LncRNA HIF1A-AS2 accelerates malignant phenotypes of renal carcinoma by modulating miR-30a-5p/SOX4 axis as a ceRNA

Objective: Several reports have proposed that lncRNAs, as potential biomarkers, participate in the progression and growth of malignant tumors. HIF1A-AS2 is a novel lncRNA and potential biomarker, involved in the genesis and development of carcinomas. However, the molecular mechanism of HIF1A-AS2 in renal carcinoma is unclear. Methods: The relative expression levels of HIF1A-AS2 and miR-30a-5p were detected using RT-qPCR in renal carcinoma tissues and cell lines. Using loss-of-function and overexpression, the biological effects of HIF1A-AS2 and miR-30a-5p in kidney carcinoma progression were characterized. Dual luciferase reporter gene analysis and Western blot were used to detect the potential mechanism of HIF1A-AS2 in renal carcinomas. Results: HIF1A-AS2 was upregulated in kidney carcinoma tissues when compared with para-carcinoma tissues (P < 0.05). In addition, tumor size, tumor node mestastasis stage and differentiation were identified as being closely associated with HIF1A-AS2 expression (P < 0.05). Knockdown or overexpression of HIF1A-AS2 either restrained or promoted the malignant phenotype and WNT/β-catenin signaling in renal carcinoma cells (P < 0.05). MiR-30a-5p was downregulated in renal cancers and partially reversed HIF1A-AS2 functions in malignant renal tumor cells. HIF1A-AS2 acted as a microRNA sponge that actively regulated the relative expression of SOX4 in sponging miR-30a-5p and subsequently increased the malignant phenotypes of renal carcinomas. HIF1A-AS2 showed a carcinogenic effect and miR-30a-5p acted as an antagonist of the anti-oncogene effects in the pathogenesis of renal carcinomas. Conclusions: The HIF1A-AS2-miR-30a-5p-SOX4 axis was associated with the malignant progression and development of renal carcinoma. The relative expression of HIF1A-AS2 was negatively correlated with the expression of miR-30a-5p, and was closely correlated with SOX4 mRNA levels in renal cancers.


Introduction
Malignant tumors greatly affect human health 1,2 . Renal cell carcinoma (RCC) accounts for 2%-3% of adult malignant tumors, and the prognoses of patients with advanced kidney carcinomas is very poor, especially for those individuals with distant metastasis 3 . The treatment of renal malignancies therefore represents an ongoing challenge. Various studies have reported possible mechanisms and causes of kidney carcinomas [4][5][6][7][8][9][10][11] ; however, the precise mechanism of RCC remains unclear.
The microRNAs (miRNAs) are 18-25 nucleotide non-coding RNAs, which can regulate the tumorigenesis and progression of many tumors [42][43][44][45][46][47][48][49] . The miRNAs also play a role in the evolution of species, and miR-30a-5p has been reported to play a role in many diseases, including cholangiocarcinoma 42 ; it can also act as a negative regulator, contributing to the evolution of renal carcinomas.
In the present study, HIF1A-AS2 was shown to be highly expressed in renal cancer tissues and cells, with miR-30a-5p being present only in small amounts. HIF1A-AS2 expression was closely correlated with differentiation across tumor node metastasis (TNM) stages. HIF1A-AS2 was found to facilitate renal cancer progression, while miR-30a-5p suppressed this process. Mechanistically, the upregulated expression of HIF1A-AS2 may inhibit the relative expression of miR-30a-5p, to subsequently increase the expression of SOX4 at a posttranscriptional level. Furthermore, HIF1A-AS2 functioned in a ceRNA-dependent manner to sponge miR-30a-5p to tightly regulate SOX4 expression. HIF1A-AS2 also acted as a significant tumor regulator and potential therapeutic target. The HIF1A-AS2-miR-30a-5p-SOX4 axis was involved in the progression of renal carcinomas, which highlights its possible application in clinical diagnosis and therapy.

Patient samples
Our study included kidney carcinoma patients who received tumorectomy. We quick-froze the kidney carcinoma tissues and paired normal peritumoral specimens in liquid nitrogen after resection. We received written informed consent from each patient. Our experimental protocol was approved by the Institutional Ethics Review Board of the First Affiliated Hospital of Soochow University (Approval No. 2019110).

Cell lines and cell culture
The cells were cultured in an incubator at 37 °C and 5% CO 2 . The 786-O, ACHN, OS-RC-2, and 293T cell lines were obtained from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. A total of 1% antibiotics (100 U/ mL penicillin and 100 μg/mL streptomycin sulfate) and 10% fetal bovine serum (FBS) were added into Minimal Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and RPMI 1640. The 786-O, OS-RC-2, ACHN, and 293T cells were cultivated in RPMI 1640, MEM, or DMEM.

