Long Non-Coding RNA MIR4435-2HG Promotes Glycolysis and Tumor Immunity in Pancreatic Cancer Through Absorption of microRNA-582-5p to Target GPX8


 Objective

Long non-coding RNA MIR4435-2HG (MIR4435-2HG) has been clarified as a promoter in cancers, but its involvement in pancreatic cancer (PC) was not thoroughly probed.
Methods

MIR4435-2HG levels in PC clinical tissue and cell lines were analyzed. The relation between MIR4435-2HG levels with clinicopathological characteristics and survival of PC patients was evaluated. After transfection with plasmids altering MIR4435-2HG, microRNA (miR)-582-5p or glutathione peroxidase-8 (GPX8), the biological functions, glycolysis and tumor immunity of PANC-1 cells were observed. In vivo tumor growth was observed in nude mice.
Results

MIR4435-2HG was highly expressed in PC tissues and cell lines which was negatively correlated with clinicopathological characteristics and prognosis of PC patients. MIR4435-2HG or GPX8 inhibition or miR-582-5p elevation restrained the growth, glycolysis and immunity of PANC-1 cells. miR-582-5p down-regulation or GPX8 up-regulation reversed the effect of silenced MIR4435-2HG on PANC-1 cells. In vivo experiments further confirmed the in vitro results.
Conclusion

Our study highlights that MIR4435-2HG promotes glycolysis and tumor immunity in PC through absorption of micR-582-5p to target GPX8.


Introduction
About 86023 pancreatic cancer (PC) cases were diagnosed in China in 2020, with an estimated agestandardized incidence rate of 3.9%. Early PC is usually clinically asymptomatic, and the disease becomes obvious only after the tumor invades or metastasizes. Typical symptoms include abdominal pain or middle back pain, weight loss and obstructive jaundice [1]. People with any risk factors such as smoking, obesity, genetics, diabetes, diet, inactivity are at higher risk of PC [2]. Methods used to diagnose PC include ultrasonography, EUS, CT, MR, endoscopic retrograde cholangiopancreatography, and thin needle aspiration [3]. Unfortunately, the cure rate of PC is only 9%, and if without treatment, the median survival of patients with metastatic PC is only 3 months [4]. In respect to effective treatments of PC, huge efforts are largely required to decipher the molecular mechanism of the disease.
Long noncoding RNA (lncRNA) has undergone signi cant changes in the progression of cancer. In particular, lncRNAs exert multiple functions in PC and have valuable implications in the progression of PC [5]. Studies have listed various lncRNAs that could mediate metabolic reprogramming of PC cells. For instance, LncRNA plasmacytoma variant translocation 1 [6] and MACC1 antisense RNA 1 [7] could repress the progression of PC through impeding glycolysis of cancer cells. Regarding to MIR4435-2HG, it has been corroborated that MIR4435-2HG enhances metastatic activity of gastric cancer (GC) cells [8] and lung cancer cells [9], while MIR4435-2HG-oriented mechanism in PC was insu ciently studied.
LncRNA can be used as a miRNA decoy to regulate gene expression, and any disruption of the regulatory loop may ultimately lead to cancer initiation and development [10]. In a varied range of cancers, miR-582-5p is usually found to act suppressively to decrease the malignant behaviors of cancer cells. Tian Y et al. have concluded that miR-582-5p obstructs bone metastasis of prostate cancer cells [11], while Liu J et al. have discovered the inhibitory impacts of miR-582-5p on non-small cell lung cancer (NSCLC) cells [12].
Glutathione peroxidases (GPXs) could reduce the damage associated with intracellular oxidative stress and have been found to be closely related to tumorigenesis [13]. As a non-selenium congener of GPXs family, GPX8 has become a key player in biological environments, far beyond the detoxi cation of hydroperoxides [14]. Indeed, GPX8 serves essentially for regulating aggressive breast cancer [15] and predicting prognosis of GC [16]. With regard to MIR4435-2HG-mediated miR-582-5p/GPX8 network in PC, little few research has been initiated. Innovatively, our research was implemented to unveil the action of MIR4435-2HG/miR-582-5p/GPX8 axis as to the aspects of growth, glycolysis and immunity of PC cells.

Ethics statement
The study was approved by the committee of West China Hospital of Sichuan University, and all subjects had signed a written consent form. Animal treatments were processed in coincidence with Animal Care and Use Guidelines of Animal Ethic Committee in West China Hospital of Sichuan University.

