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Anti-Cancer Agents in Medicinal Chemistry

Year 2020
ISSN: 1875-5992 (Online)
ISSN: 1871-5206 (Print)

linc01559 Served as a Potential Oncogene and Promoted Resistance of

Hepatocellular Carcinoma to Oxaliplatin by Directly Sponging miR-6783-
3p
Shunbin Dong1, Ying Fu2, Kaibo Yang1, Xing Zhang1, Runchen Miao1, Yunxiang Long1,3 and
Chang Liu1,*

1
Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061,
Shaanxi, China; 2Department of Oncology, Liaocheng People's Hospital, Liaocheng, 252000, Shandong Province,
China; 3Xi'an Jiaotong University Health Science Center, Xi’an, 710061, Shaanxi Province, China

Abstract: Background: Oxaliplatin (L-OHP)-based chemotherapy, such as FOLFOX4 (5-fluorouracil, leucovorin, and L-
OHP), improves the prognosis of patients with late-stage Hepatocellular Carcinoma (HCC). However, the development of
resistance to L-OHP leads to failure of chemotherapy. The aim of this study was to investigate the role of linc01559 and
miR-6783-3p in regulating resistance to L-OHP.
Methods: Quantitative reverse transcription-polymerase chain reaction was used to determine the expression profile. The
Cell Counting Kit-8 test and wound healing assay were also used. Dual-luciferase reporter gene assay, RNA pull-down
assay, and RNA immunoprecipitation were used to evaluate the interaction between linc01559 and miR-6783-3p.
Result: linc01559 expression was associated with response to FOLFOX4, as well as miR-1343-3p and miR-6783-3p
expression in vivo. A nomogram, including linc01559 and miR-1343-3p, precisely and accurately predicted the overall
survival of patients with HCC. Regarding the in vitro tests, linc01559 showed higher expression in L-OHP-resistant cell
lines, whereas miR-6783-3p was downregulated. Knockdown of linc01559 led to decreased proliferation and migration
ability, and increased expression of miR-6783-3p; however, it did not influence the expression of miR-1343-3p. We also
found that linc01559 directly interacted with miR-6783-3p. Furthermore, linc01559 and miR-6783-3p regulated the
viability of L-OHP-resistant cells following treatment with L-OHP.
Conclusion: linc01559 promoted the proliferation of HCC by sponging miR-6783-3p. This suggests that linc01559/miR-
6783-3p may be key factors in regulating resistance and response to L-OHP. Moreover, they may be potential therapeutic
targets for improving sensitivity to L-OHP in patients with HCC.
Keywords: linc01559, miR-6783-3p, miR-1343-3p, oxaliplatin resistance, nomogram, hepatocellular carcinoma

1. INTRODUCTION
In 2018, Hepatocellular Carcinoma (HCC) accounted for 841,080 new cases and 781,631 deaths in 185 countries [1]. It is a
deadly cancer associated with a poor prognosis in most cases, with an 18.4% 5-year survival rate based on the SEER database
_______________________________________________
*Address correspondence to this author at the Department of Hepatobiliary Surgery, The First Affiliated Hospital of Medical College, Xi'an Jiaotong
University, Xi’an 710061, China; Tel.: +86-29-85323900; Fax: +86-29-85324695; Email: [email protected]
(Surveillance, Epidemiology and End Results database) [2]. Moreover, the incidence of HCC is increasing in developing
countries and threatens the health of the population worldwide [3]. Early diagnosis is one of the efficient approaches to dealing
with HCC [4]. In this situation, researchers are encouraged to identify novel biomarkers that may predict the prognosis of HCC
or provide therapeutic targets for the development of novel drugs [5].
In the past decade, the important roles of long non-coding RNA (lncRNAs), which contain >200 base pairs and do not
encode proteins, in regulating biological processes (e.g., epithelial-mesenchymal transition, autophagy, drug resistance, etc.)
have been realized [6-8]. Meanwhile, numerous lncRNAs were found to be significantly associated with the prognosis of
cancers [9]. Among them, linc01559 was reported to be aberrantly highly expressed in pancreatic cancer tissues compared with
healthy pancreatic tissues, significantly contributing to the poor prognosis [10]. We observed that linc01559 was also associated
with prognosis in HCC; however, it is not differentially expressed between HCC and healthy liver tissues. Further experiments
on this puzzling phenomenon showed that linc01559 was highly expressed in patients with oxaliplatin (L-OHP)-resistant HCC.
Therefore, we hypothesized that linc01559 may be involved in the process of resistance to L-OHP in patients with HCC.
With the help of online bioinformatics tools (i.e., Starbase V2.0), we found that miR-1343-3p and miR-6783-3p may be
targets of linc01559 [11, 12]. Regarding miR-1343-3p, it was reported to be negatively associated with vascular invasion in
lung cancer. Furthermore, Zhou et al. stated that miR-1343-3p was a potential suppressor by directly targeting TEAD4 [13]. In
addition, miR-6783-3p was found to also suppress the progression of lung cancer [14]. Currently, the roles of miR-1343-3p and
miR-6783-3p in HCC have not been reported.

