Hypoxia‐induced miR‐3677‐3p promotes the proliferation, migration and invasion of hepatocellular carcinoma cells by suppressing SIRT5

Abstract Hepatocellular carcinoma (HCC), with life‐threatening malignant behaviours, often develops distant metastases and is the fourth most common primary cancer in the world, having taken millions of lives in Asian countries such as China. The novel miR‐3677‐3p is involved in a high‐expression‐related poor prognosis in HCC tissues and cell lines, indicating oncogenesis functions in vitro and in vivo. Initially, we confirmed the inhibition of proliferation, migration and invasion in miR‐3677‐3p knock‐down MHCC‐97H and SMMC‐7721 cell lines, which are well known for their high degree of invasiveness. Then, we reversed the functional experiments in the low‐miR‐3677‐3p‐expression Hep3B cell line via overexpressing miR‐3677‐3p. In nude mice xenograft and lung metastasis assays, we found suppressor behaviours, smaller nodules and low density of organ spread, after injection of cells transfected with shRNA‐miR‐3677‐3p. A combination of databases (Starbase, TargetScan and MiRgator) illustrated miR‐3677‐3p targets, and it was shown to suppress the expression of SIRT5 in a dual‐luciferase reporter system. To clarify the conclusions of previous ambiguous research, we up‐regulated SIRT5 in Hep3B cells, and rescue tests were established for confirmation that miR‐3677‐3p suppresses SIRT5 to enhance the migration and invasion of HCC. Interestingly, we discovered hypoxia‐induced miR‐3677‐3p up‐regulation benefited HCC malignancy and invasiveness. In conclusion, the overexpression of miR‐3677‐3p mediated SIRT5 inhibition, which could increase proliferation, migration and invasion of HCC in hypoxic microenvironments.


| INTRODUC TI ON
Hepatocellular carcinoma ranks as the fourth most frequently diagnosed cancer in global surveys, and it was the third highest cause of death in China in a 2018 national health survey. 1 With its early local and systemic metastatic features, increasing amounts of research have been focused on HCC. 2,3 Regardless of the many efforts made towards the treatment of malignancies, HCC remains incurable owing to its high metastatic potential, with most deaths from HCC being attributable to its aggressiveness and growing resistance to the existing targeted medicines. 4,5 Hypoxic environments have been confirmed in many types of cancers; they are closely related to metastasis, apoptosis and invasion, [6][7][8] and therefore, identifying pathways and downstream targets of hypoxic conditions might lead to new therapeutic candidates for metastatic HCC. Angiogenesis, 9,10 reprogramming of metabolism, 11,12 extracellular matrix remodelling 13,14 and the promotion of inflationary cell chemotaxis are all triggered by hypoxia. 15,16 However, the characteristics of HCC, especially under hypoxic conditions, have not been fully understood.
A number of microRNAs (miRNAs), including miR-21 [17][18][19] and miR-221, [20][21][22] have been widely studied in regard to invasive HCC. miR-876 23 and miR-1468 24 along with others have been reported in various articles. Therefore, there is great potential for identifying an underlying miRNA that can be used to develop treatments for HCC and assist in early detection of HCC and the control of metastasis. [25][26][27] miRNAs play an independent role in the pathogenesis and progression of almost all types of cancers, such as non-small cell lung cancer, 28,29 breast cancer, [30][31][32] gastric carcinoma [33][34][35] and bladder tumours. 36,37 They are responsible for modulation of their cellular differentiation, as well as in regulating the transcription of their downstream targets.
A novel miRNA, miR-3677-3p, has never been reported in any article about neoplasms, including HCC. With the support of advanced analysis of a considerable amount of HCC samples, we identified miR-3677-3p and investigated its function in HCC and in the hypoxia-regulated axis in HCC.
Sirtuin 5 (SIRT5) was initially discovered in 1999, and the five human sirtuins, SIRT1 to SIRT5, are widely expressed in foetal and adult tissues. 38 The various members of the sirtuin family have different cellular locations, with SIRT1, SIRT6 and SIRT7 in the nucleus, while SIRT3, SIRT4 and SIRT5 are localized in mitochondria, and some researchers have compared traditional histone deacetylases (HDACs) to sirtuins. 39 All members of this family play similar but different roles in gene expression and molecular modifications.
Previous studies have claimed that SIRT5 differential expression could lead to suppressed cellular growth, endoplasmic reticulum stress (ER stress), senility and apoptosis in various cancers. [40][41][42][43][44][45][46] Although the research into the function of SIRT5 in HCC just began in 2018, 47 several experiments have shown multiple roles of SIRT5 in HCC. However, there has not yet been any final verdict reached on whether SIRT5 has a positive or negative role in HCC, although it is expressed at only low levels in tumour specimens. 41,46,48,49 Thus, we investigated its phenomenon of restraint in functional and rescue experiments. Therefore, in this study, we evaluated the potential mechanism of the metastatic and invasive promoter, miR-3677-3p, in HCC and found that it targeted SIRT5 in hypoxia conditions.

