Knockdown of long noncoding RNA 01124 inhibits the malignant behaviors of colon cancer cells by regulating miR-654-5p/HAX-1

doi:10.21203/rs.3.rs-1445265/v2

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Knockdown of long noncoding RNA 01124 inhibits the malignant behaviors of colon cancer cells by regulating miR-654-5p/HAX-1

https://doi.org/10.21203/rs.3.rs-1445265/v2

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Background

Previous studies have shown that long noncoding RNAs (lncRNAs) play a key role in cancer, including colon cancer (CC). However, the exact role of long noncoding RNA 01124 (LINC01124) in CC and its mechanisms of action remain unknown. In the present study, we aimed to investigate the functional effects and the possible mechanism of LINC01124 in CC.

Results

we demonstrated that LINC01124 was upregulated in colon cancer tissues and cell lines. Further functional studies showed that knockdown of LINC01124 significantly suppressed the proliferation, migration and invasion of colon cancer cells in vitro and in vivo. Moreover, in subsequent mechanistic experiments, LINC01124 was found to act as a sponge to suppress microRNA 654-5p, which targeted HAX-1. Downregulation of LINC01124 decreased the expression of HAX-1, and overexpression of a miR-654-5p inhibitor attenuated the sh-LINC01124-induced inhibition of CC cell proliferation, migration and invasion.

Conclusion

Collectively, all the results revealed a role for the LINC01124/miR-654-5p/HAX-1 axis in the tumorigenesis and progression of CC, suggesting that LINC01124 may be a therapeutic target for colon cancer treatment.

colon cancer

LINC01124

proliferation

miR-654-5p

HAX-1

Colon cancer (CC) is the third most common malignancy worldwide ¹, with more than 1.2 million newly diagnosed patients resulting in 551 thousand deaths each year worldwide ². High morbidity and mortality make colon tumors a serious threat to human health. The lack of early diagnosis is one of the most important reasons for the high incidence of CC ³. Another important reason is that malignant CCs often possess the characteristics of rapid progression and invasion, which can result in a poor prognosis ^4,5. Therefore, the identification of key molecules in the development of CRC tumors may provide breakthroughs in the targeting and biomarker selection of antitumor drugs for this malignant disease.

Long noncoding RNA (lncRNA) is non-protein coding RNA transcript with a length greater than 200 nt that can be localized in the cytoplasm or nucleus ^6–8. At present, there are more lncRNA genes than protein-coding genes ^9,10. Many studies have reported that lncRNAs are involved in the regulation of a variety of processes, and their dysfunction can lead to many diseases, including tumors ¹¹. LncRNAs are also involved in the regulation of tumor proliferation, metastasis, autophagy and apoptosis by acting as oncogenes or tumor suppressor genes ^11,12. The dysregulation of many lncRNAs, such as lncRNA CCAT1, lncRNA POU6F2-AS2, and lncRNA ROR1-AS1, has been closely related to colon progression ^13–15. A previous study also confirmed that LINC01124 significantly inhibited the proliferation, migration, and invasive ability of NSCLC cells ¹⁶. However, the potential effect and mechanism of LINC01124 on colon cancer remain unclear. Therefore, we decided to explore the potential functions of LINC01124 in colon cancer.

HAX-1, located at chromosome 1 (1q21.3), is reported to play an important role in variety of tumors ^17–20. For example, HAX-1 was revealed to be overexpressed in hypopharyngeal squamous cell carcinoma and could promote cancer growth and migration ¹⁹. HAX-1 was found to be targeted by miR-100 to regulate the sensitivity of breast cancer cells to cisplatin ¹⁸. Moreover, in colorectal cancer, HAX-1 had been reported to be targeted by miR-654-5p to regulate the malignancy behaviors ²⁰.

