lncRNA H19 acts as a ceRNA to regulate the expression of CTGF by targeting miR-19b in polycystic ovary syndrome

The etiology of polycystic ovary syndrome (PCOS) is complex and the pathogenesis is not fully understood. Some studies have shown that dysregulation of ovarian granulosa cells may be related to abnormal follicles and excessive androgen in women with PCOS. Our team has also confirmed the high expression status of H19 in PCOS patients in the early stage. However, the relationship between H19 and miR-19b in the development of PCOS is still unknown. Therefore, we used bioinformatics to predict the binding sites of human H19 and miR-19b, and of miR-19b and CTGF genes. After the silencing and overexpression of H19, real-time polymerase chain reaction (PCR) was used to detect the expressions of H19, miR-19b, and CTGF. Western blotting was used to detect CTGF protein. Proliferation of KGN cells after H19 silencing was detected by CCK8. Flow cytometry was used to detect the apoptosis of KGN cells after H19 silencing. After the overexpression of H19, it was found that the expression of miR-19b gene decreased and the expression of CTGF increased, whereas silencing of H19 did the opposite. In addition, H19 could promote cell proliferation and decrease cell apoptosis. Finally, luciferase reporter assays showed that the 3′-end sequences of lncRNA H19 and CTGF contained the binding site of miR-19b. In conclusion, our study indicated that lncRNA H19 acted as a ceRNA to bind to miR-19b via a “sponge” to regulate the effect of CTGF on KGN cells, which may play a vital role in PCOS.


Introduction
Polycystic ovary syndrome (PCOS) is a highly heterogeneous and complex disease that is one of the most common reproductive endocrine diseases in gynecology (1). It affects both birth and metabolism. Its incidence is rising, and the number of patients is large, reaching 5-10% of women of childbearing age (2). The etiology and pathogenesis of PCOS have become hotspots in many disciplines such as obstetrics and gynecology, endocrinology, and reproductive science. Although the exact cause of PCOS is still not fully understood, the survival and proliferation of granulosa cells may be the cause of PCOS (3). There is evidence that dysfunction of granulosa cells may lead to the formation of abnormal follicles in PCOS patients, but the specific mechanism remains to be determined. For PCOS, a large number of studies have confirmed that CTGF is highly expressed in the ovaries of PCOS patients and PCOS animal models (4).
Experiments have confirmed that CTGF is involved in PCOS granulosa cell proliferation, and high expression of CTGF promotes granulosa cell proliferation and inhibits apoptosis (5).
Noncoding RNA (ncRNA) is an RNA that does not encode a protein in the body. In the last decade, with the development of sequencing technology, most RNA transcripts do not encode proteins. Depending on the number of RNAs, it can be divided into short non-coding RNAs (such as miRNAs) and long non-coding RNAs (such as lncRNA, circRNA, etc.) (6). MicroRNAs (miRNAs) are a class of highly conserved, non-coding, short-stranded RNAs that have approximately 20-23 nucleotides in length. miRNAs regulate post-transcriptionality by base pairing with the 3 0 untranslated region (UTR) of the target mRNA (7). Horizontal mRNA transcription and translation affect biological function. lncRNAs are a class of RNAs that are more than 200 nucleotides in length and have limited protein-coding potential (8). Studies have shown that lncRNAs have many functions in various pathophysiological processes. The dysfunction of IncRNAs plays a key role in many diseases. The function of lncRNA is complex. In 2011, Karreth et al. (9) proposed a theory called competitive endogenous RNA (ceRNA) hypothesis that lncRNAs, mRNAs, and pseudogenes can competitively bind to microRNA response elements (MRE) and lead to mutual regulation of expression, which provides a new gene regulation mechanism.
In recent years, in order to more deeply explore the etiology of PCOS from the epigenetics, researchers are beginning to pay attention to the relationship between non-coding RNAs and PCOS. Huang et al. (10) reported that lncRNA H19 competes with STAT3 for binding to miR-19, which in turn affects STAT3 expression. Zhong et al. (11) found that in PCOS, miR-19b is lowly expressed in ovarian tissue and granulosa cells, and miR-19b can regulate the proliferation of granulosa cells. However, there are no reports on the interaction between H19 and miR-19b in PCOS. Therefore, this study focused on the relationship between H19 and miR-19b in KGN cells, its effect on gene CTGF, and explored the role of H19 in the development of PCOS.

