Overexpression of Long Non-Coding RNA Linc01315 Predicts Poor Prognosis in Breast Cancer

Background LncRNAs have been shown to play critical roles in regulating tumorigenesis and tumor progression. Using LncRNAs to predict prognosis and therapeutic response to cancer treatment has been caused for concern, but the predictive value of lncRNAs remains to be explored and underlying mechanisms have not been completely understood. Methods The Linc01315 expression level was detected in 282 breast cancer tissues by using quantitative RT-PCR. The association between Linc01315 expression level and clinicopathological features of these breast cancer patients was further analyzed. Multiple regression analysis was used to evaluate Linc01315 predictive value of patients’ prognosis. Results Our study revealed that Linc01315 expression level was significantly correlated with vessel invasion (P = 0.028) and tumor subtype (P = 0.039). The Kaplan–Meier survival curves demonstrated that patients with lower Linc01315 expression level had significantly longer disease free survival (DFS) (P = 0.002) and overall survival (OS) (P=0.019). Multiple regression analysis showed that Linc01315 level could be an independent predictive factor for DFS (hazards ratio = 0.613, 95% confidence interval = 0.375-1.003; P = 0.049) and OS (hazards ratio = 0.439, 95% confidence interval = 0.228-0.845; P = 0.014). Further analysis showed that low Linc01315 level patients with endocrine therapy could benefit patients DFS (P=0.037) and OS (P=0.025). Conclusion Our results demonstrate that Linc01315 expression level is significantly correlated with breast cancer patients’ prognosis. Linc01315 may represent an independent prognostic marker and therapeutic target in breast cancer.


INTRODUCTION
Breast cancer is the most common malignancy in women. The overall survival and prognosis of some patients with breast cancer remains poor despite therapeutic improvements in cancer systemic treatment over the last several decades. Thus, there is still a great challenge in exploring the underlying mechanisms of breast carcinogenesis and progression, so as to identify promising prognostic molecular markers that can benefit the effect of appropriate therapeutic regimen.
Previous studies have shown that TCGA lncRNome information could be used to screen and find several lncRNA candidates that associated with cancer genomic alterations, and might be correlated with patient prognosis in breast cancer (17,18). Linc01315 located on chromosome 12 which has been reported to be upregulated in triple-negative breast cancer (19). Moreover, down-regulated expression of Linc01315 could suppress colorectal carcinoma progression by sponging miR-205-3p (20). In this study, we investigated Linc01315 expression level in the cohort of 282 breast cancer patients from Fudan University Shanghai Cancer Center and found that high Linc01315 expression level had a significantly worse clinical outcome, and Linc01315 might be an independent prognostic predictor in breast cancer.

Patients' Samples
A series of 282 primary breast cancer tissues of stage I to III invasive carcinoma were collected at Fudan University Shanghai Cancer Center. The exclusion criteria were: (i) advanced breast cancer, (ii) neo-adjuvant chemotherapy received prior to surgery, (iii) recurrent breast cancer diagnosed upon surgery, and (iv) noninvasive ductal carcinoma. Patients' clinical data and tumor pathology are summarized (Table 1)

RNA Isolation and Quantitative RT−PCR
Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. DNase I was used to digest genome DNA before cDNA Synthesis. qRT-PCR was performed to quantitate the mRNA expression levels. To analyze Linc01315 expression, we used the SYBRGreen method with primers listed below: Linc01315 forward 5′ CTGCTGAGCGATGAAGTGGA 3′ and Linc01315 reverse 5′ CTACAGCTGGAGGGAAACCG 3′. Their relative expression were quantified to GAPDH by 2 −△Ct values.

