let‐7g sensitized liver cancer cells to 5‐fluorouracil by downregulating ABCC10 expression

Patients with advanced liver cancer may benefit from 5‐fluorouracil (5‐FU) therapy. However, most of them eventually faced drug resistance, resulting in a poor prognosis. The present study aims to explore the potential mechanism of let‐7g/ABCC10 axis in the regulation of 5‐FU resistance in liver cancer cells. Huh‐7 cells were used to construct 5‐FU resistant Huh‐7/4X cells. CCK8, flow cytometry, and TUNEL staining were used to detect the characterization of Huh‐7 cells and Huh‐7/4X cells. Double luciferase report, PCR, and western blot analyses were used to detect the regulatory effects between let‐7g and ABCC10. The levels of biomarkers related to cell cycle progression and apoptosis were detected by western blot assays. The role of let‐7g in 5‐FU sensitivity of liver cancer cells was evaluated in nude mice. Compared with LX‐2 cells, the expression of let‐7g was decreased in Hep3B, HepG2, Huh‐7, and SK‐Hep1 cells, with the lowest expression in Huh‐7 cells. The sensitivity of Huh‐7 cell to 5‐FU was positively correlated with let‐7g expression. Transfection of let‐7g mimics inhibited the viability of Huh‐7/4X cells by prolonging the G1 phase, with the downregulation of ABCC10, PCNA, Cyclin D1, and CDK4. Meanwhile, let‐7g promoted apoptosis to increase 5‐FU sensitivity of Huh‐7/4X by downregulating ABCC10, Bcl‐XL as well as upregulating Bax, C‐caspase 3, and C‐PARP. Dual‐luciferase assay further confirmed that let‐7g inhibited ABCC10 expression by binding to the ABCC10 3’‐UTR region. Furthermore, let‐7g increased the sensitivity of Huh‐7/4X to 5‐FU in vitro and in vivo, which can be reversed by ABCC10 overexpression. In conclusion, let‐7g sensitized liver cancer cells to 5‐FU by downregulating ABCC10 expression.

detect the regulatory effects between let-7g and ABCC10.The levels of biomarkers related to cell cycle progression and apoptosis were detected by western blot assays.The role of let-7g in 5-FU sensitivity of liver cancer cells was evaluated in nude mice.Compared with LX-2 cells, the expression of let-7g was decreased in Hep3B, HepG2, Huh-7, and SK-Hep1 cells, with the lowest expression in Huh-7 cells.The sensitivity of Huh-7 cell to 5-FU was positively correlated with let-7g expression.Transfection of let-7g mimics inhibited the viability of Huh-7/4X cells by prolonging the G1 phase, with the downregulation of ABCC10, PCNA, Cyclin D1, and CDK4.Meanwhile, let-7g promoted apoptosis to increase 5-FU sensitivity of Huh-7/4X by downregulating ABCC10, Bcl-XL as well as upregulating Bax, C-caspase 3, and C-PARP.Dual-luciferase assay further confirmed that let-7g inhibited ABCC10 expression by binding to the ABCC10 3'-UTR region.
Furthermore, let-7g increased the sensitivity of Huh-7/4X to 5-FU in vitro and