RT-PCR
Total RNA was extracted from the specimens and renal cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) based on the product description. The cDNA was synthesized from whole RNA using the Prime Script RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). SYBR Premix Ex Taq II (Takara) was used to detect the relative expression levels of HIF1A-AS2 using RT-qPCR and the CFX96 sequence detection system (Bio-Rad, Hercules, CA, USA). Supplementary  Table S1 shows the main primer sequences. The endogenous controls were glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and U6 small nuclear RNA. A relative quantification method (2 -ΔΔCt ) was used to calculate the expressions, which were normalized to endogenous controls.

Cell proliferation assays
Cell proliferation was detected using the CCK-8 assay (Beyotime, Shanghai) based on the product description. SiRNAs or plasmids were used to transfect cells, which had been incubated for 24 h in 96-well plates. A microplate reader (Bio-Rad) was used to measure the absorbance in each well at 0, 24, 48 and 72 h after transfection.

The 5-ethyl-2′-deoxyuridine (EdU) incorporation assay
An EdU Apollo DNA in vitro kit (Ribobio, Guangzhou, China) used the EdU incorporation assay based on the product descriptions. Briefly, cells transfected with siRNA or plasmid were incubated for 2 h at 37 °C, then treated with 100 μL of 50 μM EdU per well, followed by fluorescence microscopy to visualize the cells.

Cell migration assay
The cells were transferred to 6-well plates, and were cultured in an incubator until 90%-100% confluent, followed by siRNA or plasma transfection of the cells. A 200 μL sterilized pipet tip was then used to generate clean lines in 6-well plates. Cell images were then captured using a digital camera. After 24 h, the images of cells were again captured using a digital camera.

Flow cytometry assay
SiRNAs or plasmid vectors were respectively transfected in kidney carcinoma cells. After 48 h of transfection, cells were collected and resuspended in fixation fluid, which included 5 μL annexin V-FITC, 10 μL propidium iodide, and 195 μL cell suspension. Flow cytometry (Beckman Coulter, San Jose, CA, USA) was used to detect cell apoptosis.

Western blot analysis
Total protein was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking in 5% nonfat milk, the membranes were incubated overnight for 16 h in 4 °C with the primary antibody. The membranes were then incubated for 1-2 h with a secondary antibody, and an enhanced chemiluminescence ECL kit (Beyotime, Shanghai, China) was used to visualize the bands. β-Actin, tubulin, or GAPDH were used as internal standards. The antibodies used are listed in Supplementary Table S2. Luciferase reporter assays TCF (T cell factor) transcription factor activity was used to measure canonical Wnt signaling pathway activity. TOP or FOP flash and Renilla-luciferase plasmids were used to transfect renal cells. The luciferase activity was analyzed using a DLR assay system (Promega, Madison, WI, USA). PmirGLO Dual-luciferase vectors (Fubio, Shanghai, China) were used to clone the binding and mutant sequences. HIF1A-AS2 or SOX4 wild type (WT) or mutant type (MUT) was constructed and co-transfected along with miR-30a-5p mimics or normal control (NC), then transfected with Lipofectamine 3000 and incubated for 48 h. A microplate reader was used to measure the luciferase activities.

Animal experiments
The 5-week-old male BALB/c nude mice were divided into 2 groups, with each group comprised of 6 mice. LV-NC and LV-HIF1A-AS2 were made by Gene Pharma (Shanghai). A total of 2 × 10 6 OS-RC-2 cells were injected into the mouse dorsal flank regions, and tumor growth was measured every 5 days. The formula, a × b 2 /2 (a: long diameter; b: short diameter) was used to calculate tumor volume. Finally, mice were sacrificed after 30 days, and each subcutaneous tumor was weighed. The animal experimental protocol was approved by the Ethics Committee of Soochow University (Approval No. ECSU-20190002018).

Statistical analysis
Every experimental assay was conducted at least in triplicate. The data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using SPSS statistical software for Windows, version 20.0 (SPSS, Chicago, IL, USA). The relative expression analyses of HIF1A-AS2 were conducted using the paired sample t-test. Analysis of variance was used to analyze the CCK-8 assay data. Other data analyses used the independent samples t-test. P < 0.05 was regarded as statistically significant.