LINC01124 is located on chromosome 2q31.1 and contains one exon. In the current study, we investigated the role of LINC01124 in the progression of CC and explored the underlying mechanisms of LINC01124. The results showed that LINC01124 was upregulated in CC tissues and cell lines. Knockdown of LINC01124 suppressed cell proliferation, migration and invasion in vitro and in vivo. In addition, miR-654-5p was verified to be a target miRNA of LINC01124, and we showed that sh-LINC01124 inhibited the progression of CC by modulating miR-654-5p-HAX-1 signaling. All these findings suggested that LINC01124 may be an underlying biomarker and a potential therapeutic target for colon cancer.

Cell culture

A human normal colonic epithelial cell line (NCM460) and 5 human colon cancer cell lines (LoVo, SW620, HT29, HCT116 and SW480) were purchased from Nanjing Cobioer Biotechnology Co. Ltd. (Nanjing, Jiangsu, China). All of the CRC cell lines were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) medium supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin. All cells were maintained in an incubator at 37°C with 5% CO₂ in a humidified atmosphere ²¹.

Transfection and lentivirus transduction

The miR-654-5p inhibitor and its negative control (Ctrl) were purchased from RiboBio, and oligonucleotide transfection was carried out using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). A short hairpin RNA (shRNA) targeting LINC01124 was designed by GenePharma (Shanghai, China) and cloned into the Prnat-u6.1/Neo plasmid (Biovector, Beijing, China). To establish a cell line with stable silencing of LINC01124, the plasmid carrying sh-LINC01124 or sh-Ctrl was cotransfected with packaging vectors to produce pseudotyped lentiviruses designated Lv-sh-LINC01124 and Lv-sh-Ctrl. The lentiviruses were concentrated by ultracentrifugation and then used to infect CC cells ²¹.

Quantitative real-time PCR

After transfection, total RNA was extracted from cells by TRIzol extraction (Invitrogen; Thermo Fisher Scientific, Inc.). Then, RNA samples were reverse transcribed into cDNA using a PrimeScript RT reagent kit (Takara). RT‑qPCR analyses were performed with SYBR Green (Takara). The results were normalized to the expression of glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH). Relative gene expression levels were calculated using the 2^−ΔΔct method ¹⁶.

Cell proliferation analysis

For CCK-8 assays, 2000 cells were seeded into 96-well plates in 100 μl of complete medium and cultured for 1, 2, 3, 4, and 5 days. At each time point, 10 μL of the CCK‐8 solution (Dojindo, Kumamoto, Japan) was added into each well, and the absorbance was then measured at 450 nm using a microplate reader after 2 h at 37°C. The cell viability was measured by the absorbance. For colony formation assays, cells were seeded into 12-well plates at a density of 500/well and incubated for 10-12 days. Then, colonies were stained with 0.1% crystal violet (Sigma) and counted ²².

Cell cycle analysis

HCT116 and SW480 cells were washed with cold PBS and then fixed with ice-cold 70% ethanol at 4°C overnight. On the second day, the cells were washed with PBS, and intracellular DNA was labeled with propidium iodide (PI, Sigma-Aldrich; Merck KGaA) at 4°C for 30 min and analyzed using BD FACSCalibur flow cytometry (BD Bioscience, San Jose, CA, USA). ModFit software (Verity Software House Inc., Topsham, ME, USA) was used to analyze the proportions of cells in the GO/G1, S, and G2/M phases ²¹.

Wound healing assay

Cells (1x10^5/well) were seeded into 6-well plates, and before starting the assay, cells were starved in fetal bovine serum (FBS)-free culture medium overnight. Then, a wound was made using a 200 µl pipette tip. Next, the cells were incubated with 2% FBS medium. The wound was imaged at 0 h and 36 h ²².

