) among the controls. Pearson's chi-squared test was used to 3

calculate the frequency distribution of the demographic characteristics and genotype 4

results of the MALAT1 polymorphisms. The association between the genotypes and 5

risk of CRC was estimated by crude and adjusted odds ratios (ORs) and 95% 6

confidence intervals (CIs). The time-dependent ROC curve analysis was used to 7

calculate the best cutoff value. The different time periods of the follow-up were 8

evaluated through the performances of different scores by plotting (t, AUC[T])(24). 9

All P-values were two-sided and statistical significance level was set at P < 0.05. All 10

statistical analyses were carried out using SAS 9.2 software (SAS Institute, Cary, NC, 11

USA). 12

Results 13

MALAT1 rs664589 G allele increased the risk of CRC 14

Detailed information of the CRC cases and controls was reported previously(25). Two 15

SNPs (rs664589 and rs3200401) located in MALAT1 were genotyped. As shown in 16

Table 1, the distribution of alleles among controls were in agreement with the HWE 17

(P = 0.7474 for rs664589, P = 0.7225 for rs320401). Compared with the rs664589 CC 18

genotype, CG genotype elevated the risk of CRC (adjusted OR = 1.35, 95% CI = 19

1.08-1.69), and the increased risk was more pronounced in the GG genotype (adjusted 20

OR = 3.57, 95% CI = 1.60–7.97). In addition, we found that rs664589 CG/GG 21




genotypes were associated with higher risk of CRC compared with the rs664589 CC 1

genotype in the dominant genetic model (CG/GG vs. CC, adjusted OR = 1.45, 95% CI 2

= 1.16–1.80). 3

Stratified analysis showed that CG/GG genotypes profoundly increased the risk of 4

CRC among younger (adjusted OR = 1.48, 95% CI = 1.10-1.99) and male subgroups 5

(adjusted OR = 1.57, 95% CI = 1.19-2.08). Moreover, we found a similar result in 6

subgroups of colon (adjusted OR = 1.48, 95% CI = 1.12-1.94), rectum (adjusted OR = 7

1.43, 95% CI = 1.11-1.85), lower grade (adjusted OR = 1.96, 95% CI = 1.47-2.61), 8

deepest invasion (adjusted OR = 1.96, 95% CI = 1.44-2.67), lymph node metastasis 9

(adjusted OR = 2.41, 95% CI = 1.86-3.11), distant metastasis (adjusted OR = 3.63, 95% 10

CI = 2.43-5.42), TNM phase III (adjusted OR = 2.12, 95% CI = 1.60-2.81), and TNM 11

phase IV (adjusted OR = 3.63, 95% CI = 2.43-5.42) patients (Table s1). However, no 12

significant association was observed between rs3200401 genotypes and the risk of 13

CRC. 14

MALAT1 rs664589 G allele elevated MALAT1 expression and predicted poor 15

overall survival for CRC patients 16

Here, MALAT1 expressions in our TMA (consisting 807 pairs of CRC tissues and 17

adjacent non-tumor tissues from 1078 CRC patients) were assessed by using the 18

RNAscope 2.0 HD-Brown Manual Assay (Advanced Cell Diagnostics, Newark, CA, 19

USA). A total of 720 CRC patients were enrolled for final analysis as some of them 20

lost in antigen retrieval process. Higher expression of MALAT1 in CRC tissues than 21

that in the matched non-tumors was observed in 274 of 332 cases (Xuzhou cohort) 22




and 305 of 388 cases (Nanjing cohort), respectively (Fig. 1A-B). Moreover, patients 1

with rs664589 CG/GG genotype presented higher MALAT1 levels than those carrying 2

the rs664589 CC genotype (Fig. 1C-D). In addition, the elevated MALAT1 levels 3

decreased overall survival in CRC patients (Fig. 1E). 4

We then detected expression of MALAT1 mRNA in CRC cells and adjacent tissues in 5

patients with the CC (n=77), CG (n=18) or GG (n=1) genotype at rs664589. Results 6

showed that patients with rs664589 CG/GG genotype presented higher MALAT1 7

mRNA levels than those carrying the rs664589 CC genotype in cancer tissues, but not 8

in adjacent tissues (Fig. s2 A). 9

We further evaluated whether rs664589 polymorphism affects the overall survival of 10

