| Objective:To explore the association and possible molecular mechanism of HOTAIR and trastuzumab resistance in breast cancer in vitro and in vivo study.Methods:(1) Trastuzumab-resistant breast cancer cell lines SK-BR-3-TR were developed by continuous culture of SK-BR-3-TS cell lines in the presence of trastuzumab with gradually increased dose (0.5-1.0-2.0-4.0-8.0μg/ml)for 6 months and 4μg/ml trastuzumab for 1 month.(2) HOTAIR expression was detected in the SK-BR-3-TS and SK-BR-3-TR cell lines by qRT-PCR. MTT assay was used to detect the cell proliferative abilities of the two cell lines. Flow cytometry and transwell invasion assay were used to observe the tumor cell apoptotic and invasive abilities, respectively. The EMT-associated genes and proteins of TGF-β, Snail, Vimentin and E-cadherin and the HER2 receptor signal pathway activity were detected by qRT-PCR and western blot techniques respectively.(3) The methylation levels of PTEN, CNK1B (P27kip1) and TGF-β genes in the two cell lines were detected by methylation-specific PCR (MSP) technique.(4) The correlation between HOTAIR and trastuzumab resistance and its molecular mechanisms was reversely verified by siRNA interference experiments in SK-BR-3-TR cell lines.(5) The bearing tumor models with BALB/c nude mice injected with SK-BR-3-TS, SK-BR-3-TR and si-HOTAIR-SK-BR-3-TR respectively were established and the tumor growth was observed. The protein expression of TGF-β, Snail, E-cadherin, PTEN and Ki67 was detected in the three models by IHC technique.Results:(1) Trastuzumab-resistant breast cancer cell lines SK-BR-3-TR were successfully induced and cultured. The expression of HOTAIR was significantly up-regulated compared with SK-BR-3-TS (2.216+0.332 V 0.326+0.05, p=0.0006).(2) The cell proliferative and invasive abilities were significantly enhanced, whereas, the apoptotic ability was reduced in the SK-BR-3-TR cell lines compared with SK-BR-3-TS by MTT assay, transwell invasion assay and flow cytometry techniques, respectively(p<0.05).(3) The gene and protein expression of TGF-P, Snail and Vimentin were up-regulated, meanwhile, E-cadherin down-regulated in SK-BR-3-TR cell lines. HER2, PI3K, Akt, mTOR and MAPK did not change significantly compared with SK-BR-3-TS, whereas, phosphorylated level of Akt and MAPK was up-regulated, and the expression level of PTEN and p27 were down-regulated detected by qRT-PCR and western blot techniques respectively.(4) The methylation level of PTEN gene was up-regulated, meanwhile, the methylation level of TGF-p was down-regulated, and the methylation level of CNK1B (P27kip1) gene did not change significantly in SK-BR-3-TR cell lines compared with SK-BR-3-TS.(5) HOTAIR expression was down-regulated in SK-BR-3-TR cell lines by siRNA interference experiment. The siHOTAIR-SK-BR-3-TR cells restored sensitivity to trastuzumab detected by MTT assay. Cell proliferation activity and invasion ability were reduced and the proportion of apoptotic cells increased compared with SK-BR-3-TR cell lines. The gene and protein expression of TGF-P, Snail and Vimentin was down-regulated, meanwhile, E-cadherin expression was up-regulated in siHOTAIR-SK-BR-3-TR cell lines. HER2, PI3K, Akt, mTOR and MAPK did not change significantly compared with SK-BR-3-TR, but phosphorylated level of Akt and MAPK was down-regulated and PTEN and p27 levels were up-regulated detected by qRT-PCR and western blot techniques respectively.(6) PTEN gene methylation level was down-regulated, meanwhile, TGF-β gene methylation level was up-regulated, and CNK1B (P27kip1) did not chang significantly in siHOTAIR-SK-BR-3-TR cell lines compared with SK-BR-3-TR by MSP detection.(7) The tumor bearing models with nude mice injected respectively with SK-BR-3-TS, SK-BR-3-TR and si-HOTAIR-SK-BR-3-TR were successfully established. The growth rate of SK-BR-3-TR tumor bearing models was significantly faster than that of SK-BR-3-TS and si-HOTAIR-SK-BR-3-TR models (p<0.05). The positive expression ratio of TGF-β and Snail(16.7% and 33.3%,respectively) was lower in siHOTAIR-SK-BR-3-TR models than that of SK-BR-3-TR model (66.7% and 83.3%,respectively), but the difference was not statistically significant (chi square tests and Fisher’s exact test. p=0.242). Meanwhile, the positive expression ratio of E-cadherin and PTEN protein was higher in siHOTAIR-SK-BR-3-TR models than that of SK-BR-3-TR model, but there was also no statistically significant difference between the two groups (p=0.242).Conclusion:HOTAIR promotes the trastuzumab resistance in breast cancer cell lines SK-BR-3-TR in vitro and in vivo. It’s the main molecular mechanism of HOTAIR promoting trastuzumab resistance that HOTAIR contributes the tumor cells to EMT by its epigenetically demethylated regulation to TGF-β gene and enhances the intrinsic activity of PI3K/Akt/ mTOR pathway by its epigenetically methylated regulation to PTEN gene. |
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