LINC01088/miR-22/CDC6 Axis Regulates Prostate Cancer Progression by Activating the PI3K/AKT Pathway

Background Prostate cancer (PCa) harms the male reproductive system, and lncRNA may play an important role in it. Here, we report that the LINC01088/microRNA- (miRNA/miR-) 22/cell division cycle 6 (CDC6) axis regulated through the phosphatidylinositide 3-kinases- (PI3K-) protein kinase B (AKT) signaling pathway controls the development of PCa. Methods lncRNA/miRNA/mRNA associated with PCa was downloaded and analyzed by Gene Expression Omnibus. The expression and correlation of LINC01088/miR-22/CDC6 in PCa were analyzed and verified by RT-qPCR. Dual-luciferase was used to analyze the binding between miR-22 and LINC01088 or CDC6. Cell Counting Kit-8 and Transwell were used to analyze the effects of LINC01088/miR-22/CDC6 interactions on PCa cell viability or migration/invasion ability. Localization of LINC01088 in cells was analyzed by nuclear cytoplasmic separation. The effect of LINC01088/miR-22/CDC6 interaction on downstream PI3K/AKT signaling was analyzed by Western blot. Results LINC01088 or CDC6 was upregulated in prostate tumor tissues or cells, whereas miR-22 was downregulated, miR-22 directly targets both LINC01088 and CDC6. si-LINC01088 inhibits the PCa process by suppressing the PI3K/AKT pathway. CDC6 reverses si-linc01088-mediated cell growth inhibition and reduction of PI3K and AKT protein levels. Conclusion Our results demonstrate that the LINC01088/miR-22/CDC6 axis functions in PCa progression and provide a promising diagnostic and therapeutic target.


Introduction
Prostate cancer (PCa) is a cancer that affects men and is very common worldwide. It accounts for 21% of all new cases of cancer in men. Unfortunately, it causes significant illness and death in those affected [1,2]. Early systematic screening has a very positive effect on the prevention and treatment of prostate cancer, and the available set of PC biomarkers is growing [3]; there is still a significant unmet need for new and more effective treatments.
Cell division cycle 6 (CDC6) is a novel cancer target that regulates the DNA replication process and is considered an early indicator of malignancy [19]. CDC6 has been recognized as a cancer diagnosis biomarker in various cancers [20,21]. For instance, Mahadevappa et al. found the prognostic significance of CDC6 in breast cancer [22]. However, only few studies have investigated the CDC6 expression in PCa [23]; therefore, CDC6 requires further investigation.
The AR and frequent activation of PI3K signaling are key factors in the progression of PCa [24]. The PI3K/AKT signaling pathway, which is cross-regulated through several reciprocal inhibitory loops with AR pathways, is the driver of prostate tumor development [25,26]. Recent research by Tan et al. showed that PI3K/AKT axis inactivation suppresses prostate tumorigenesis [27]. Dai et al. confirm that inhibition of the PI3K/AKT signaling pathway facilitates PCa cell autophagy [28]. PI3K and AKT inhibitors will soon be introduced as antitumor drugs and are currently in preclinical and clinical development [29][30][31].
This study is aimed at understanding the interaction and mechanism of the LINC01088/miR-22/CDC6 axis and exploring its possibility as a biomarker for systematic screening of PCa.

Materials and Methods
2.1. Samples. Ten pairs of fresh frozen PCa and adjacent tissues were obtained from the Longgang District People's Hospital of Shenzhen (Shenzhen, China), along with pathological information. All patients were histologically confirmed to have a prostate tumor. The experiments was approved by the institutional ethics committees of Longgang District People's Hospital of Shenzhen (Shenzhen, China; approval no. 2022071) and conducted in accordance with the Declaration of Helsinki.

Microarray Raw Data
Analyses. Datasets were from Gene Expression Omnibus. The series (GSE104749) dataset were utilized to analyze lncRNA/mRNA expression profiles in PCa and categorized as follows: control (4 benign prostatic hyperplasia (BPH) fine-needle aspiration biopsy tissues), and PCa (4 PCa fine-needle aspiration biopsy tissues). The GEO series (GSE45604) dataset was utilized to analyze miRNA expression profiles in PCa and categorized as follows: normal (10 normal prostate tissues) and PCa (10 PCa tissues). Expression of dysregulated RNA was identified to meet the jfold changej ≥ 1:5 criteria. GEPIA2 was used for The Cancer Genome Atlas (TCGA) survival analysis. Candidate genes and cancer subtypes were chosen as the inputs to generate survival curves for disease-free survival. Statistical significance was defined as P < 0:05.