2. METHODS
2.1. Patients
We recruited 362 patients with HCC at the Liaocheng People’s Hospital and First affiliated hospital of Xi`an Jiaotong
University from 2010 to 2015. All patients were diagnosed through enhanced computed tomography, magnetic resonance
imaging, and liver puncture. The recruitment of patients was based on the Performance Status (PS) test, Barcelona Clinic Liver
Cancer (BCLC) criteria, and Child–Pugh liver function criteria. All patients had BCLC stage B (PS0 and Child–Pugh A–B) or
stage C (PS1–2 and Child–Pugh A–B) disease. All patients were treated with L-OHP (85mg/m2, intravenous drip for 2h, day 1),
calcium folinate (200mg/m2, intravenous drip, d1–2); 5-fluorouracil (400mg/m, intravenous drip, day 1–2) and 5-fluorouracil
(600mg/m, continuous intravenous drip for 22h, d1–2) every 2 weeks (four treatment cycles in total). Treatment efficacy was
evaluated after two consecutive treatment cycles using computed tomography and magnetic resonance imaging. Progressive
disease and stable disease denoted efficient, while partial response and complete response denoted drug resistance. Our study
obtained tissues from patients with HCC, adjacent healthy tissues, and corresponding clinical manifestations. All recruited
patients understood the basic concept of our study, and individually provided written informed consent. This study was
approved by the Ethics Committee of Liaocheng People’s Hospital and First affiliated hospital of Xi`an Jiaotong University.
Patients with HCC were subsequently allocated into the linc01559 high and low expression groups.
2.2. Cell Culture
HCC cell lines MHCC97L and Huh7 were purchased from the Type Culture Collection of the Chinese Academy of
Sciences (Shanghai, China). The cells were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640) medium
supplemented with 10% fetal bovine serum under the condition of 5% carbon dioxide at 37°C. The normal liver line Lo2 was
purchased from the Type Culture Collection of the Chinese Academy of Sciences and cultured in DMEM medium (Dulbecco’s
Modified Eagle’s Medium) supplemented with 10% fetal bovine serum. The culture conditions were identical to those used for
the HCC cell lines.
2.3. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
Total RNA from HCC tissues and HCC cell lines was extracted using the TRIzol reagent (Invitrogen, USA). The
PrimeScript™ RT reagent Kit (Takara, Japan) was used to transcribe the target RNA into complementary DNA (cDNA) for
further experiments. We used SYBR Premix Ex Taq II (Takara) to perform PCR reactions for the evaluation of linc01559
expression. The expression of linc01559 was subsequently normalized to that of GAPDH (glyceraldehyde‐ 3‐phosphate
dehydrogenase).
For the evaluation of microRNA expression, we used the Taqman MirNA (Carlsbad, USA) reverse transcription kit and
PrimeScript™ RT Master Mix (TaKaRa) to transcribe the target RNA into cDNA. Subsequently, we used the Taqman MirNA
assay kit and SYBR® Select Master Mix (Applied Biosystems, Carlsbad, CA, USA) to perform the PCR reactions. U6 was used
as the control. Finally, we used the 2−ΔΔCt method to calculate the fold change in expression. The paired t-test was used to
evaluate the difference.
2.4. Cell Transfection
Control lentivirus, lentivirus for the downregulation of linc01559, and lentivirus for the upregulation and downregulation of
miR-1343-3p and miR-6783-3p were acquired from Genchem (Shanghai, China). Subsequently, we used qRT-PCR to evaluate
the transfection efficiency.
2.5. Cell Proliferation Assays
We used the Cell counting Kit-8 (CCK-8; Apexbio, USA) to measure the proliferation ability of transfected cell lines
compared with that of MHCC97L and Huh7 cell lines. Target cells were resuspended at a concentration of 2×103, followed by
seeding and incubation for 24, 48, 72, 96, and 120h. Next, CCK-8 solution (10µl) was added and the cells were incubated for
2h. The absorbance was measured at the wavelength of 490nm. The paired t-test was used to evaluate the difference.
