Atractylenolide II reverses the influence of lncRNA XIST/miR‐30a‐5p/ROR1 axis on chemo‐resistance of colorectal cancer cells

Abstract This investigation was conducted to elucidate whether atractylenolide II could reverse the role of lncRNA XIST/miR‐30a‐5p/ROR1 axis in modulating chemosensitivity of colorectal cancer cells. We totally collected 294 pairs of colorectal cancer tissues and adjacent normal tissues and also purchased colorectal cancer cell lines and human embryonic kidney cell line. 5‐fluorouracil, cisplatin, mitomycin and adriamycin were designated as the chemotherapies for colorectal cell lines, and atractylenolides were arranged as the Chinese drug. The expressions of XIST, miR‐30a‐5p and ROR1 were quantified with aid of qRT‐PCR or Western blot, and luciferase reporter gene assay was implemented to determine the relationships among XIST, miR‐30a‐5p and ROR1. Our results demonstrated that XIST and ROR1 expressions were dramatically up‐regulated, yet miR‐30a‐5p expression was down‐regulated within colorectal cancer tissues (P < 0.05). The overexpressed XIST and ROR1, as well as under‐expressed miR‐30a‐5p, were inclined to promote viability and proliferation of colorectal cells under the influence of chemo drugs (P < 0.05). In addition, XIST could directly target miR‐30a‐5p, and ROR1 acted as the targeted molecule of miR‐30a‐5p. Interestingly, atractylenolides not only switched the expressions of XIST, miR‐30a‐5p and ROR1 within colorectal cancer cells but also significantly intensified the chemosensitivity of colorectal cancer cells (P < 0.05). Finally, atractylenolide II was discovered to slow down the viability and proliferation of colorectal cancer cells (P < 0.05). In conclusion, the XIST/miR‐30a‐5p/ROR1 axis could be deemed as pivotal markers underlying colorectal cancer, and administration of atractylenolide II might improve the chemotherapeutic efficacy for colorectal cancer.


| INTRODUC TI ON
Colorectal cancer (CRC), a malignancy stemming from epithelium or gland of colorectal mucosa, stood as the third most common cancer around the world. 1 The worldwide prevalence of new CRC cases has risen swiftly to 1.2 million per year, with approximately 0.5 million deaths per year. 2 The hazard factors for CRC were acknowledged as family history, inflammatory bowel disease, smoking, excessive consumption of alcohol, intake of substantial red meat, obesity and diabetes. For CRC patients of stage I-II, surgery was prioritized without risky parameters, 3 while the patients of advanced CRC were still faced with neoplastic recurrence and metastasis that was accompanied by dramatically reduced 5-year survival. 4 Furthermore, the appearance of drug resistance confined the efficacy of chemotherapies and even led to chemotherapeutic failure. Thus, how to seek for novel biomarkers for early-stage CRC and enhance chemosensitivity of CRC has been increasingly pivotal.
Accumulating investigations have documented that expressions of lncRNAs metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), HOX antisense intergenic RNA (HOTAIR), colon cancer-associated transcript 1 (CCAT1), colorectal neoplasia differentially expressed (CRNDE) and X-inactive specific transcript (XIST) were elevated with aggravation of CRC, whereas lncRNAs maternally expressed gene 3 (MEG3) and RP11-462C24.1 were lowly expressed when CRC became invasive and metastatic. [5][6][7][8][9][10][11] Among them, the XIST was a principal gene for modulating inactivation of X chromosome within mammals, and it could merely be transcribed from inactivated X chromosome. 12 This lncRNA mattered much for neoplastic pathologies, because of its responsibility for altering genetic expressions by affecting the stability of heterochromatins. 13 For instance, XIST was indispensable for maintaining the long-term survival of hematopoietic stem cells, 14 and knock-down of XIST restrained growth and metastasis of glioma cells. 15 Besides, the up-regulated expression of XIST tended to differentiate NSCLC tissues from normal tissues, 16 and the receiver operating characteristic curve suggested a highly diagnostic value of XIST for NSCLC sufferers. 17 Despite the carcinogenic predisposition of XIST, the specific molecular network of XIST that contributed to CRC development remained far from complete.
With starbase software (version 2.0) adopted to herald the target miRNAs of XIST, 18 the predicted miR-30a has been reported to obviously weaken the progression of multiple neoplasms, including NSCLC, hepatic carcinoma, pancreatic cancer and melanoma. [19][20][21] Additionally, miR-30a was also validated to symbolize the poor prognosis of CRC patients, when it was down-regulated. 22 Further in vivo and in vitro experiments also argued that miR-30a could prohibit migration and invasion of cancer cells by regulation of downstream signalling pathways or target genes. [23][24][25] Taking receptor-tyrosinekinase-like orphan receptor 1 (ROR1) for instance, its high expression appeared positively correlated with malignant traits and poor prognosis of CRC patients, 26 and silencing of it could remarkably delay the invasive and migratory pace of chemoresistant cancer cells. 27 To sum up, miR-30a-modifying ROR1 might be involved in the aetiology of diverse neoplasms, yet whether this combined action could act on progression and chemosensitivity of CRC required further researches.
As already reported, combined treatments of traditional Chinese medicine (TCM) and chemotherapy for post-operative cancer patients not merely relieved the toxicity and resistance generated because of chemotherapy but also raised the patients' own immunity. [28][29][30][31] Within this study, white atractylodes rhizome, originated from a perennial herb that pertained to atractylodes macrocephala koidz, was adopted as the candidate TCM treatment regimen, with ingredients featured by anti-inflammatory and anti-neoplastic functions. 32 For instance, atractylenolide I (AO-I) 33 could fight against tumours by inducing apoptosis of leukaemia cells and it acted against inflammation partly by bringing down levels of IL-1, TNFα and proteolysis-inducing factor. 34,35 Nevertheless, few investigations were carried out to explore whether AO would attenuate chemoresistance in cancers, especially CRC, through the modification of certain signalling pathways. In response, this investigation was intended to validate the role of XIST/miR-30a/ROR1 axis and AO in regulating the chemosensitivity of CRC cells.