Material and Methods
Cell recovery and culture A thermostatic water bath was preheated to 37°C, and the table top was wiped and ultra-cleaned with 75% ethanol beforehand. Then, an ultraviolet light was turned on to illuminate the table for 30 min. The cryotube was quickly placed into a 37°C constant temperature water bath, and shaken continuously, so that the cell cryopreservation medium in the cryotube was melted rapidly and taken out. The tube was centrifuged at 800 g for 5 min at 4°C; the supernatant was discarded, 1-mL of medium was added, and the cells were shaken as appropriate to resuspend the cells. The cells were then placed in culture dishes, which were placed in a carbon dioxide incubator at 37°C for constant temperature culture for 48 h, and passed 2-3 generations to prove that the cells were viable. The culture solution was continuously changed according to the growth of the cells. The cells were rinsed in the cell culture flask 1-2 times with PBS water. A 1.5-mL trypsin-EDTA solution was added to the flask to lightly wash the cell culture dish. The trypsin-EDTA solution was discarded and the cells digested in a 37°C carbon dioxide incubator for 3 min. If cells were found to be in a circular state, 1-mL of medium was added for further digestion. The cell solution was repeatedly pipetted with a 1-mL pipette to mix the cells, and the culture solution of each flask was replenished, transferred to a new flask, and the culture solution was added in a diluted ratio.

Real-time PCR
One millileter of Trizol was added to the cells, and the tube was shaken and mixed at 4°C for 5 min. Chloroform (0.2 mL) was added, the tube was vigorously shaken for 15 s, and then allowed to stand at 4°C for 3 min. An equal volume of isopropanol was added, mixed, and left to stand at -20°C for 20 min; the tube was then centrifuged at 12,000 g for 15 min at 4°C, and the supernatant removed. Centrifugation was repeated at 8000 g for 5 min at 4°C, and 1.5 mL of the sample solution was measured for concentration in an ultra-micro UV analyzer. Reverse transcription was performed as described in the Bestar TM qPCR RT Kit (DBI, Germany).

Cell transfection
KGN cells (80%) were removed and the old medium was aspirated. The cells were washed one or two times with PBS, trypsin solution was added, and then aspirated when the cells were separated and round pellets appeared. Fresh complete medium was added, and the cells were pipetted well to prepare a single-cell suspension. The cell density was adjusted to 8 Â 10 4 /mL and 0.25 mL of siRNA or 0.2 mg of plasmid was added to 50 mL of serumfree medium, gently mixed, and allowed to stand at room temperature for 5 min. Lipofectamine TM 2000 (0.25 mL; Thermo Fisher Scientific, USA) was added to 50 mL of serum-free medium, gently mixed, and allowed to stand at room temperature for 5 min. The diluted siRNA or plasmid was mixed with Lipofectamine TM 2000, gently mixed, and allowed to stand at room temperature for 20 min to form a plasmid/Lipofectamine TM 2000 complex. siRNA (100 mL) or plasmid/Lipofectamine TM 2000 complex was added to the wells of the corresponding group of culture plates at a final concentration of 50 nM; after 6 h, the cells were replaced with complete medium for 48 h. The samples were subjected to qPCR and western blot detection to screen for optimal sequences.

Cell proliferation
Cell viability was determined using Cell Counting Kit-8 (CCK8) (Dojindo, Japan) according to the instructions of the manufacturer. In brief, KGN cells during logarithmic growth period were seeded into 96-well microtiter plates at a density of 5 Â 10 3 cells/well. Then, cells were transfected with indicated plasmids or oligonucleotides. After transfection for 48 h, 10 mL of CCK-8 reagent was added to each well and incubated for another 4 h at 37°C and 5% CO 2 . Absorbance at 450 nm was measured to assess relative cell viability using a microplate reader (Bio-Rad, USA).

Apoptosis
Cell apoptosis was examined using an Annexin V-FITC apoptosis detection kit (Abcam, UK) following the manufacturer's instructions. KGN cells at a density of 1 Â10 5 cells/well were rinsed twice with cold PBS solution and resuspended in 1 Â binding buffer. Afterwards, 5 mL of Annexin V-FITC and 10 mL of propidium iodide (PI) were introduced and incubated for 10 min in the dark. Finally, apoptotic cells were determined using a flow cytometer (BD Biosciences, USA)

Luciferase reporter assay
Bioinformatics analysis using Starbase online software (Sun Yat-sen University, China) first proved the existence of putative binding sites between H19 and miR-19b. For luciferase assay, partial fragments of H19 containing the assumed miR-19b binding sites were amplified and sub-cloned into psiCHECK-2 vector (Promega, USA) to generate wild-type H19 plasmid (H19-WT). Then, a Site-Directed Mutagenesis Kit (Thermo Fisher Scientific) was used to generate mutated H19 plasmid (H19-MUT). H19-WT and H19-MUT constructs were co-transfected into KGN cells along with miR-19b mimics or miR-NC (negative control). About 48-h post-transfection, relative luciferase activity was determined using the dual-luciferase reporter assay system (Promega).

