LncRNA SNHG22 Sponges miR-128-3p to Promote Progression of Colorectal Cancer through Upregulating E2F3

Background: Long non-coding RNA (lncRNA) termed small nucleolar RNA host gene 22 (SNHG22) has been reported as a crucial regulator in several types of human cancers. In this study, we aimed to evaluate the function and mechanism of SNHG22 in colorectal cancer (CRC) progression. Methods: Quantitative RT-PCR (qRT-PCR) was used to detect the expression of SNHG22 in adenoma, tumor tissues (TTs), and adjacent nontumorous tissues (ANTs). The biological behaviors of SNHG22 in CRC cell lines were explored both in vitro (CCK-8 assay, ow cytometry, wound scratch, and transwell assays) and in vivo (nude mouse xenograft model). The interaction between SNHG22 and miR-128-3p, and the target genes of miR-128-3p were explored by online tools, qRT-PCR, western blot, and dual-luciferase reporter assay. Results: SNHG22 expression was gradually upregulated in ANTs, adenoma, and TTs. High expression levels of SNHG22 were signicantly related to advanced clinicopathological factors and worse survival in patients with CRC. SNHG22 knockdown markedly prohibited CRC cell proliferation, migration, and invasion; and drove cell apoptosis in vitro; and hindered tumor growth in vivo. Mechanistic investigation showed that SNHG22 could bind to microRNA-128-3p (miR-128-3p) and attenuate its inhibitory effects on the expression levels and activity of E2F3. Rescue experiments exhibited that miR-128-3p inhibition or E2F3 upregulation can offset the functions of SNHG22 knockdown in CRC cells. Conclusion: Our ndings support the existence of an interactive regulatory network of SNHG22, miR-128-3p, and E2F3 in CRC cell lines, indicating that the SNHG22/miR-128-3p/E2F3 axis is a novel diagnostic and therapeutic target in CRC. expression levels of E3F3. Further functional rescue experiments validated that SNHG22 regulates CRC proliferation and invasion by competitively sponging miR-128 ‐ 3p and restoring the activity of E2F3. In summary, our results revealed that SNHG22, as an oncogenic lncRNA in CRC, is upregulated and related to the dismal survival of CRC patients. Functional and mechanistic analyses show that SNHG22 promotes CRC tumorigenesis and metastasis via sponging miR-128-3p, leading to elevated expression of E2F3. These ndings may provide new insights into the development of novel therapeutics for CRC. positive SNHG22 E2F3 CRC tissues from Zhengzhou cohort (R = P COAD, colon adenocarcinoma;