| Cell Counting Kit-8 (CCK-8)
A CCK-8 kit (#CK04-05, Dojindo Molecular Technologies Maryland, USA) was used for the determination of cell viability. The cells were seeded in 96-well plates at a density of 3500 cells per well in 10% foetal bovine serum (FBS) Dulbecco's modified Eagle medium (DMEM) for 0, 24, 48 or 72 hours. The total cell numbers were assessed after incubation with 10 μL of CCK8 for another 0.5 hours. The optical density (OD) measured by a microplate reader at 450 nm was reported as the means ± SD. The experiments were repeated three times.

| Haematoxylin and eosin (H&E) staining
The whole staining process consists of five steps: dewaxing, dyeing, dehydration, transparency and sealing. After dewaxing, we stained the slides in haematoxylin for 10 minutes. 50 Then, after washing away the haematoxylin and allowing the colour to develop for another 2 minutes, we placed the slide for 10 seconds in 1% hydrochloric acid alcohol, so the colour faded to red. Next, we washed the slides for 2 minutes in water and put them into the red eosin dye for 4 minutes. The slides were rinsed in water, dehydrated and sealed with a coverslip.

| Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Levels of SIRT5, HIF-1a, miR-3677-3p and beta-actin were determined using RT-qPCR. The total RNA was extracted with TRIzol (Invitrogen Inc, Carlsbad, CA, USA) following the manufacturer's instructions, and then the RNA was reverse transcribed into cDNA (#k1622, Thermo Fisher, Shanghai, China). The PCR program was performed following the kit's protocol (for reverse transcription, 42°C for 5 minutes, 70°C for 62 minutes and 4°C at standby; for qPCR, 95°C for 10 minutes, 95°C for 5 seconds for cDNA or 95°C for 15 seconds for the miRNA RT product, 60°C for 30 seconds and 72°C for 30 seconds for 40 cycles within the last 3 steps). Beta-actin and U6 were used as internal references for mRNA and miRNA, respectively. The fold changes were calculated by means of relative quantification (2 −ΔΔCT method). IgG secondary antibody (#ab7072, 1:6000, Abcam) or goat anti-rabbit IgG secondary antibody (#ab6721, 1:6000, Abcam Inc) at room temperature for one and a half hours. Next, the membrane was rinsed three times with PBST and exposed to western HRP substrate (#WBLUF0110, Merck-Millipore,Darmstadt, Germany) under dark conditions. Relative protein levels of the target genes were expressed as the ratio of grey values of the target protein bands to those of the β-actin band.  with 5% CO 2 . After a 36 hours incubation, the cells were fixed and stained with 0.1%-0.5% crystal violet. For the migration assay, the same procedure was followed omitting the Matrigel. The stained cells were counted under the microscope to determine the average number of cells in five randomly selected fields.

