LncRNA HOTAIR enhances breast cancer radioresistance through facilitating HSPA1A expression via sequestering miR-449b-5p

Abstract

Background: Breast cancer (BRCA) is the leading cause of cancer-related death in women worldwide. Pre- and postoperative radiotherapy play a pivotal role in BRCA treatment but its efficacy remains limited and plagued by the emergence of radiation resistance, which aggravates patient prognosis. The long noncoding RNA (lncRNA)-implicated mechanisms underlying radiation resistance are rarely reported. The aim of this study was to determine whether lncRNA HOX transcript antisense RNA (HOTAIR) modulated the radiosensitivity of breast cancer through HSPA1A.

Methods: A Gammacell 40 Exactor was used for irradiation treatment. Bioinformatic tools and luciferase reporter assay were adopted to explore gene expression profile and demonstrate the interactions between lncRNA, miRNA and target mRNA 3'-untranslated region (3'-UTR). The expression levels of certain genes were determined by real-time PCR and western-blot analyses. in vitro and in vivo functional assays were conducted by cell viability and tumorigenicity assays.

Results: The levels of oncogenic lncRNA HOTAIR were positively correlated with the malignancy of BRCA but reversely correlated with the radiosensitivity of breast cancer cells. Moreover, the expression levels of HOTAIR were positively associated with those of heat shock protein family A (Hsp70) member 1A (HSPA1A) in clinical BRCA tissues and HOTAIR upregulated HSPA1A at the mRNA and protein levels in irradiated BRCA cells. Mechanistically, miR-449b-5p restrained HSPA1A expression through targeting the 3'-UTR of HSPA1A mRNA, whereas HOTAIR acted as a competing sponge to sequester miR-449b-5p and thereby relieved the miR-449b-5p-mediated HSPA1A repression. Functionally, HOTAIR conferred decreased radiosensitivity on BRCA cells, while miR-449b-5p overexpression or HSPA1A knockdown abrogated the HOTAIR-enhanced BRCA growth under the irradiation exposure both in vitro and in vivo.

Conclusions: LncRNA HOTAIR facilitates the expression of HSPA1A by sequestering miR-449b-5p post-transcriptionally and thereby endows BRCA with radiation resistance.

Key points: Therapeutically, HOTAIR and HSPA1A may be employed as potential targets for BRCA radiotherapy. Our findings shed new light into the mechanism by which lncRNAs modulate the radiosensitivity of tumors.

Keywords: Breast cancer; HOTAIR; HSPA1A; miR-449b-5p; radioresistance.

Figure 1

HOTAIR endows breast cancer with radiation resistance. (a–c) The expression profile of HOTAIR in different cancer types and the corresponding normal tissues (a), the relative HOTAIR levels in breast cancer tissues and normal breast tissues () Tumour, () Normal (b) and the relationship between HOTAIR levels and overall survival of BRCA patients () Tumour, () Normal (c) were obtained from the GEPIA database. () Low HOTAIR TPM, () High HOTAIR TPM (d) The relative abundance of HOTAIR in different BRCA cells was determined by real‐time PCR. (e and f) CCK‐8 assay was used to measure the cell viability of T47D (e) () 0 Gy, () 10 Gy and SKBR‐3 (f) after 0 or 10 Gy irradiation treatment. () 0 Gy, () 10 Gy (g) Colony formation assay was used to measure the proliferative ability of T47D and SKBR‐3 after 6 Gy irradiation treatment. (h and i) MDA‐MB‐231 and MCF‐7 cells were transfected with pcDNA3.1/pcDNA3.1‐HOTAIR 12 hours before the irradiation treatment, CCK‐8 assay was used to measure the cell viability after 10 Gy irradiation treatment. () pcDNA3.1, () pcDNA3.1‐HOTAIR (j and k) MDA‐MB‐231 and MCF‐7 cells were transfected with siRNA Ctrl/si‐HOTAIR 12 hours before the irradiation treatment, CCK‐8 assay was used to measure the cell viability after 10 Gy irradiation treatment. ; () siRNA Ctrl, () HOTAIR siRNA; (l and m) MDA‐MB‐231 cells were transfected with pcDNA3.1/pcDNA3.1‐HOTAIR (l) or siRNA Ctrl/si‐HOTAIR (), pcDNA3.1, () pcDNA3.1‐HOTAIR (m) 12 hours before the irradiation treatment, CCK‐8 assay was used to measure the cell viability after 0, 15, 20 and 25 Gy irradiation treatment. () siRNA Ctrl, ( ) HOTAIR siRNA (n) MCF‐7 cells were transfected with pcDNA3.1 + siRNA Ctrl/pcDNA3.1‐HOTAIR/si‐HOTAIR 12 hours before the irradiation treatment, then colony formation assay was used to measure the proliferative ability after 6 Gy irradiation treatment. Data are shown as mean ± SD of three independent experiments. Statistical significant differences are indicated: *, P < 0.05; **, P < 0.01; Student's t‐test.