2.6. Dual-Luciferase Reporter Assay
We constructed a linc01559-wild-type (linc01559-Wt) plasmid, linc01559-Mut (mutated binding site to miR-1343-3p and
miR-6783-3p respectively), and miR-1343-3p and miR-6783-3p inhibition plasmid. MHCC97L and Huh7 were suspended at a
concentration of 1.0×106 cells and seeded into a six-well plate. Lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen) was
used for the transfection. The experiment groups were as follows: (1) control plasmid+miR-6783-3p, linc01559-Wt+miR-6783-
3p, and linc01559-Mut+miR-6783-3p; (2) control plasmid+miR-1343-3p, linc01559-Wt+ miR-1343-3p, and linc01559-Mut+
miR-1343-3p. Subsequently, relative luciferase activity was measured for each group and normalized to the Renilla luciferase
activity.
2.7. RNA Pull-Down Assay
miR-6783-3p-Wt (Wild type of miR-6783-3p) and miR-6783-3p-mut (Mutated miR-6783-3p) were used for the RNA pull-
down assay. The Pierce™ RNA 3' End Desthiobiotinylation Kit (Thermo Fisher Scientific, USA) was used to label the 3’
single strand RNA with biotin. After transfection, cells were cultured for 48 h, and cell lysates were collected for further
experiments. Streptavidin-Dyna beads (Dyna beads M-280 streptavidin) (Thermo Fisher Scientific, Invitrogen) and Yeast
tRNA (stock 10mg/ml) (Thermo Fisher Scientific, Ambion) were incubated with the cell lysates to be used as the input. After
pull-down using a magnetic stand, the TRIzol reagent (Thermo Fisher Scientific, Ambion) was used for RNA isolation. Biotin-
labeled RNA and input RNA were used for further qRT-PCR to detect the expression of linc01559.
2.8. RNA Immunoprecipitation (RIP)
The relationship between miR-6783-3p and linc01559 was determined using the Immunoprecipitation (RIP) kit (Millipore
Inc., Bedford, MA, USA). Following cell lysis and centrifugation, the supernatant was collected and a part of the cell extract
was removed to be used as input. The remaining cell extracts were coprecipitated with rabbit anti‐AGO2 antibody (ab186733,
1:50; Abcam) or rabbit anti‐human IgG antibody (ab109489, 1:100; Abcam) to be used as the negative control. In brief, each
coprecipitation reaction system was pre‐incubated with 50μl of magnetic beads, which were subsequently suspended. In
accordance with the experimental grouping, antibody (5μg) was incubated with magnetic beads for binding. Next, the complex
of magnetic beads‐antibody was resuspended, followed by incubation at 4°C overnight with 100μl of cell extract. Afterwards,
the magnetic‐bead‐protein complex was collected to extract and quantify the RNA.
2.9. Isolation of Cytoplasmic and Nuclear RNA
After RNA extraction using the RNeasy Mini Kit (Qiagen, Leusden, Netherlands), its purity and concentration were
determined with the NanoDrop ND‐1000 ultraviolet spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA).
Next, the extracted RNA was reversely transcribed into cDNA utilizing the PrimeScript™ RT reagent Kit (Takara
Biotechnology Ltd., Dalian, Liaoning, China) and miRNA using the One Step PrimeScript™ miRNA cDNA Synthesis Kit
(Takara Biotechnology Ltd.). The qRT‐PCR was subsequently performed using the SYBR Premix Ex Taq II kit (TaKaRa,
Shiga, Japan) on an ABI 7500 quantitative PCR instrument (Applied Biosystems). The expression of RNA and miRNA was
quantified using the 2−ΔΔCt method. U6 and glyceraldehyde‐3‐phosphate dehydrogenase were regarded as the internal standards
of miR-6783-3p and the remaining genes, respectively.
2.10. Statistical Analysis
The R 3.3.1 software was used to perform the statistical analysis. One-way analysis of variance was employed to evaluate
the results of the qRT-PCR analysis. The “ROCit” package of R was used to plot the receiver operating characteristic curve.
Analysis of variance for repeated measurement design data was used to evaluate the results of the CCK-8 assay. The
relationship between the expression of linc01559, miR-6783-3p, and miR-1343-3p was determined using Pearson’s correlation.
A P<0.05 indicated statistical significance.

