Linc00460/miR-641 promoted promotes proliferation and autophagy of breast cancer

This manuscript aimed to investigate the oncogenic role of linc00460 in breast cancer both in vivo and in vitro and then specify its downstream microRNAs to build a ceRNA network. As for in vivo tests, we used subgroup analysis and nomogram for survival analysis. As for in vitro tests, qRT-PCR, CCK-8 and Dual luciferase reporter gene were used. Linc00460 expressed highly in breast cancer. The nomogram indicated that linc00460 was associated with worse prognosis. Linc00460 might negatively regulate miR-641 to promote the proliferation and autophagy of breast cancer cell. In conclusion, linc00460 could be a risk factor for breast cancer by regulating miR-641; and it has the potential to be a novel biomarker.

. Patients with presence of estrogen receptors, progesterone receptors or HER2 tended to have better prognosis based on the bene ts of hormone therapy or targeting therapy of HER2.
However, approximately 15%-25% of breast cancer did not express the ER, PR and HER2 3 . This molecular subtype was de ned as triple-negative breast cancer (TNBC). This made treatment become more di cult because most therapies target one of the three receptors. And the recurrence and metastasis risks for TNBC patients were signi cantly higher than other breast cancer patients [5][6][7] . Therefore, it is important to nd an e cient therapy targets for TNBC in order to improve their prognoses.
Long noncoding RNAs (lncRNA) have attracted great attention in recent years according to their various biological functions in cancers [8][9][10] . LncRNAs represent a group of RNA transcripts which are longer than 200 nucleotides but cannot be translated into proteins 11  The linc-00460 and miR-641 lentivirus and relative negative control lentivirus were synthesized by GenChem (Shanghai, China). Transfection was thereby performed according to the manufacturer's instructions.

Real-time PCR for detection of linc00460and miR-641
Total RNA was extracted by Trizol reagent (TAKARA, Japan). Breast cancer and para cancer tissues' total RNA was extracted by Trizol reagent (Carlsbad, USA).
As for linc00460, SYBR Premix Ex Taq II (Takara, Japan) was used for PCR reactions. GAPDH was used as the control.
As for miR-641, Taqman MirNA reverse transcription Kit (Carlsbad, USA) and PrimeScriptTM RT Master Mix (Takara, Japan) were used for reverse transcription. Then qPCR process was conducted by Taqman MirNA assay kit and SYBR® Select Master Mix. The relative expression of LINC00460 and miR-641 were normalized to GAPDH and U6 respectively, by using the 2 −ΔΔCt method.

Cell proliferation assay
Cell proliferation ability was evaluated by cell counting kit-8 (CCK-8) assay (Dojindo, Japan). Treated cells were seeded into 96-well plates with density of 1000 cells per well. 10 ul CCK8 reagent was added to each cell sample before absorbance measurement. The absorbance of each cell sample at 450 nm was measured at 0,1,2 days after CCK8 reagent added.

Protein extraction and western blot
RIPA buffer and BCA (Sigma Aldrich, Cambridge, MA) were used to extract and validate the total protein.
Proteins were transferred onto the PVDF membrane after electrophoresis. After blocking the membrane for 2 hours, primary antibodies were incubated over night at 4℃. Secondary antibody was incubated for 1 hour at room temperature.

Autophagy
Autophagy virus (GFP-RFP-LC3) was synthesized by Genchem (Shanghai, China). Autophagy virus was transfected into target cells and cultured for at least one day; then, approximately 1 × 10 5 transfected cells were added into a 96-well plate. Confocal microscope was used to observe the green and red intensity.

Statistical analysis
The presenting Data was the mean ± SD from at least three independent experiments. Student's t-test was adopted to compare the difference between two groups. P value less than 0.05 was de ned as statistically signi cant. All statistical analysis was calculated by R 3.3.5.
2 linc00460 could negatively regulate the expression of miR-641 According to Table 1, we noticed that the expression of miR-641 and miR-491 statistic signi cantly correlated to the expression of linc00460. Furthermore, we down-regulated the expression of linc00460( Fig. 2A) in MDA-MB-231 cell line and found the correspondingly increased expression of miR-641 and miR-491 (Fig. 2B). Following dual luciferase reported gene assay indicated that only miR-641 had the potential to interact with linc00460( Fig. 2C and 2D). * indicates that the P value was smaller than 0.05.
After that, we have up-and down-regulated miR-641 in MDA-MB-231 cell line. (Fig. 2E and 2F) The result showed that the exotic regulation of miR-641 wouldn't in uence the expression of linc00460, suggesting that miR-641 could be the downstream target of linc00460 ( Fig. 2G and 2H).

