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Abstract

BACKGROUND:

Breast cancer has become a major threat to women’s physical and mental health. Many long noncoding RNAs (lncRNAs) have been reported to exert effect in the progression of breast cancer. However, the regulatory mechanism of HAND2-AS1 remains unclear. Therefore, this study aimed to elucidate the role of HAND2-AS1 in breast cancer.

METHODS:

The expression of HAND2-AS1, miR-1275 and SOX7 was assessed using RT-qPCR and Western blot analysis. CCK-8, Transwell and luciferase reporter assays were used to investigate their regulatory mechanisms.

RESULTS:

Downregulation of HAND2-AS1 was detected in breast cancer and was associated with adverse clinical features and prognosis. Furthermore, overexpression of HAND2-AS1 restrained cell viability, migration and invasion in breast cancer. In addition, reciprocal suppression between HAND2-AS1 and miR-1275 was identified in breast cancer cells. Further, SOX7, a target of miR-1275, strengthened the effect of HAND2-AS1 in breast cancer.

CONCLUSION:

LncRNA HAND2-AS1 sponging miR-1275 restrains proliferation and metastasis of breast cancer cells by regulating SOX7 expression.

Keywords

HAND2-AS1 breast cancer miR-1275 SOX7

1. Introduction

Breast cancer is a common malignant tumor that seriously affects women’s physical and mental health and even life-threatening. Moreover, its incidence is usually related to heredity [1], Surgery and radiotherapy alone can cure most breast cancer patients before tumor metastasis. Once metastasis occurs, active treatment can only cure a small number of patients [2]. In addition, the prognosis of breast cancer patients is mainly related to the extent of tumor invasion and pathological type. Patients with triple-negative breast cancer have the worst prognosis [3]. The 5-year survival rate of patients at stage I is 88%. However, the survival rate of patients at stage IV is only 15% [4]. Besides that, postoperative recurrence is another major threat to the health of breast cancer patients [5]. However, due to clinical and molecular heterogeneity, the molecular mechanism of breast cancer recurrence is unclear [6]. Now, a more accurate prediction of clinical outcomes and recurrence is a huge challenge for breast cancer treatment [7]. Therefore, it is still urgent to explore and develop effective breast cancer treatments.

Long non-coding RNAs (lncRNAs) are involved in the regulation of various biological processes in human diseases and cancers [8]. It has become the hottest research area for messenger RNA (mRNA) and microRNA post functional genomics. Moreover, many lncRNAs have been found to play important roles in the pathogenesis of breast cancer. Li et al. has found the carcinogenic potential and diagnostic significance of lncRNA LINC00310 in breast cancer [9]. LncRNA p10247 has been demonstrated to be high expressed in breast cancer and was correlated with metastasis [10]. Inversely, lncRNA CASC2 was found to be downregulated in breast cancer and restrained cell growth and metastasis by modulating the miR-96-5p/SYVN1 pathway [11]. Recently, the dysregulation of lncRNA HAND2-AS1 has been investigated in some malignant tumors other than breast cancer. You et al. found that HAND2-AS1 was downregulated in papillary thyroid cancer and may play a role in its progression [12]. Moreover, lncRNA HAND2-AS1 was found to repress HIF1 $\alpha$ -mediated energy metabolism and inhibit osteosarcoma progression [13]. Besides that, lncRNA HAND2-AS1 has been reported to inhibit proliferation and promote apoptosis of chronic myeloid leukemia cells by sponging miR-1275 [14]. However, the function mechanism of lncRNA HAND2-AS1/miR-1275 remains unclear in breast cancer.