3. RESULTS
3.1. Expression Profile of linc01559, miR-1343-3p, and miR-6783-3p in HCC Tissues
We found that linc01559 was differentially expressed in the tissues of 362 patients with HCC versus healthy liver tissue
(Fig. 1A). Moreover, the expression of miR-1343-3p and miR-6783-3p in HCC tissues was lower than that measured in normal
liver tissue (Fig. 1B and 1C). According to Fig. (1D) 1, the expression of linc01559 was associated with response to FOLFOX4
(5-fluorouracil, leucovorin, and L-OHP). The scattered plot shows that linc01559 expression was negatively associated with the
expression of miR-1343-3p and miR-6783-3p (Fig. 1E and 1F).
Fig. (1). (A) Expression of linc01559 in the tissues of 362 patients with HCC and healthy liver tissue. (B) Expression of miR-
6783-3p in the tissues of 362 patients with HCC and healthy liver tissue. (C) Expression of miR-1343-3p in the tissues of 362
patients with HCC and healthy liver tissue. (D) Expression of linc01559 in the tissues of 362 patients with FOLFOX4
responsive or resistant HCC tissue. (E) Association of linc01559 expression with miR-1343-3p expression in patients with
HCC. (F) Association of linc01559 expression with miR-6783-3p expression in patients with HCC.
3.2. Survival Analysis of linc01559, miR-1343-3p, and miR-6783-3p
The log-rank test and Kaplan–Meier plot show that high expression of linc01559 was associated with poorer prognosis (Fig.
2A), while high expression of miR-1343-3p was associated with better prognosis (Fig. 2B). However, miR-6783-3p was not
significantly associated with the prognosis (Fig. 2C). In contrast, sex, BCLC classification, and response to FOLFOX4 were
associated with the prognosis (Fig. 2D-F). Subsequently, we constructed a nomogram including linc01559, miR-1343-3p, sex,
BCLC classification, and response to FOLFOX4 (Fig. 2G). The C-index was 0.73 (95% confidence interval: 0.65-0.77,
P<0.00). The calibration curves for the prediction of 6-month and 1-year survival revealed that the nomogram could be used to
predict survival (Fig. 2H and 2I).
Fig. (2). (A) K-M plot for the influence of linc01559 on overall survival. (B) K-M plot for the influence of miR-1343-3p on
overall survival. (C) K-M plot for the influence of miR-6783-3p on overall survival. (D) K-M plot for the influence of sex on
overall survival. (E) K-M plot for the influence of BCLC classification on overall survival. (F) K-M plot for the influence of
response to FOLFOX4 on overall survival. (G) Nomogram, including nc01559, miR-1343-3p, sex, BCLC classification, and
response to FOLFOX4 for the prediction of 1-year and 3-year survival in patients with HCC. (H&I) Calibration curves for the
rates of 1-year and 3-year survival.
linc01559 and miR-6783-3p were aberrantly expressed in HCC cell lines and regulated cell proliferation. As shown in Fig.
(3A), linc01559 was highly expressed in the MHCC97L and Huh7 cell lines compared with the Lo2 cell line. The expression of
miR-6783-3p was downregulated in HCC cell lines (Fig. 3B). However, miR-1343-3p expression was not significantly
different between the HCC cell lines and Lo2 cell line (Fig. 3C). In addition, we found that linc01559 was expressed more
abundantly in the cytoplasm than in the nucleus (Fig. 3D and 3E).
According to the results of the CCK-8 assays, downregulation of linc01559 resulted in increased cell proliferation ability
(Fig. 3F and 3G). Notably, upregulation and downregulation of miR-6783-3p resulted in decreased (Fig. 3H and 3I) and
increased (Fig. 3J and 3K) cell proliferation ability, respectively. However, upregulation and downregulation of miR-1343-3p
did not influence the cell proliferation ability (Fig. 3L and 3M).
3.3. linc01559 and miR-6783-3p were Associated with Resistance to L-OHP