CRC patients. As shown in Fig. s2 B-C and Table s2, rs664589 CC genotype 11

markedly increased the overall survival of CRC patients (CG/GG vs. CC, adjusted HR 12

= 2.01, 95% CI = 1.68-2.40). Similar results were observed in the stratified subgroup, 13

except for depth of invasion at T1, T2, and TNM stage I (Table s3). Multivariate Cox 14

regression analysis showed that MALAT1 was an independent predictive factor for 15

CRC (Table s4). 16

rs664589 G allele potentiated the aggressiveness phenotypes of CRC 17

To further explore the potential molecular mechanisms that underlie the 18

above-mentioned results, we then constructed SW480 and SW620 cells with different 19

rs664589 genotypes (CC, CG, and GG) using CRISPR-cas9 strategy (Fig. S1 A-D). 20

We initially examined the effects of rs664589 polymorphism on cell phenotypes. As 21

shown in Fig. 2 A-D, cells carrying G allele exhibited elevated proliferation, 22




migration and invasion capacity in vitro in both SW480 and SW620 cells, especially 1

in GG homozygotes. We next injected SW480 and SW620 cells with different 2

rs664589 genotypes (CC, CG and GG) into the left flank of nude mice. We found that 3

cells with G allele developed larger subcutaneous xenografts and more distant 4

metastases (liver and lung tissue) in vivo (Fig.2 E-F). 5

MALAT1 with rs664589 C allele was suppressed by miR-194-5p 6

We then applied the lncRNASNP and RNAfold databases to explore whether 7

biological functions of MALAT1 were notably affected by genetic variants. As 8

displayed in Fig. s3 A-B, rs664589 G/C SNPs altered the secondary structure of 9

MALAT1. In addition, rs664589 variant was predicted to locate in the binding site of 10

miR-194-5p, miR-1792, and miR-4717. We then performed RNA pull-down assay to 11

confirm the prediction. Results showed that the binding affinity of miR-194-5p to 12

MALAT1 GG genotype was decreased when compared with MALAT1 rs664589 CC 13

genotype (Fig. 3 A-B). Next, the luciferase reporter gene plasmid (rs664589 C or G 14

allele) as well as miR-194-5p mimics or inhibitors were introduced into CRC cells 15

(Fig. 3 C). As illustrated in Fig.3 D, SW480 cells with rs664589 C allele and 16

miR-194-5p mimics exhibited reduced luciferase activity compared with negative 17

controls in a concentration-dependent manner, which was attenuated after 18

co-transfection with miR-194-5p inhibitors. However, no significant alternations were 19

observed in CRC cells co-transfected with rs664589 G allele and miR-194-5p mimics 20

or inhibitors. Similar results were also observed in SW620 cells (Fig. 3E). We also 21

found that cells carrying rs664589 G allele showed high levels of MALAT1 22




expression (Fig. 3F-G). Furthermore, the expression levels of MALAT1 in cells with 1

CC/CG genotypes, rather than GG genotype, were up-regulated when transfected with 2

miR-194-5p inhibitors (Fig.3 H-I). We then detected miR-194-5p levels in CRC cells 3

with different rs664589 genotypes. Results showed that levels of miR-194-5p were 4

quite similar across cells with different rs664589 genotypes (Fig. s3 C-D), which 5

indicated that MALAT1 with rs664589 C allele is a target for miR-194-5p. 6

To determine the role of miR-194-5p in CRC, the CRC cells were transfected with 7

miR-194-5p inhibitors. We found that miR-194-5p inhibitor significantly increased 8

the migration and invasion ability of CRC cells with rs664589 CC/CG genotypes (Fig. 9

2 C-D and Fig. 3 J-K). 10

miR-194-5p regulates MALAT1 in nucleus 11

In order to locate where MALAT1 was regulated, we detected the location of 12

MALAT1 in both CRC cells and fresh paraffin-embedded CRC sections using the 13

RNAscope 2.0 HD-Brown Manual Assay. We found that MALAT1 was localized in 14

the nucleus of CRC tissues and cells (Fig. s4 A-C). We also investigated whether 15

