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Contribution of OCC-1 gene in Wnt signaling regulation
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2 Alternative Splicing of OCC-1 Gene Generates Three Splice Variants and a
3 Novel Exonic MicroRNA which Regulate the Wnt Signaling Pathway
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5 Hadi Najafia, Bahram M. Soltani a*, Sadat Dokanehiifarda, Shirzad Nasiri b, Seyed Javad Mowla a
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8 a Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
9 b Tehran University of Medical Sciences, Shariati Hospital, Tehran, Iran
10 *Corresponding author Tel.: +98-2182884703; fax: +98-2182884717.
11 E-mail address: [email protected]
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16 Running title: Contribution of OCC-1 gene in the Wnt signaling
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18 Keywords: Colorectal cancer / Wnt pathway / Alternative splicing / microRNA / OCC-1 / APPL2
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Contribution of OCC-1 gene in Wnt signaling regulation
1 Abstract
2 Wnt signaling pathway is hyperactivated in most of the colorectal cancers (CRC). Finding new regulators
3 of this pathway represents the potential of cancer diagnosis or treatment. OCC-1 was initially reported as
4 an up-regulated gene in colon carcinoma, without knowing its mechanism of action. Here, two novel
5 transcript variants and an exonic microRNA originated from OCC-1 gene are reported, showing positive
6 effects on Wnt activity. Upregulation of the known OCC-1 variant (assigned as OCC-1A/B) was limited
7 to CRC and its overexpression increased survival of CRC-originated SW480 cells (Wnt+) while, resulted
8 in apoptosis of Wnt-suppressed SW480 cells or HeLa cells (Wnt-), detected by PI staining.
9 Immunocytochemistry showed that OCC-1A/B-encoded peptide was localized to the nucleus, where its
10 overexpression resulted in Wnt signaling upregulation, detected by TOP/FOP flash assay. Non-coding
11 portion of OCC-1A/B transcript had a suppressive effect on Wnt activity and had a negative correlation
12 with APPL2 neighboring gene expression. Unlike OCC-1A/B, the novel OCC-1C splice variant had no
13 expression alteration in CRC and it seemed to encode a smaller peptide with cytoplasmic localization. A
14 60-nucleotide fragment containing AUG start codon is spliced out to produce OCC-1D non-coding RNA
15 variant. The 60-nucleotide RNA was validated as precursor of a novel microRNA, we named as miR-ex1.
16 Both OCC-1D and miR-ex1 were coordinately up-regulated in CRC. MiR-ex1 functional analysis revealed
17 that it is targeting APC2 tumor suppressor gene and is an activator of Wnt signaling pathway. Overall,
18 OCC-1 gene is now introduced as a novel Wnt signaling regulator and as a potential therapy target.
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Contribution of OCC-1 gene in Wnt signaling regulation
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2 1. Introduction
3 Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females
4 [1]. Both environmental and genetic factors contribute to CRC development. While contribution of
5 genetic factors is estimated to be 35%, over 25% of this contribution is unknown yet [2]. Genetic
6 alteration of Wnt signaling components, which results in signaling pathway overactivation, is a crucial
7 genetic event in CRC development [3-6]. For example, mutations of APC and β-catenin occur in a high
8 proportion of sporadic CRC (up to 80% and 10%, respectively) [7]. Other than mutations, there are other
9 side players which modulate Wnt signaling pathway [8]. Finding new players in Wnt signaling pathway
10 will likely yield a better understanding of Wnt signaling involvement in CRC. These new factors would
11 be promising prognostic marker or therapeutic targets. There was a preliminary data suggesting that
12 human locus 12q23.3 might be a novel CRC susceptible locus [9]. In this locus, Overexpressed in
13 Colorectal Carcinoma-1 (OCC-1) gene (officially named C12orf75; NCBI gene ID: 387882) was initially
14 reported as an up-regulated gene in CRC. OCC-1 gene consists of 11 exons (Fig.1 A) and its genomic
15 DNA spans a region of ~164 kb on 12q23.3 (hg38: nt 105,235,250-105,398,725). Current human