Statistical analysis
All experiments were performed three times. Statistical analysis was conducted using SPSS 20.0 (IBM Corp., USA), and results are reported as means ± SD. Student's t-test or one-way analysis of variance was carried out to estimate significant group differences. Po0.05 represented a statistically significant result.

H19 overexpression recombinant plasmid and H19 silencing site
Total RNA was extracted from KGN cells, and then reversed transcribed into cDNA. PCR amplified H19 by designed primers, and the product was electrophoresed, and the amplified product was 2362 bp ( Figure 1A). H19 was connected to pcDNA3.0+ vector to construct H19-pcDNA 3.0+ recombinant vector and transfer it to DH5a competent cells. Monoclonal colonies were selected and restriction enzymes were used to digest the recombinant vector for PCR identification ( Figure 1B).
Three silent sites, 1552, 1617, and 1712, were selected to silence H19, and the corresponding siRNA was designed and transfected into KGN cells. Compared with the control group, the expression of H19 was significantly decreased after silencing of H19 by siH19-1552, siH19-1617, and siH19-1712 (Po0.05). Among the three silent sites, the expression of siH19-1617 was significantly more pronounced after silencing H19, and the silencing effect was the best of the three ( Figure 1C).

Overexpression of lncRNA H19 in KGN cells promoted the expression of CTGF
After transfecting the constructed H19 overexpression plasmid into KGN cells, H19, miR-19b, and CTGF were detected at the RNA level and protein expression by qRT-PCR and Western blot in the presence of H19 overexpression.
Compared with the pcDNA3.0+NC group, the expression of H19 transfected with the H19 overexpression plasmid group was significantly increased (Po0.05) (Figure 2A). Compared with the pcDNA3.0+NC group, miR-19b transfected with the H19 overexpression plasmid group was significantly decreased (Po0.05) ( Figure 2B). Compared with the pcDNA3.0+NC group, CTGF transfected with the H19 overexpression plasmid group was significantly increased (Po0.05) ( Figure 2C). The expression of CTGF protein was consistent with the expression of CTGF at the RNA level. After H19 overexpression, the expression of CTGF protein was also significantly increased ( Figure 2D).

Overexpression of H19 promoted KGN cell proliferation and inhibited KGN cell apoptosis
Compared with the control group, KGN cells in the overexpressed group had enhanced proliferative capacity at 48 and 72 h, and the apoptotic ability was weakened ( Figure 3, Supplementary Figure S1). The results indicated that high expression of H19 had an effect on promoting proliferation and inhibiting apoptosis in KGN cells.

Silencing lncRNA H19 in KGN cells inhibited the expression of CTGF
Compared with the corresponding siRNA control group, the expression of miR-19b in KGN cells transfected with siH19-1617 was significantly increased (Po0.05). The CTGF reduction of KGN cells transfected with siH19-1617 was significant (Po0.05) compared to the corresponding siRNA control group. The CTGF protein results were consistent with the results of qRT-PCR. After silencing H19, the expression of CTGF protein was decreased ( Figure 4A-C).

Low expression of H19 inhibited KGN cell proliferation and promoted KGN cell apoptosis
Compared with the siRNA control group, after transfection of siH19-1617 to KGN cells, the proliferation of KGN cells was weakened at 48 and 72 h, and apoptosis was enhanced ( Figure 4D-E, Supplementary Figure S2). The experimental results showed that the low expression of H19 inhibited the proliferation of KGN cells and promoted the apoptosis of KGN cells.

Dual luciferase reporter gene
Compared with the control group, the miR-19b mimics co-transfected with wild-type H19 or 3 0 UTR CTGF inhibited the fluorescence activity of H19 and CTGF (Po0.05). After mutation of the binding site sequence, miR-19b mimics had no effect on the fluorescence activity of the mutant H19 and CTGF (P40.05). The results of the dual luciferase reporter gene showed that the binding mode between lncRNA H19 and miR-19b and between miR-19b and CTGF were both directly combined targeted adsorption ( Figure 5).