MALAT1 was regulated by miR-194-5p in the nucleus in an Ago2-dependent manner 16

as previously reported(26). Western blot analysis showed that Ago2 was expressed in 17

the nucleus and cytoplasm fraction of wild-type CRC cells (Fig. 4A). We then 18

performed RNA immunoprecipitation in the nuclear fraction of CC wild-type CRC 19

cells using Ago2 antibody, in which the results showed that both MALAT1 and 20

miR-194-5p were preferentially enriched with Ago2 rather than igG antibody (Fig. 4 21

B-C). These findings suggested that MALAT1 with C allele might be a target by 22




miR-194-5p in the nucleus through an AGO2-dependent manner. 1

Discussion 2

In this study, we evaluated the relationship between functional genetic variants in 3

MALAT1 and CRC. We demonstrated that rs664589 CG/GG genotypes significantly 4

increased the risk of CRC and deteriorated prognosis compared with CC genotype. 5

Mechanistically, miR-194-5p could not bind to MALAT1 with rs664589 G allele in 6

the nucleus, leading to increase of MALAT1 expression, as well as ultimately 7

promoting CRC development (Fig. 5A). 8

Long non-coding RNA MALAT1, also known as metastasis associated with lung 9

adenocarcinoma transcript-1, was first shown to render lung cancer metastasis (27). 10

Further evidence confirmed the oncogenic role of MALAT1 in various types of 11

cancers (28-31). Here, we demonstrated that high levels of MALAT1 were associated 12

with poor survival outcomes of CRC patients, corroborating the earlier 13

findings(13,16,32). 14

Besides, lncRNAs are also critically associated with cancer susceptibility, in which 15

genetic variation plays a vital role. Zheng et al. showed that the change of LINC00673 16

rs11655237 G>A might create a target site for miR-1231, which may affect the 17

degradation of PTPN11, and ultimately contribute to pancreatic cancer(33). Ling et al. 18

reported that the rs6983267 variant might affect expression of lncRNA CCAT2, that 19

could regulate the CRC development by interacting with TCF7L2(34). However, to 20

our knowledge, no studies have still focused on the association between the SNPs in 21

the MALAT1 and the risk of CRC. Considering this factor and the important role of 22




MALAT1 in CRC, two potential functional SNPs that predicted by the LncRNASNP 1

database were finally analyzed in our study. Additionally, we found that rs664589 G 2

allele significantly increased the risk and deteriorated prognosis of CRC. 3

Accumulating evidences have shown that secondary structure of lncRNAs might be 4

important because of their function (35,36), while SNPs in lncRNAs might influence 5

the folding structure(37,38). We found that the secondary structure was dramatically 6

changed along with the selected SNPs by using SNPfold algorithm, suggesting that 7

rs664589 might affect the increased risk of CRC by altering the secondary structure of 8

MALAT1. 9

Previous studies showed that lncRNAs and miRNAs may negatively regulate each 10

other through forming a reciprocal repression-regulatory loop(39,40). Wu et al. 11

reported that rs11752942 G allele markedly attenuates the expression level of 12

lincRNA-uc003opf.1 by binding miRNA-149*, thereby reducing the risk of 13

esophageal squamous cell carcinoma (ESCC)(41). Fan et al. found that rs8506 G>A 14

polymorphism affected the expression of lincRNA-NR_024015 by disrupting the 15

binding site for hsa-miR-526b, thereby contributing to non-cardia gastric cancer 16

progression(42). It also has been reported that MALAT1 could directly bind to 17

miRNAs (e.g., miR-206(43) and miR-133(44)), and then in turn regulate the 18

expression level of MALAT1 (39,40,44-46). The classic mechanism for miRNAs is 19

that they mediate post-transcriptional gene silencing by promoting cytoplasmic 20

mRNA decay in an Ago2-dependent manner. Gagnon et al. reported that around 75% 21

of miRNAs in the cytoplasm are shuttled into the cell nucleus, and the majority of 22




them can bind to nuclear Ago2(47). Similar finding have been reported by Yan et al., 1