16 GENCODE release (version 25) revealed that it transcribes five different splicing RNA variants, two of
17 them (OCC-1A and OCC-1B) are similarly spliced (with the same 6 exons) but different in lengths of
18 their 5´- and 3´- ends [9]. Recently, a 63-amino acid (aa) protein has been attributed to these transcripts
19 with a suggested function in stemness and adipogenesis [10]. However, the exact function of OCC-1 gene
20 is not clearly known yet. APPL2 gene (NCBI gene ID: 55198), also known as DIP-13β (DCC interacting
21 protein 13-β), is located upstream of OCC-1 gene with ~ 970 bp overlapping region. APPL2 protein
22 belongs to APPL protein family [11] whose structure and function is well known and they have diverse
23 set of functions [12]. Downstream region of OCC-1 is vacant of any coding gene with the exception of
24 the processed ST13 pseudogene 3 (ST13P3) (NCBI gene ID: 144638). This pseudogene is just inferred
25 from homology [13] without any reported transcript. Nevertheless, the OCC-1 downstream region
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Contribution of OCC-1 gene in Wnt signaling regulation
1 contains several reported ESTs that are not well characterized yet. Some of these ESTs belong to Cancer
2 Susceptibility Candidate 18 (CASC18) (NCBI gene ID: 101929110) gene which is recently classified as a
3 long non-coding RNA in human genome [14].
4 Here, three novel transcripts of OCC-1 gene are introduced, two of them including a novel miRNA are
5 shown to be associated with Wnt signaling pathway. We also show that OCC-1 gene affects the
6 transcription level of its neighboring gene APPL2.
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Contribution of OCC-1 gene in Wnt signaling regulation
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3 2. Results
4 2.1. Identification of two novel splice variants of OCC-1 gene and a novel exonic microRNA
5 The previously reported variants of OCC-1 (OCC-1A and OCC-1B), contain common central region but
6 differ in their 5' and 3'-ends. A single accession number (NM_001145199) represents both variants in the
7 GenBank, and here we have referred them as OCC-1A/B transcript variant. By using OCC-1 specific
8 primers against a cDNA library (originated from U87 cell line), two novel OCC-1 splice variants were
9 discovered, designated as OCC-1C (GenBank acc. #: AB735447) and OCC-1D (GenBank acc. #:
10 AB735446) (Fig. 1B). Conventional donor and acceptor splice sites between the exons 6 and 7 of the
11 gene have been shifted inside (pointed by vertical arrows in Fig. 1B), causing 36 nucleotides deletion of
12 OCC-1A/B to produce OCC-1C variant (Fig. 1B). Using two cryptic splice sites within the third exon of
13 OCC-1 gene, 60 nucleotides including OCC-1A/B start codon is spliced out, generating OCC-1D variant
14 (Fig. 1B).
15 Three additional splice variants (here we refer them as: OCC-1E, OCC-1F and OCC-1G) have also been
16 recently reported in NGS data however, the primers that have been used for the amplification of OCC-
17 1A/B, OCC-1C and OCC-1D were not suitable for the amplification of other variants (Fig. 1C). Both
18 OCC-1C and OCC-1D are also detectable in the recent NGS databases (S1).
19 Interestingly, the DNA segment corresponding to the spliced out 60 nucleotides RNA fragment
20 represented a saddle-like conservation plot, which is a prominent characteristics of miRNA precursors
21 (Fig. 1D) and RNA fold software predicted a stem-loop structure for it (Fig. 1E). This stem-loop
22 (nominated as pre-mir-ex1; acc. #: HF679086) had multiple characteristics of a putative miRNA
23 precursor (S2). Furthermore, pre-mir-ex1 and its predicted mature form, miR-ex1, were PCR amplified
24 from a U87 cell line cDNA preparation. PCR products were cloned in TA vectors and sequencing of
25 several colonies indicated that 19 nucleotides long mature miRNA is produced. The identity of miR-ex1
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Contribution of OCC-1 gene in Wnt signaling regulation
1 (acc. # LT601573) was confirmed by alignment of the sequencing results with the sequence of miR-ex1
2 precursor (Fig. 1F). MiR-ex1 expression was also detected in other human cell lines (S4-3C) and its
3 sequence is detectable in recent NGS database (S2). To date, no identical miRNA has been reported in the
4 miRbase database for miR-ex1.