Introduction
Colorectal cancer (CRC) represents one of the most common malignancies worldly. Every year, over 1.3 million people are diagnosed with CRC in the world, of which nearly 0.7 million succumb to this disease. 1 Only about fty percent of patients with CRC will be alive at ve years after diagnosis, 2 although some improvements in early diagnosis and systemic therapies have been made. The pathogenesis and progression of CRC is a complex process, and the potential mechanism remains unclear yet.
Long non-coding RNAs (lncRNAs) are a class of non-encoding RNA longer than 200 nucleotides. Recent researches have indicated that many lncRNAs exert an important role in almost all physiological and pathological processes, including embryonic stem cell, carcinogenesis, and cancer metastasis. 3,4 The lncRNAs have been reported to function as oncogenes or tumor suppressor gene, participating in CRC tumorigenesis and progression. For example, a report from Xu et al. showed that lncRNA MIR17HG drove tumor development and metastasis in colorectal cancer cells via enhancing the expression of NF-κB/RELA. 5 HOTAIR enhanced CRC cells migration and drug resistance via miR-203a-3p dependent Wnt/ ß-Catenin signaling pathway. As an oncogene, LncRNA H19 accelerated cell proliferation and metastasis in CRC by acting on Wnt signaling. 6 Small nucleolar RNAs (snoRNA), a subgroup of ncRNAs with 60-300 nucleotides in length, contributes to the tumorogenesis and metastasis in diverse human cancers. Most snoRNAs are encoded in the introns of snoRNA host genes (SNHG). 7 Speci cally, many SNHGs are shown in the carcinogenesis and progression in CRC. For example, overexpressed SNHG7 is shown tumor tissues (TTs) as compared with adjacent noncancerous tissues (ANTs) in CRC, and this high expression is related to aggressive pathological characteristics, such as tumor size, TNM stage, and distant metastasis, as well as dismal survival. 8,9 SNHG22, located on chromosome 8q21.1, has 2157 nucleotides in length. 10 Several reports have found that SNHG22 contributed to cell growth, migration, invasion, and chemotherapy resistance in epithelial ovarian carcinoma (EOC), papillary thyroid cancer (PTC) and clear cell renal cell carcinoma (ccRCC). [11][12][13] Besides, upregulated SNHG22 was shown in TTs as compared with that in the ANTs, and whose high expression indicated poor prognosis in the three types of human malignancies. [11][12][13] To our knowledge, the biological role and expression patterns of SNHG22 have not been examined in human CRC.
In the current study, we investigated SNHG22 expression in adenoma and CRC tissues and evaluated its clinical signi cance. Moreover, the biological behaviors of SNHG22 in the migratory and invasive phenotype of CRC cell lines were explored in vitro as well as in vivo. We also investigated the underline mechanisms involved in the pro-oncogenic effects of SNHG22 on CRC in detail.

Tissue samples and Cell
We obtained 93 paired CRC tissues and matched ANTs from patients who underwent surgery at the First A liated Hospital, Zhengzhou University. Additional fresh specimens were collected from colorectal adenoma (CRA) patients (n = 33) who underwent colorectoscopy. The diagnosed with CRA or CRC according to histopathologic evaluation. All specimens were snap-frozen in liquid nitrogen quickly and stored at − 80 °C until required. The study was approved by the Ethics Committee of Zhengzhou University. Patients with any history of other cancers, receiving preoperative radiotherapy or chemotherapy, were excluded. The clinical characteristics of all patients are listed in Supplementary  Table S1.

Cell proliferation
Cell proliferation ability was examined using a cell counting kit-8 (CCK-8, Beyotime Institute of Biotechnology, Shanghai, China). The absorbance in each well was measured at 0, 1, 2, and 3 d after transfection by a microplate reader (Bio-Rad, Hercules, CA, USA).

Flow cytometry
Cell cycle distribution was detected using ow cytometry. After CRC cells were harvested, washed, and resuspended in DNA staining solution, we analyzed the fractions in the G0/G1, S, and G2/M phases. Cell apoptosis was detected using an Annexin VFITC/ propidium iodide (PI) Apoptosis Detection kit (MultiSciences Biotech Co., Ltd., Hangzhou, China). After CRC cells (5 × 10 4 /well) were seeded and resuspended in 12well plates, Annexin VFITC (5 µl) and PI (100 µl) were added to each reaction system for 6 minutes. Immediately, ow cytometric assays were conducted on a ow cytometry (EPICS, Beckman, CA, USA).

Wound-scratch
Cell migration ability was examined using a wound-scratch assay. Brie y, CRC cells were seeded into a 96-well plate and grown to con uence. To produce a clear line in the wells, we utilized a sterile 20 µL pipette tip. Cells were photographed to record the wound width (0 h). After 24 h, pictures were retaken.
Migration distance was counted at the time of 0 h and 24 h.

Cell invasion
Transwell inserts used for invasion assay were pre-coated with Matrigel (BD Falcon, San Jose, CA).
Brie y, 2 × 10 5 cells in 200 µL serum-free medium were suspended into the upper chamber, and 700µL medium with 15% FBS was added into the lower chamber. Following 24 h incubation, the chamber membranes were xed in 4% paraformaldehyde and stained with crystal violet for 18 minutes. The cells onto the membrane were counted in ve random elds.