We evaluated the expression profile of linc01559 in L-OHP-resistant cell lines (MHCC97L-R and Huh7-R). The results
showed that linc01559 was highly expressed in the MHCC97L-R and Huh7-R cell lines compared with their normal
counterparts (Fig. 4A and 4B). In addition, higher expression of linc01559 in the cytoplasm was detected in L-OHP-resistant
cells (Fig. 4C and 4D) than in HCC cell lines (Fig. 3B and 3C).
In MHCC97L-R and Huh7-R cells, downregulation of linc01559 significantly decreased cell viability following treatment
with L-OHP (Fig. 4E and 4F). Furthermore, upregulation of the expression of miR-6783-3p led to decreased cell viability (Fig.
4G and 4H); downregulation of miR-6783-3p led to increased cell viability following treatment with a higher dosage than
16μg/ml and 8μg/ml L-OHP (Fig. 4I and 4J).
Fig. (3). (A) qRT-PCR results for linc01559 in the MHCC97L and Huh7 cell lines. (B) qRT-PCR results for miR-6783-3p in
the MHCC97L and Huh7 cell lines. (C) qRT-PCR results for miR-1343-3p in the MHCC97L and Huh7 cell lines. (D)
Expression of linc01559 in the cytoplasm and nucleus of MHCC97L cells. (E) Expression of linc01559 in the cytoplasm and
nucleus in Huh7 cells. (F) CCK-8 test for the knockdown of linc01559 in MHCC97L cells. (G) CCK-8 test for the knockdown
of linc01559 in Huh7 cells. (H) CCK-8 test for the upregulation of miR-6783-3p in MHCC97L cells. (I) CCK-8 test for the
upregulation of miR-6783-3p in Huh7 cells. (J) CCK 8 test for the downregulation of miR-6783-3p in MHCC97L cells. (K)
CCK-8 test for the downregulation of miR-6783-3p in Huh7 cells.
Fig. (4). (A, B) qRT-PCR results for linc01559 in L-OHP-resistant cell lines compared with HCC cell lines. (C) Expression of
linc01559 in the cytoplasm and nucleus of MHCC97L-R cells. (D) Expression of linc01559 in the cytoplasm and nucleus of
Huh7-R cells. (E) Cell viability detected using the CCK-8 assay for the knockdown of linc01559 in MHCC97L-R cells treated
with L-OHP. (F) Cell viability detected using the CCK-8 assay for the knockdown of linc01559 in Huh7-R cells treated with L-
OHP. (G) Cell viability detected using the CCK-8 assay for the down regulation of miR-6783-3p in MHCC97L-R cells treated
with L-OHP. (H) Cell viability detected using the CCK-8 assay for the dowm regulation of miR-6783-3p in Huh7-R cells
treated with L-OHP. (I) Cell viability detected using the CCK-8 assay for the up regulation of miR-6783-3p in MHCC97L-R
cells treated with L-OHP. (J) Cell viability detected using the CCK-8 assay for the up regulation of miR-6783-3p in Huh7-R
cells treated with L-OHP.
3.4. linc01559 Directly Interacted with miR-6783-3p
We hypothesized that miR-6783-3p and miR-1343-3p may be targets of linc01559 (Fig. 5A). We found that downregulation
of linc01559 increased the expression of miR-6783-3p (Fig. 5B). However, the slight changes observed in the expression of
miR-1343-3p were not statistically significantly (Fig. 5C).
According to Fig. (5D and 5E), the luciferase reporter assays showed that miR-6783-3p mimics decreased the luciferase
activity of linc01559-Wt in the MHCC97L and Huh7 cells. In contrast, the luciferase activity of linc01559-Mut was not
significantly influenced, indicating that linc01559 may directly interact with miR-6783-3p. Subsequently, we transfected the
MHCC97L and Huh7 cells with the miR-6783-3p inhibitor, and found that the luciferase activity of linc01559-Wt was
significantly increased (Fig. 5F and 5G). The RNA pull-down assay showed that the expression of miR-6783-3p was higher in
the linc01559-WT group versus the linc01559-Mut group (Fig. 5H and 5I). The RIP results indicated that linc01559 was
enriched in the miR-6783-3p-Wt group compared with the miR-6783-3p-mut group and negative control in MHCC97L and
Huh7 cells (Fig. 5J and 5K).