Survival analysis for linc00460 and miR-641 in breast cancer
As shown in Table 2, we found that high expression of linc-00460 was associated with worse overall survival (Fig. 3A); and high expression of miR-641 was associated with better overall survival (Fig. 3B).
And advanced stage was associated with worse prognosis (Fig. 3C); while ER ( Fig. 3D) and PR (Fig. 3E) were associated with better prognosis; However, the in uence of Her-2 on overall survival was not statistically signi cant (Fig. 3F) ( Table 2). In Table3, we found that advanced stage and high expression of linc-00460 were associated with worse prognosis. Therefore, we selected those independent risk factors to build a nomogram to predict the 1- year and 3-year survival rate for breast cancer patients. The nomogram indicated that M stage contribute the most to the prognosis of breast cancer (Fig. 4A); and linc00460 was an important risk factor as well. The calibration curve for 3-year overall survival prediction and 5-year (Fig. 4B) overall survival prediction ( Fig. 4C) and C-index (0.763 ± 0.061) suggested that the nomogram could precisely and correctly predict the survival rate. cell line (Fig. 5A); and up regulation of miR-641 could inhibit the proliferation of MDA-MB-231 cell line, while the down regulation of miR-641 promoted the proliferation (Fig. 5B and Fig. 5C). As for wound healing assay, we found that linc00460 downregulation leads to decreased wound closure (Fig. 5D). However, down regulation of miR-641 resulted in increased wound closure (Fig. 5E) whereas up regulation of miR-641 inhibit wound closure (Fig. 5F). Down regulation of miR-641 can reverse the inhibitory effect of linc00460 inhibition in breast cancer proliferation (Fig. 5G).
6 linc00460 and miR-641 promoted autophagy in breast cancer We found that linc00460 knock down leads to decreased expression of LC3 II and increased expression of LC3 I, indicating that autophagy is suppressed (Fig. 6A). The average green luciferase intensity is signi cantly increased, indicating the autophagy is inhibited (Fig. 6B). Overexpression of miR-641 resulted in the suppression of autophagy as well (Fig. 6C). However, miR-641 knock down induces the expression of LC3 I and decrease the expression of LC3 II (Fig. 6D).

Discussion
The oncogenic role of linc00460 has been investigated in several cancers, including esophageal carcinoma, ovarian cancer, gastric cancer and lung cancer [17][18][19] ; and we were the rst to report its oncogenic function in breast cancer. Our group has found the aberrant high expression of linc00460 in breast cancer tissues, and further survival analysis showed that high expression of linc00460 led to the worse prognosis. Therefore, we assumed that linc00460 might have the potential to be a novel biomarker for breast cancer. Besides, we noticed the positively association of linc00460 expression with the presence of Her-2 status in vivo, which required more experiments to clarify the exact mechanisms. Based on current studies, linc00460 was involved in the progression of EMT process, which resulted in the invasive behavior of cancer cell 18 . In this context, it was more evident that linc00460 could regulate the malignant behavior of breast cancer. In addition, the most widely investigated molecular pathways for linc00460 was ceRNA. Xing H et al illustrated that linc00460 could sponge miR-539 and then induced the malignancy for meningioma 20 . Meanwhile, Ye J et al has reported that miR-302c-5p could be one of linc00460's targets using dual luciferase reporter gene 16 .
As for survival analysis, we have built the nomogram including T stage, M stage, ER, PR, Her-2 and linc004600, to clearly and distinctly present the in uence of every risk factor on breast cancer overall survival. For example, we could infer from the nomogram that linc00460 contributed more to the overall survival than miR-641 did. In addition, we could predict the 3-and 5-year survival rate exploiting the nomogram. For example, if there were a stage I, ER (-), PR (-), Her-2(-), linc00460(+) patient; according to the nomogram, the total score would be14.9, therefore the 3-year survival rate would roughly be 90%, and the 5-year survival rate would be around 35%. In short, linc00460 has the potential to be novel biomarkers in predicting the prognosis of breast cancer patients. And this conclusion needs a larger cohort for validation before it could be interpreted for clinical practices.
With the help of online informatics tool, Starbase V2.0, we were able to identify the potential downstream target, which was miR-641, for linc00460. To date, it is well known that miR-641 is located in the intronic region of AKT2, which is a vital oncogene for the apoptosis and chemotherapy response of carcinomas 21 . In glioma, miR-641 plays an important role in suppressing the activation of PI3K/AKT pathway, which is closely associated with tumor progression and development 21 . Moreover, Chen J et al has found that the overexpression of miR-641 was relevant of erlotinib resistance in lung cancer 22 . And miR-641 might regulate erlotinib resistance through negatively regulating NF1 and then inducing the expression of ERK pathway 22  Compliance with the ethical standards

Con icts of interest
The authors have declared that they have no con icts of interest.

Ethics approval and consent to participate
The work was approved by the Ethics Committee of First a liated hospital of xi`an jiaotong university . Each patient recruited was received and signed the Informed consent forms.