Quantitative RT-PCR (qRT-PCR)
Total RNA was extracted from frozen tissues and exponentially growing cells using TRIzol (Invitrogen, Carlsbad, CA, USA). Nuclear and cytoplasmic RNA was separated using the PARIS Kit (Thermo Fisher Scienti c, Waltham, Mass., USA). The cDNA was synthesized from total RNA (1000 ng) using a reverse transcriptase cDNA synthesis kit (Toyobo). The expression levels were measured using SYBR Premix Ex Taq II (Takara) on the CFX96 sequence detection system (Bio-Rad). The primer sequences were shown in Supplementary Table S2. qRT-PCR data were calculated by the 2 −ΔΔCT methods using actin and U6 as the internal control. 14

Western blot
Total proteins were extracted using RIPA buffer (Thermo Fisher Scienti c). Proteins (20 µg) were separated with 10% SDS-PAGE gels and then transferred to polyvinylidene di uoride (PVDF) membranes (Millipore, USA). After blocking, the membranes were blotted with the primary antibody against E2F3 (ab50917, Abcam, England) for 16 h and then incubated with secondary antibodies for 2 h. Immunoreactive bands were detected using chemiluminescence (Pierce Chemical Co., Rockford, IL). βactin (ab8227, Abcam, England) acted as a loading control.

Luciferase reporter
The predicted binding sites of miR-128-3p with SNHG22-3' untranslated region (UTR) or E2F7-3'UTR were obtained from Starbase 3.0. The mutant type (mut) SNHG22 and E2F7 3′ UTR luciferase reporter vectors were available by using a Mutagenesis Kit (QIAGEN, California, USA). The binding and mutant sequences were respectively cloned into the psiCHECK2 vector (Promega, Madison, WI, USA). CRC cells and 293 T cells in 96-well plates were co-transfected with the wt/mut plasmid and miR-128-3p or NC using Lipofectamine RNAi MAX (Invitrogen, USA). After transfection for 36 h, cells were collected and tested using a Dual-Luciferase Assay System (Promega, Madison, WI) following the manufacturer's instructions. PRL-TK was used as internal control.

RNA immunoprecipitation (RIP)
RIP was carried out using the EZ-Magna RIP Kit (Millipore) and an Ago2 antibody (Abcam). CRC cell lines (2 × 10 7 ) were lysed in RIP buffer and centrifuged at 10 000 g for ve min. Cell lysis was incubated with magnetic beads conjugated with anti-Argonaute (Ago2; ab32381, Abcam) or IgG for 3 hours. After extensive washing using an elution buffer at 65 °C for 10 minutes, the puri ed RNA was analyzed by qRT-PCR.

Animal experiments
Animal experimental procedures were approved by the Animal Ethics Committee of Zhengzhou University (He' nan, China). A total of 15 male immune-de cient BALB/c nude mice (Four-week-old; Shanghai Experiment Animal Centre; Shanghai, China) were maintained under speci c pathogen-free conditions and randomly subdivided into three subgroups. The Lv-shNC, Lv-sh#1, and Lv-sh#2 vectors stably transfected LoVo cells were harvested. Cells (6 × 10 6 ) were injected subcutaneously into the dorsal ank regions of each mouse. Tumor growth was measured every 5 days and was calculated according to the formula volume = (width 2 × length)/2. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH (Bethesda, MD).

