Chronic oxymatrine treatment induces resistance and epithelial‑mesenchymal transition through targeting the long non-coding RNA MALAT1 in colorectal cancer cells

Abstract

A major reason for colorectal cancer (CRC) chemoresistance is the enhanced migration and invasion of cancer cells, such as the cell acquisition of epithelial-mesenchymal transition (EMT). Long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been considered as a pro-oncogene in multiple cancers. However, the precise functional mechanism of lncRNA MALAT1 in chemoresistance and EMT is not well known. In the present study, we focused on the effect of oxymatrine on CRC cells and further investigated the role of MALAT1 in oxymatrine-induced resistance and EMT process. The human CRC cell line HT29 was exposed to increasing doses of oxymatrine to establish stable cell lines resistant to oxymatrine. The established HT29 oxymatrine resistant cells showed an EMT phenotype including specific morphologic changes, enhanced migratory and invasive capacity, and downregulation of E-cadherin protein expression. Subsequently, high-throughput HiSeq sequencing and RT-qPCR showed that lncRNA MALAT1 was significantly upregulated in the oxymatrine resistant cells (P<0.01), while knockdown of MALAT1 partially reversed the EMT phenotype in HT29 resistant cells. Furthermore, oxymatrine treatment suppressed the migration and invasion ability of CRC cells, however, this effect was significantly reversed by overexpression of MALAT1. Finally, we investigated the clinical role of MALAT1 and found that high lncRNA MALAT1 expression level is associated with poor prognosis in CRC patients receiving oxymatrine treatment (P<0.01). In conclusion, we demonstrate that lncRNA MALAT1 is a stimulator for oxymatrine resistance in CRC and it may provide therapeutic and prognostic information for CRC patients.

Figure 1.

Chronic treatment of oxymatrine induces resistance to oxymatrine in HT29 cells. (A) The established HT29 oxymatrine resistant cells showed elevated cell viability compared with the HT29 parental cells when incubated with culture medium containing 2 mg/ml concentration of oxymatrine. (B) The concentration-effect curve indicated that the IC₅₀ value of oxymatrine on HT29 oxymatrine resistant cells was significant higher than that on HT29 parental cells. (C) CCK-8 assay indicated that the cell proliferation rate of HT29 oxymatrine resistant cells significantly decreased when compared with HT29 parental cells. Error bars represent median ± SD (standard deviation).

Figure 2.

Acquisition of oxymatrine resistance induces EMT in CRC cells. (A) The HT29 oxymatrine-resistant cells induced specific morphologic changes consistent with EMT, such as spindle-shaped cells with loss of polarity, increased intercellular separation and pseudopodia (arrows). (B) A significant increased migratory and invasive capacity was observed in HT29 oxymatrine resistant cells compared with parental cells. (C) Western blot assay showed that the E-cadherin protein level was suppressed while vimentin protein level was upregulated in HT29 oxymatrine resistant cells. Error bars represent **P<0.01.

Figure 3.

The heat map shows expression of the 78 lncRNAs that showed >2-fold difference between oxymatrine resistant HT29 cells (6 samples) and parental cells (6 samples). The heat map was generated with an R package using normalization across rows (cells). P, HT29 parental cells; R, HT29 oxymatrine resistant cells.

Figure 4.

(A-F) Concentrations of the 6 identified lncRNAs in HT29 oxymatrine resistant cells and parental cells using RT-qPCR assay. The boxes/line/whiskers represent interquartile range, median and 10–90% of data, and each experiment was performed in triplicate.

Figure 5.

Knockdown of MALAT1 partially reverses EMT of HT29 oxymatrine resistant cells. (A) MALAT1 was silenced in CRC cell lines by transfection of small interfering RNA. (B) Western blotting showed that transfection of siMALAT1-1 promoted E-cadherin while suppressed vimentin expression level in HT29 cells. (C) Knockdown of MALAT1 partially reversed the obtained migratory and invasive ability in HT29 oxymatrine resistant cells. (D) CCK-8 assay indicated that MALAT1 had no effect on proliferation of HT29 cells. Error bars represent median ± SD; *P<0.05; **P<0.01.

Figure 6.

Oxymatrine inhibits the migration and invasion of CRC cells through targeting MALAT1. (A) HT29 and SW480 cells were incubated with medium containing 1 mg/ml of oxymatrine for 36 h. RT-qPCR indicated that oxymatrine treatment significantly suppressed the expression of MALAT1 in both types of CRC cells. (B) Overexpression of MALAT1 markedly promoted the migratory and invasive capacity of HT29 cells. (C) The metastatic capacity of HT29 cells was suppressed when treated with oxymatrine for 24 h at the concentration of 1 mg/ml, however, pMALAT1 partially rescued the inhibitory effect of oxymatrine on cell migration and invasion. Error bars represent median ± SD; *P<0.05; **P<0.01.

Figure 7.

High MALAT1 expression is associated with poor survival in CRC patients receiving oxymatrine treatment. (A) RT-qPCR showed that MALAT1 was significantly upregulated in CRC tissues when compared with adjacent non-cancerous tissues. (B) The MALAT1 expression level was analyzed using RT-qPCR and expressed as log₂-fold-change (CRC/normal), and the log₂-fold-changes were presented as follows: >1, overexpression (38 cases); <1, underexpression (6 cases); the remainder was defined as unchanged (14 cases). (C) The proportion of patients that experience recurrence was significantly higher in the high MALAT1 expressing group than in the low MALAT1 expressing group. (D) Kaplan-Meier survival analysis showed that patients with high MALAT1 expression was associated with poor overall survival.