in which their results showed that miR-150-5p might regulate lncRNA MIAT in the 2

nucleus in an Ago2-dependent manner(26). Furthermore, Leucci et al. showed that 3

miRNA-9 might target MALAT1 for degradation in the nucleus(48). In the present 4

study, we found that Ago2 was expressed in the nucleus and cytoplasm fraction of 5

sw480 and sw620 cells. Moreover, RNA immunoprecipitation showed that both 6

MALAT1 and miR-194-5p were detected in Ago2 immunoprecipitation in the nuclear 7

fraction. Besides, miR-194-5p was found be expressed in nucleus fraction of tumor 8

cells based on RNALocate database(49), which was documented to inhibit the growth 9

of CRC cells by targeting MAP4K4/c-Jun/MDM2 signaling pathway(50). We also 10

found that knockdown of miR-194-5p significantly increased the migration and 11

invasion ability of wild-type CRC cells. Taken together, it suggested that miR-194-5p 12

might function as a tumor suppressor through regulating MALAT1 expression in 13

nucleus. 14

In conclusion, we found that MALAT1 with rs664589 G allele altered the binding 15

affinity to miR-194-5p in the nucleus, leading to increase of MALAT1 expression, as 16

well as ultimately promoting CRC development. 17

18




Acknowledgments: This work was financially supported by National Natural Science 1

Foundation of China (81472938, 81861138017, and 91643109 to R. Chen), the 2

Thousand Young Talents Plan of China to R. Chen, the Fund of the Distinguished 3

Talents of Jiangsu Province to R. Chen( BK20150021), the Fund of the Distinguished 4

Professor of Jiangsu Province to R. Chen, the Natural Science Foundation of Jiangsu 5

Province (BK20151418 to X. Li), the Six talent peaks project in Jiangsu Province to R. 6

Chen (2016-WSN-002), the Open Research Fund of the State Key Laboratory of 7

Bioelectronics, Southeast University to R. Chen, the Fundamental Research Funds for 8

the Central Universities to R. Chen, X. Li and S.wu,the Postgraduate 9

Research&Practice Innovation Program of Jiangsu Province (KYCX17_0187 to 10

S.wu,), the National Institute of Environmental Health Sciences (NIEHS) (R01 11

ES10563, R01 ES07331 and R01 ES020852 to M. Aschner). 12

13




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10

11




Table 1 Association between MALAT1 polymorphisms and colorectal cancer risk. 1

Genotype Cases Controls

P Adjusted OR a n % n %

rs664589 858 79.6 999 85 0.0002 1

CC

CG 195 18.1 168 14.3

1.35(1.08-1.69)

GG 25 2.3 8 0.7

3.57(1.60-7.97)

CG/GG 220 20.4 176 15 0.0007 1.45(1.16-1.80)

G allele

0.1136

0.0783 0.00005

HWE

0.0008

0.7474

P trend

0.00009

rs3200401

CC 751 69.7 856 72.9 0.1923 1

CT 294 27.3 292 24.9

1.15（0.95-1.39）

TT 33 3.1 27 2.3

1.40（0.83-2.35）

HWE

0.5191

0.7225

a Adjusted for age, gender in the logistic regression model. 2

3

4




Figure Legends 1

Fig. 1 rs664589 G allele upregulates MALAT1 expression 2

(A). In situ hybridization (ISH) staining for MALAT1 in CRC cells and adjacent 3

tissues (Representative ISH staining images were obtained at magnification of 100x 4

and 400x). 5

(B). The distribution of MALAT1 expression score in CRC cells and adjacent tissues 6

(n=332, 388, 720 in Xuzhou, Nanjing, and combined cohort, respectively,). P-values 7

were calculated with the Wilcoxon signed-rank test. 8

(C, D). Representative ISH staining images of MALAT1 with indicated r664589 9

genotypes are shown (C) (n=647, 142, 18 in CC, CG, GG genotype, respectively,). 10

The staining scores were calculated (D). (*** P < 0.001, ** P < 0.01, * P < 0.05, 11