Statistical
Analysis. Mean ± SD values were used to present the data, and GraphPad Prism 9 was utilized for statistical analysis. Initially, one-way ANOVA was performed, followed by a Bonferroni post hoc test to establish the presence of a statistically significant change overall. Subsequently, a Student's t-test was employed to examine the difference between any two groups. Statistical significance was defined as P < 0:05.

LINC01088 Is Upregulated in PCa.
We conducted a comprehensive inquiry into the potential role of a recently discovered long noncoding RNA (lncRNA) in the advancement of prostate cancer (PCa). Our investigation primarily involved lncRNA chip data, which revealed that 45 differentially expressed transcripts lncRNAs, were identified in PCa specimens compared to the control group. Of these transcripts, LINC01088 exhibited the most significant log-fold change (logFC) value, thereby implying its potential as a target for further research (Figure 1(a)). Additionally, we validated the overexpression of LINC01088 in PCa tissues (Figure 1(b)), which was linked with decreased disease-free survival in PCa patients, and associated with diverse clinicopathological features of PCa (Figure 1(c)). The expression of LINC01088 was notably heightened in PCa cells in

Mediators of Inflammation
PCa Control   Mediators of Inflammation comparison to RWPE-2 cells (Figure 1(d)), particularly in PC3 and LNCAP cells, highlighting the significant role of LINC01088 in PCa progression. Subsequent findings illustrated that siRNA-LINC01088-1 was the most potent inhibitor of LINC01088 among the three siRNAs in PC3 and LNCAP cells, and we used it as an antagonist for further molecular mechanistic studies (Figure 1(e)). Our findings indicate that the reduction of LINC01088 expression results in a diminished cellular viability, as well as decreased migratory and invasive capacity of both PC3 and LNCAP cell lines (as depicted in Figures 1(f)-1(i)). Moreover, based on our observations, LINC01088 is preferentially localized in the cytoplasmic region of these cells (Figure 1(j)), implying that its effect on prostate cancer pathophysiology may be mediated by a competing endogenous RNA (ceRNA) mechanism.

LINC01088 Directly Targeted miR-22.
Based on miRNA chip data analysis results, 64 differentially expressed hsa-miRNAs were identified with 5 upregulated and 59 downregulated hsa-miRNAs using 10 PCa and 10 normal prostate samples (Figure 2(a)), the joint analysis of LncBase v3 database and miRNA chip data showed that miR-22 was the only intersection among the down-regulated miRNAs (Figure 2(b)) and miR-22 was downregulated in both tissues and cells of PCa (Figures 2(c) and 2(d)). Moreover, suppressing the expression of LINC01088 increased the expression of miR-22 (Figure 2(e)). Subsequent experiments confirmed that LINC01088 directly targets miR-22 (Figure 2(f)). Here, we confirmed that LINC01088 can adsorb miR-22 to regulate PCa development.

CDC6 Directly Targeted miR-22.
Given that miRNAs possess the capacity to regulate transcription and translation via the binding to the 3′UTR of mRNAs [33], we analyzed PCa-related differentially expressed mRNAs in the GEO dataset. The mRNA chip data analysis showed that 1,067 oncogenes were upregulated (Figure 3(a)). The combined analysis of the TargetScan database and TarBase V.8 database showed that 20 genes had overlapped, among which 2 genes had potential binding sites, namely, VASH1 and CDC6 (Figure 3(b)). A previous investigation revealed that VASH1 was downregulated in PCa [34], while the augmented expression of CDC6 conspicuously truncated the disease-free survival of PCa patients (Figure 3(c)). Therefore, we designated the CDC6 gene as a plausible target for subsequent verification. Our finding substantiated that CDC6 was highly expressed within PCa tissues and cells (Figures 3(d) and 3(e)). In addition, our dual-luciferase assay validated that CDC6 directly targeted miR-22 (Figure 3(f)). Following the suppression of LINC01088, the expression of CDC6 was downregulated within PC3 and LNCAP cells (Figure 3(g)).

LINC01088 Is
Positively Correlated with CDC6. The association between LINC01088 and CDC6 was verified by constructing an overexpression plasmid for CDC6 (ov-CDC6). Results indicated that CDC6 were upregulated in ov-CDC6-transfected PC3 and LNCAP cells, confirming the effectiveness of CDC6 overexpressed plasmid (Figure 3(h)). In addition, after cotransfection of PC3 and LNCAP cells with ov-CDC6 and si-LINC01088, excessive CDC6 reversed the inhibition of LINC01088 expression by   Mediators of Inflammation si-LINC01088. (Figure 3(i)). Thus, miR-22 directly targets both LINC01088 and CDC6, and LINC01088 is positively correlated with CDC6.
3.5. The LINC01088/miR-22/CDC6 Axis Affects PCa Development through PI3K/AKT Signaling. Next, to identify the key regulatory signaling pathway, we analyzed enriched signaling pathways associated with the differentially expressed genes using the DAVID database. Among the top 10 enriched pathways, the PI3K/AKT pathway associated with 45 participating differential genes, which is the highest number of genes, was selected (Figure 4(a)). Based on Western blotting, knockdown of LINC01088 in PC3 and LNCAP cells resulted in decreased CDC6, p-PI3K, and p-AKT protein levels (Figure 4(b)). ov-CDC6 reversed the effects of LINC01088 (Figure 4(c)). Moreover, si-LINC01088 can inhibit the  Mediators of Inflammation