Transwell invasion assay

The Transwell chamber (8-μm pore size, Corning, Cambridge, MA, USA) was used to perform the invasion assay. Cells (2x10^5/well) were cultured in the upper chamber with Matrigel (BD Biosciences), and complete medium containing 20% FBS was added to the lower chamber. After incubation for 36 h at 37°C, cells adhering to the lower surface of the Transwell membrane were fixed in 20% methanol and stained with 0.1% crystal violet. The number of invaded cells was analyzed ²².

Tumorigenesis assay in vivo

For the subcutaneous xenograft assay, 5x10⁵ cells infected with sh-Ctrl or sh-LINC01124 were subcutaneously inoculated into the flanks of 5-week-old male athymic nude BALB/c mice. The tumor volumes were examined by using calipers every three days. After 5 weeks, the mice were sacrificed by euthanasia. The tumors were then removed and weighed ²¹. All animal studies were performed in strict accordance with the recommendations in the guidelines for the Animal Care and Use Committee of Traditional Chinese Medicine University of Guangzhou. Permit number: 20190228035.

Luciferase reporter assay

The binding sequences of miR-654-5p and LINC01124 were predicted through online websites (http://www.mircode.org/ and https://cm.jefferson.edu/ran22/Interacti-

ve/). The potential binding sequences of miR-654-5p and LINC01124 (WT) and the mutant (Mut) miR-654-5p binding sequences in LINC01124 were inserted into a pmirGL3-basic vector (Promega Corporation, Madison, USA) to construct dual luciferase reporter plasmid. Then, cells were cotransfected with the WT (or Mut) plasmid and a Ctrl mimic or miR-654-5p mimic. After 48 h, luciferase activity was detected using a Dual Luciferase Reporter Gene Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s protocol. The relative luciferase activity was normalized to Renilla luciferase activity ²².

Western blot

Protein samples were isolated with lysis buffer (RIPA) containing protease inhibitors. After quantification with a BCA kit (Beyotime, China), total protein (25 µg) was separated by 8-15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skimmed milk for 60 min, the membranes were incubated with the primary antibodies anti-HAX-1 (ab137613, 1:500) and anti-GAPDH (#5174, CST, 1:1000), overnight, followed by incubation with fluorescence-conjugated secondary antibodies (1:1,000) for 30 min. Bands were detected by a two-color infrared laser imaging system (Odyssey; Li-Cor, Lincoln, NE, USA) ²².

Statistical analysis

All data in this study were obtained from experiments repeated at least three times and are presented as the mean ± standard deviation (SD). Two independent sample t-tests or one-way ANOVA for multiple comparisons were performed using SPSS v22.0 (IBM, Armonk, NY, USA). **p < 0.01 or *p < 0.05 was considered to indicate a statistically significant difference ²².

The expression of LINC01124 was upregulated in colon cancer tissues and cell lines.

To investigate the role of LINC01124 in colorectal cancer, we first analyzed the expression levels of LINC01124 in the online public TCGA database (http://gepia.cancer-pku.cn/index.html). As shown in Fig. 1A, the expression of LINC01124 was significantly upregulated in colorectal cancer tissues. Next, we analyzed the expression of LINC01124 in CC cell lines. As illustrated in Fig. 1B, the expression of LINC01124 in all CC cell lines (LoVo, SW620, HT29, HCT116 and SW480) was significantly higher than that in the normal colonic epithelial cell line (NCM460). HCT116 and SW480 cells were chosen for downstream experiments based on their high expression. Our results indicated that LINC01124 was overexpressed in CC cell lines.

LINC01124 knockdown inhibited cell proliferation in vitro.

Loss-of-function experiments were performed by silencing LINC01124 to explore the regulatory effect of LINC01124 on CC cell progression. We knocked down LINC01124 in HCT116 and SW480 cells. RT-qPCR results showed that the expression of LINC01124 was significantly downregulated in these cells by transfecting sh-LINC01124 (Fig. 2A). We then performed CCK-8 and colony formation assays to evaluate cellular proliferation. The results demonstrated that LINC01124 knockdown clearly inhibited cell proliferation (Fig. 2B-2D and Supplementary Fig. 1). More importantly, FACS analysis indicated that after transfection with sh-LINC01124, fewer cells entered S phase and G2/M phase, while more cells were arrested in G0/G1 phase (Fig. 2E). Collectively, all these findings showed that knockdown of LINC01124 inhibited CC cell proliferation in vitro.