| INTRODUCTION
Cancer remains a global health challenge, with its incidence expected to exceed 1 million cases by 2025 (Llovet et al., 2021).Hepatocellular carcinoma (HCC) causes the sixth most common cancer-related death worldwide with a very low 5-year survival rate (Wong et al., 2012).Recent research reports that the abnormal expression of miRNAs contributes to the development and progression of HCC (Chen, Zhang, et al., 2022;Wang et al., 2010).Additionally, accumulating evidence has indicated that several dysregulated miRNAs or lncRNAs were important regulatory factors in HCC drug resistance (Wei et al., 2019;Yao et al., 2021).
The let-7 family is significantly downregulated in HCC (Boyerinas et al., 2010), and nine of its members have been found in humans (Reinhart et al., 2000).As one of 9 members in let-7 family, let-7g was reported to inhibit the invasion and migration of liver cancer cells (Ji et al., 2010;Lan et al., 2011).However, its roles in chemoresistance of liver cancer remain unknown.Dysregulation of miRNAs can affect the function of drug transporters and the tumor microenvironment, thereby affecting chemotherapy sensitivity to cancer cells (Mondal & Meeran, 2021).Chronic inflammation caused by liver injury or infection may promote HCC (Chen, Cheng, et al., 2022).let-7g acts synergically with interferon/Ribavirin to inhibit hepatitis C virus replication (Chou et al., 2016).Currently, 5-fluorouracil (5-FU) is one of the most important drugs in the chemotherapy for patients with liver cancer (Zhang et al., 2018).However, the majority of HCC patients ultimately acquire chemotherapy resistance after 5-FU treatment, which poses a major challenge to clinical treatment efficiency (Wang, Chu, et al., 2021).Studies have shown that let-7g can sensitize liver cancer cells to 5-FU (Tang et al., 2014), but its mechanism remains unclear.
Adenosine triphosphate binding box subfamily C (ABCC) is a member of the largest membrane protein superfamily (Meng et al., 2022).ABCC10 can actively transport a wide range of cytotoxic chemotherapy drugs outside the cell, such as taxanes, 5-FU, and other nucleoside analogs, leading to the occurrence of multi-drug resistance (Dabrowska & Sirotnak, 2017).Targeting these ABC transporters may give a new sight to overcome drug resistance in cancer chemotherapy (Zhou et al., 2008).Upregulation of ABC transporters in HCC occurs prior to chemotherapy and is associated with downregulation of miRNA (Borel et al., 2012).
Therefore, this study aims to explore whether let-7g can enhance the sensitivity of liver cancer cells to 5-FU by regulating the expression of ABCC10, in order to give new insights into the chemotherapy resistance of HCC.
Huh-7/4X cells were constructed as described formerly (Duz & Karatas, 2020).Briefly, the primary parental Huh-7 cell line was cultured with 5-FU to create the 5-FU-resistant Huh-7 subline.The cells were first incubated in a 25 cm 2 flask at a concentration of 1 × 5-FU for 48 h.The medium was then changed to a fresh medium without 5-FU.The cells were allowed to recover for 1-2 weeks until they began to grow exponentially.Cells become resistant to 1 × 5-FU after 6-8 treatments and are called "Huh-7/1X."Then, 2X-and 4X-resistant strains were constructed sequentially.The upregulation of let-7g or ABCC10 was stimulated by transfection of let-7g mimics or pcDNA3.1-ABCC10using Lipofectamine 2000 (Invitrogen) for 48 h in cells. in vivo, which can be reversed by ABCC10 overexpression.In conclusion, let-7g sensitized liver cancer cells to 5-FU by downregulating ABCC10 expression.

| Cell cycle assay
The cells were suspended to separate into individual cells.Cells were added with precooled 100% ethanol (1.2 mL) at a final concentration of 75% and placed at 4°C overnight for fixation.The cells were washed and suspended with PBS to remove ethanol.Cells were added with 150 μL propyl iodide (1 μg/μL, MB2920, Meilune) and stained for 30 min.Cells were analyzed by a flow cytometer (A00-1-1102, Beckman) to measure the cell cycle.

| Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
One milliliter trizol was added to the cell culture and fully ground in a homogenizer.The total RNA was extracted according to the manual.Next, miRNA (CW2141, CWBIO) and mRNA (CW2569, CWBIO) kits were used to reverse transcribe cDNA using total mRNA as a template.Quantitative PCR was performed by an UltraSYBR mixture (CW2601, CWBIO) kit and RCP instrument (PIKOREAL96, Thermo).Finally, 2 −ΔΔCt was used to analyze the ratio of the target gene level of each sample to that of the control sample by using U6 or actin as internal parameters.Primers used in this study have been shown in Table 1.

Gene
Sequence Length

| Western blot
Cells were added with 200 μL RIPA lysate (AWB0136, Abiowell), gently scraped down to collect the suspension, and ultrasonically broken for 1.5 min.Tissue (0.025 g) was added into 300 μL RIPA cracking solution and repeatedly ground in a biological sample homogenizer, then placed on ice for cracking for 10 min.The supernatant was obtained by centrifugation at 12,000 rpm for 15 min at 4°C.The protein concentration was determined by the BCA method.

| Statistics
All the quantitative assays were performed in triplicate at least.Statistical analysis of data in this study was carried out by GraphPad Prism 8.0 statistical software.Data were expressed as mean ± SD.Normality and homogeneity of variance were tested.An unpaired t test was used between groups.One-way anova was used to compare differences among multiple groups.The Pearson coefficient was used for correlation analysis.p < .05indicated a significant difference.

| let-7g expression was positively correlated with 5-FU sensitivity of liver cancer cells
Compared with LX-2 cells, the expression of let-7g was decreased in Hep3B, HepG2, Huh-7, and SK-Hep1 cells (Figure 1a), with the lowest expression in Huh-7 cells (Figure 1a).We then examined the viability of Huh-7 cells in response to 5-FU at different concentrations.
With the increase of 5-FU concentration, the activity of Huh-7 cells decreased obviously (Figure 1b).Moreover, we found that let-7g expression was positively correlated with Huh-7 cell sensitivity to 5-FU at 100 μM (Figure 1c).