| Follow-up survey
The CRC patients were surveyed in the form of outpatient review and phone call during the follow-up period (ie 5 years). Immediately after surgery, the subjects were strictly monitored every 3-6 months. The inspection items consisted of: (a) physical examination, especially anal examination; (b) examination of blood carcinoembryonic antigen and indicators that have ever increased; (c) X-ray inspection for lung, B-mode ultrasound for abdomen and pelvic cavity, as well as magnetic resonance imaging for brain; and (d) enhanced computed tomography (CT) examination for chest pelvic, enteroscopy and whole body bone scan per year. Local recurrence and distant metastasis were determined when any of liver, lung, bone and brain showed metastatic symptoms.

| Cell culture
The purchased CRC cell lines (ie SW480, HCT116, Lovo and SW620; Shanghai Institutes for Biological Sciences, China) and human embryonic kidney cells (HEK293T; American Type Culture Collection, Manassas, VA, USA) were cultured within RPMI medium (Gibco, Grand Island, NY, USA) that contained 100 mL/L foetal bovine serum in 5% CO 2 and saturated humidity at 37°C.
The nutrient solution was changed every 2-3 days, and cells at the logarithmic phase were digested by pancreatin and passaged before performing the following experiments.

| Cell transfection
Cells at the density of 5 × 10 4 /well were inoculated into 24-well plates, with the culture medium free from antibiotics. When cells grew to

| Colony formation assay
Cells at the logarithmic phase were seeded into 6-well plates at a concentration of 400-500 per well, and they were cultivated in 5% CO 2 at 37°C for 2-3 weeks. The culture would be terminated when clonal

| Cell apoptosis assay
The cells in each treatment group were digested with 0.25% pancreatin and were then washed with pre-cooled PBS at 4°C. Exactly  Furthermore, Taqman ® MicroRNA Reverse Transcription Kit and Taqman ® Universal Master Mix II kit were employed to determine the expressions of miR-30a-5p. The GAPDH was taken as the internal reference for XIST and ROR1, and U6 was set as the internal reference for miR-30a-5p. The relative expressions of genes were normalized through comparative CT value method (ie 2 −△△Ct method), and the primers for XIST, miR-30a-5p and ROR 1 were enlisted in Table 1.