Immunohistochemistry (IHC)
After sections were blocked in 3% hydrogen peroxide, they were ready for IHC. The slides were incubated with polyclonal antibodies to Ki-67 (1: 800, Abcam) using the DAKO Envision system (DAKO, Carpinteria, California) as described previously. 15

SNHG22 is upregulated in CRC tissues and cell lines and correlates with poor prognosis in CRC patients
To investigate SNHG22 expression levels in CRC, we conducted the qRT-PCR analysis to examine the SNHG22 levels in TT and ANT colon samples (93 pairs) and human CRA samples (n = 32). Results showed that the expression of SNHG22 in TTs colon tissues was signi cantly higher than those in adenoma tissues and ANTs (Fig. 1A). Additionally, correlation analysis suggested that high SNHG22 expression was signi cantly related to advanced T stage (P < 0.001; Fig. 1B), lymph node metastasis (P = 0.0042; Fig. 1C), distant metastasis (P < 0.001; Fig. 1D), advanced clinical stage (P = 0.042; Fig. 1E), and poor disease-free survival (DFS) and overall survival (OS; P = 0.006 and 0.008, respectively; Fig. 1F-G). Interestingly, data from TCGA also con rmed our results (Fig. 1H-I Table 1). High SNHG22 expression was also con rmed in CRC cell lines (LS174T, LoVo, Caco2, SW480, HT-29, and SW620) as compared with FHC cell lines (Fig. 1J). These results exhibited that SNHG22 was indeed highly expressed in CRC tissues and cell lines, and was associated with poor survival of CRC cases. Also, the coding potential of SNHG22 was calculated using the online assessment tools (CPAT and LNCipedia). The results showed that the coding probability of SNHG22 is very low (Supplementary Fig. S1).

Upregulation of SNHG22 stimulates proliferation, migration, invasion and inhibits apoptosis of CRC cells in vitro
To further investigate the role of SNHG22 in the progression of CRC cell, we used Caco2 and LS147T cell lines with low SNHG22 expression levels in the following gain-of-function experiments. SNHG22 expression was signi cantly enhanced after transfecting with pcDNA/SNHG22 in Caco2 and LS147T cells ( Supplementary Fig.s2). Functionally, the upregulation of SNHG22 promoted the proliferation, G2/M phase arrest, but decreased the rate of early apoptotic cells in both cell lines ( Fig. 2A-F). Furthermore, wound-healing and transwell assays demonstrated a marked enhancement in the proliferation and invasive ability of Caco2 and LS147T cells with SNHG22 overexpression (Fig. 2G-J). These data implicated that overexpression of SNHG22 promoted CRC cell proliferation, migration, invasion, but inhibited apoptosis.

Knockdown of SNHG22 inhibits cell proliferation, migration, invasion, but stimulates apoptosis of CRC cell lines in vitro
To determine whether SNHG22 silencing could affect the biologic function of CRC cells, we used two siRNAs targeting the SNHG22 (si#1 and si#2) for the knockdown experiments. QRT-PCR assay con rmed the knockdown e ciency of both siRNAs in CRC cell LoVo (Supplementary Fig.s3). The CCK-8 and ow cytometry assay demonstrated a marked decrease in the proliferation, G2/M phase arrest, but an increase in the percentage of early apoptosis in LoVo cells (Fig. 3A-C). Further wound-healing and transwell assays showed that silencing SNHG22 attenuated the migratory and invasive ability of LoVo cells ( Fig. 3D-E). These data implicated that knockdown of SNHG22 inhibited growth, migration, invasion of CRC cell lines.