| Tumour formation and lung metastasis model in nude mice
An MHCC-97H cell suspension was prepared after digestion by trypsin-EDTA (HyClone, GE, Buckinghamshire, England). A mixture of PBS and Matrigel (#356234, BD, California, USA) at a ratio of 1:1 was prepared for suspending cells transfected with sh-NC and sh-miR-3677-3p at a final concentration of 3 × 10 6 cells/150 μL.
A total of 16 male 4-week-old nude mice (SLAC Laboratory Animal Co. Ltd., Shanghai, China) were divided into two groups, a xenograft group and a lung metastasis group, with eight mice per group. The mice were allowed to adapt for one week, and then they were anaesthetized with chloral hydrate.
The xenograft group was inoculated subcutaneously in the left posterior cervical subcutaneous site with the prepared cells. The mice were kept in the same specific pathogen-free environment and were observed and measured weekly after the injections.
Tumour length and width were recorded, and the gross tumour volume was calculated according to the international common formula, volume = (length × width 2 )/2. On the 28th day, the mice were sacrificed by chloral hydrate and the tumours were removed and weight.
For the lung metastasis model, the same procedure was followed except the cells were injected slowly into the tail vein at 3 ×

| Statistical analysis
The statistical analysis was conducted by using SPSS 19.0 (IBM, Armonk, USA) and GraphPad 6.0 (GraphPad Software, San Diego, USA). Mean ± standard deviation was used for measurement data.
Two groups' data were compared using t tests, and multiple-groups data were compared using one-way analysis of variance (ANOVA).
Fischer's least significant difference (LSD) method was adopted for paired comparisons. Enumeration data are presented as percentages and were analysed using chi-square tests. Values of P < 0.05, P < 0.01 or P < 0.001 were defined as varying levels of statistical significance.

3677-3p had a poor prognosis
According to databases analyses, we found many miRNAs with abnormal expression in deceased or living samples (OncomiR: http://www. F I G U R E 1 HCC with high expression of miR-3677-3p has a poor prognosis Comparing differences in the expression levels of miR-3677-3p between (A) HCC and matched nontumour tissues; (B) aggressive and non-aggressive tumour tissues; and (C) HCC cell lines and the immortalized hepatic cell line LO2, U6 was used as internal control. D, Overall survival was compared between HCC patients with high-expression levels of miR-3677-3p and those with low levels of miR-3677-3p. *P < .05, **P < .01, ***P < .001 oncom ir.org/). In HCC tissues, miR-3677-3p was expressed at a high level and it was correlated with a poor prognosis. Its significant upregulation in 374 tumour samples was obvious ( Figure S1A,B from Starbase: http://starb ase.sysu.edu.cn/ and OncomiR). Then, we tested its expression in liver cancer specimens from our medical institution, which consisted of 40 pairs of adjacent normal and liver tumour tissues, and 42 invasive and 71 non-aggressive tumours. The expression of miR-3677-3p was increased in the tumours relative to the normal tissue, and it was also increased in the aggressive tumours relative to the non-aggressive tumours as expected ( Figure 1A,B). Similarly, the results of relative expression in HCC cell lines indicated that the MHCC-97H and SMMC-7721 cell lines, well known for being invasive, expressed miR-3677-3p at high levels. miR-3677-3p was expressed at lower levels in noninvasive cell lines like Hep3B, although even in noninvasive HCC cell lines, its expression level was remarkably elevated relative to normal hepatic cells ( Figure 1C).
Considering the poor prognostic features of HCC, we investigated the correlation between the prognosis and the expression level of this miRNA. We found higher expression of miR-3677-3p was correlated with a low survival rate ( Figure 1D). In addition, the TCGA database (OncoLnc: http://www.oncol nc.org/) corroborated the correlation between miR-3677-3p expression, using the median as the cut-off, with a lower survival percentage over 10 years ( Figure S1C).