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7 2.2. Differential expression pattern of novel OCC-1 splice variants
8 In order to analyze the expression pattern of OCC-1 splice variants in human cells, specific primers at
9 unique exon-exon junctions were designed and PCRs (for 35 cycles) were carried out on cDNAs of
10 different human cell lines. OCC-1A/B transcript was detected in SW480, KYSE, AGS, 5637, SK-MEL3,
11 MCF-7, HeLa, HUH-7, K562, U87-MG and HEK293-T cell lines as well as stem- and progenitor cells
12 such as NT-2, Human Cardiomyocytes (HCM1 & 2) and undifferentiated Cardiac Stem Cells (undiff.
13 CSCs). While, OCC-1C and OCC-1D transcripts were detected in several cell lines, they were not
14 expressed in the tested NT-2, HCM and cardiac stem- or progenitor cell lines (Fig. 2A).
15 Since, preliminary reports attributed a non-coding RNA function to the OCC-1 gene [9], OCC-1
16 transcript variants expression status was analyzed in 42 paired tumor and normal colorectal tissues in
17 combination with expression analysis of its neighboring genes located on 12q23.3 chromosomal region
18 (Fig. 2B). Compared to the normal pairs, APPL2 gene expression level was significantly downregulated
19 in the tumors (p<0.0043) (Fig. 2C). In the same pairs, the expression level of OCC-1C variant was
20 unchanged in CRC samples. However, the expression level of OCC-1A/B and OCC-1D showed ~5 and
21 ~12 folds increase in tumor samples, respectively. Similar to the level of OCC-1D variant, miR-ex1
22 expression level showed ~12 folds upregulation in CRC samples compared to their non-tumor paired
23 samples (Fig. 2D). CASC18 showed no expression in CRC and other epithelial tissues, including
24 esophageal, gastric and bladder tissue sample pairs using real time PCR (data not shown). In order to
25 investigate if miR-ex1 and OCC-1D production are coordinated, their expression pattern was compared in
26 CRC tissues, using real time PCR. Results suggested a positive correlation between these RNA variants
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Contribution of OCC-1 gene in Wnt signaling regulation
1 (S4-4), supporting simultaneous formation of OCC-1D and miR-ex1. Further, the absolute expression
2 level of OCC-1 splice variants was measured and represented as “RNA copies per 100 ng of total RNA”
3 in HT-29, HCT116 and SW480 cell lines, representing stages I, II and IV of CRC, respectively. Real time
4 analysis of OCC-1 transcript variants in these cell lines indicated no correlation of OCC-1C expression
5 with CRC staging. However, OCC-1A/B was increased and OCC-1D was decreased in the cell lines with
6 higher level of malignancy (Fig. 2E).
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9 2.3. Coding potential of OCC-1 transcripts and their effects on fibroblast morphology
10 A 63-amino acid peptide has been recently attributed to the OCC-1A/B transcript. Sequence analysis of
11 OCC-1C variant indicated that 36 nucleotides (corresponding to the 12 amino acids) have been eliminated
12 from the middle of OCC-1A/B variant without changing the translation frame, and potentially encodes an
13 unknown 51 amino acids peptide (Fig. 3A). The coding potential of OCC-1 variants and subcellular
14 localization of related peptides were further analyzed using FLAG-tagged constructs, transfected in
15 SW480 cell line. ICC results indicated that OCC-1A/B peptide was localized in the nucleus (Fig. 3B; top).
16 OCC-1C seemed was capable of producing a novel peptide (GenBank Acc. #: BAM34484.1), which was
17 localized in the cytoplasm of transiently transfected SW480 cells (Fig. 3B; middle).
18 Overexpression of the FLAG-tagged OCC-1A/B construct resulted in size and number reduction of the
19 transfected fibroblast cells as well as some morphological changes (Fig. 3C; top left). These cells
20 demonstrated increased cell death, detected by flow cytometry (Fig. 3C; top right). The cells that were
21 transfected by the FLAG-tagged OCC-1C construct did not show such alterations compared to the cells
22 transfected by scrambled control vector (Fig. 3C; middle and bottom). In all of these experiments, real
23 time PCR confirmed the overexpression of exogenous OCC-1A/B and OCC-1C variants (data not shown).