Precious studies have shown that miR-1275 was upregulated in head and neck squamous cell carcinoma and glioma, promoting cell migration, invasion and proliferation [15, 16]. In addition, lncRNA miR143HG was proposed to suppress the development of bladder cancer by modulating miR-1275/AXIN2 axis [17]. These studies indicate that the interaction between lncRNAs and miR-1275 is involved in tumorigenesis. Besides that, miR-1275 is involved in human cancers via regulating target genes, such as IGF2 [18]. In this study, SOX7 was selected as a target for miR-1275, which has not been confirmed in previous studies. It is well known that SOX7 can be used as an inhibitor of malignant tumors, such as hepatocellular carcinoma and lung adenocarcinoma [19, 20]. In particularly, SOX7 was downregulated and exerted tumor suppressive effect in breast cancer [21]. But the functional mechanism of lncRNA HAND2-AS1/miR-1275/SOX7 is still unknown in breast cancer. Therefore, this study aims to confirm their relationship and explain its regulatory mechanisms in breast cancer.

2. Materials and methods

2.1 Clinical tissues

Experimental tissues were obtained from 88 patients with breast cancer in Linyi Cancer Hospital. The clinicopathological features of these patients, including age, tumor size, lymph node metastasis, ER status, PR status and Her-2 status, are shown in Table 1. Informed consents were obtained from all breast cancer patients. Furthermore, breast cancer patients did not receive any treatment prior to surgery. This study was approved by the Institutional Ethics Committee of Linyi Cancer Hospital.

Table 1
Relationship between HAND2-AS1 expression and clinic-patholo- gical characteristics in breast cancer patients

Characteristics	Cases	HAND2-AS1		$P$ -value
		High	Low
Age (years)				0.08
$\geqslant$ 50	30	16	14
$<$ 50	58	10	48
Tumor size				0.02*
$<$ 2 cm	27	10	17
$\geqslant$ 2 cm	61	16	45
Lymph node metastasis				0.01*
No	28	9	19
Yes	60	17	43
Her-2 status				0.31
Positive	52	18	34
Negative	36	8	28
ER status				0.42
Positive	35	7	28
Negative	53	19	34
PR status				0.08
Positive	36	8	28
Negative	52	18	34

Statistical analyses were performed by the $\chi^{2}$ test. ${}^{*}P<$ 0.05 was considered significant.

2.2 Cell culture and transfection

Human breast epithelial cell line MCF10A and breast cancer cells MDA-MB-231, MDA-MB-468, MCF-7 and UACC812 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The growth conditions for these cells were 5% CO ${}_{2}$ , 37 ${}^{\circ}$ C and DMEM medium (10% FBS). HAND2-AS1 pcDNA3.1 plasmid (2 $\mu$ g), HAND2-AS1 siRNA (50 nM), miR-1275 mimic (30 nM), miR-1275 inhibitor (30 nM), miR-NC (30 nM), and SOX7 siRNA (50 nM) were transfected into MDA-MB-231 and MCF10A cells using Lipofectamine 2000 (Invitrogen, CA, USA). They were all purchased from RiboBio (Guangzhou, China).

2.3 RNA isolation, reverse transcription and RT-qPCR

Total RNA isolation was performed using TRIZOL reagent (Invitrogen, USA). The cDNA solution was synthesized using PrimeScript RT reagent kit (Takara, Dalian, China). RT-qPCR assay was performing on ABI 7500 Sequence Detection system by Real-time PCR Mixture assays (Takara). The internal reference for HAND2-AS1, SOX7 or miR-1275 is GAPDH or U6. The expression level was quantified by the 2 ${}^{-\vartriangle\vartriangle cq}$ method. The primer sequences used in RT-qPCR were as follows: HAND2-AS1 forward, 5’-GGA GTC ACA GGC AGT CGT AGA-3’ and reverse, 5’-GAA GGC ACA GAT CAT TCA TGG-3’; miR-1275 forward, 5’-CTC TGT GAG AAA GGG TGT GG-3’ and reverse, 5’-TCT GCC TTG GGG AAA ATA AG-3’; and U6 forward, 5’-GCT TCG GCA GCA CAT ATA CTA AA-3’ and reverse, 5’-GCT TCA CGA ATT TGC GTG TCA T-3’. SOX7 forward, 5’-CCT CAT GCT CTG AAG ATT GCC-3’ and reverse, 5’-CCT CAT GCT CTG AAG ATT GCC-3’; GAPDH forward, 5’-ACA ACT TTG GTA TCG TGG AAG G-3’ and reverse, 5’-GCC ATC ACG CCA CAG TTT C-3’.