Discussion
Our aim was to identify a novel lncRNA with potential therapeutic value in the treatment of PCa. Initially, we investigated the oncogenic function of LINC01088 and CDC6 and the tumor-suppressing role of miR-22 in PCa. LINC01088 and CDC6 expression were both upregulated in PCa tissues and cells, unlike miR-22, promoting PCa cell growth in vitro. We discovered that LINC01088 sponges miR-22, CDC6 is the target of miR-22, and LINC01088 is coordinately upregulated with CDC6 in PCa progression. Furthermore, LINC01088 activates the PI3K/AKT pathway.
LINC01088 is an influential regulatory factor in various cancers [8,9]. For instance, Peng et al. discovered that LINC01088 promotes glioma progression [35], and Liu et al. observed that LINC01088 promotes proliferation and migration of non-small-cell lung cancer [8]. In a similar vein, our research showed that LINC01088 is upregulated in PCa, and its overexpression significantly reduces the disease-free survival of PCa patients. LINC01088 promotes PCa cell viability rate and migration/invasion via the PI3K/ AKT signaling, indicating that LINC01088 has an oncogenic role in promoting PCa development. When LINC01088 was suppressed, the growth of PCa cells is restricted. Furthermore, LINC01088 plays a role as a ceRNA, a miRNA sponging factor, in cancers. For instance, Zang et al. found that LINC01088 inhibits the tumorigenesis of ovarian epithelial cells by sponging miR-24-1-5p [36], and Li et al. found that LINC01088 plays a ceRNA role by sponging miR-22 in the progression of colorectal cancer [9]. In our study, we demonstrated that LINC01088 sponges miR-22 and negatively regulative miR-22 in PCa. Moreover, suppressing the expression of LINC01088 promoted the expression of miR-22 in PCa cells.
miR-22 regulates the target gene to affect posttranscription or translation in PCa progression [13,14]. Bioinformatics predicted that miR-22 targets CDC6, and the target mechanisms were proved by dual-luciferase reporter

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Mediators of Inflammation assay. Furthermore, CDC6 expression was negatively correlated with miR-22 expression in PCa tissues and cells.
Notably, CDC6 promotes PCa progression [37,38]. Moreover, LINC01088 is coordinately upregulated with miRNA target genes in the ceRNA axis. In our study, LINC01088 was coordinately upregulated with CDC6, si-LINC01088 reduced CDC6 expression in PCa cells, and CDC6 reversed si-LINC01088-mediated PI3K and AKT protein expression reduction in PCa cells. In addition, the slow growth of PCa cells caused by the inhibition of LINC01088 expression was significantly alleviated by the overexpression of CDC6. These results suggest that LINC01088 exerts oncogenic effects through the miR-22/ CDC6 axis. The PI3K/AKT pathway directly controls PCa [39,40]. Shorning et al. found the PI3K-AKT signaling and PCa at the intersection of AR-MAPK-Wnt [41]. In the present study, at the time of suppression of cell viability rate, promotion of cell apoptosis, and suppression of migration/invasion, PI3K/AKT phosphorylation levels were reduced. LINC01088, miR-22, and CDC6 control the PI3K/AKT pathways to regulate PCa progression. si-LINC01088 or miR-22 suppressed PCa cell growth and decreased PI3K/ AKT phosphorylation levels. CDC6 promoted cancer cell growth and enhanced PI3K/AKT signaling and reversed the effect of si-LINC01088 on PCa cells.
Interlaboratory variability may also affect the development of therapeutic strategies targeting the LINC01088/ miR-22/CDC6 axis. Despite the potential limitations, this study provides a foundation for further research on the LINC01088/miR-22/CDC6 axis as a potential therapeutic target for PCa treatment. Future studies can build upon these findings and investigate the molecular mechanisms underlying the regulatory interactions among LINC01088, miR-22, and CDC6 in PCa. Therefore, it is important to conduct validation studies to confirm the therapeutic potential of this axis across multiple laboratories and patient populations before initiating clinical trials.
In conclusion, this study confirms the LINC01088/miR-22/CDC6 axis as a potential therapeutic target, providing more directions and theoretical basis for the treatment of PCa.