24 Bioinformatics analysis predicted nuclear localization for OCC-1A/B peptide while, cytoplasmic
25 localization for OCC-1C peptide (S3-C).
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Contribution of OCC-1 gene in Wnt signaling regulation
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2 2.4. Tumor-specific up-regulation of OCC-1A/B variant and its cell type-specific survival
3 promotion
4 Compared to the results of CRC samples, transcription level of OCC-1A/B variant was measured in 10
5 paired samples of esophageal, gastric and bladder tumor tissues (T) versus their marginal non-tumor (N)
6 sections. While, a distinct upregulation of OCC-1A/B transcript was evident in CRC specimens, a
7 significant downregulation was detected in other cancer types (Fig. 4A). Wnt activity was compared in
8 these cancer types using qRT-PCR against c-Myc (as a Wnt pathway target gene [15]) and results
9 indicated the highest transcription level of c-Myc gene in the CRC specimens (Fig. 4A).
10 In order to investigate the OCC-1A/B overexpression effect on the cell cycle progression, OCC-1A/B
11 cDNA was overexpressed in cell lines with different Wnt signaling activity (Wnt- and Wnt+). Colorectal
12 originated SW480 (Wnt+) and non-colorectal HeLa (Wnt-) cells were transiently transfected using OCC-
13 1A/B cDNA construct. The cell cycle was promoted in SW480 cells (Fig. 4B) while, it was arrested in
14 HeLa cells (Fig. 4D), detected by flow cytometry. Consistently, Annexin V/PI results indicated reduced
15 early and late apoptosis rate in the transfected SW480 cells (Fig. 4C) while, increased apoptosis rate in
16 the transfected HeLa cells (Fig. 4E). When, Wnt signaling was suppressed in the SW480 cells using
17 XAV939 small molecule (10 µM), cell cycle was arrested (Fig.4F) and apoptosis rate was increased (Fig.
18 4G) following the transfection of OCC-1A/B cDNA in these cells.
19 Consistently, OCC-1A/B overexpression ended in increased SW480 cells viability while, resulted in
20 reduced cell viability in the Wnt- HeLa and in the Wnt-suppressed SW480 cells, detected by MTT assay
21 (Fig. 4H). Finally, qRT-PCR expression analysis of interferon-β gene indicated that observed cell death
22 induction in HeLa and Wnt-suppressed SW480 cells, has not been due to the interferon response (S6).
23 All data of OCC-1A/B-transfected cells were compared to that of scrambled- and non-transfected controls
24 and p-value < 0.05 was considered as statistical significance level.
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Contribution of OCC-1 gene in Wnt signaling regulation
1 2.5. Suppressive effect of OCC-1 RNA on transcription level of APPL2 gene provides a putative
2 CRC progression index
3 A significant negative correlation (Pearson correlation = -0.699; p-value < 0.0001) was obtained between
4 OCC-1A/B and APPL2 expression levels in 42 paired CRC specimens (Fig. 5A & B). In some cases,
5 samples with high level of OCC-1A/B expression had very low or undetectable APPL2 expression even in
6 40 cycles of PCR (Pearson correlation = -1.0) (Fig. 5A). The alteration of APPL2 gene expression was
7 further investigated under the OCC-1A/B protein coding ORF or OCC-1-cDNA overexpression
8 conditions in SW480 cell line. To this aim, about 300 bp of OCC-1A/B upstream sequence corresponding
9 to its ORF and 5’-UTR sequences was PCR amplified and cloned in pCMV-Tag4 expression vector (Fig
10 5C, fragment a-b). Also, 1366 bp cDNA sequence of OCC-1 gene (including its long 3´-UTR) was
11 cloned in the same vector (Fig. 5C, fragment a-c). While, overexpression of OCC-1-cDNA strongly
12 suppressed APPL2 transcription, overexpression of its ORF region did not show such an effect (Fig. 5C).