Statistical Analysis
All statistical analyses were performed using SPSS 22.0 software (SPSS Inc, Chicago, IL, USA). Comparisons among groups were done by the independent sample t-test. The Wilcoxon matched pairs test was used to examine Linc01315 expression in breast cancer tissues versus adjuvant normal tissues. Pearson's c2 test was performed to evaluate the correlation between Linc01315 expression and breast cancer clinicopathological characteristics. In survival analysis, Kaplan-Meier analysis and the Cox proportional hazards model were used to examine whether Linc01315 expression level impacted prognosis. Two-tailed P-values were adopted, P-value 0.05 was considered as statistically significant. We used SPSS 22.0 software (SPSS Inc, Chicago, IL, USA) to draw survival curve.

Linc01315 Expression in Breast Cancer Cell Lines
Linc01315 expression levels were examined in seven breast cancer cell lines and one normal breast cell MCF10A. As shown in Figure 1

Relationship Between Linc01315 Expression Level and Clinicopathological Characteristics
Expression of Linc01315 in 44 paired of breast cancer tissues and their adjacent normal tissues were then detected. The data showed that a little bit higher of Linc01315 was in normal tissues than in breast cancer tissues, but the difference was not significant (P=0.1038) (Supplementary Figure 1). To identify the clinical relevance of Linc01315 in breast cancer, association between Linc01315 expression level and clinicopathological features such as age, ER/PR, Her-2, Ki67, vessel invasion, tumor size, lymph node status, tumor grade and TNM stage were examined ( Table 1). According to the ROC curve assessment which was constructed using patient Linc01315 levels to establish a sensitivity-specificity relationship of the measured prognostic marker, the expression of Linc01315 in tumor tissues was divided into 167 low expression and 115 high expression. Chi square test was used to determine the cutoff value of Linc01315 in this study. Linc01315 expression level in breast cancer was significantly correlated with TNM stage (p=0.003), vessel invasion (p = 0.028) and tumor subtype (p = 0.039). TNM stage I-II patients seemed to have lower Linc01315 expression. Vessel invasion negative patients show significant correlation with low Linc01315 expression level. Moreover, Her-2 positive subtype patients tended to have high Linc01315 expression level, while luminal A and B subtype patients seemed to have low Linc01315 expression ( Table 1). The expression levels of Linc01315 were higher in Luminal A and Her-2 subtype than in Luminal B and basal like subtype (Supplementary Figure 2).

Association Between Linc01315 Expression and Patient Prognosis
Furthermore, analyzing the disease free survival (DFS) and overall survival (OS) of these 282 breast cancer patients revealed that patients with lower Linc01315 expression level had significantly longer DFS (P = 0.002) and OS (P=0.019) (Figures 2A, B). Interestingly, the tumor stage, lymph node stage, TNM stage and vessel invasion also showed significant correlation with patients DFS and OS, as patients with lower tumor stage (P = 0.036,     Figure 3). ER positive breast cancer patients receive endocrine therapy to block estrogen action in the tumor, consequently stop or slow the development of hormonesensitive tumors. Additionally, we investigated the prognosis of patients treated with endocrine therapy (n = 131). The 131 ER positive patients received Tamoxifen or Aromatase inhibitor for 5 years based on menopausal status at diagnosis. We found low Linc01315 level patients with endocrine therapy could benefit patients DFS (P=0.037) and OS (P=0.025) ( Figures 5A, B). Nevertheless, Her-2 negative patients with high Linc01315 expression showed poor DFS (P=0.005) and OS (P=0.002) ( Figures 3C, 4C). Ki67 gene encodes a nuclear protein that is associated with and may be necessary for cellular proliferation. Ki67 positive patients with lower Linc01315 expression seemed to benefit DFS (P=0.019) and OS (P=0.009) ( Figures 3D, 4D).