Knockdown of SNHG22 inhibits tumor xenograft growth of CRC in vivo
We then conducted a tumor xenograft assay to con rm the roles of SNHG22 in the growth of CRC in vivo. As exhibited in Fig. 4A-C, compared with the Lv-siNC group, the size, volumes, and weight of tumors in mice of the Lv-si#1 and Lv-si#2 group were remarkably reduced (P < 0.01). Furthermore, qRT-PCR assay veri ed the decreased levels of SNHG22 expression in tumors from the Lv-si#1 and Lv-si#2 group, respectively, compared with control tumors (Fig. 4D). The expression levels of Ki-67 staining by IHC were decreased in tumors from the Lv-si#1 and Lv-si#2 groups compared to that in tumors from the control group ( Fig. 4E-F).
3.5 SNHG22 functions as a ceRNA to regulate miR-128-3p expression in CRC Because subcellular localization of lncRNA could indicate its function, results from the lnclocator 16 indicated that SNHG22 was preferentially localized in the cytoplasm ( Supplementary Fig. s4). Further cellular fractionation experiments also con rmed this in CRC cells (Caco2, LS147T, and LoVo; Fig. 5A). Based on the online softwares, such as Targetscan7.1, StarBase v3.0, miRcode, and miRanda, we found that SNHG22 contained complementary binding sites to miR-128-3p seed regions (Fig. 5B). To test this hypothesis, we rst performed RIP experiments with anti-Ago2 in Caco2, LS147T cells, and observed enrichment of SNHG22 with the Ago2 antibody (Fig. 5C). To further explore whether SNHG22 was regulated by miR-128-3p, we constructed dual-luciferase reporters containing wt or mut putative binding sites of SNHG22 transcripts, respectively. Markedly lower luciferase activity was discovered when SNHG22 wt and miR-128-3p mimic were co-transfected into 293T cells. However, no statistical changes in the luciferase activity were observed in the reporter with the mutated binding sites. These results were also con rmed in Caco2, LS147T cells (Fig. 5D). Furthermore, qRT-PCR assay showed that the upregulation of SNHG22 dramatically decreased, while silencing SNHG22 boosted, the expression levels of miR-128-3p in CRC cells (P < 0.01, Fig. 5E-5F). Expression of SNHG22 had a signi cant inverse relationship with miR-128-3p expression in human CRC tissues from the Zhengzhou cohort (Spearman's R = -0.3857, P < 0.001; Fig. 5G). The ndings demonstrated that SNHG22 acted as a sponge of miR-128-3p in CRC.

MiR-128-3p targets and regulates E2F3
We then performed an analysis of 5 independent algorithmic programs to de ne putative targets, and 25 common predicted targets were identi ed (Fig. 6A). Among these, E2F3 was selected because reports have indicated that E2F3 could enhance CRC progression and be associated with tumor status based on TCGA-COAD dataset ( Fig. 6B and supplementary g. s5A-B). We showed that the up-regulation of miR-128-3p dramatically decreased, while silencing miR-128-3p enhanced, the expression levels of E2F3 mRNA and protein in Caco2 and LS147T cells (Fig. 6C-F). Moreover, Spearman's correlation analysis found a signi cant inverse relationship between the expression of E2F3 mRNA and miR-128-3p in human CRC tissues from the Zhengzhou cohort (Fig. 6G). To determine if E2F3 is an authentic target of miR-128-3p, we generated luciferase reporter gene constructs wherein the 3′-UTR sequences of E2F3 were fused with the renilla luciferase coding sequence (Fig. 6H). We demonstrated that the up-regulation of miR-128-3p signi cantly decreased, while miR-128-3p silencing increased, the luciferase activity of E2F3 wt 3′-UTR (Fig. 6I). Nevertheless, changing miR-128-3p expression showed no signi cant effects on mut construct (Fig. 6I). In addition, the expression of E2F3 mRNA had a signi cant positive relationship with SNHG22 in human CRC tissues from the Zhengzhou cohort (Fig. 6J). Our data suggested that E2F3 could be a direct target of miR-128-3p in CRC cells.

SNHG22 exerts its function by inhibiting the miR-128-3p/E2F3 axis in CRC cells
We then conducted functional rescue experiments to examine whether miR-128-3p/E2F3 axis mediated the biological roles of SNHG22 in CRC cells. Overexpression of SNHG22 or E2F3 stimulated the proliferative, migratory, and invasive capacity of Caco2 cells, and these in uences were reversed by the upregulation of miR-128-3p in caco2 cells. In contrast, knockdown of SNHG22 or E2F3 signi cantly weakened the proliferation, migratory, and invasive capacity of Lovo cells; nevertheless, these inhibitory effects could be attenuated by co-transfection with miR-128-3p inhibitors (Fig. 7A-F). These results showed that miR-128-3p/E2F3 reversed the stimulating effects of SNHG22 on the proliferative, migratory, and invasive capacity of CRC cells.

