A total of 69 patients with advanced HCC, who were admitted to The Sixth People's Hospital of Qingdao, Qingdao, China, between September 2015 and May 2018, were enrolled. During biopsy, adjacent (5 cm around tumors) healthy and tumor tissues were collected from each participant. Blood (5 ml) was also extracted from each patient using EDTA tubes before treatment under fasting conditions for the isolation of plasma. Inclusion criteria: i) Patients with expected survival time >12 months; ii) patients at advanced stage (stages III and IV), who were not suitable for surgical resection; and iii) patients who were treated with different doses of carboplatin according to their conditions. Exclusion criteria: i) Patients with other diseases; ii) patients who were treated before admission; iii) patients who failed to cooperate with researchers; and iv) patients who died during this study. Blood was also extracted under fasting conditions at 3 and 6 months after the beginning of carboplatin treatment to extract plasma. The patients included 38 males and 31 females, with a mean age of 48.2±5.1 years (age range, 30–66 years). There were 38 patients at stage III and 31 patients at The American Joint Committee on Cancer stage IV (12). All patients signed informed consent. The Ethics Committee of The Sixth People's Hospital of Qingdao approved this study.

HCC cell lines SNU-398 and SNU-182 were purchased from American Type Culture Collection (ATCC). A mixture composed of 90% RPMI 1640 medium (ATCC) and 10% FBS (ATCC) was used to cultivate cells at 37°C in a 5% CO₂ incubator.

In order to detect CASC11, total RNA was extracted from in vitro cultivated cells, plasma and tissue sample using RNAzol^® RT RNA Isolation reagent (GeneCopoeia, Inc.) and RT was performed using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.), through the following conditions: 25°C for 10 min, 52°C for 20 min and 80°C for 10 min. Mixtures for qPCR were prepared using the qScript One-Step RT-qPCR kit (Quantabio). In order to detect miR-21, the ReliaPrep™ miRNA Cell and Tissue Miniprep system (Promega Corporation) was used to extract miRNAs, the miScript II RT kit (Qiagen GmbH) was used to carry out RT, and qPCR mixtures were prepared using the mirVana qRT-PCR miRNA Detection kit (Thermo Fisher Scientific, Inc.). All PCR reactions were performed on the ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.). PCR conditions were: 95°C for 1 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 45 sec. Primers of lncRNA CASC11, miR-21, and endogenous controls GAPDH and U6 were obtained from Sangon Biotech Co., Ltd. CASC11 forward, 5′-GGACACCAACTATTGCTTCA-3′ and reverse, 5′-TCCAGGCTCCAAATGTAG-3′; GAPDH forward, 5′-GTCTCCTCTGACTTCAACAGC-3′ and reverse, 5′-ACCACCCTGTTGCTGTAGCCA-3′; miR-21 forward, 5′-TAGCTTATCAGACTGATG-3′ and reverse primer (universal primer) and U6 primers were from the RT-qPCR miRNA Detection kit (Thermo Fisher Scientific, Inc.). According to the 2^−ΔΔCq method (13) of quantification, lncRNA CASC11 expression was normalized to that of GAPDH and miR-21 expression was normalized to that of U6.

lncRNA CASC11 overexpression vectors and empty vectors, as well as lncRNA CASC11 small interfering (si)RNA (5′-UUCUUCACCACCUCCAGUUGC-3′) and negative control siRNA (5′-UGAACGUACGGGCAUGUCAGC-3′) were purchased from Sangon Biotech Co., Ltd. Negative miRNA control (5′-UGAACGUACGGGCAUGUCAGC-3′) and miR-21 mimic (5′-UAGCUUAUCAGACUGAUGUUGA-3′) were purchased from Sigma-Aldrich; Merck KGaA. Lipofectamine^® 2000 reagent (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect 1×10⁶ SNU-398 and SNU-182 cells in each well of a 6-well cell culture plate (2 ml medium per well) with vectors at a dose of 10 nM and siRNA or miRNA at a dose of 35 nM. The duration of incubation with transfection reagent and vector was 6 h. Cells without transfections were considered as the control cells. The expression of CASC11 and miR-21 was detected by RT-qPCR. Subsequent experiments were performed 48 h later.