DISCUSSION
Increasing efforts to achieve effective personalized treatment have emphasized the importance of applicable molecular markers to predict patient prognosis and therapeutic effect.
Multiple studies have indicated that LncRNAs can function as tumor suppressors or oncogenes through regulating coding genes expression and epigenetic modification (21). Molecular mechanisms of lncRNAs in various cancers may represent the key points for therapeutic intervention. Linc01315 is located in chromosome 22q. LINC01315 has three variants, NR_120595, NR_120596, and NR_120597, and the primers we designed could recognize these three isoforms. A previous study speculated that Linc01315 could promote the proliferation, migration and invasion of nasopharyngeal carcinoma (NPC) cells, and reduced the apoptosis of NPC cells, resulting in facilitating the tumorigenesis of NPC (22). The study demonstrated that Linc01315 might be used for the early diagnosis and treatment of NPC. In our study, we identified that Linc01315 expression level might be associated with breast cancer patients' prognosis. High Linc01315 expression in breast cancer significantly correlated with worse DFS and OS (Figures 2A, B). Multiple regression analysis displayed that the Linc01315 level could be an independent predictive factor for DFS and OS in breast cancer ( Table 2).
To our knowledge, about 70% of breast cancer patients presenting ER/PR positive (23). ER mediates the effects of estrogen on the development and progression of breast cancer, and it acts as an important therapeutic target for treatment. Endocrine therapy resistance in breast cancer is a major challenge in the treatment of hormone receptor positive breast cancer. Several lncRNAs have been reported to play crucial roles in the modulation of endocrine therapy responses in breast cancer cells. Previous study showed that lncRNA UCA1 could confer tamoxifen resistance by activating the mTOR signaling pathway (24) and Wnt/b-Catenin signaling pathway (25). LncRNA HOTAIR was found to interact with ER, and then enhance its transcriptional activity and promote tamoxifenresistant breast cancer progression (26). Our study showed that Linc01315 expression level in breast cancer was significantly correlated with tumor subtype, the expression level of Linc01315 was higher in Luminal A and Her-2 subtype than in Luminal B and basal like subtype ( Table 1 and Supplementary Figure 2). However, previous study showed that the expression level of Linc01315 was higher in triple negative breast cancer(TNBC) tissues compared with non-TNBC tissues, and high Linc01315 expression associated with better DFS in TNBC patients (19). Considering samples selection bias, we may need to expand the sample size, and design a prospective trial in the further study. Further analysis also revealed that ER/PR positive patients with lower Linc01315 expression displayed longer DFS and OS ( Figures 3A, B; Figures 4A, B), and low Linc01315 level patients treated with endocrine therapy could benefit patients DFS and OS ( Figures 5A, B). The results indicate that Linc01315 may be associated with endocrine therapy resistance in breast cancer, and represent a promising therapeutic target for cancer treatment.
In the past decade, lncRNA research is booming. Therapeutics based on targeting lncRNAs has evolving rapidly. It has been well demonstrated that multiple RNA-based therapies, such as small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), antisense oligonucleotides (ASOs), as well as CRISPR-Cas13a based RNA editing, showed promising results in preclinical research. In the ASOs scenario, different chemical modifications have been developed to enhance the stability of this kind of drug, also the delivery efficiency to tumor cells. ASOs are single stranded chemically modified DNA/RNA synthetic molecules complementary to target mRNA, causing mRNA degradation through RNase H cleavage or stabilizing the targets. We have demonstrated the efficiency of ASOs in the treatment of triplenegative breast cancer cells targeting LINC02273 in vivo (12). Therefore, our study could also utilize this tool for future translational study although it may need more mechanism and animal studies. In conclusion, our data demonstrate that Linc01315 expression level is significantly correlated with breast cancer patients' prognosis. Lower Linc01315 expression level may benefit patients DFS and OS, especially in ER/PR positive patients treated with endocrine therapy. Linc01315 also has prognostic effect in Her-2 negative or Ki67 positive breast cancer patients. It is plausible that Linc01315 may represent an independent prognostic marker and therapeutic target in breast cancer.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
This study was approved by the Ethical Committee of Fudan University Shanghai Cancer Center for Clinical Research. Written informed consents were obtained from all the patients.

AUTHOR CONTRIBUTIONS
YaC and JW designed the research study. JX, SH, and Y-zC conducted the experiments. JX, SH and JC collected clinical data. JX, JC and Z-mS analyzed the data. JX wrote the manuscript with contribution from all authors. All authors contributed to the article and approved the submitted version.