| Western blotting
The tissues and cells were added with 100 μL lysis buffer RIPA

| Association of XIST and miR-30a-5p expressions with clinicopathological features of CRC patients
As illustrated in Figure 1A, XIST expression within CRC tissues was around 2.98 folds of that within paracarcinoma normal tissues (P < 0.05), while expression of miR-30a-5p within CRC tissues achieved only 37% of that within the normal tissues (P < 0.05).
Moreover, the included CRC patients were grouped into ones with highly expressed XIST (>2.56) and ones with lowly expressed XIST (≤2.56) based on the median level of XIST expression. The same population was also categorized into highly expressed miR-30a-5p group (>1.68) and ones with lowly expressed miR-30a-5p group (≤1.68) in line with the median level of miR-30a-5p expression. It was exhibited that overexpressed XIST and under-expressed miR-30a-5p were

| Comparison of chemo-resistance among CRC cell lines
With HEK293T cell line as the control, markedly raised XIST expression and lowered miR-30a-5p expression were determined within SW480, Lovo, HCT116 and SW620 cell lines (P < 0.05) ( Figure 1C). F I G U R E 1 The association of XIST and miR-30a-5p expressions with onset and prognosis of colorectal cancer patients, and colorectal cancer cells were selected regarding their resistance to chemotherapies. A, XIST and miR-30a-5p expressions were compared between colorectal cancer tissues and paracarcinoma tissues. *P < 0.05 when compared with paracarcinoma tissues. B, Expressions of XIST and miR-30a-5p were compared among HEK293T, SW480, HCT116, Lovo and SW620 cell lines. *P < 0.05 when compared with HEK293T. C, Lowly expressed XIST and highly expressed miR-30a-5p were correlated with more favourable overall survival of colorectal cancer patients than highly expressed XIST and lowly expressed miR-30a-5p respectively. D, The sensitivities of SW480, HCT116, Lovo and SW620 cell lines were compared in response to 5-fluorouracil, mitomycin, cisplatin and adriamycin

| Regulatory contribution of XIST and miR-30a-5p to chemosensitivity of CRC cells
Among the 3 si-XISTs adopted, it was indicated that si-XIST-3 presented a far stronger capacity to inhibit XIST expression than si-XIST-1 and si-XIST-2 (P < 0.05), so si-XIST-3 was prepared for the following experiments ( Figure 2A). After transfection of pcDNA-XIST or si-XIST3, the expression of XIST was, respectively, brought up and down with statistical significance (P < 0.05) ( Figure 2A).
Nonetheless, transfection of si-XIST2 or miR-30a-5p mimic hindered the survival rate of Lovo and SW480 cell line, when compared with NC group (P < 0.05).

| ROR1 mediated the role of miR-30a-5p in regulating chemosensitivity of CRC cells
It was indicated in Figure 5A,B that ROR1 expression within human CRC tissues and cell lines (ie SW480, HCT116, Lovo and SW620) surpassed that within paracarcinoma tissues and HEK293T cell line (P < 0.05). What was more, ROR1 expression was negatively correlated with miR-30a-5p expression among the CRC tissues included (r s = −0.270, P < 0.001) ( Figure 5C).
In addition, miR-NC+pCMV1-ROR1 group appeared to boost the resistance of Lovo cell line to drugs when compared with miR-30a-5p mimic group (P < 0.05), though its contribution to chemosensitivity was below that of miR-NC group (P < 0.05) ( Figure 5F).
Meanwhile, we observed that the viability and proliferation of CRC cells were encouraged the apoptotic condition of the cells was suppressed after co-transfection of miR-NC and pCMV1-ROR1, with miR-30a-5p mimic group as the reference (P < 0.05) ( Figure 6).
Correspondingly, miR-NC+pcDNA-ROR1 group potently prohibited apoptosis of CRC cells and improved their viability and proliferation more significantly than miR-30a-5p mimic group and miR-NC group (P < 0.05).

| AO-II promoted chemosensitivity of CRC cells by modifying XIST/miR-30a-3p/ROR 1 axis
After treating CRC cells with AO-I, AO-II and AO-III ( Figure 7A As shown in Figure 8A By contrast, AO-II generated higher apoptotic percentages than NC group (P < 0.05) ( Figure 8E).