Fig. (5). (A) Predicted binding site of linc01559 for miR-6783-3p and miR-1343-3p. (B) qRT-PCR results for miR-6783-3p in
linc01559-knockdown cell lines. (C) qRT-PCR results for miR-1343-3p in linc01559-knockdown cell lines. (D, E) Dual-
luciferase reporter gene assay for miR-6783-3p and linc01559 in MHCC97L and Huh7 cells. (F, G) Dual-luciferase reporter
gene assay for miR-6783-3p inhibitor and linc01559 in MHCC97L and Huh7 cells. (H, I) RNA pull-down assay for miR-6783-
3p and linc01559 in MHCC97L and Huh7 cells. (J, K) RIP assay for miR- 6783-3p and linc01559 in MHCC97L and Huh7
cells.
4. DISCUSSION
Systemic chemotherapy has become a common palliative treatment strategy for liver cancer, and its therapeutic effect on the
control of local advanced cancer foci is gradually recognized [15]. Early diagnosis is difficult due to the occult onset and rapid
progress of primary liver cancer. Therefore, most patients have reached the local late stage or distant metastasis at the time of
diagnosis, losing the opportunity for surgical treatment. In this setting, chemotherapy is the main treatment option for dealing
with liver cancer [16, 17]. Qin et al. conducted the EACH study, showing that use of the FOLFOX4 regimen has obvious
advantages (i.e., satisfactory safety and improved overall survival) [18]. Consequently, use of the L-OHP-based treatment in
patients with late-stage HCC has been well accepted in routine clinical practice.
L-OHP, as a third-generation platinum drug, mainly forms a cross bond with DNA, leading to its destruction. Moreover, it
blocks DNA replication and transformation, thus inhibiting cell proliferation [18, 19]. However, resistance to L-OHP leads to
failure of systemic chemotherapy in patients with late-stage HCC [20]. This study was designed to identify key factors in the
regulation of resistance to L-OHP from the lncRNA perspective. The role of lncRNAs in the treatment of HCC has been
thoroughly investigated in the past decade [21]. An increasing number of studies focused on the regulatory effect of lncRNAs
on chemosensitivity. For example, lncRNA nuclear receptor 2F-anti-sense 1 could sponge miR-363, therefore decreasing the
expression of adenosine triphosphate binding cassette subfamily C member 1 to induce resistance to L-OHP [22]. We have
found that linc01559 was not differentially expressed in tissues obtained from 362 patients with HCC versus healthy liver
tissue. However, it was highly expressed in patients with HCC who were resistant to L-OHP. The in vitro experiments revealed
that linc01559 promoted the proliferation ability of HCC cells. The oncogenic role of linc01559 has been previously reported in
pancreatic cancer and renal cell carcinoma [10, 23]. We hypothesized that linc01559 may play important roles in regulating
resistance response to L-OHP. Further in vitro study revealed that linc01559 was highly expressed in L-OHP-resistant HCC cell
lines. Moreover, knockdown of linc01559 decreased the viability of L-OHP-resistant cells following treatment with L-OHP.
These results shed light on the potential positive regulatory role of linc01559 in resistance response to L-OHP.
Based on the currently available evidence, linc01559 is mainly located in the plasma [10]. It has been reported that
cytoplasmic lncRNAs mainly sponged microRNAs and thereby modulate relevant genes. In this study, we confirmed that
linc01559 was mainly expressed in the plasma of patients with HCC. Interestingly, we also noticed that linc01559 was highly
expressed in L-OHP-resistant cells versus HCC cells. This may indicate that linc01559 was activated to interact with
microRNAs in response to the development of resistance to L-OHP. Subsequently, we used the Starbase online database and
found that miR-6783-3p and miR-1343-3p are potential targets of linc01559. Yao et al. found that miR-6783-3p may be a
tumor suppressor by targeting the Wnt/β-catenin signaling pathway [14]. Of note, miR-1343-3p has shown low expression in an
array of cancers [24, 25]. Further investigation through the dual-luciferase reporter gene assay, RIP, and RNA pull-down assay
confirmed the interaction between linc01559 and miR-6783-3p. These results provided robust evidence that linc01559 exerts
biological functions by sponging miR-6783-3p. Furthermore, we found a potential association of miR-6783-3p with resistance
to L-OHP. Upregulation of miR-6783-3p expression led to decreased cell viability in L-OHP-resistant cells. Downregulation of
miR-6783-3p led to increased cell viability following treatment with higher dosages than 16μg/ml and 8μg/ml L-OHP.