Figure 2

HOTAIR upregulates the stress‐inducible oncogene HSPA1A in BRCA cells. (a and b) The abundance of HSPA1A in different cancer types and the corresponding normal tissues (a) and the relative HSPA1A levels in breast cancer tissues and normal breast tissues () Tumour, () Normal (b) were obtained from the GEPIA database. () Tumour, () Normal (c) The relationship between HSPA1A levels and overall survival of BRCA patients was explored by searching the Kaplan‐Meier Plotter database. () Low, () High (d) The expression of HSPA1A at mRNA level in 20 cases of clinical BRCA tissues and their paired peritumor tissues (P < 0.001, Wilcoxon's signed‐rank test) was examined by real‐time PCR analysis. (e) The correlation of HSPA1A and HOTAIR mRNA levels in 20 cases of clinical BRCA tissues was measured by real‐time PCR and analyzed by Pearson's correlation (R = 0.5391, P < 0.05). (f–h) The levels of HSPA1A mRNA (f) and protein (g and h) in irradiated BRCA cell lines MDA‐MB‐231 and MCF‐7 after the transfection of pcDNA3.1/pcDNA3.1‐HOTAIR were tested by RT‐PCR and western blot analysis, respectively. The relative expression levels of HSPA1A in three independent experiments were statistically analyzed by measuring the gray value of each band using Image J software. (i–k) The levels of HSPA1A mRNA (i) and protein (j and k) in irradiated BRCA cell lines MDA‐MB‐231 and MCF‐7 after the transfection of siRNA Ctrl/si‐HOTAIR were tested by RT‐PCR and western blot analysis, respectively. The relative expression levels of HSPA1A in three independent experiments were statistically analyzed by measuring the gray value of each band using Image J software. Data are shown as mean ± SD of three independent experiments. Statistical significant differences are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's t‐test.

Figure 3

miR‐449b‐5p suppresses the expression of HSPA1A by targeting the mRNA 3′‐UTR of HSPA1A. (a) A schematic representation for the predicted binding site of miR‐449b‐5p in the 3′‐UTR of HSPA1A mRNA. The HSPA1A mRNA 3′‐UTR fragment containing the wild‐type or mutant of miR‐449b‐5p binding site was synthetized and inserted into the downstream of the luciferase reporter gene in pGL3‐control vector. (b) A schematic model depicting the interaction between HOTAIR and miR‐449b‐5p via complementary base‐pairing was obtained from the bioinformatic prediction of Bielefeld Bioinformatics Service. The mutant sites were indicated. (c) Real‐time PCR analysis was performed to evaluate the expression levels of miR‐449b‐5p in 20 paired of clinical BRCA tissues and the corresponding normal tissues (P < 0.001, Wilcoxon's signed‐rank test). (d) The correlation between HSPA1A mRNA and miR‐449b‐5p levels was determined by real‐time PCR in 20 clinical BRCA tissues (P < 0.01, R = −0.6204, Pearson's correlation coefficient). (e and f) The effects of miR‐449b‐5p on reporters of pGL‐HSPA1A‐wt and pGL‐HSPA1A‐mut in irradiated MDA‐MB‐231 (e) and MCF‐7 (f) cell lines were measured by luciferase reporter gene assays. (g and h) The effects of anti‐miR‐449b‐5p on reporters of pGL‐HSPA1A‐wt and pGL‐HSPA1A‐mut in irradiated MDA‐MB‐231 (g) and MCF‐7 (h) cell lines were examined by luciferase reporter gene assays. Data are shown as mean ± SD of three independent experiments. Statistical significant differences are indicated: *, P < 0.05; **, P < 0.01; Student's t‐test.

Figure 4

miR‐449b‐5p suppresses the expression of HSPA1A by targeting the mRNA 3′‐UTR of HSPA1A. (a and b) The BRCA cell lines MDA‐MB‐231 (a) and MCF‐7 (b) were transfected with miR‐449b‐5p or control 12 hours before the irradiation treatment. Then, 36 hours after the irradiation, total RNA and protein were extracted. The mRNA and protein levels of HSPA1A were detected by real‐time PCR and western blot analysis, respectively. (c and d) The BRCA cell lines MDA‐MB‐231 (c) and MCF‐7 (d) were transfected with the miR‐449b‐5p inhibitor or control 12 hours before the irradiation treatment. A total of 36 hours after the irradiation, total RNA and protein were extracted. The mRNA and protein levels of HSPA1A were detected by real‐time PCR and western blot analysis, respectively. Data are shown as mean ± SD of three independent experiments. Statistical significant differences are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's t‐test.