| D ISCUSS I ON
The main approaches for treating CRC covered surgical treatment, chemotherapy, radiotherapy and TCM treatment, among which chemotherapy, as a valid systemic therapy, was especially beneficial to patients who did not meet the surgical indications.
Although novel anticancer drugs and combined chemotherapies have sprung up, the chemotherapeutic efficacy for CRC was reinforced indistinctively, owing to the gradual production of drug resistance and accompanying toxicity. The primary drug resistance and multidrug resistance appeared as the tough obstacle to tumour chemotherapy, therefore, reversing the drug resistance of F I G U R E 5 The mediation of ROR1 for the contributions of XIST and miR-30a-5p to chemosensitivity of colorectal cancer cells. A, The expression of ROR1 was compared between colorectal cancer tissues and paracarcinoma tissues. *P < 0.05 when compared with paracarcinoma tissues. B, The expression of ROR1 was determined within HEK293T, SW480, HCT116, Lovo and SW620 cell lines. *P < 0.05 when compared with HEK293T. C, Among the incorporated colorectal cancer tissues, ROR1 expression was positively correlated with XIST expression, yet it displayed negative relevance to miR-30a-5p. D, The expression of ROR1 was detected after transfections of pcDNA-XIST, s-XIST, miR-30a-5p mimic and miR-30a-5p inhibitor, and the expressions of XIST and miR-30a-5p were also determined after transfections of pCMV1-ROR1 and si-ROR1. *P < 0.05 when compared with NC. E, ROR1 was subjected to target of miR-30a-5p in certain sites, and the luciferase activity of cells was compared among miR-30a-5p mimic+pmirGLO-ROR1-Wt, miR-30a-5p mimic+pmirGLO-ROR1-Mut and miR-30a-5p mimic+pmirGLO groups. *P < 0.05 when compared with pmirGLO+miR-30a-5p mimic group. F, The sensitivity of colorectal cancer cells was compared when responding to 5-fluorouracil, mitomycin, cisplatin and adriamycin among the miRNA-NC, miR-30a-5p mimic and miR-NC+pCMV1-ROR1 groups. *P < 0.05 when compared with NC Incremental evidence indicated that the XIST investigated here was tightly linked with occurrence and progression of tumours, and it counted in maintaining the normal state of genome. 16,36 For example, highly expressed XIST was present concurrently with larger tumour volume, metastatic lymph node, distant metastasis and advanced TNM staging of patients with gastric cancer. 37,38 Furthermore, knockout of XIST exhibited a role in inhibiting progression of glioblastoma by reducing cell proliferation, migration and invasion, as well as inducing cell apoptosis. 15 Analogously, XIST expression was up-regulated within CRC tissues, as was illustrated in Figure 1. In F I G U R E 8 The reversal function of atractylenolide II for chemosensitivity of colorectal cancer cells via modifying XIST/miR-30a-3p/ROR 1 axis. A, The expressions of XIST, miR-30a-3p and ROR 1 within colorectal cancer cells were determined after additions of atractylenolide II. *P < 0.05 when compared with NC. B, The viability of colorectal cancer cells was evaluated after supplementation of atractylenolide II. *P < 0.05 when compared with NC. C, The proliferative capability of colorectal cancer cells was appraised after being treated with atractylenolide II. *P < 0.05 when compared with NC. D, The expressions of ki-67 and PCNA were determined within colorectal cancer cells under the influence of atractylenolide II. *P < 0.05 when compared with NC. E, The apoptosis of colorectal cancer cells was evaluated under the action of atractylenolide II. *P < 0.05 when compared with NC addition, it was reported that XIST expression was negatively correlated with the median lethal concentration of paclitaxel in treating ovarian cancer cells, suggesting that cell lines with down-regulated XIST expression exhibited descending sensitivity to treatment of paclitaxel. 39 The XIST also displayed up-regulated expression within CRC cells that presented resistance to 5-fluorouracil. 40 Within our investigation, the function of XIST in CRC was expanded that XIST could favour resistance of CRC cells to 5-fluorouracil, mitomycin, adriamycin and cisplatin (Figure 2), and this phenomenon was conjectured to the result of XIST' facilitating proliferation and viability of CRC cells (Figure 3).
