High long non-coding RNA NORAD expression predicts poor prognosis and promotes breast cancer progression by regulating TGF-β pathway

21 cases of cancer tissue and 10 cases of paracancerous tissue were collected from May 2017 to September 2018 in the Taihe Hospital Affiliated to Hubei Medicine University. Paraffin specimens were removed from 18 cases of breast cancer and 10 cases of hyperplasia of breast tissue.

Tissue and cells were collected and cleaved, and an appropriate amount of Trizol solution was added (1 ml Trizol solution was added to the cells of the 6-cm culture plate). The cells were left to fully contact for 1 min, and the cells were transferred to the non-RNA enzyme centrifuge tube. Extraction: 1/5 volume of trichloromethane was added and shaken violently for 1 min. After fully mixing, it was left at room temperature for 3 min. 37 °C, 12,000 RPM centrifuge for 10 min. Transfer the upper RNA layer to the new RNA-free enzyme centrifuge tube. Precipitation: isopropyl alcohol was added, mixed, and left at room temperature for 10 min. 4 °C, 12,000 RPM centrifuge for 10 min. Precipitated RNA could be observed at the bottom. Discard the supernatant and retain the precipitate. Wash: gently add 1 ml of freshly prepared 75% ethanol. Upside-down washing precipitation and pipe wall. 4 °C, 12,000 RPM centrifugal for 5 min. Discard the supernatant and retain the precipitate. Dry at room temperature. Dissolve: add the appropriate amount of DEPC to treat the water and dissolve the RNA. Measure RNA concentration. RNA was detected by agarose gel electrophoresis test.

The cells were washed twice in a cold PBS solution and a mixture of protease inhibitors was added to RIPA lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% deoxycholic acid sodium, 1% Triton x-100 and 0.1% SDS). The whole protein was subjected to SDS-PAGE electrophoresis under 30 μg/pore. Transfer the film by wet transfer. Sealing fluid for 5% skimmed milk powder solution containing 0.1% Tween 20, immersed membrane in sealing fluid, 37 °C shaking bed 1 h incubation. Primary antibodies against special proteins diluted with PBST to the appropriate concentration, incubation at 37 °C shake on the bed overnight. PBST was used to wash the membrane 3 times, 5 min each time, and the non-specific binding on the membrane was washed. Then, the membrane was incubated for 40 min with the HRP-labeled second antibody diluted with PBST at 37 °C shaking bed. Wash the film 3 times with PBST for 5 min each. Protein expression was detected by ECL reagent using a chemical image luminescence system.

1 × 10⁶ cells were spread in the 6-well plate. Scratched with 10 μl pipette tip. The migration progress was detected 24 h later and photographed.

The cells were starved for 24 h, then the 5 × 10⁵ (invasion) cells in serum-free medium were spread to cell compartments with 8 mm holes, and the culture medium containing 10% FBS was added to the pore plate below. For the invasion–determination experiment, the room was coated with Matrigel and stained for 48 h. The cells were stained with crystal violet for 16 h after incubation. Randomly selected areas were photographed and the stained cells were statistically analyzed.

The MDA-MB-231 breast cancer cells were transfected with lenti-control (sh-nc) or lenti-shRNA NORAD (sh-NORAD) (GeneChem, Shanghai, China) and selected for puromycin resistance (10 μg/ml). The 4 weeks of BALB/c female mice were bred, and MDA-MB231 cells were cultured. Then, 5 × 10⁶ cells/200 μl PBS/nude mice were injected into the buttocks of the mice, 5 in each of the experimental group and the control group. After the injection of the cells, the tumor’s long diameter and short diameter were observed once a week, and the mice were killed after 6 weeks. The tumor mass was completely removed and photographed. In this part, the calculation formula of tumor volume is L × S/2, L is the tumor’s long diameter and S is the tumor’s short diameter. And the tumor weight was also weighted.

Aberrant lncRNA-NORAD expression has been found in several cancers, we first detected lncRNA-NORAD expression levels in two breast cancer cell lines (MDA-MB231and MCF-7) and the final results indicated that lncRNA-NORAD were higher than that in the normal mammary cell line (MCF10A) (Fig. 1a). We further detected lncRNA-NORAD in breast cancer tissues and adjacent normal breast tissues, and the results showed that lncRNA-NORAD was higher in breast cancer tissues than normal tissues (Fig. 1b). The whole patients were divided into two groups, the low- and high-expression groups, according to the lncRNA-NORAD levels in the tumor. And survival analysis indicated that the high-expression groups had worse survival compared to the low-expression groups with the Kaplan–Meier method (Fig. 1c). This reinforced our hypothesis that lncRNA-NORAD might be an oncogenic gene that participated in the breast cancer progression.

In order to investigate the influences of lncRNA-NORAD on cell proliferation, breast cancer cells MCF-7 and MDA-MB-231 were stably transfected with si-NORAD or si-nc. The expression level of lncRNA-NORAD in MCF-7 and MDA-MB-231 cells was significantly suppressed at least 50% of si-NORAD groups compared with that of negative control group si-nc (Fig. 2a, c). Meanwhile, the cell viability of MCF-7 and MDA-MB-231 cells, that determined by MTT assays, was significantly reduced in experiments (Fig. 2b, d). In conclusion, inhibiting lncRNA-NORAD expression could significantly inhibit breast cancer cell viability and proliferation.

We further try to investigate the influences of lncRNA-NORAD on cell migration and invasion in breast cancer cells. MCF-7 and MDA-MB-231 were stably transfected with si-NORAD or si-nc. Lnc-NORAD knockdown by si-NORAD led to both migration and invasion inhibition in the MCF-7 and MDA-MB-231 cells (Fig. 3a, b).

In order to uncover the mechanisms of lnc-NORAD promote the development of breast cancer, we analyzed lnc-NORAD impact on TGF-β by western blot. When lncRNA-NORAD was knocked down in the MCF-7 and MDA-MB-231 breast cancer cells, obviously down-regulated TGF-β expression levels were observed. And the downstream factors such as Smad2 and RUNX2 were also suppressed (Fig. 4a, b). These results robustly indicated that lncRNA-NORAD likely regulated the TGF-β signaling pathway and thus involved in the progression of breast cancer.

We also compared the growth of tumors in the animals. The growth of the tumors formed from the injected cells was apparently smaller in the si-NORAD group during the period (Fig. 5a). And si-NORAD effectively suppressed the expression of lncRNA-NORAD in the MDA-MB231 transfected cells (Fig. 5b). In addition, the results of in vivo xenograft assays showed that tumor volume and weight of both si-NORAD groups were much smaller compared with those of si-nc group (Fig. 5c, d). These results suggested that inhibiting the expression of lncRNA-NORAD in breast cancer cells could efficiently suppress tumorigenesis in vivo. Through combining our above discoveries in cancer cells and tissue samples, we concluded that lncRNA-NORAD might be an important oncogene, and this lncRNA might function significant roles in cancer progression.