13 Since, both constructs shared a-b sequence, the effects of full length OCC-1-cDNA on APPL2 expression
14 level might be attributed to the non-coding segment of this transcript (b-c fragment). Using a
15 complementary anti-sense RNA against OCC-1A/B variant, resulted in increased expression level of
16 APPL2 gene (Fig. 5C). The effects of these constructs were compared to the scrambled sequence cloned
17 in the same vector (pCMV-tag4). A strong negative correlation (Pearson correlation = -0.828; p-value <
18 0.0001) is calculated between the APPL2 and OCC-1A/B expression in this experiment (Fig. 5D).
19 Since, OCC-1 and APPL2 negative correlation was supported by several experiments (presented in Fig. 2
20 and Fig. 5), OCC-1/APPL2 index was deduced from a significant number of tumor samples (n= 28 ) and
21 results were categorized based on the CRC progression (S7). Analysis of the categorized data indicated a
22 high positive correlation between OCC-1/APPL2 index and the higher stages of CRC.
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Contribution of OCC-1 gene in Wnt signaling regulation
1 2.6. Linking OCC-1 gene to the Wnt signaling pathway
2 OCC-1A/B and miR-ex1 functional effect on Wnt signaling was further investigated using diverse
3 experimental procedures. In comparison with the scrambled-transfected cells, OCC-1A/B-ORF
4 overexpression led to the Wnt signaling upregulation, detected by TOP/FOPflash assay. However, OCC-
5 1A/B-cDNA construct overexpression led to the Wnt signaling attenuation (Fig. 6A). In order to
6 discriminate the coding and non-coding RNA functions of OCC-1 gene against Wnt signaling, mutated
7 OCC-1-cDNA (harboring ATG CTG mutation) was also transfected in SW480 cells. Wnt signaling
8 pathway was strongly suppressed in these cells compared to the scrambled control (Fig. 6A). Wnt
9 signaling upregulation effect of OCC-1A/B-ORF overexpression was further supported via qRT-PCR
10 against Cyclin-D1 and c-Myc genes which are targeted by this pathway (Fig. 6B).
11 In comparison with the mock-transfected cells, miR-ex1 overexpression led to the Wnt signaling
12 upregulation, and miR-ex1 downregulation led to the Wnt signaling suppression, detected by
13 TOP/FOPflash assay (Fig. 6C). Wnt signaling upregulation effect of miR-ex1 overexpression was further
14 supported via qRT-PCR against Cyclin-D1 and c-Myc genes (Fig. 6D). Co-transfection of OCC-1A/B-
15 ORF or miR-ex1 with FOPflash construct did not change the luciferase expression in these experiments.
16 Results of TOP/FOPflash assay are presented as ratio of the TOP over FOP luciferase signals.
17 APC2 gene with a pivotal role in Wnt pathway was predicted as the main target gene for miR-ex1 (S5).
18 Moreover, RT-PCR analysis indicated that miR-ex1 overexpression caused APC2 mRNA level reduction
19 (Fig. 6E). MiR-ex-1 overexpression effect on APC2 protein was also investigated through ELISA.
20 Compared to the multiple controls, results indicated that APC2 protein level has been reduced following
21 the miR-ex1 overexpression in SW480 cells. Consistently, miR-ex1 downregulation resulted in subtle
22 (non-significant) APC2 protein level elevation (Fig. 6F). Direct interaction of miR-ex1 and APC2
23 transcripts was also investigated through cloning of APC2, 3´-UTR sequence downstream of the
24 luciferase ORF. Compared to controls, luciferase activity was significantly downregulated in the cells
25 overexpressing miR-ex1, supporting the direct interaction of miR-ex1 with 3´-UTR sequence of APC2.
26 Neither overexpression nor downregulation of miR-ex1 affected luciferase activity level of a construct
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Contribution of OCC-1 gene in Wnt signaling regulation
1 made of luciferase ORF fused to non-target TrkC 3 -UTR sequence (Fig. 6G). According to flow
2 cytometry data, overexpression of OCC-1A/B or miR-ex1 constructs induced cell cycle progression, while
3 downregulation of OCC-1A/B or miR-ex1 caused an elevation in proportion of the cells in sub-G1 stage
4 (Fig. 6H and J). Consistently OCC-1A/B-ORF or miR-ex1 overexpression led to the increased cell
5 viability of transfected cells, detected by MTT assay (Fig. 6I and K).