LINC01124 knockdown reduced tumor growth in vivo.

We further investigated the function of LINC01124 in vivo. We subcutaneously injected sh-LINC01124 or control SW480 cells into the flanks of nude mice. From the first week, we measured tumor volumes at indicative time points. After 5 weeks, we sacrificed the mice and removed the tumors. As shown in Fig. 3A and Fig. 3B, knockdown of LINC01124 significantly inhibited tumor growth in vivo. Moreover, the tumors in the sh-LINC01124 group were smaller than those in the control group (Fig. 3C). These findings indicate that knockdown of LINC01124 can reduce CC progression in vivo.

LINC01124 knockdown inhibited cell migration and invasion.

We assessed the effect of LINC01124 depletion on cell migration and invasion by using wound healing assays and Transwell assays. As expected, LINC01124 knockdown significantly suppressed the migration and invasion of HCT116 and SW480 cells (Fig. 4A and 4B). Moreover, the expression of the metastasis-related protein VIM was significantly downregulated, while another metastasis-related protein, CDH1, was upregulated in HCT116 cells transfected with sh-LINC01124 (Fig. 4C). Collectively, all findings suggest that LINC01124 may act as an oncogene in CC.

LINC01124 knockdown inhibited the progression of CC via the miR-654-5p/HAX-1 axis.

LncRNAs have been demonstrated to act as competing endogenous RNAs for miRNAs. Thus, two different mRNA target prediction algorithms, miRcode and RNA22, were used to predict the potential miRNAs that directly bind to LINC01124. Among all potential targets, we identified miR-654-5p, whose expression was downregulated in CC cells ²⁰, as the most promising candidate. The potential binding sequences of miR-654-5p and LINC01124 are shown in Fig. 5A. Overexpression of miR-654-5p significantly suppressed the expression of LINC01124 (Fig. 5B) and overexpression of LINC01124 significantly suppressed the expression of miR-654-5p (Supplementary Fig. 2). To confirm the association of LINC01124 and miR-654-5p, the expression levels of these two RNAs were analyzed using TCGA data, as showed in the Supplementary Table 1, Table 2 and Supplementary Fig. 3, there is an obvious negative correlation between the LINC01124 and miR-654-5p, r = 0.556966. Moreover, a luciferase reporter assay indicated that overexpression of miR-654-5p suppressed the luciferase activity in HCT116 and SW480 cells transfected with the WT-LINC01124 vector (Fig. 5C). It has been reported that miR-654-5p targets HAX-1 to regulate the malignant behaviors of colorectal cancer cells ²⁰; thus, we detected the expression of HAX-1 by Western blotting in HCT116 and SW480 cell lines transfected with sh-LINC01124 or cotransfected with sh-LINC01124 and miR-654-5p inhibitor. As shown in Fig. 5D, the expression of HAX-1 was decreased when LINC01124 was knocked down; however, the opposite expression pattern of HAX-1 was observed when sh-LINC01124 and miR-654-5p inhibitor were cotransfected. Altogether, these findings demonstrated that LINC01124 directly bound to miR-654-5p in CC.

After confirming the direct interaction between miR-654-5p and LINC01124, rescue experiments were performed. The sh-LINC01124 and miR-654-5p inhibitor were cotransfected into both HCT116 and SW480 cell lines. By assessing cell proliferation, migration and invasion using colony formation, wound healing and Transwell assays, we found that the miR-654-5p inhibitor partly reversed the suppressive effect of sh-LINC01124 on cell proliferation, migration and invasion (Fig. 5E-5G). Taken together, our findings suggested that LINC01124 acted as an oncogene via inhibition of miR-654-5p by targeting HAX-1.