Huh-7 cells to 5-FU
To explore the role of let-7g, let-7g abundance was changed by transfecting Huh-7 cells with let-7g mimics (Figure 2a).CCK-8 assays demonstrated that let-7g mimic synergistically represses the viability of Huh-7 cells with 5-FU (Figure 2b).Flow cytometry showed that treatment with 5-FU arrested more Huh-7 cells in the G1 phase, and let-7 g upregulation exacerbated this effect (Figure 2c).In addition, levels of PCNA, Cyclin D1, and CDK4 proteins were downregulated after treating with let-7g mimics or/ and 5-FU in Huh-7 cells, compared with control groups (Figure 2d).What is more, we found that incubation with let-7g mimics or/and 5-FU promoted apoptosis in Huh-7 cells (Figure 2e), which was accompanied by changes in apoptotic markers including Bax, C-caspase 3, C-PARP, and Bcl-XL (Figure 2f).These findings indicated that let-7g sensitized Huh-7 cells to 5-FU in vitro.

| let-7g inhibited the expression of ABCC10
To decipher the potential role of let-7g, candidate targets of this miRNA were explored.Bioinformatics analysis revealed the presence of binding sites in the 3′-UTR of ABCC10 (Figure 3a).We found that let-7g mimics significantly inhibited ABCC10 expression in Huh-7 cells (Figure 3b).Additionally, transfection with let-7g mimics significantly decreased the luciferase activity of the ABCC10 3′-UTR wild-type reporter gene but had no effect on that of the ABCC10 3′-UTR-mutated reporter gene (Figure 3c).These findings demonstrated that ABCC10 is a direct target of let-7g in Huh-7 cells.
3.5 | let-7g promotes 5-FU sensitivity via targeting ABCC10 in vivo Next, we investigated the effect of let-7g in vivo by establishing a xenograft tumor model.As shown in Figure 5a-c, let-7g mimic treatment reduced tumor growth compared to the control group with the downregulation of ABCC10 in tumor tissues, and transfection of pcDNA3.1-ABCC10plasmid reversed the change.In addition, the levels of PCNA, Cyclin D1, and CDK4 changed along with the response alteration of Huh-7/4X to 5-FU (Figure 5d).Subsequently, the TUNEL assay showed that let-7g mimic caused more DNA damage after 5-FU treatment with the changes in levels of Bcl-XL, Bax, C-caspase 3, and C-PARP proteins, suggesting that let-7g promoted apoptosis of Huh-7/4X when exposed to 5-FU (Figure 5e,f).Taken together, these results demonstrated that let-7g sensitized the response of liver cancer cells to 5-FU by inhibiting ABCC10 expression in vivo (Figure 6).