Discussion
PCOS is a common reproductive endocrine disease in gynecology. Anovulatory infertility caused by PCOS is a serious problem for women of childbearing age (12). PCOS not only affects female fertility but also affects multiple organs in the body (13,14). Currently, there is no effective drug to cure PCOS completely, and many patients suffer from PCOS for life. The pathogenesis of polycystic ovary syndrome is complex, and in-depth study of its specific molecular regulation mechanism can provide a useful target for the diagnosis and treatment of PCOS.
In our previous study, we found that lncRNA H19 was highly expressed in peripheral blood of patients with PCOS (15). There are many studies on CTGF in PCOS  that confirm that CTGF is highly expressed in PCOS (16), but the reason why is unknown. Is there an upstream regulatory factor of the highly expressed CTGF? Is noncoding RNA involved in the regulation of CTGF expression? What role does lncRNA H19 play in the regulation of CTGF expression? These issues require in-depth study.
Follicular dysplasia can be observed in patients with PCOS, and granulosa cells around the follicle play a key role in follicular development (17). An important clinical feature of PCOS is infertility caused by abnormal follicular development in women of childbearing age, which is also the main cause of PCOS in patients (17). During growth and development of the oocyte, the oocyte is closely related to the surrounding granulosa cells (18). The nutrients during the growth and development of the oocyte are provided by the surrounding granulosa cells, and the granulosa cells provide growth regulators to promote oocyte maturation (19). The ovaries of PCOS patients contain twice as many follicles at various stages of development. The number of granulosa cells in the ovary of anovulatory PCOS patients is increased compared to the number of granulosa cells in normal ovaries. These conclusions are also confirmed in the PCOS animal model. The relationship between CTGF and granulosa cell proliferation phenotype involved in this experiment has been clarified. High expression of CTGF can promote granulosa cell proliferation and inhibit granulosa cell apoptosis (20).
The experimental results of our KGN cell study showed that overexpression of H19 promoted the expression of CTGF and inhibited the expression of miR-19b. By inhibiting the expression of H19 by siRNA, the expression of CTGF can be inhibited and the expression of miR-19b can be promoted. CTGF protein level also confirmed the results of changes in CTGF at the RNA level. We studied the effects of lncRNA H19 on proliferation and apoptosis of KGN cells by CCK8 and flow cytometry. With overexpression of H19, KGN cells increased proliferation and decreased apoptosis; with silencing of H19, KGN cells decreased proliferation and increased apoptosis. The results showed that lncRNA H19 can affect the downstream molecular CTGF in KGN cells, and abnormally expressed CTGF can participate in the pathogenesis of PCOS granulosa cell proliferation and ovarian fibrosis. lncRNA H19 ultimately affected the pathogenesis of  PCOS by regulating the expression of CTGF. We found that the direction of regulation of lncRNA H19 was consistent with that of CTGF. With overexpression of lncRNA, H19 and CTGF were also highly expressed. The regulation of lncRNA H19 was opposite to that of miR-19b. After overexpression of lncRNA H19, miR-19b expression was decreased. After silencing lncRNA H19, the situation also satisfied the above. This regulatory trend was consistent with ceRNA. In ceRNA, the regulation trend of lncRNA and mRNA was the same, and the regulation trend of lncRNA and miRNA was the opposite. This provides a basis for our subsequent research on ceRNA mechanisms.
In order to solve the hypothesis that lncRNA H19 directly binds to miR-19b via ceRNA mechanism to regulate CTGF, we verified by dual luciferase assay that miR-19b directly targeted lncRNA H19 and CTGF. Our study showed that lncRNA H19 can bind to miR-19b, and the binding ability of miR-19b and CTGF was reduced, which reduced the inhibition of CTGF gene expression by miR-19b, thereby promoting the expression of CTGF. In conclusion, lncRNA H19 affected the expression of CTGF by competitively binding miR-19b, and this abnormally expressed CTGF was involved in KGN miR-19b cells proliferation.
In this study, we found that was an intermediate molecule in the lncRNA H19/miR-19b/CTGF regulatory axis. lncRNA H19 regulated CTGF expression via the 'intermediate bridge' miR-19b. miR-19b acted as a 'middleman' in this ceRNA regulatory axis. This is the first time that the proliferation of granulosa cells from the direction of ceRNA regulation mechanisms was explored. In previous studies, the researchers found that miRNAs can regulate the downstream target gene mRNA, which affects granulosa cell proliferation. In this experiment, we further explored the upstream molecules, finding that lncRNA-miRNA-mRNA regulatory axis regulated the proliferation of granulosa cells.
In summary, this experiment confirmed the role of lncRNA H19 as a ceRNA in KGN cells, which regulated the expression of CTGF through the lncRNA H19/miR-19b/CTGF regulatory axis, and finally played an important role in the pathogenesis of PCOS. However, it should be noted that the ceRNA regulatory network is complex (21,22). Each molecule that can act as a ceRNA (such as lncRNA, circRNA, etc.) contains multiple MREs, which can simultaneously bind to multiple different miRNAs, and the miRNAs ultimately regulate different downstream mRNAs. The regulatory axis has complex biological functions (23). lncRNA H19 is one of the long non-coding RNAs, which was one of the first discovered lncRNAs. Experiments have been carried out to show the new role of lncRNA H19 in ceRNA regulatory networks (24), further confirming the complexity and diversity of ceRNA regulatory networks. Therefore, in PCOS, further exploration of lncRNA H19 and biological phenotypes will help us to understand the pathogenesis of PCOS from a macro perspective.

Supplementary Material
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