CONCLUSION
In conclusion, the results of this study showed that linc01559 promoted the proliferation of HCC cells by sponging miR-
6783-3p. We infer that linc01559/miR-6783-3p may be a key factor in regulating resistance to L-OHP by targeting miR-6783-
3p. Furthermore, it may be a potential therapeutic target in improving sensitivity to L-OHP in patients with HCC.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was approved by the Ethics Committee of Liaocheng People’s Hospital and first affiliated hospital of Xi`An
Jiaotong University (the ethical approval number: 2017-66), China.

HUMAN AND ANIMAL RIGHTS

No animals were used in this study. All human research procedures were in accordance with the standards set forth in the
Declaration of Helsinki https://www.wma.net/policiespost/wmadeclaration-of-helsinki-ethicalprinciplesfor-medicalresearch-
involvinganimals-subjects/principles of 1975, as revised in 2008 (http://www.wma.net/en/20activities/10ethics/10helsinki/).

CONSENT FOR PUBLICATION

All recruited patients understood the basic concept of our study, and individually provided written informed consent.

AVAILABILITY OF DATA AND MATERIALS

The data that support the findings of this study are available from the corresponding author, C.L., upon reasonable request.
FUNDING
This manuscript is funded by National Natural Science Foundation (81972236)

CONFLICT OF INTEREST
The author declares no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS
Declared none.

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