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Contribution of OCC-1 gene in Wnt signaling regulation
1 3. Discussion
2 Wnt signaling pathway is the most altered signaling pathway in CRC [4, 16]. The human locus 12q23.3 is
3 enriched for cancer-related genes including APPL2, OCC-1, CASC18 and ST13P3. APPL2 is interactive
4 with Deleted in Colorectal Carcinoma (DCC) gene [17] and is considered as a positive regulator of Wnt
5 signaling pathway [18]. OCC-1 has been considered as a differentially upregulated gene in CRC [9] and
6 ST13P3 is a pseudogene of the CRC-related gene ST13 [13]. Here, we intended to investigate the
7 association of 12q23.3 locus and CRC through functional analysis of OCC-1 novel transcripts against
8 Wnt signaling pathway components.
9 OCC-1 gene is reported to be transcribed in two highly similar transcript variants that here we assigned
10 them as OCC-1A/B variant. Here for the first time, two novel splice variants of OCC-1 gene assigned as
11 OCC-1C, OCC-1D and a novel miRNA named miR-ex1 are introduced (Fig. 1). OCC-1 was initially
12 reported as a non-coding RNA gene [9] however, it (OCC-1A/B variant) encodes a protein called AGD3
13 with an attributed role in differentiation of adipocytes [10]. Later, it was reported that AGD3 protein is
14 expressed in several areas of rat brain and binds to the insulin receptor substrate 4 (IRS4) protein,
15 promoting insulin signaling [19].
16 OCC-1C lacks an internal 36 nucleotides (12aa) at the middle of AGD3 protein coding ORF (Fig. 1B and
17 3A). In another event, AUG start codon of the gene has been spliced out to create OCC-1D non-coding
18 RNA variant (Fig. 1B). Spliced out short RNA segment had most of the miRNA precursor features [20]
19 including 60 nt length, hairpin secondary structure and saddle-like conservation pattern (Fig. 1D and E).
20 Therefore, production of an OCC-1-originated miRNA was investigated following a protocol we have
21 used for other miRNA discoveries [21-23]. Briefly, predicted miRNA precursor and its mature form
22 (miR-ex1) were PCR amplified from cDNA library of U87 cell line (S4-3). Then, sequencing of the
23 amplification products indicated that at least 19-nucleotide long miR-ex1-5p has been produced in this cell
24 line (Fig. 1F). Recently, RNA-seq databases have introduced miR-ex1-3p which supports our findings
25 (S2-3).
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Contribution of OCC-1 gene in Wnt signaling regulation
1 OCC-1A/B variant was expressed in different cell lines however, OCC-1C and OCC-1D variants were not
2 detected in the tested stem or progenitor cell lines (Fig. 2A). It remains to be tested if these variants are
3 involved in stemness of the cells. These results also indicate that novel OCC-1 transcripts are not aberrant
4 transcripts, which often are produced during the cancer progression [24-26].
5 OCC-1D and miR-ex1 expression level changes were about three fold higher than OCC-1A/B expression
6 level change in CRC specimens (Fig. 2D) which candidate these variants as distinct markers for CRC
7 progression with potential clinical application. Bioinformatics analysis predicted a coding ORF for OCC-
8 1 gene which is known to encode AGD3 protein [10]. Deduced protein sequence alignment indicated
9 that 12 aa have been deleted from the middle of this ORF to form OCC-1C peptide (Fig. 3A).
10 Distinguished detection of OCC-1A/B and OCC-1C peptides using immunostaining is laborious due to
11 their small size and high similarity of amino acid sequences. Therefore, their coding potential and
12 subcellular localization was investigated using exogenous expression of FLAG-tagged OCC-1A/B or
13 OCC-1C sequences, followed by ICC experiment and using monoclonal antibody against the tag (Fig.
14 3B). As a result, SW480 cells expressing FLAG-tagged OCC-1A/B showed concentrated nuclear
15 fluorescent signal for OCC-1A/B encoded peptide, while OCC-1C-related signal was localized in the
16 cytoplasm of transfected SW480 cells (Fig. 3B). Consistent to our results, PSORTII bioinformatics tool
17 predicted nuclear and cytoplasmic localization of OCC-1A/B and OCC-1C peptides, respectively (S3-C).