Cells were harvested for an MTT assay when the overexpression rates of lncRNA CASC11 and miR-21 reached 200% and the lncRNA CASC11 knockdown rate reached 50%. Briefly, cell suspensions were prepared and cell density was adjusted to 3×10⁴ cells/ml. Cells suspensions were transferred to a 96-well plate (0.1 ml/well). Carboplatin [C₄H₆(CO₂)₂Pt(NH₃)₂; Sigma-Aldrich; Merck KGaA] was added at doses of 200 µg/ml at 4 h after seeding. Cells were cultured under normal conditions (37°C, 5% CO₂) for 24 h, followed by the addition of MTT (10 µl/well). Cells were cultured for an additional 4 h, followed by the addition of DMSO 10 µl/well) and the measurement of optical density values at 570 nm.

The values of the mean ± SD were calculated from data of 3 biological replicates. Pearson's correlation analysis was performed to analyze the correlation between CASC11 and miR-21 levels. Comparisons of lncRNA CASC11 and miR-21 expression levels between two different types of tissues were performed with paired Student's t-test. Comparisons among multiple groups were carried out using one-way ANOVA and Tukey's post hoc test. Any difference with a P<0.05 was considered to be statistically significant.

Expression of lncRNA CASC11 and miR-21 was detected by RT-qPCR. Compared with those in adjacent healthy tissues, the expression levels of CASC11 (Fig. 1A) and miR-21 (Fig. 1B) were significantly higher in tumor tissues (both P<0.05).

Pearson's correlation analysis was performed to assess the correlation between the expression levels of lncRNA CASC11 and miR-21 in tumor tissues and adjacent healthy tissues. As shown in Fig. 2A, the expression levels of lncRNA CASC11 and miR-21 were significantly and positively correlated in tumor tissues (P<0.001). In contrast, the expression levels of these molecules were not correlated in adjacent healthy tissues (P=0.76; Fig. 2B).

In vitro overexpression experiments were performed to further investigate the association between lncRNA CASC11 and miR-21 in HCC cell lines. Compared with the control and negative control groups, the overexpression of lncRNA CASC11 led to upregulation of miR-21 in SNU-398 and SNU-182 cells (Fig. 3A; all P<0.05). In contrast, the overexpression of miR-21 had no significant effect on the expression of lncRNA CASC11 in these cells (Fig. 3B; all P>0.05).

The plasma levels of lncRNA CASC11 and miR-21 were detected by RT-qPCR at three time-points, including before treatment and at 3 and 6 months after treatment with carboplatin. The plasma levels of lncRNA CASC11 (Fig. 4A) and miR-21 (Fig. 4B) were significantly increased at 3 and 6 months after treatment (all P<0.05), compared with the levels before treatment. The plasma levels of lncRNA CASC11 (Fig. 4A) and miR-21 (Fig. 4B) were also significantly higher at 6 months after treatment (both P<0.05), compared with the levels at 3 months.

SNU-398 and SNU-182 cells were treated with 50, 100 and 200 µg/ml carboplatin for 24 h, followed by the detection of lncRNA CASC11 expression by RT-qPCR. As shown in Fig. 5, carboplatin treatment resulted in a dose-dependent upregulation of CASC11 in these cells.

The cell viability assay was performed under carboplatin treatment only (200 µg/ml). It is a limitation of this study that the cell viability assay was performed only under conditions of carboplatin treatment and no-treatment controls were not included. Overexpression of lncRNA CASC11 and miR-21 promoted, whereas lncRNA CASC11 knockdown inhibited, the viability of carboplatin-treated HCC cells, compared with the control groups (all P<0.05). In addition, miR-21 overexpression attenuated the effects of lncRNA CASC11 knockdown on cell viability (P<0.05; Fig. 6).