MARCKSL1-2 reverses docetaxel-resistance of lung adenocarcinoma cells by recruiting SUZ12 to suppress HDAC1 and elevate miR-200b

Abstract

Background: Long non-coding RNAs (lncRNAs) are implicated in the development of multiple cancers. In our previous study, we demonstrated that HDAC1/4-mediated silencing of microRNA-200b (miR-200b) enhances docetaxel (DTX)-resistance of human lung adenocarcinoma (LAD) cells.

Methods and results: Herein, we probed the function of LncRNA MARCKSL1-2 (MARCKSL1-transcript variant 2, NR_052852.1) in DTX resistance of LAD cells. It was found that MARCKSL1-2 expression was markedly reduced in DTX-resistant LAD cells. Through gain- or loss- of function assays, colony formation assay, EdU assay, TUNEL assay, and flow cytometry analysis, we found that MARCKSL1-2 suppressed the growth and DTX resistance of both parental and DTX-resistant LAD cells. Moreover, we found that MARCKSL1-2 functioned in LAD through increasing miR-200b expression and repressing HDAC1. Mechanistically, MARCKSL1-2 recruited the suppressor of zeste 12 (SUZ12) to the promoter of histone deacetylase 1 (HDAC1) to strengthen histone H3 lysine 27 trimethylation (H3K27me3) of HDAC1 promoter, thereby reducing HDAC1 expression. MARCKSL1-2 up-regulated miR-200b by blocking the suppressive effect of HDAC1 on the histone acetylation modification at miR-200b promoter. Furthermore, in vivo analysis using mouse xenograft tumor model supported that overexpression of MARCKSL1-2 attenuated the DTX resistance in LAD tumors.

Conclusions: We confirmed that MARCKSL1-2 alleviated DTX resistance in LAD cells by abolishing the inhibitory effect of HDAC1 on miR-200b via the recruitment of SUZ12. MARCKSL1-2 could be a promising target to improve the chemotherapy of LAD.

Keywords: Docetaxel-resistance; HDAC1; Lung adenocarcinoma; MARCKSL1–2; SUZ12; miR-200b.

Fig. 1

Down-regulation of lncRNA MARCKSL1–2 is associated with DTX resistance and poor prognosis in LAD patients. a MARCKSL1–2 expression was detected by RT-qPCR in the DTX sensitive- (CR + PR; n = 28) and insensitive- (SD + PD; n = 32) LAD tissues. b MARCKSL1–2 expression in normal lung tissues (n = 20) and LAD tissues (n = 60) was determined by RT-qPCR. c Kaplan-Meier analysis of the association between progression-free survival (PFS) and MARCKSL1–2 level in LAD patients. d Kaplan-Meier analysis of the association between overall survival (OS) and MARCKSL1–2 level in LAD patients. e RT-qPCR tested the expression of MARCKSL1–2 in DTX-resistant LAD cells relative to parental H1299 or SPC-A1 cells. f-g Subcellular fractionation analyzed MARCKSL1–2 distribution in LAD cells and matched DTX-resistant cells. h IF analysis of MARCKSL1–2 distribution in parental and DTX-resistant LAD cells. Scale bar = 20 μm. ^**P < 0.01

Fig. 2

Effects of MARCKSL1–2 on the proliferation and apoptosis of DTX-sensitive or -resistant LAD cells under increasing dose of DTX treatment. H1299 cells were transfected with shRNAs targeting MARCKSL1–2, and H1299/DTX cells were transfected with pcDNA/MARCKSL1–2. a-d Colony formation and EdU assays (Scale bar = 100 μm) measured the impact of MARCKSL1–2 inhibition or overexpression on the proliferation capacity of H1299 cells treated with different doses of DTX (0, 5 and 10 μg/L) and H1299/DTX cells treated with different doses of DTX (0, 50 and 100 μg/L). e-h Flow cytometry analyses and TUNEL assays (Scale bar = 100 μm) detected the apoptosis rate of H1299 cells and H1299/DTX cells under different conditions. ^*P < 0.05, ^**P < 0.01. n.s.: no significance