2.4 CCK-8 assay

First, the transfected cells were incubated in 96-well plates for 24 hours (at 37 ${}^{\circ}$ C, 5% CO ${}_{2}$ ). Then, the transfected cells (3 $\times$ 10 ${}^{3}$ /well) were incubated for 24, 48, 72 and 96 h. Next, these cells were incubated with 10 ml of CCK-8 (Dojindo, Kumamoto, Japan) for 4 hours. The absorbance at 450 nm was observed with a microplate reader (Molecular Devices).

2.5 Colony formation

The transfected cells were digested by trypsin, and then collected and seeded into 6-well plates at a density of 2 $\times$ 10 ${}^{3}$ cells per well, followed by incubation for 2 weeks in DMEM at 37 ${}^{\circ}$ C. The medium was replaced every 3 days. Cells were treated with 4% paraformaldehyde and stained with moderate gentian violet solution. Finally, cell colonies were counted and photographed by a digital camera (Olympus, Tokyo, Japan).

2.6 Wound healing assays

A Wound-healing assay was performed to examine the migratory ability of the transfected cells. The cells were washed with serum free media and treated with mitomycinc (10 $\mu$ g/ml) for 30 min. Transfected cells were cultured in 12-well plates for 24 h. The cell monolayer was scratched using a 20- $\mu$ l pipette tip. Finally, the migration was observed and photographed at 24 h after wounding. The distance between the two edges of the scratch was measured.

2.7 Transwell assay

First, cell invasion was detected in the upper chamber with Matrigel. After 30 min, transfected cells (3 $\times$ 10 ${}^{3}$ cells/well) were added to the Transwell upper chamber. Low chamber was added with DMEM medium in 24-well plates. After 24 h, the moving cells were stained with 0.1% crystal violet. Cell migration assay is similar to cell invasion except that Matrigel is not used. Observation and photographing were performed by a light microscope.

2.8 Western blot analysis

Protein sample extraction was performed by RIPA lysis buffer (Beyotime). Next, 25 $\mu$ g of protein was separated by 10% SDS-PAGE. Protein samples were transferred to PVDF membranes and blocked with 5% skim milk. Then, the protein samples were incubated with SOX7 (Rabbit polyclonal, 1:1000; cat. no. ab94397; Abcam, Shanghai, China) and GAPDH (Rabbit monoclonal; dilution, 1:1000; cat. no. ab 181602; Abcam) primary antibodies at 4 ${}^{\circ}$ C overnight. After washing, the protein sample was incubated with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:5,000; cat no. ab190492; Abcam). Protein bands were assessed by ECL kit (Beyotime) and quantified using Image Lab Software (Bio-Rad, Kidlington, UK).

2.9 Dual luciferase reporter assay

Reporter plasmids of HAND2-AS1 (wt-HAND2-AS1 and mut-HAND2-AS1) and SOX7 (wt-SOX7 and mut-SOX7) were obtained from RiboBio (Guangzhou, China). Next, the reporter plasmids were transfected into MDA-MB-231 cells with miR-1275 mimics (RiboBio, Guangzhou, China). After 48 h, luciferase activity was assessed by the dual-luciferase reporter assay system (Promega, USA).

Figure 1.

HAND2-AS1 was involved in pathogenesis of breast cancer. (A) The mRNA HAND2-AS1 expression in breast cancer tissues. (B) The expression of HAND2-AS1 at different breast cancer subtypes (luminal A, luminal B, normal-like and basal-like). (C) The mRNA HAND2-AS1 expression in breast cancer cell lines. (D, E) High HAND2-AS1 expression patients showed longer overall survival (OS) and (recurrence-free survival) RFS. * $P<$ 0.05, ** $P<$ 0.01.

Figure 2.