P-values were calculated by one-way analysis of variance (ANOVA)). 12

(E). Kaplan-Meier curves depicting overall survival according to MALAT1 13

expression (Log-rank P <0.001). 14

Fig. 2 MALAT1 rs664589 G allele provokes aggressive phenotypes of CRC cells 15

(A-B). Cell colony formation assay of SW480 (A) and SW620 (B) with indicated 16

rs664589 genotype (n=9 from three independently differentiated clones in each 17

genotype, ***P<0.001, compared with CC genotype, one-way analysis of variance 18

(ANOVA), Error bars, S.D.). 19

(C-D). Cell migration and invasion assay of SW480 (C) and SW620 (D) with 20

indicated rs664589 genotype (n=9 from three independently differentiated clones in 21

each genotype, ***P<0.001, compared with CC genotype, one-way ANOVA, Error 22

bars, S.D.). 23




(E-F). CRC cells with indicated genotype were implanted into the left flank of nude 1

mice. Luciferase activity in xenografts, liver, and lung tissues were determined (n=9 2

from three independently differentiated clones in each genotype, *** P < 0.001, ** P 3

< 0.01, * P < 0.05, compared with CC genotype, one-way ANOVA, Error bars, S.D.). 4

Fig. 3 MALAT1 rs664589 G allele failed to bind miR-194-5p 5

(A-B). Heat map of binding miRNA levels in sw480(A) and sw620(B) cells based on 6

the RNA pull-down assay. The low and high levels of binding miRNA were expressed 7

by green color and red color, respectively (n=3 each genotype). 8

(C). Schematic image of binding interaction between miR-194-5p and MALAT1 9

rs664589 C and G allele. 10

(D, E). The psi-CHECK-2-MALAT1-C-allele or G-allele construct as well as 11

miR-194-5p inhibitor were co-transfected into SW480 (D) and SW620 (E) cell lines. 12

Relative luciferase activities in the indicated cells were determined (n=9 from three 13

independently differentiated clones in each genotype, *P < 0.05, compared with the 14

psi-CHECK-2-MALAT1-C-allele constructs co-transfected with miRNA control, 15

two-tailed t-test, Error bars, S.D.) 16

(F, G). Levels of MALAT1 in SW480 (F) and SW620 (G) cells carrying different 17

rs664589 genotypes (n=9 from three independently differentiated clones in each 18

genotype, *** P < 0.001, ** P < 0.01, compared with CC genotype, one-way analysis 19

of variance (ANOVA), Error bars, S.D., normalized to GAPDH) 20

(H, I). Levels of MALAT1 in SW480 (H) and SW620 (I) cells co-transfected with 21

miRNA control and miRNA-194-5p inhibitor (n=9 from three independently 22




differentiated clones in each genotype, *** P < 0.001, compared with CC genotype, 1

one-way ANOVA, Error bars, S.D., normalized to GAPDH). 2

(J, K). Cell migration and invasion ability in SW480 (J) and SW620 (K) cells treated 3

with miR-194-5p inhibitor (n=9 from three independently differentiated clones in 4

each genotype, NS means no statistical significance, compared with CC genotype, 5

one-way ANOVA, Error bars, S.D.). 6

Fig. 4 miR-194-5p regulates MALAT1 in nuclear 7

(A). Ago2 expression distribution in wild-type CRC cells. 8

(B, C). MALAT1(B) and miR-194-5p(C) levels bound to Ago2 in nuclear fractions of 9

wild-type CRC cells (n=3 for each group, *** P < 0.001, compared with igG, 10

two-tailed t-test, Error bars, S.D.) 11

Fig. 5 Schematic model of regulations among MALAT1 rs664589 polymorphisms, 12

miR-194-5p, and MALAT1 involved in CRC development. 13

The rs664589 C>G polymorphism may alter the binding affinity of the miR-195-5p to 14

the rs664589 mutation region, thereby increasing MALAT1 expression, as well as 15

ultimately facilitating a high level of proliferation, migration, and invasion ability of 16

CRC cells. 17




Published OnlineFirst July 16, 2019.Cancer Res Shenshen Wu, Hao Sun, Yajie Wang, et al. metastasismiR-194-5p contributing to colorectal cancer risk, growth and MALAT1 rs664589 polymorphism inhibits binding to

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