| DISCUSSION
Previous studies have depicted the key role of let-7g in the development of hepatocellular carcinoma.A study reported that low let-7g expression in tumors predicts poor survival in patients with HCC, partly because let-7g is able to inhibit HCC metastasis by targeting COL1A2 (Ji et al., 2010).Some other studies revealed that let-7g acts as a tumor suppressor in the development of HCC by regulating E-cadherin (Abdel Wahab et al., 2022) or c-Myc (Lan et al., 2011).Moreover, re-expression of let-7g can significantly inhibit the malignant characteristics of liver cancer cells (Chen et al., 2014).Similar to the results of the above studies, we found that let-7g expression was lower in Huh-7 cells compared with LX-2 cells.Furthermore, let-7g expression was positively correlated with Huh-7 cell sensitivity to 5-FU, which revealed that let-7g expression was positively correlated with liver cancer cell sensitivity to 5-FU.
Previous studies have reported the roles of let-7 family in the regulation of drug sensitivity.For instance, let-7g acted as a ceRNA mediator of LMCD1-AS1/P4HA2 axis to regulate the response of liver cancer cells to aspirin (Wang et al., 2019).let-7c enhanced the apoptosis of liver cancer cells exposed to sorafenib by downregulating Bcl-2 protein Mcl-1 expression (Shimizu et al., 2010).Additionally, low let-7g expression predicted acquired chemoresistance for patients with late stage of epithelial ovarian cancer (Biamonte et al., 2019).However, whether let-7g is associated with liver cancer progression remains unclear.In the present study, we found let-7g sensitized the response of Huh-7/4X cells to 5-FU by prolonging the G1 phase and promoting apoptosis, which provides new insights into the role of let-7g in the regulation of 5-FU sensitivity of liver cancer.
The expression of ABC transporters always increased significantly in resistant cells after exposure to higher doses of 5-FU (Duz & Karatas, 2020;Wang, Wang, et al., 2021;Xie et al., 2017;Zhao et al., 2018).A previous study reported that NEK2 mediates cisplatin resistance by regulating ABCC10 expression in HCC patients (Wu et al., 2017).In addition, ABCC10 inhibitor administration reversed FoxM1-induced 5-FU resistance of colorectal cancer in vivo (Xie et al., 2017).Huang et al. reported that miR-98 promotes paclitaxel resistance of endometrial cancer cells via the MRP-7/ABCC10 axis (Huang et al., 2021).Consistent with the above reports, our findings confirmed that ABCC10 overexpression reversed 5-FU sensitization of Huh-7/4X cells induced by let-7g in vitro and in vivo.Together with the findings that ABCC10 is a direct target gene of let-7g in liver cancer cells, we demonstrated the important role of the let-7g/ABCC10 axis in the regulation of 5-FU sensitivity of liver cancer, indicating that let-7g/ABCC10 might be the promising target for 5-FU resistance in liver cancer.
In conclusion, our findings demonstrated that let-7g/ ABCC10 axis is an important regulator of 5-FU sensitivity in liver cancer.This novel signaling axis regulating 5-FU sensitivity might shed new light on the treatment of liver cancer.

F
I G U R E 1 Correlation analysis of let-7g expression and liver cancer cell sensitivity to 5-FU.(a) The expression of let-7g in LX-2, Hep3B, HepG2, Huh-7, and SK-Hep1 cells was detected by qPCR.(b) CCK-8 assay indicated the viability of Huh-7 cells exposed to a gradient concentration of 5-FU.(c) Pearson analyses the correlation between let-7g expression and Huh-7 cell sensitivity to 5-FU at 100 μM.*p < .05.

F
I G U R E 3 let-7g suppress the expression of ABCC10.(a) The web tool miRmap predicted the binding site of let-7g in ABCC10 3′-UTR.(b) The expression of ABCC10 in each group was detected by western blot.(c) The regulation between let-7g and ABCC10 was detected by a dual-luciferase experiment.The grouping of blots cropped from different gels was displayed, and full-length blots are presented in Data S1.*p < .05.F I G U R E 4 Overexpression of ABCC10 reversed the promotion effect of let-7g-mimic on 5-FU sensitivity of Huh-7/4X cells.(a)The expression of let-7g in each group was detected by qPCR.(b) The expression of ABCC10 in each group was detected by western blot.(c) The viability of Huh-7/4X cells in different groups was detected by CCK-8 assays.(d) The cell cycle phase distribution of Huh-7/4X cells in each group was detected by flow cytometry.(e) The expression levels of PCNA, Cyclin D1, and CDK4 were tested by western blot.(f) Apoptosis rate of each group was detected by TUNEL staining (200×).(g) The expression levels of Bcl-XL, Bax, C-caspase 3, and C-PARP were detected by western blot.The grouping of blots cropped from different gels was displayed, and full-length blots are presented in Data S1.*p < .05.

F
I G U R E 5 let-7g increased 5-FU sensitivity of liver cancer cells by down-regulating ABCC10 in vivo.(a) Excised tumors from the xenograft model were displayed and their tumor weights were measured.(b) The tumor growth curve of each treatment group.(c) The expression of ABCC10 in representative tumors of each group was detected by western blot.(d) The expression levels of PCNA, Cyclin D1 and CDK4 in representative tumors of each group were tested by western blot.(e) Apoptosis rate in representative tumors of each group was detected by TUNEL staining (200×).(f) The expression levels of Bcl-XL, Bax, C-caspase 3 and C-PARP in representative tumors of each group were detected by western blot.The grouping of blots cropped from different gels were displayed, and full-length blots are presented in Supplementary File 1. *P<0.05.F I G U R E 6 Schematic diagram of let-7g/ABCC10 axis involved in 5-fluorouracil sensitivity of liver cancer cells.let-7g sensitized liver cancer cells to 5-FU by downregulating ABCC10 expression and thus reducing the extracellular transport of the drug.