HAND2-AS1 restrained cell proliferation and metastasis in breast cancer. (A) HAND2-AS1 expression in MDA-MB-231 and MCF10A cells with its overexpression vector or siRNA. (B, C, D) Cell proliferation and colony formation in MDA-MB-231 and MCF10A cells with HAND2-AS1 overexpression vector or siRNA. (E, F) Cell migration in MDA-MB-231 and MCF10A cells with HAND2-AS1 overexpression vector or siRNA detected by Transwell and Wound healing assays. (G) Cell invasion in MDA-MB-231 and MCF10A cells with HAND2-AS1 overexpression vector or siRNA detected by Transwell assay. * $P<$ 0.05, ** $P<$ 0.01.

Figure 3.

Reciprocal suppression between HAND2-AS1 and miR-1275 was identified in breast cancer. (A) The binding sites between HAND2-AS1 with miR-1275. (B) Luciferase reporter assay. (C) RNA pull-down experiment was performed to determine the binding between HAND2-AS1 and miR-1275. (D) MiR-1275 expression regulated by HAND2-AS1 overexpression vector or siRNA in MDA-MB-231 cells. (E) HAND2-AS1 expression regulated by miR-1275 mimic or inhibitor (30 nM). (F) A negative correlation between HAND2-AS1 and miR-1275 was identified in breast cancer tissues. (G) MiR-1275 expression in MDA-MB-231 cells with its mimics or inhibitor (30 nM). (H, I, J) Cell proliferation, migration and invasion in MDA-MB-231 cells with miR-1275 mimics or inhibitor. * $P<$ 0.05, ** $P<$ 0.01.

Figure 4.

SOX7 targeted by miR-1275 regulated the effect of HAND2-AS1 in breast cancer. (A) The binding sites between SOX7 with miR-1275. (B) The mRNA SOX7 expression in breast cancer tissues. (C) SOX7 expression regulated by miR-1275 mimic or inhibitor. (D) Luciferase activity assay. (E, F) SOX7 expression regulated by different transfections in MDA-MB-231 and MCF10A cells. (G) SOX7 expression regulated by HAND2-AS1. (H, I) Cell proliferation, migration and invasion in MDA-MB-231 cells with different vectors. * $P<$ 0.05, ** $P<$ 0.01.

2.10 RNA pull-down assay

Biotinylated HAND2-AS1 was transfected into MDA-MB-231 cells. A biotinylated lncRNA not complementary to miR-1275 was employed as a negative control. After 48 hours, the cell lysate was incubated with M-280 streptavidin magnetic beads (Invitrogen), followed by RT-qPCR assay to measure miR-1275 level.

2.11 Statistical analysis

Data were analyzed using Student’s t-test, one-way ANOVA, Univariate Kaplan-Meier method with log-rank test and Chi-squared test in SPSS 17.0 or Graphpad Prism 6 software. Data are shown as mean $\pm$ SD. Differences are considered as significant at $P<$ 0.05.

3. Results

3.1 HAND2-AS1 was involved in pathogenesis of breast cancer

First, we detected abnormal expression of HAND2-AS1 in breast cancer. We found that HAND2-AS1 was downregulated in breast cancer tissues compared to normal tissues ( $P<$ 0.01, Fig. 1A). Moreover, expression of HAND2-AS1 in different breast cancer subtypes (luminal A, luminal B, normal-like and basal-like) was detected. We found that HAND2-AS1 expression was lower in luminal A, luminal B and basal-like compared to normal tissues ( $P<$ 0.05, Fig. 1B). Similarly, HAND2-AS1 expression in MDA-MB-231, MDA-MB-468, MCF-7 and UACC812 cells was lower than in MCF10A cells ( $P<$ 0.05, Fig. 1C). Among them, MDA-MB-231 cells showed the lowest expression of HAND2-AS1, which was selected for further experiments. In addition, the relationship between HAND2-AS1 expression and clinicopathological features of breast cancer patients was analyzed. The median relative expression level of HAND2-AS1 in breast cancer tissues was used as a cut-off point to separate low and high expression of HAND2-AS1. Abnormal expression of HAND2-AS1 was found to be correlated with tumor size and lymph node metastasis in breast cancer patients ( $P<$ 0.05, Table 1). Furthermore, breast cancer patients with low HAND2-AS1 expression showed shorter overall survival (OS, $P<$ 0.05, Fig. 1D) and recurrence-free survival (RFS, $P<$ 0.05, Fig. 1E). These results reveal that HAND2-AS1 is involved in the pathogenesis of breast cancer.