Due to limitations in the early diagnosis of colon cancer, its 5-year survival rate is still less than 30% in many low-income countries ^23,24. An increasing number of methods are being used to explore the pathogenesis of cancers, and accumulating evidence suggests that lncRNAs have important biological functions ^25–27. LncRNAs were once considered to be “transcriptional noise”, but now lncRNAs have been proven to be involved in the regulation of tumorigenesis and progression as tumor suppressor genes or oncogenes ^28–30.

For the past few years, an increasing number of researchers have focused on the effect of lncRNAs on colon cancer. For example, some scholars have proven that lncRNA ROR1-AS1 can promote proliferation in colon cancer by suppressing the expression of DUSP5/CDKN1A ¹⁴, lncRNA NEAT1 can regulate invasion and migration in colon cancer through the miR-185-5p/IGF2 axis ³¹, lncRNA LINC00460 silencing suppresses EMT in colon cancer through downregulation of ANXA2 ³², lncRNA POU6F2-AS2 can promote drug resistance in colon cancer by regulating miR-377/BRD4 ¹⁵, etc. LINC01124, which is located on chromosome 2q31.1, acts as a tumor suppressor in NSCLC. The expression level of LINC01124 was downregulated in tumor tissues compared with paired normal lung tissues, and LINC01124 could significantly inhibit the proliferation, migration, and invasive ability of NSCLC cells ¹⁶. However, the role of LINC01124 in colon cancer remains unclear. Here, we report for the first time that LINC01124 is upregulated in colon cancer tissues and cell lines. The results showed that the downregulation of LINC01124 suppressed the proliferation, migration and invasion of CC cell lines in vitro and in vivo. This function of LINC01124 in CC was the opposite of that in NSCLC cells.

Many studies have revealed some potential biological mechanisms of lncRNAs, and the theory of competing endogenous RNAs (ceRNAs) is one of the commonly accepted theories ³³. CeRNAs can directly bind to miRNAs and affect the expression of target mRNAs, thus affecting the biological process of cells ^34–36. LINC01124 has not been reported to function as a ceRNA until now, so we hypothesized that LINC01124 could regulate CC progression by binding to miRNAs. By using an online database, we found that miR-654-5p had a higher score on binding to LINC01124, which has neem reported to suppress cell proliferation, migration and invasion in colon cancer ²⁰. The luciferase reporter assay confirmed our prediction that LINC01124 can bind to miR-654-5p directly. Further experiments showed that the downregulation of LINC01124 significantly decreased the levels of HAX-1, which is the target gene of miR-654-5p. Additionally, the miR-654-5p inhibitor rescued the effects of sh-LINC01124 on CC cell proliferation, migration and invasion. All these findings suggested that the miR-654-5p/HAX-1 axis might be involved in the anti-CC effect of sh-LINC01124.

Taken together, our data for the first time showed that LINC01124 was upregulated in colon cancer tissues and cell lines. Downregulation of LINC01124 was involved in malignant behaviors in colon cancer. Additionally, we identified for the first time miR-654-5p as a target of LINC01124. In conclusion, our findings reveal that the LINC01124/miR-654-5p/HAX-1 axis can play an important role in colon cancer, suggesting a novel diagnostic and therapeutic target for colon cancer.

ACKNOWLEDGMENTS

This study was supported by Guangdong Province Traditional Chinese Medicine Bureau (20173029), Guangzhou Science and Technology Innovation Commission (#201704020171), and Guangzhou Municipal Health and Health Committee (#2019A011014).

CONFLICTS OF INTEREST

The authors of the study declare no potential conflicts of interest.

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No competing interests reported.

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Knockdown of long noncoding RNA 01124 inhibits the malignant behaviors of colon cancer cells by regulating miR-654-5p/HAX-1

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