Discussion
Accumulating evidence has recently revealed that dysregulation of lncRNAs plays an essential role in the development and invasion of diverse human cancers, including CRC. 17,18 Here, we showed that SNHG22 expression was signi cantly elevated in CRC tissues and cell lines. High levels of SNHG22 expression were signi cantly correlated with unfavorable clinicopathological characteristics and worse survival in patients with CRC. Functionally, ectopic SNHG22 could drive cell proliferation, migration, invasion, and prohibit cell apoptosis in CRC cell lines. Knockdown of SNHG22 prohibited xenograft tumor growth in vivo. We further con rmed that SNHG22 executed tumor-promoting function through sponging miR-128-3p and enhancing the expression and activity of E2F3. To the best of our knowledge, our results are the rst to clarify that SNHG22 acts as an oncogene in CRC, and can be used as a potential therapeutic target for this disease.
The current anatomic-based TNM staging system is not able to precisely distinguish the risk of recurrence/distant metastasis in CRC patients. So, it is essential to nd the novel prognostic biomarkers. Dysregulation of lncRNAs in various types of the human tumor was associated with excellent or dismal survival, making them promising prognostic biomarkers. 19,20 For example, SNHG7 expression is enhanced in CRC tissues as compared with noncancerous tissues, and this high expression is related to aggressive pathological characteristics, such as TNM stage, lymphatic metastasis, and distant metastasis, as well as poor prognosis. 8,9 Upregulation of PVT1 was an indicator of dismal survival in several cancers, including CRC. 20 Some researchers have reported the upregulated expression of SNHG22 in EOC, PTC, and ccRCC, and whose expression indicated poor prognosis in the three types of human malignancies. [11][12][13] In this study, we rst explored the expression patterns of SNHG22 in ANT, CRA, and CRC tissues, and found that SNHG22 expression was gradually upregulated in ANTs, CRA, and TTs. The high expression level of SNHG22 was associated with advanced T stage, node involvement, metastasis, and poor survival in CRC patients. So, SNHG22 may serve as an independent prognostic indicator in patients with CRC.
Reports have found that being as oncogenes, several SNHG family members may play critical roles in the processes of tumor advancement. Initially, SNHG22 was shown to be highly expressed in EOC, and could stimulate EOC cell proliferation, invasion, and chemotherapy resistance in EOC. 11 SNHG22 was then determined to be highly expressed in PTC and ccRCC. 12,13 We also checked the effects of SNHG22 on cell phenotype of CRC. We did prove that SNHG22 could promote cell proliferation, invasion, migration, and inhibit cell apoptosis of CRC in vitro. Knockdown of SNHG22 in vivo prohibited xenograft tumor growth. All these results in this report implicate that SNHG22 may act as a critical oncogenic lncRNA in CRC.
Most cytoplasmic lncRNAs exert their effects on the regulation of gene expression through function as ceRNAs, in which lncRNAs regulate miRNAs by competitively binding their target sites on protein-coding mRNA molecules. 21,22 For example, Acting as a ceRNA, MIR17HG bases its biological behaviors on a sequence-speci c interaction with miRNA-17-5p, thus attenuating their roles in mRNA targets by titrating the amount of miRNA. 5 Chen et al. found that lncRNA UICLM promoted the invasion of CRC cell lines by sponging miR-215. 29 Based on computer analyses and experimental assays, we demonstrated that SNHG22 was preferentially localized to the cytoplasm. Furthermore, we searched and con rmed the miRNAs that SNHG22 could sponge in CRC, and the dual-luciferase reporter assay indicated that SNHG22 could interact with miR-128-3p and hinder its biological roles. Previous studies have reported that miR-128-3p could function as a tumor suppressor in several types of human cancer, [23][24][25][26][27] involving in cell proliferation, cell cycle, and chemosensitivity. Concerning CRC, it is suggested that the miR-128-3p enhance the chemosensitivity of oxaliplatin-resistant CRC cells by targeting Bmi1 and MRP5. 