3.2 HAND2-AS1 restrained cell proliferation and metastasis in breast cancer

To explore the functional role of HAND2-AS1 in breast cancer, gain-loss functional experiments were performed in MDA-MB-231 and MCF10A cells with HAND2-AS1 plasmid or siRNA. RT-qPCR was used to assess transfection efficiency. It showed that HAND2-AS1 plasmid significantly increased its expression, while HAND2-AS1 siRNA reduced its expression in MDA-MB-231 and MCF10A cells ( $P<$ 0.01, Fig. 2A). Functionally, cell proliferation was found to be restrained by HAND2-AS1 overexpression and was promoted by knockdown of HAND2-AS1 in MDA-MB-231 cells ( $P<$ 0.05, Fig. 2B). However, HAND2-AS1 had little effect on cell proliferation in MCF10A cells (Fig. 2C). Furthermore, colony formation also showed the same results in MDA-MB-231 and MCF10A cells ( $P<$ 0.01, Fig. 2D). Transwell and Wound healing assays showed that overexpression of HAND2-AS1 hindered cell migration, whereas downregulation of HAND2-AS1 promoted cell migration in MDA-MB-231 cells ( $P<$ 0.01, Fig. 2E and F). However, cell migration was not affected by HAND2-AS1 in MCF10A cells. In addition, cell invasion was also inhibited by upregulation of HAND2-AS1 and was promoted by downregulation of HAND2-AS1 ( $P<$ 0.01, Fig. 2G). And no significant changes in cell invasion were observed in MCF10A cells with HAND2-AS1 plasmid or siRNA. These results suggest that HAND2-AS1 acts as a tumor suppressor in breast cancer by inhibiting cell proliferation and metastasis.

3.3 Reciprocal suppression between HAND2-AS1 and miR-1275 was identified in breast cancer

Next, the downstream target of HAND2-AS1 was searched in the starBase database (http://starbase.sysu. edu.cn/) to further explain its regulatory mechanisms in breast cancer progression. HAND2-AS1 was predicted to have a binding site with miR-1275 (Fig. 3A). Dual-luciferase reporter assay was then performed to confirm this hypothesis. MiR-1275 mimic was found to reduce the luciferase activity of HAND2-AS1-WT, but did not affect the luciferase activity of HAND2-AS1-MUT ( $P<$ 0.01, Fig. 3B). RNA pull-down analysis showed substantial enrichment of miR-1275 in the Bio-HAND2-AS1 group compared to the control group (Fig. 3C). We found that overexpression of HAND2-AS1 reduced miR-1275 expression, and knockdown of HAND2-AS1 accelerated miR-1275 expression in MDA-MB-231 cells ( $P<$ 0.01, Fig. 3D). Similarly, upregulation of miR-1275 hindered the expression of HAND2-AS1, while downregulation of miR-1275 promoted HAND2-AS1 expression in MDA-MB-231 cells ( $P<$ 0.01, Fig. 3E). Correspondingly, a negative correlation between HAND2-AS1 and miR-1275 expressions was identified in breast cancer tissues ( $P<$ 0.01, Fig. 3F). Based on these results, we consider that HAND2-AS1 functions as a miR-1275 sponge in breast cancer. Besides that, miR-1275 mimic or inhibitor was transfected into MDA-MB-231 cells to explore its role in breast cancer. RT-qPCR showed that miR-1275 expression was increased by miR-1275 mimics and decreased by miR-1275 inhibitor ( $P<$ 0.01, Fig. 3G). Functionally, CCK-8 showed that overexpression of miR-1275 promoted cell proliferation, while miR-1275 downregulation inhibited proliferation of MDA-MB-231 cells ( $P<$ 0.05, Fig. 3H). Transwell assay suggested that cell migration and invasion were promoted by overexpression of miR-1275 and was restrained by downregulation of miR-1275 ( $P<$ 0.01, Fig. 3I and J). These results indicate that miR-1275 functions as an oncogene in breast cancer.