Clinical samples
Ninety-six PC patients were enrolled, and cancer tissue and normal tissues were surgically obtained. Patients were con rmed as PC through the WHO standard histopathological diagnosis. No radiotherapy or chemotherapy was performed before and after the operation. After the operation, the patients were followed up every six months by outpatient reviews, and the median follow-up was 60 months. Detection of lactate dehydrogenase (LDH) NK cell cytotoxicity was analyzed by the LDH kit (C0016; Beyotime, Shanghai, China). Cells (1 × 10 4 ) were allowed to grow in 96-well plates for 4 h and the release of LDH was measured [25].
Tumor xenografts in nude mice SPF-free BALB/c male nude mice (3-5 weeks old, 20 ± 5g) were reared at 25 ± 3°C with a humidity of 45-50%. The mice were randomly divided into 10 groups, each with 6 mice. Cells were resuspended in 50% Matrigel (354234; Corning, NY, USA) to 1 × 10 7 cells/mL, and the cell suspension (0.5 mL) was subcutaneously into the left armpit of mice which had been anesthetized with pentobarbital sodium (50 mg/kg). Tumor size was measured with a vernier caliper every 5 days, a total of 6 times. After 30 d, the mice were anesthetized with pentobarbital sodium (50 mg/kg) and euthanized to take tumors. Tumors were weighed and tumor volume (mm 3 ) was measured: (length × width 2 )/2 [26].
The primer sequences are shown in Table 1. Statistical analysis SPSS 22.0 software (IBM, NY, USA) was applied for statistical analysis. Each experiment was repeated 3 times. Data were displayed as mean ± standard deviation and assessed by t-test or one-way analysis of variance (ANOVA) and Tukey's post-hoc test. Chi-square test and Kaplan-Meier analysis were adopted to determine the relationship between MIR4435-2HG expression and patients' pathological features and survival, respectively. Pearson test was allowed to evaluate the correlation of MIR4435-2HG, miR-582-5p and GPX8. P < 0.05 presented statistical signi cance.

Results
Negative correlation between MIR4435-2HG expression and patients' prognosis in PC MIR4435-2HG suppression can inhibit cancer cell development [8,30]. In PC, MIR4435-2HG expression was determined in clinical tissues and cells, and the outcomes re ected that MIR4435-2HG was excessively expressed in cancer tissues (Fig. 1A) and cell lines (Fig. 1B). PANC-1 cells had the most Page 7/20 obvious difference from hTERT-HPNE cells in the level of MIR4435-2HGs, so it was used for subsequent cell experiments.
Because cancer cells have more material and energy requirements, the level of glycolysis increases, which is manifested in the increase of glucose uptake and lactate production [31]. Therefore, by testing the glucose consumption and lactate production, it was captured that lowering MIR4435-2HG expression reduced glycolysis and hindered cancer cells from obtaining energy (Fig. 2G).
LDH is not only related to glycolysis pathway, but can also promote tumor immunity by changing tumor microcircles. High LDH predicts the reduction of NK cytotoxicity [32]. MICA and MICB are ligands on the tumor surface that could promote NK cytotoxicity. Tumor immunity is usually related to the shedding of MIC on the cell membrane [33]. Detection of LDH, MICA and MICB found that after interference with MIR4435-2HG, NK cytotoxicity, and MICA/B expression were enhanced (Fig. 2H, I).
Tumor formation in nude mice displayed that PANC-1 cells carrying silenced MIR4435-2HG had inhibited ability to increase tumor volume and weight (Fig. 2J, K).
In vivo, the ability of PANC-1 cells to promote tumor growth was restrained by up-regulated miR-582-5p, causing reduced tumor volume and weight (Fig. 4J, K).
Targeting relation between miR-582-5p and GPX8 GPX8 knockdown can effectively inhibit the growth of tumors [35]. Through RT-qPCR and Western blot, GPX8 mRNA and protein levels were discovered to be increased in cancer tissues and cell lines (Fig. 5A-D).

GPX8 knockdown impedes the biological functions of PC cells
For clarifying GPX8's effect on PC, sh-NC and sh-GPX8 were transfected into PANC-1 cells, and the transfection results were checked by RT-qPCR and Western blot (Fig. 6A, B). Afterwards, it was further analyzed that GPX8 depletion in PANC-1 cells blocked cell growth, prevented cells from obtaining energy for glycolysis, improved NK cytotoxicity ( Fig. 6C-J). In vivo results further con rmed the tumor suppressor effect of knocked down GPX8 in PC (Fig. 6K, L).

miR-582-5p inhibition or GPX8 overexpression reverses the effect of silenced MIR4435-2HG on inhibiting PC development
Finally, rescue experiments were set up to verify that MIR4435-2HG can mediate GPX8 through miR-582-5p to affect PC progression. Similarly, we veri ed the transfection e ciency of the cells by RT-qPCR and Western blot (Fig. 7A, B). Subsequently, it was disclosed that both miR-582-5p down-regulation and GPX8 up-regulation can reverse the inhibitory effect of silenced MIR4435-2HG on PANC-1 cell growth, glycolysis and immunity (Fig. 7C-L).