18 Nevertheless, protein atlas database (http://www.proteinatlas.org/search/agd3) has reported both nuclear,
19 cytoplasm and cell membrane localization for AGD3 protein (S3-B) without taking to account the
20 presence of OCC-1C encoded peptide. Also, challenging our results, others have used a polyclonal
21 antibody for detection of an endogenously expressed AGD3 homologue in rat brain cells and reported its
22 cytoplasmic localization [19]. Since, OCC-1A/B and OCC-1C peptides are small and they only differ in
23 12 aa, the use of polyclonal antibody may explain our different results.
24 Furthermore, unlike the OCC-1C, OCC-1A/B overexpression resulted in morphological alteration of
25 transfected fibroblast cells (Fig. 3C; left). Nuclear localization of OCC-1A/B peptide proposed a
26 regulatory effect for this peptide which motivated us to follow its effect on biology of the cell.
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Contribution of OCC-1 gene in Wnt signaling regulation
1 OCC-1A/B transcription level is increased in the CRC samples, while it is decreased in other tested
2 epithelial cancers, compared to the normal paired samples (Fig. 4A). Similar effect has been reported for
3 some other genes such as TGF-β which is downregulated in gastric [27] and upregulated in other cancers
4 [28, 29] including CRC [30]. This CRC-specific effect was further supported by flow cytometry results
5 of OCC-1A/B cDNA overexpression; while OCC-1A/B overexpression promoted cell cycle in SW480
6 (Wnt+ CRC-originated cell line), it increased cell death rate in HeLa ( Wnt-) and Wnt-suppressed SW480
7 cell lines (Fig. 4B-G).
8 Transfection of OCC-1A/B -cDNA in a Wnt-suppressed SW480 cell lines, resulted in induction of early
9 and late apoptosis in these cells (Fig. 4G). To investigate if such an induced apoptosis rate is specific to
10 OCC-1A/B but not the interferon responses [31], we analyzed the expression of interferon-β (IFN-β) gene
11 which is associated with apoptosis [32]. Results indicated that IFN-β remained unchanged during all
12 transfection procedures (S6), implying that detected apoptosis is associated to OCC-1A/B expression in
13 these cells. The mechanism(s) of distinctive OCC-1A/B apoptotic effect in Wnt- cells remains to be
14 investigated. Consistently, TOP/FOPflash luciferase assay [33] indicated that OCC-1A/B variant
15 overexpression has resulted in increased Wnt signaling activity in SW480 cell line (Fig. 6A). This result
16 emphasized on the colorectal-specific regulatory role of the OCC-1A/B variant. Wnt signaling activation
17 caused by OCC-1A/B overexpression and high level of OCC-1A/B expression in CRC samples proposed
18 it as a novel CRC-specific oncogene.