| Clinical significance of miR-3677-3p in HCC patients
To further confirm that expression of miR-3677-3p was associated with the clinical features of HCC patients, we divided 160 HCC patients into high and low groups according to the median value  inhibited after miR-3677-3p knock-down ( Figure 2D).

| miR-3677-3p promotes metastasis, invasion and proliferation of HCC
Next, overexpression of miR-3677-3p, tested by qPCR, was used to support the results that miR-3677-3p promotes cell growth, metastasis and invasion ( Figure S2A). The Hep3B cell line was chosen for these experiments, and we found miR-3677-3p did indeed enhance proliferation, invasive and migration ( Figure 3).

| Knocking-down miR-3677-3p blocked the growth of xenografts and lung metastasis in nude mice
To confirm the tumour-promoting effects of miR-3677-3p in vivo, we used tumour xenograft models. The group with miR-3677-3p showed decreased expression, verified by post-transfection sh-miR-3677-3p qPCR, illustrated a weakened capacity of the HCC xenografts ( Figure S2B and Figure 4A). Measurement of the injected tumour nodules suggested significant suppression of tumour volume in the sh-miR-3677-3p group ( Figure 4B). Not only the weight of the nodules but also the weight of the mice themselves confirmed that the tumour sites in the negative group were larger than the sh-group and the mice had no statistical differences in regards to weight, sex or age ( Figure 4C and Figure S2C).
Models of lateral vein injection were used, and they showed fewer and smaller foci in the lungs of nude mice via microscopic evaluation ( Figure 4D). Taken together, these results suggest that miR-3677-3p promotes oncogenesis and metastatic behaviours of HCC in vivo.

| SIRT5 plays an inhibitory role in HCC
As  Figure S2D).
We overexpressed SIRT5 in HCC cells ( Figure S2E). CCK-8, EDU, and Transwell invasion and migratory assays showed it had an inhibitory effect ( Figure 6). To investigate the SIRT5 expression in

| Up-regulation of miR-3677-3p or silencing of SIRT5 enhances HCC cell proliferation, metastasis and invasion
Next, compared with the NC groups, a significant decrease in SIRT5 protein level with miR-3677-3p inhibitor and rescue with miR-3677-3p inhibitor + si-SIRT5 groups ( Figure 7A) was tested, while the siRNA efficiency was tested primarily in the MHCC-97H cell line in the high-miR-3677-3p group, which was associated with several malignant characteristics, such as tumour-node-metastasis (TNM) SIRT5 is a well-known gene that leads to histone acetylation and has been reported to be related to hypoxia in some low-quality reports. However, in two cerebral ischaemia model articles, the author reported that 'genetic deletion of SIRT5 decreased infarct size, improved neurological function and blunted systemic inflammation following stroke'. 52,53 Beyond the fact that SIRT5 accelerates the damage of ischaemia, we may make a bold hypothesis that in the hypoxic condition of HCC, there is a failure of SIRT5 to be induced, which facilitates carcinogenesis.

| CON CLUS ION
In conclusion, the aforementioned results demonstrated that miR3677-3p could promote cell division, migration and invasion of HCC through the direct down-regulation of SIRT5 in a hypoxic condition, which provides novel insights for developing therapeutic approaches to HCC. However, this study remains at the stage before clinical application and the mechanism of hypoxia and whether HIF-1a regulates miR-3677-3p directly are not yet established. Therefore, further studies of direct regulation between HIF-1a and miRNAs or SIRT5 need to be broadened, and we will expound more upon the function of miR-3677-3p in different tumour microenvironments.

ACK N OWLED G EM ENT
This work was supported by a grant from the National Natural Science Foundation of China (81572847). We must thank for all participants enrolled in the present study and the complete samples database in laboratory of department of hepatobiliary surgery in First Affiliated Hospital of Xi'an Jiaotong University. Besides, we feel grateful for Center for Translational medicine in the same institution.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest. Formal analysis (equal); Project administration (lead); Resources (lead); Supervision (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.