Discussion
PC remains a deadly disease with relatively low survival rate. Through an exploration of PC-related molecular mechanism, it was con rmed that silencing MIR4435-2HG hindered growth, glycolysis and tumor immunity of PC cells through serving as a decoy of miR-582-5p to down-regulate GPX8 (Figure 8).
Initially, our report realized that MIR4435-2HG was excessively expressed in cancer tissues of PC patients, and was correlated with tumor size, LNM, TNM stage, tumor differentiation and survival time of patients. Next, using the PC cell line, PANC-1, it was proved that silencing MIR4435-2HG enhanced proliferation, wound healing and invasion, and decreased cell apoptosis rate in vitro, and repressed tumor formation in vivo. Accordingly, a prediction analysis has demonstrated that the higher the MIR4435-2HG expression, the poorer the overall survival of patients with CRC [36]. In the case of GC, the association has been found between high MIR4435-2HG expression and patients' tumor stage, and the promoting role of MIR4435-2HG is re ected to augment aggressiveness of GC cells in vitro and in vivo [8]. As to hepatocellular carcinoma, MIR4435-2HG expression maintains a high level which is related to tumor size, and moreover, MIR4435-2HG overexpression triggers proliferation of cancer cells [37]. Consistent with this, a report of Haiyun Q et al. offers explanation that enriched MIR4435-2HG presents a negative correlation with LNM of patients with lung cancer, and cancer cells inhibiting MIR4435-2HG are characterized by impaired proliferation and invasion capacities [9]. Also, MIR4435-2HG down-regulation could reduce glucose consumption and lactate production, as well as increase cytotoxicity and levels of MICA and MICB, thereby inhibiting glycolysis and tumor immunity. As far as we know, the actual role of MIR4435-2HG in tumor cell glycolysis and immunity has rarely been described, which represents the innovative concept of our research.
Afterwards, miR-582-5p which was lowly expressed in PC tissues and cell lines, was discovered to be negatively regulated by MIR4435-2HG. By gene expression regulation, the tumor suppressor role of miR-582-5p was re ected in restraining cell outgrowth in vitro and in vivo, preventing glycolysis and reducing tumor immunity. On the other hand, miR-582-5p inhibition decelerated MIR4435-2HG silencing-mediated PC cell development. In fact, suppressed levels of miR-582-5p have been detected in NSCLC, and mechanistically, enforced levels of miR-582-5p in tumor cells indicate obstacles for cell growth [38]. Besides, our study results are supported by the report of Jin Y et al. which proves that elevating miR-582-5p expression in GC cells utmost weakens the proliferative property and strengthens apoptosis [39]. In particular, lowly expressed miR-582-5p has been once examined in human endometrial cancer, and treatment with restored miR-582-5p in tumor cells accounts for proliferation blockade and apoptosis promotion [40]. In salivary adenoid cystic carcinoma cells expressing up-regulated miR-582-5p, it could be noticed that the malignant phenotypes are all suppressed in vitro, and the contribution of miR-582-5p is further veri ed in vivo [41]. In an article related to bladder cancer, reduced miR-582-5p is tested in cancer samples, and miR-582-5p induction barricades cell activities in vitro and could even delays tumor formation in vivo [42]. In terms of glycolysis and immunity, miR-582-5p-related actions were seldom discussed, which shall be further probed.
In addition,our research found the targeting relation between miR-582-5p and GPX8, and further tested that GPX8 knockdown depressed the development of PC cells while GPX8 overexpression resulted in reversal of silenced MIR4435-2HG-induced effects on PC. As clinically analyzed, GPX8 high expression is an independent indicator of prognosis of breast cancer [16]. Concerning to the actual function of GPX8 in cancers, Khatib A et al. have explained that loss of GPX8 decelerates the formation and growth rate of breast cancer tumors in mice [15]. At present, the lack of studies regarding to GPX8-regulated glycolysis and immunity still awaits for deep analysis.
Shortly, our report supports that depleting of MIR4435-2HG elevates miR-582-5p and reduces GPX8 expression, so as to preventing cell growth, glycolysis and tumor immunity in PC. Our study analysis may offer a new niche for developing targeted therapy for PC. But, the downstream and upstream mechanisms of MIR4435-2HG/miR-582-5p/GPX8 axis in PC were not explored, which is the limitation of our research.

Declarations
Funding none Ethics approval and consent to participate The study was approved by the committee of West China Hospital of Sichuan University, and all subjects had signed a written consent form. Animal treatments were processed in coincidence with Animal Care and Use Guidelines of Animal Ethic Committee in West China Hospital of Sichuan University.

Con ict of interest
The Authors declare no con icts of interest directly related to the contents of this article.        Tumor volume and weight in nude mice. Measurement data were expressed as mean ± standard deviation,* P < 0.05 vs the si-MIR4435-2HG + inhibitor-NC group, ^ P < 0.05 vs the si-MIR4435-2HG + oe-NC group.

Figure 8
MIR4435-2HG promotes glycolysis and tumor immunity in PC through absorption of miR-582-5p to target GPX8.