Fig. 3

MARCKSL1–2 inhibits DTX resistance in LAD cells by regulating HDAC1 or miR-200b. a-b. HDAC1 and miR-200b expression was assessed using RT-qPCR in H1299 and SPC-A1 cells with or without MARCKSL1–2 interference. c-d By using RT-qPCR, the expression of HDAC1 or miR-200b was assessed in H1299/DTX and SPC-A1/DTX cells transfected with pcDNA3.1 or pcDNA/MARCKSL1–2. e-f Luciferase reporter assays evaluated the luciferase activity of miR-200b or HDAC1 promoter in LAD cells with MARCKSL1–2 depletion and in DTX-resistant LAD cells with MARCKSL1–2 overexpression. g-h The IC₅₀ value of indicated LAD cells to DTX treatment was assessed by CCK-8 assays. i-j The IC₅₀ value of DTX in indicated LAD cells was assessed by CCK-8 assays. ^**P < 0.01

Fig. 4

MARCKSL1–2 interacts with SUZ12 in LAD cells. a-b ChIP assay detected the changes in histone-H3 acetylation level at miR-200b promoter in SPC-A1/H1299 cells under shMARCKSL1–2 suppression and that in SPC-A1/DTX/H1299/DTX cells upon MARCKSL1–2 overexpression. c RNA pull-down assay followed by mass spectrometry analysis examined the proteins binding with MARCKSL1–2. d RNA pull- down plus Western blot detected the existence of SUZ12 in the pull-down complexes of MARCKSL1–2. e-f RIP assays followed by AGE or RT-qPCR examined the enrichment of MARCKSL1–2 in SUZ12 groups. g-h. RT-qPCR and Western blot detected the influence of MARCKSL1–2 on SUZ12 expression in indicated LAD cells. ^**P < 0.01. n.s.: no significance

Fig. 5

MARCKSL1–2 epigenetically inhibits HDAC1 expression by recruiting SUZ12 to HDAC1 promoter. a-b The impact of SUZ12 on the mRNA and protein levels of HDAC1 assessed by RT-qPCR and Western blot in indicated LAD cells. c-d ChIP assay plus AGE or RT-qPCR was used to evaluate the enrichment of HDAC1 promoter in SUZ12 groups. e ChIP assay analyzed the impact of SUZ12 on the H3K27me3 modification at HDAC1 promoter in indicated LAD cells. f-g ChIP assay was conducted to evaluate the changes in enrichment of HDAC1 promoter recognized by SUZ12 in SPC-A1/H1299 cells with MARCKSL1–2 inhibition and in DTX-resistant LAD cells with MARCKSL1–2 upregulation. h The influence of MARCKSL1–2 silence or overexpression on the level of H3K27me3 modification at HDAC1 promoter assessed by ChIP experiments. ^**P < 0.01

Fig. 6

MARCKSL1–2 affects DTX resistance of LAD cells by regulating HDAC1/miR-200b axis. H1299 cells were transfected with shCtrl, shMARCKSL1–2#1, shMARCKSL1–2#1 + sh/HDAC1 or shMARCKSL1–2#1 + miR-200b mimics. H199/DTX cells were transfected with pcDNA3.1, pcDNA/MARCKSL1–2, pcDNA/MARCKSL1–2 + pcDNA/HDAC1 or pcDNA/MARCKSL1–2 + miR-200b inhibitor. Then, rescue assays were conducted on these groups of cells. a-d. Colony formation and EdU assays detected the proliferation of indicated H1299 and H1299/DTX cells. e-h. Flow cytometry analyses and TUNEL assays examined the apoptosis of indicated H1299 and H1299/DTX cells. ^**P < 0.01. n.s.: no significance

Fig. 7

MARCKSL1–2 suppresses tumor growth and DTX resistance in vivo. a Tumors resected from mice in four different groups (pcDNA3.1 or pcDNA3.1/MARCKSL1–2-transfected H1299/DTX cells with or without DTX treatment). b-c Tumor volume and weight in four different groups. ^*P < 0.05, ^***P < 0.001