19 OCC-1A/B ORF encompasses only 14% of the 1366 nucleotides long transcript and the rest is non-
20 coding. On the other hand, non-coding RNAs may modulate the neighboring genes expression in Cis or
21 Trans [34]. Therefore, OCC-1D variant and also OCC-1A/B long UTR sequence may affect the flanking
22 genes expression. Real time analysis of OCC-1 variants and its neighboring genes in CRC specimens
23 showed a strong negative correlation between OCC-1A/B and APPL2 gene expression level (Fig. 2D, 5A
24 and B). Neighboring co-expressed genes often share regulatory elements [35, 36]. However, no such
25 element was detected for these two genes. In order to investigate if OCC-1A/B peptide, alone or along
26 with its long non-coding 3´-UTR are responsible for the negative correlation with APPL2 gene,
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Contribution of OCC-1 gene in Wnt signaling regulation
1 corresponding sequences of OCC-1A/B were cloned and overexpressed in SW480 cell line in a gain of
2 function strategy. Results indicated that OCC-1A/B 3´-UTR is responsible for the observed negative
3 correlation (Fig. 5C and D). Regardless of the mechanism(s), APPL2 downregulation under the
4 overexpression of OCC-1 cDNA, suggest that OCC-1 non-coding RNA may function as an upstream
5 regulator of APPL2 gene expression. Similar effects on nearby genes are now reported for many other
6 non-coding RNA genes [37]. When, OCC-1A/B ORF (without 3'-UTR) and OCC-1A/B full length cDNA
7 (having 3'-UTR) were equally transfected in SW480 cells, real time PCR detected higher level of OCC-
8 1A/B ORF RNA than that of OCC-1A/B full length cDNA. Transfection of a full length cDNA with
9 impaired ATG start codon resembled the results of wild type cDNA transfection (Fig. S10). 3'-UTR in
10 OCC-1A/B is encoded by two exons which are unified through splicing out of the last intron. Such
11 transcripts are rare and would normally be expected to undergo nonsense-mediated mRNA decay [38]
12 that may justify lower level of OCC-1A/B full length cDNA. According to the observed negative
13 correlation, a CRC progression index was deduced between the OCC-1A/B and APPL2 genes’ expression
14 levels, which could be used as an indicator of CRC progression with potential clinical application (S-7).
15 The highest level of this ratio is specific to stage 4 of CRC in which OCC-1 is overexpressed while,
16 APPL2 transcripts are not detectable. It remains to be tested if downregulation of OCC-1 or upregulation
17 of APPL2 is capable of suppressing CRC.
18 While OCC-1 versus APPL2 expression level (Fig. 5 and S7) provided a strong CRC progression index,
19 still it does not associate 12q23.3 human chromosome locus to CRC. More direct evidence came from
20 our discovery of OCC-1-originated miR-ex1 which was capable of targeting APC2 transcripts (Fig. 6E-
21 G). MiR-ex1 or OCC-1A/B overexpression resulted in promotion of Wnt signaling and consistently, their
22 knockdown attenuated this pathway (Fig. 6A-D). Furthermore, their knockdown resulted in increased cell
23 death (Fig. 6H and J). Taken together, OCC-1-originated transcripts (Fig. 1) in a cell type-specific fashion
24 (Fig. 4) can modulate Wnt signaling pathway (Fig. 6) directly through targeting of APC2 transcripts or
25 other nuclear components of Wnt signaling pathway (Fig. 3), or indirectly through regulation of APPL2
26 gene expression (Fig. 5).
15
Downloaded from rnajournal.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press
Contribution of OCC-1 gene in Wnt signaling regulation
1 Accordingly, here a model is suggested which functionally links human chromosome 12q23.3 co-located
2 OCC-1 and APPL2 genes together and also to Wnt signaling pathway (Fig. 7). Since OCC-1A/B peptide
3 is localized to the nucleus (Fig. 3) and its overexpression resulted in Wnt signaling upregulation (Fig. 6),
4 it may activate Wnt signaling through TCF activation which is shown as a nonsolid arrow in the model,
5 denoting unknown mechanism (Fig. 7). While, APPL2 is a positive regulator of Wnt signaling pathway
6 [39] its transcription is suppressed by OCC-1 non-coding RNA portion by unknown mechanism (Fig.
7 5C), shown by solid red line and question marks (Fig 7). That may justify when OCC-1A/B full length
8 cDNA is overexpressed in the SW480 cell line, its non-coding RNA function attenuates Wnt signaling
9 upregulation which is induced by OCC-1A/B coding function (Fig. 6A). Non-coding RNA function of
10 OCC-1 gene is more highlighted when only ATG start codon of the gene is point-mutated and its
11 suppression effect on Wnt signaling is stronger than wild type OCC-1 cDNA sequence (Fig. 6A). That
12 means, OCC-1 upregulates Wnt signaling pathway through both; its nuclear localized peptide (Fig. 3) and
13 also through production of miR-ex1 (Fig. 1 and 6) which is shown by red arrows in the model. Overall,
14 Wnt signaling pathway is extended in our model by introducing OCC-1 as a new r