25 In addition, the nanocomplexes loaded with miR-128-3p could elevate chemotherapy roles through dualtargeting silence the activity of PI3K/AKT and MEK/ERK pathway in CRC cell lines. 24 We also con rmed the anti-carcinogenic effects of miR-128-3p on CRC cell lines in this study, and the function of miR-128-3p is inhibited after sponging by SNHG22.
As a ceRNA, the function of lncRNA depends on the miRNA target gene. Using ve online databases, we predicted E2F3 as a potential target of miR-128-3p. Recent studies have uncovered that E2F3 plays an essential role in the regulation of cell proliferation, apoptosis, and chemoresistance of human cancer cells. 28,29 Initially, we performed luciferase reporter assays to validate this hypothesis. Furthermore, overexpression of miR-128-3p inhibited, while silencing of miR-128-3p elevated, the mRNA and protein expression levels of E3F3. Further functional rescue experiments validated that SNHG22 regulates CRC proliferation and invasion by competitively sponging miR-128-3p and restoring the activity of E2F3.
In summary, our results revealed that SNHG22, as an oncogenic lncRNA in CRC, is upregulated and related to the dismal survival of CRC patients. Functional and mechanistic analyses show that SNHG22 promotes CRC tumorigenesis and metastasis via sponging miR-128-3p, leading to elevated expression of E2F3. These ndings may provide new insights into the development of novel therapeutics for CRC. The raw data used and analyzed in the current study are available from the corresponding author upon a reasonable request.  Transwell assay analysis of the migration and invasion of Caco2 and LS147T cells transfected with the indicated vectors. CRC, colorectal cancer; NC, negative control; n.s., not signi cant. Data are presented as means ± SD from triplicate experiments. *P < 0.05; **P < 0.01; ***P<0.001 relative to the control. SNHG22 functions as a ceRNA to regulate miR-128-3p expression in CRC A, Subcellular localization of SNHG22 in CRC cell lines (Caco2, LS147T and LoVo). Actin and U6 served as a cytoplasmic and nuclear localization marker, respectively. B, The predicted binding sites of miR-128-3p to the SNHG22 sequence. C, RIP assay was performed with an antibody against Ago2 in Caco2 and LS147T cell lines transfected with NC mimic and miR-128-3p mimic. D, Luciferase activity of 293T, Caco2 and LS147T cell lines cotransfected with miR-128-3p mimic (or NC mimic) and luciferase reporters containing SNHG22 wt or SNHG22 mut transcript were analyzed. E-F, qRT-PCR assay analysis of the expression levels of miR-128-3p in CRC cells after transfection with the indicated vectors. G, A negative correlation between SNHG22 and miR-128-3p expression in human CRC tissues from the Zhengzhou cohort (Spearman's correlation analysis, R = -0.3857, P <0.001). RIP, RNA immunoprecipitation; Ago2, Argonaute2; CRC, colorectal cancer; NC, negative control; wt, wild-type; mut, mutant-type; n.s., not signi cant. Data are presented as means ± SD from triplicate experiments. *P < 0.05; **P < 0.01; ***P<0.001 relative to the control. containing wt or mut E2F3 transcript. The relative luciferase activity was normalized to the Renilla luciferase activity. J, A positive correlation between SNHG22 and E2F3 expression in human CRC tissues from Zhengzhou cohort (R = -0.4047, P <0.001). COAD, colon adenocarcinoma; CRC, colorectal cancer; NC, negative control; wt, wild-type; mut, mutant-type; n.s., not signi cant. Data are presented as means ± SD from triplicate experiments. *P < 0.05; **P < 0.01; ***P<0.001 relative to the control. Figure 7