LncRNAHIF1A-AS2 was mainly distributed in the cytoplasm
The cellular localizations of lncRNAs were used to assess possible functions and then revealed their potential mechanisms of action, including chromatin remodeling and translational regulation. Using the fractionation indicators of 18S RNA and U6, FISH was used to show that HIF1A-AS2 was mainly localized to the cytoplasm of kidney cell lines [ Figure 1I  Cell proliferation was also detected using EdU assays. As shown [ Figure 2D
The quantity of Edu-positive cells in the miR-30a-5p mimic group was decreased by 51.  Figure 3D (a-c)] after transfection with the miR-30a-5p inhibitor. These results showed that higher miR-30a-5p expression suppressed renal carcinoma cell migration and decreased miR-30a-5p accelerated renal carcinoma cell migration.
TOP/FOP flash is a method for the determination of intracellular beta-catenin-mediated transcription activity. TOP/FOP flash reporter assays were used to detect whether HIF1A-AS2 functioned via the WNT/β-catenin signaling pathway. Data illustrated that decreasing HIF1A-AS2 expression downregulated Wnt-activity in the 786-O and OS-RC-2 cells (Figure 5G).

Silencing of SOX4 reversed the malignant renal carcinoma cell phenotype promotion of HIF1A-AS2 overexpression
We wished to confirm whether HIF1A-AS2 regulated malignant phenotypes in a SOX4-dependent manner in renal carcinomas cells.
Together, our results indicated that HIF1A-AS2 accelerated malignant renal carcinoma cell phenotypes in a SOX4dependent manner.

Knockdown of HIF1A-AS2 decreased tumorigenicity of kidney carcinoma cells
Xenograft models were further used to determine whether HIF1A-AS2 regulated tumorigenicity of kidney carcinoma   cells. We found that knockdown of HIF1A-AS2 decreased the tumorigenicity of kidney carcinoma cells in vivo ( Figure  7A-7F). Tumors collected from mice are shown and measured ( Figure 7A). Downregulation of HIF1A-AS2 expression was significant when compared to the control group of kidney carcinoma cells in vivo ( Figure 7B). We found that LV-HIF1A-AS2 downregulated SOX4, β-catenin, Met, C-myc, cyclinD1, Fra-1, VEGF, and the expression of kidney carcinoma cells in vivo ( Figure 7C). Compared to the LV-NC treatment group, the tumor weights were less in the LV-HIF1A-AS2 group ( Figure 7D). Tumor growth of the LV-NC treatment group was faster than that in the LV-HIF1A-AS2 group ( Figure 7E). Immunohistochemistry assays showed that the relative protein expression level of HIF1A-AS2 was upregulated in the renal cancer tissues, and that the knockdown of HIF1A-AS2 downregulated SOX4 expression in vivo in renal carcinoma cells [ Figure 7F(a, b)]. These data showed that HIF1A-AS2 facilitated tumorigenicity of kidney carcinoma cells in vivo.
As shown in Figure 7G, HIF1A-AS2 was significantly upregulated in renal carcinoma cells and HIF1A-AS2 sponged miR-30a-5p to closely regulate SOX4 expression. Upregulated SOX4 protein facilitated transcription and translation of proteins operating through abnormal protein signaling pathways, and subsequently accelerating malignant renal carcinoma phenotypes.