3.4 SOX7 targeted by miR-1275 regulated the effect of HAND2-AS1 in breast cancer

Finally, the target gene of miR-1275 was searched in the TargetScan database (http://www.targetscan.org). It predicts that miR-1275 has a site that binds to the 3’-UTR of SOX7 (Fig. 4A). Moreover, downregulation of SOX7 was detected in breast cancer tissues compared to normal tissues ( $P<$ 0.01, Fig. 4B). In addition, RT-qPCR assay showed that miR-1275 mimics reduced SOX7 expression and miR-1275 inhibitor promoted SOX7 expression ( $P<$ 0.01, Fig. 4C). Next, luciferase reporter assay suggested that miR-1275 mimics or HAND2-AS1 siRNA obstructed the luciferase activity of SOX7. However, miR-1275 inhibitor restored a decrease in luciferase activity of SOX7 induced by HAND2-AS1 siRNA ( $P<$ 0.01, Fig. 4D). These results indicate that miR-1275 directly targets SOX7. Furthermore, SOX7 expression can be regulated by HAND2-AS1. As shown in Fig. 4D and E, knockdown of HAND2-AS1 reduced SOX7 expression, when downregulation of miR-1275 rescued this decrease in MDA-MB-231 cells ( $P<$ 0.01, Fig. 4E and F). The same result was also found in MCF10A. However, the change in SOX7 expression was not significant compared to MDA-MB-231 cells ( $P<$ 0.05, Fig. 4E and F). It demonstrated that HAND2-AS1 promoted SOX7 expression by downregulating miR-1275 in breast cancer. Next, SOX7 siRNA was transfected into MDA-MB-231 cells with HAND2-AS1 vector to confirm whether HAND2-AS1 regulates breast cancer progression by regulating SOX7 expression. We found that HAND2-AS1 vector promoted the expression of SOX7. Furthermore, miR-1275 mimics or SOX7 siRNA reduced upregulation of SOX7 induced by HAND2-AS1 vector ( $P<$ 0.01, Fig. 4G). Functionally, inhibition of cell proliferation induced by HAND2-AS1 vector was restored by miR-1275 mimics or SOX7 siRNA in MDA-MB-231 cells ( $P<$ 0.01, Fig. 4H). Similarly, the reverse effects of miR-1275 mimics or SOX7 siRNA on cell migration and invasion were also identified in MDA-MB-231 cells ( $P<$ 0.01, Fig. 4I). Overall, HAND2-AS1 was considered to act as a tumor suppressor in breast cancer by sponging miR-1275 and promoting SOX7 expression.

4. Discussion

Recently, many lncRNAs have been found to serve as tumor promoters or inhibitors in the progression of breast cancer. For example, lncRNA CASC2 was downregulated in breast cancer, and upregulation of CASC2 restrained cell proliferation and metasta- sis [22]. Here, downregulation of HAND2-AS1 was also found in breast cancer, which has not been demonstrated in previous studies. In addition, this downregulation was associated with poor clinical features and prognosis in breast cancer patients. Functionally, HAND2-AS1 overexpression restrained cell viability, migration and invasion in breast cancer. Further, HAND2-AS1 sponging miR-1275 was found to impede breast cancer progression by regulating SOX7 expression. All of these findings imply that HAND2-AS1 is involved in the development of breast cancer by acting as an inhibitor.

Consistent with our results, HAND2-AS1 has been identified to be downregulated in hepatocellular carcinoma and HPV-negative head and neck squamous cell carcinoma [23, 24]. Huang et al. proposed that downregulation of HAND2-AS1 predicted a poor prognosis in patients with colon adenocarcinoma [25]. In our study, poor prognosis in breast cancer patients was also identified to be associated with low HAND2-AS1 expression. Functionally, lncRNA HAND2-AS1 has been demonstrate to repress invasion and migration of non-small cell lung cancer cells [26]. The same effect of HAND2-AS1 was also found in breast cancer. Additionally, Yan et al. showed that HAND2-AS1 restrained cell proliferation in esophagus squamous cell carcinoma by regulating microRNA-21 [27]. Here, it was observed that HAND2-AS1 restrained breast cancer cell proliferation by sponging miR-1275. Similarly, Zhou et al. revealed that HAND2-AS1 sponging miR-1275 hindered the progression of colorectal cancer [28]. Besides that, reciprocal suppression between HAND2-AS1 and miR-1275 was identified in breast cancer cells.