Figure 5

HOTAIR facilitates the expression of HSPA1A by sequestering miR‐449b‐5P. (a) The correlation between the levels of miR‐449b‐5p and HOTAIR in 20 cases of clinical BRCA tissues were tested by real‐time PCR and assayed by Pearson's correlation (R = −0.5407, P < 0.05). (b and c) The BRCA cell lines MDA‐MB‐231 (b) and MCF‐7 (c) were transfected with pcDNA3.1 or pcDNA3.1‐HOTAIR 12 hours before 10 Gy irradiation treatment. Total RNA was extracted and polyadenylated by poly (A) polymerase 36 hours after the irradiation. The effects of HOTAIR on the expression of miR‐449b‐5p were monitored by real‐time PCR analysis. (d and e) The BRCA cell line MDA‐MB‐231 was transfected with Ctrl/50 nM of miR‐449b‐5p/ 100 nM of miR‐449b‐5p (d) or Ctrl/50 nM of miR‐449b‐5p inhibitor/100 nM of miR‐449b‐5p inhibitor (e) 12 hours before 10 Gy irradiation treatment. Total RNA was extracted 36 hours after the irradiation. The effects of miR‐449b‐5p on the expression of HOTAIR were monitored by real‐time PCR analysis. (f) The BRCA cell line MDA‐MB‐231 was cotransfected with pGL‐HSPA1A, pRL‐TK plus Ctrl/miR‐449b‐5p/miR‐449b‐5p + HOTAIR/miR‐449b‐5p + HOTAIR‐449b‐mut 12 hours before 10 Gy irradiation treatment. The luciferase activities of pGL‐HSPA1A were measured 36 hours later by luciferase reporter gene assays. (g) The BRCA cell line MDA‐MB‐231 was transfected with Ctrl/miR‐449b‐5p/miR‐449b‐5p + HOTAIR/miR‐449b‐5p + HOTAIR‐449b‐mut 12 hours before 10 Gy irradiation treatment. Total proteins were extracted 36 hours later. The protein levels of HSPA1A were detected by western blot analysis, respectively. Data are shown as mean ± SD of three independent experiments. Statistical significant differences are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significance; Student's t‐test.

Figure 6

HOTAIR enhances the proliferation of irradiated‐BRCA cells through HSPA1A in vitro. The grouping of the test was as follows: Ctrl, IR (10 or 6 Gy), IR (10 or 6 Gy) + HOTAIR, IR (10 or 6 Gy) + HOTAIR+miR‐449b‐5p and IR (10 or 6 Gy) + HOTAIR+si‐HSPA1A. (a) The expression of HSPA1A at mRNA and protein levels in MDA‐MB‐231 cells from each group was assessed by real‐time PCR and western blot analysis, respectively. (b) The cell viability of MDA‐MB‐231 cells from each group was determined by CCK‐8 assay. () Ctrl, () IR 10 Gy, () IR 10 Gy + HOTAIR, () IR 10 Gy + HOTAIR+miR‐449b‐5p, () IR 10 Gy + HOTAIR + si‐HSPA1A (c and d) The proliferative ability of MDA‐MB‐231 cells from each group was validated by colony formation assay (c) and EdU incorporation assay (d, scale bar, 30 μM), respectively. The colony formation efficiency and percentage of EdU positive cells were calculated. Data are shown as mean ± SD of three independent experiments. Statistical significant differences are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significance; Student's t‐test.

Figure 7

HOTAIR enhances the growth of irradiated‐BRCA cells through HSPA1A in vivo. MDA‐MB‐231 cells were grouped as the following treatment: Ctrl‐1, Ctrl‐2, HOTAIR, HOTAIR+miR‐449b‐5p and HOTAIR+si‐HSPA1A. The aforementioned cells were injected subcutaneously into the first mammary fat pad in the armpit of nude mice. Five days of consecutive 2 Gy IR‐treatment was applied to the tumors from Ctrl‐2, HOTAIR, HOTAIR+miR‐449b‐5p and HOTAIR+si‐HSPA1A groups when the tumors reached 100 mm³. The tumor volume was recorded during the tumorigenicity process every five days. (a) The growth curves of tumors from each group are presented. () Ctrl, () IR, () IR + HOTAIR, () IR + HOTAIR + miR‐449b‐5p, () IR + HOTAIR + si‐HSPA1A (b and c) The image of excised tumors and tumor weights of each group are presented. (d–g) The expression levels of HSPA1A mRNA and protein (d and e), HOTAIR (f) and miR‐449b‐5p (g) were quantified by real‐time PCR or western blot analysis. (h) The expression levels of Ki‐67 in tumor tissues were examined by IHC staining. Scale bar, 20 μM. Data are shown as mean ± SD of three independent experiments. Statistical significant differences are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; Student's t‐test.