Discussion
Studies have reported that lncRNAs are abnormally expressed RNAs with more than 200 nucleotides, which play special roles in various diseases, especially malignant tumor formation 12-28 . As key components in regulating gene expression and tumor progression, studies of lncRNAs have expanded our understanding of their biological behavior during diseases, especially during carcinoma [18][19][20][21][22][23][24][25][26][27][28] . In addition, reports have shown that the lncRNAs are significant biomarkers and possible treatment targets.
LncRNA HIF1A-AS2 is located on chromosome 14, NC_000014.9, and has been found to be overexpressed, to act as an oncogene in many tumor tissues, including gastric carcinoma 29 , triple-negative breast carcinoma [30][31][32][33] , bladder carcinoma 34,35 , glioblastoma multiforme 36 and osteosarcoma 38 . HIF1A-AS2 is also involved in the progression of diseases in tissues, such as human umbilical vein endothelial cells, colorectal cancer, adipose used to extract stem cells, coronary artery disease, and preeclampsia 37,39,40 . In most cases, HIF1A-AS2 is involved in the progression and tumorigenesis of carcinomas and functions as an oncogene. Knowledge of the basic structures and interactions of lncRNAs with other cellular biomolecules can provide direction for further research to reveal the mechanism of tumorigenesis and tumor progression. However, the relationship between HIF1A-AS2 and kidney carcinoma has been rarely reported. Studies have reported a mutual function between lncRNAs and miRNAs [50][51][52][53][54][55][56] , which was further confirmed by our report. LncRNAs act as miRNAs sponges or baits to titrate miRNA concentrations, thereby decreasing and preventing miRNAs from binding to specific mRNAs. Studies have also reported the roles of HIF1A-AS2 in tumors, such as HIF1A-AS2 targeting miR-548c-3p in breast cancer 31 , sponging miR-129-5p in colorectal cancer and miR-33b-5p in osteosarcoma 37,41 , sponging miR-665 in osteogenic differentiation 38 , and sponging miR-153-3p in human umbilical vein cells 56 . Our findings were consistent with these existing reports.
SOX4, the target gene of miR-30a-5p, was predicted by bioinformatics analyses in addition to our experimental studies. SOX4 belongs to the SOX transcription factor family, and binds to the A/TA/TCAAG motif to regulate target gene transcriptional activity via the high mobility group domain, which regulates various biological functions, such as embryonic development and cell progression [51][52][53][54][55] . It regulates cell differentiation, proliferation, and metastasis. SOX4 via the lnc01694/miR-340-5p/Sox4 axis regulates gallbladder cancer 51 . SOX4 is regulated by SNHGR-miR-489-3p competitive binding in acute myeloid leukemia 52 . Sox4 is targeted by lnc-NNT-AS1/miR-142-5p axis in gastric cancer 53 , and lnc-FTX/ miR-214-5p axis in the osteosarcoma 54 . SOX4 also participates in the epithelial-mesenchymal transition via lncRNA HCP5/ miR-140-5p sponging 55 . Our results further elucidated the role of HIF1A-AS2 in renal carcinoma, and expanded the knowledge of the role of HIF1A-AS2 in diverse diseases.
A correlation with miR-30a-5p and HIF1A-AS2 in renal carcinomas remains unknown. Through bioinformatics analyses, we identified a putative binding site between HIF1A-AS2 and miR-30a-5p, and found that HIF1A-AS2 acted as the sponge of miR-30a-5p, which directly bound to miR-30a-5p. Furthermore, HIF1A-AS2 overexpression led to downregulation of miR-30a-5p, and HIF1A-AS2 knockdown upregulated the relative expression level of miR-30a-5p. The MiR-30a-5p inhibitor partially reversed the effects, and miR-30a-5p mimics enhanced the effects induced by HIF1A-AS2 knockdown on renal carcinoma cells. The miR-30a-5p directly targeted SOX4 to reduce the relative protein expression levels of SOX4 in renal carcinomas. Likewise, the relative protein expression level of SOX4 was upregulated during HIF1A-AS2 overexpression, to mediate the function of HIF1A-AS2. Overexpression of HIF1A-AS2 reduced the relative expression of miR-30a-5p and subsequently increased the relative protein expression of SOX4 at the post-transcriptional level in renal cancer cells. Silencing of SOX4 reversed the malignant renal carcinoma cell phenotype promoted by overexpressed HIF1A-AS2.
We elucidated the mutual function of HIF1A-AS2 and miR-30a-5p in renal carcinomas, which could provide a novel biomarker and therapeutic target for the diagnosis and treatment of renal carcinomas. We also demonstrated that the relative expression of HIF1A-AS2 was significantly increased in renal carcinoma samples and cell lines. The relative expression of HIF1A-AS2 was positively correlated with differentiation and the TNM stage in renal carcinomas. Downregulated expression of HIF1A-AS2 inhibited renal carcinoma cell proliferation or migration, and upregulated apoptosis. Overexpression of HIF1A-AS2 showed the opposite effect. We have demonstrated that HIF1A-AS2 enhanced the development and progression of renal cancers by promoting cell proliferation, migration, and reducing apoptosis. Furthermore, HIF1A-AS2 sponged miR-30a-5p in a ceRNA-dependent manner. Mechanistically, upregulation of HIF1A-AS2 decreased the relative expression of miR-30a-5p and subsequently promoted the relative expression of SOX4 and WNT signaling at the posttranscriptional level. In summary, our study showed that HIF1A-AS2 acted as a tumor promoter by miRNA sponging, and may therefore be a potential therapeutic target in renal carcinomas.

Conclusions
The results showed that HIF1A-AS2 sponged miR-30a-5p to closely regulate SOX4 expression, and subsequently accelerated the malignant phenotypes of renal carcinoma cells and Wnt/β-catenin signaling, acting as an oncogene in the pathogenic mechanism of kidney carcinomas. Our results provide useful pathways to further explore the pathogenesis of renal carcinoma progression and development. In conclusion, the results showed that the HIF1A-AS2-miR-30a-5p-SOX4 axis played significant roles in the progression and development of renal carcinomas. HIF1A-AS2 and miR-30a-5p were the novel and important tumor biomarkers, which could be used as diagnostic biomarkers and remedial targets for malignant renal carcinomas.