More and more studies have shown that miR-1275 is involved in human cancers. For instance, miR-1275 was downregulated and repressed the growth of nasopharyngeal carcinoma [29]. However, upregulation of miR-1275 was detected in glioma, which promoted the progression of glioma [16]. These findings suggest that the dysregulation of miR-1275 has tissue specificity in human cancers. Moreover, the function of miR-1275 is still dim in breast cancer. Here, we found that overexpression of miR-1275 inhibited the progression of breast cancer. Furthermore, our rescue experiments indicated that upregulation of miR-1275 weakened the inhibitory effect of HAND2-AS1 in breast cancer. All of these results reflect that miR-1275 serves as a carcinogenic miRNA in breast cancer progression.

Further, downstream target of miR-1275 was explored in this study. SOX7 was identified as a direct target of miR-1275, which has not been investigated in other studies. We found that SOX7 expression was downregulated by miR-1275 and upregulated by HAND2-AS1 in breast cancer cells. Downregulation of SOX7 and its inhibitory effect have been extensively investigated in human cancers, such as lung cancer and colorectal cancer [30, 31]. Here, SOX7 was found to be downregulated and exert tumor suppressive effects in breast cancer [21]. In addition, knockdown of SOX7 was found to restore the inhibitory effect of HAND2-AS1 in breast cancer. It also suggests that SOX7 serves as a suppressive regulator in breast cancer, consistent with previous studies. Moreover, this is the first report that SOX7 involves in breast cancer progression through interacting with the HAND2-AS1/miR-1275 axis. However, we have only initially elucidated the regulatory mechanism of the HAND2-AS1/miR-1275/SOX7 axis in the pathogenesis of breast cancer. The specific functional mechanisms of HAND2-AS1/miR-1275/SOX7 axis and their roles in vivo have not been investigated in this study. Another drawback is that we currently have a small number of clinical samples. In the future, we will increase the number of samples and further explore the function of HAND2-AS1/miR-1275/SOX7 axis in vivo.

5. Conclusion

In conclusion, this study first proposed the downregulation of HAND2-AS1 in breast cancer. Furthermore, downregulation of HAND2-AS1 was associated with adverse clinical features and prognosis in breast cancer patients. Importantly, HAND2-AS1 restrained breast cancer progression and acted as a competing endogenous RNA to regulate SOX7 expression by sponging miR-1275.

Footnotes

Conflict of interest

The authors declare that they have no competing interests.

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Long noncoding RNA HAND2-AS1 restrains proliferation and metastasis of breast cancer cells through sponging miR-1275 and promoting SOX7

Abstract

BACKGROUND:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Clinical tissues

Table 1 Relationship between HAND2-AS1 expression and clinic-patholo- gical characteristics in breast cancer patients

2.3 RNA isolation, reverse transcription and RT-qPCR

2.4 CCK-8 assay

2.5 Colony formation

2.6 Wound healing assays

2.7 Transwell assay

2.8 Western blot analysis

2.9 Dual luciferase reporter assay

2.11 Statistical analysis

3. Results

3.1 HAND2-AS1 was involved in pathogenesis of breast cancer

3.2 HAND2-AS1 restrained cell proliferation and metastasis in breast cancer

3.3 Reciprocal suppression between HAND2-AS1 and miR-1275 was identified in breast cancer

3.4 SOX7 targeted by miR-1275 regulated the effect of HAND2-AS1 in breast cancer

4. Discussion

5. Conclusion

Footnotes

Conflict of interest

References

Table 1
Relationship between HAND2-AS1 expression and clinic-patholo- gical characteristics in breast cancer patients