Besides, a targeted relationship between XIST and miR-30a was built here, and XIST was identified to down-regulate miR-30a expression in CRC through targeting it ( Figure 4). Virtually, apart from CRC, 41 the miR-30a, located in the intron region of human chromosome 6q13, has also been suggested as a protector against progression of liver cancer, 42,43 renal clear cell carcinoma, 44 gastric cancer, 45 prostate cancer 46 and breast cancer. 47 The tumour-suppressing potential of miR-30a was speculated as the result of its obstructing epithelial-mesenchymal transition and metastasis of lung cancer, hepatoma carcinoma and breast cancer cells. 21,42,48 Consistent with the malignancies, our results also indicated miR-30a as a marker promoting the prognosis of CRC patients (Figure 1), and the intrinsic pathology was specifically embodied in its restraining viability and proliferation of CRC cells (Figure 3). Based on the experimental evidence and reasoning, it was convincing that XIST collaborating with miR-30a could modify the chemo-resistance of CRC cells (Tables 2 and 3).
In addition, this investigation ascertained that miR-30a-5p could down-regulate ROR1 expression to suppress the invasive and migratory potency of CRC cells ( Figure 5). And ROR1 was also testified as the downstream mediator of miR-30a-5p and XIST in modulating chemosensitivity of CRC cells ( Figure 6). The ROR1 studied herein was a membrane-spanning protein belonging to the receptor TA B L E 2 Correlation between XIST and miR-30a-5p expressions and baseline characteristics of colorectal cancer patients The bold value indicate a significant results with a P < 0.05.
tyrosine kinase (RTK) family, and it was aberrantly overexpressed within multiple haematological and solid malignancies, such as lymphatic leukaemia, melanoma and ovarian cancer. [49][50][51][52][53] In terms of molecular mechanisms, furthermore, the ROR1 was inclined to activate signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol 3-kinase (PI3K) and cellular-mesenchymal to epithelial transition factor (c-Met), which appeared essential to modification of tumour growth and metastasis. [54][55][56] In fact, the involvement of ROR1 in neoplasms was also subjected to the control of other miRNAs, such as miR-382 in ovarian cancer and miR-30a in breast cancer. 25,57 It was insinuated that a molecular network among miR-NAs and genes was existent underlying the aetiology of CRC, and the contribution of ROR1 to CRC progression could also be modulated by several other upstream miRNAs, which demanded in-depth researches.
Concerning the TCM drug, we observed that the AOs could attenuate the chemosensitivity of CRC cells through weakening viability and proliferation of CRC cells (Figure 7). The results followed the same principle with previous documentations, which believed that AO-I was adept at inhibiting growth of human white blood cell strain and mouse leukaemia cell lines. 34 Moreover, AO-II could serve to control the proliferation of melanoma cells, 58 and AO-III triggered depressed tumour growth and incremental release of lactic dehydrogenase. 59 The above proof all pointed to a direction that AO tended to postpone neoplastic deterioration, which suggested a linkage of AO with elevated chemosensitivity of cancer cells. We found that AO-II was equipped with the strongest ability to inhibit chemoresistance of CRC cells among the 3 AOs, and addition of them was associated with down-regulated XIST and ROR1 expressions, along with up-regulated miR-30a-5p expression (Figure 8). It was thus insinuated that the AO-II might work for incremental chemoresistance of CRC cells by fighting against the contribution of XIST/ miR-30a-5p/ROR1 axis to CRC development. As for the additional particularized mechanism related with AO-II and CRC, extra investigations were demanded.
In conclusion, this investigation demonstrated the vital role of LncRNA XIST/MiR-30a-5p/ROR1 axis in altering chemosensitivity of CRC cells, and AO-II also played a part contrary to the signalling axis. The conclusion of this investigation might help to ameliorate the chemotherapeutic efficacy for CRC.

CO N FLI C T O F I NTE R E S T
None. The bold value indicate a significant results with a P < 0.05.