• Users Online: 231
  • Print this page
  • Email this page

 
Table of Contents
ORIGINAL ARTICLE
Year : 2023  |  Volume : 66  |  Issue : 4  |  Page : 200-208

miR-22-3p ameliorates the symptoms of premature ovarian failure in mice by inhibiting CMKLR1 expression


The First College of Clinical Medical Science, China Three Gorges University; Department of Gynecology, Central People's Hospital of Yichang, Yichang, China

Date of Submission10-Jan-2023
Date of Decision15-Mar-2023
Date of Acceptance27-Mar-2023
Date of Web Publication06-Jul-2023

Correspondence Address:
Dr. Miaomiao Pan
The First College of Clinical Medical Science, China Three Gorges University, Department of Gynecology, Central People's Hospital of Yichang, Yiling Avenue, Yichang
China
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjop.CJOP-D-23-00004

Rights and Permissions
  Abstract 


Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. This study was aimed at exploring the improving effects of miR-22-3p on the symptoms of POF in mice by inhibiting chemokine-like receptor 1 (CMKLR1) expression. Female mice were intraperitoneally injected with cyclophosphamide to construct POF mice models. Lentiviral vectors containing miR-22-3p, short hairpin RNA (sh)-CMKLR1, and overexpression (oe)-CMKLR1, respectively, or in combination, were injected into the ovaries of both sides of POF mice. miR-22-3p and CMKLR1 expression in ovarian tissues of mice was assessed, and the targeting relationship between miR-22-3p and CMKLR1 was predicted and verified. Serum estradiol (E2), anti-Mullerian hormone, and follicle-stimulating hormone levels were assessed. Ovarian weight was weighed, and pathological changes and the number of primordial follicles, primary follicles, secondary follicles, and atresia follicles were observed. Apoptosis of ovarian tissues was determined. In ovarian tissues of POF mice, miR-22-3p expression was decreased while CMKLR1 expression was increased. miR-22-3p up-regulation or CMKLR1 down-regulation restored sex hormone levels, improved ovarian weight and the number of primordial follicles, primary follicles, and secondary follicles, and reduced the number of atresia follicle and ovarian granulosa cell apoptosis in POF mice. miR-22-3p targeted CMKLR1, and overexpressing CMKLR1 reversed the ameliorative effects of miR-22-3p overexpression on POF mice. Our research highlights that overexpressed miR-22-3p down-regulates CMKLR1 to ameliorate the symptoms of POF in mice. Therefore, the miR-22-3p/CMKLR1 axis could improve the symptoms of POF.

Keywords: Ameliorative effects, apoptosis, chemokine-like receptor 1, follicle counts, microRNA-22-3p, ovarian weight, premature ovarian failure, sex hormone levels


How to cite this article:
Pan M. miR-22-3p ameliorates the symptoms of premature ovarian failure in mice by inhibiting CMKLR1 expression. Chin J Physiol 2023;66:200-8

How to cite this URL:
Pan M. miR-22-3p ameliorates the symptoms of premature ovarian failure in mice by inhibiting CMKLR1 expression. Chin J Physiol [serial online] 2023 [cited 2023 Sep 26];66:200-8. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/200/380669




  Introduction Top


Premature ovarian failure (POF) is usually utilized to describe women less than 40 years of age, who present with hypergonadotropic hypogonadism, amenorrhea, as well as infertility.[1] The incidence of POF is elevated year by year, which seriously influences the patients' physical and mental health and enhances the economic burden on families and society.[2],[3] Reasons for POF involve some genetic diseases, autoimmunity disorders, and environmental factors.[4] Despite achievements in human infertility diagnosis and treatment, POF is still classified as being idiopathic in 50%–80% of cases, strongly indicating a genetic source for the disease.[5]

MicroRNAs (miRs) can mediate posttranslational silencing of the genes associated with the modulation of differentiation, proliferation, development, apoptosis, tumorigenesis, and hematopoiesis.[6] miRs are found to exert regulatory functions in oocyte maturation and ovarian follicular development, and miRs are differentially expressed in the plasma of POF patients and normal cycling women.[7] Extensive evidence has shown that miRs play a role in the pathology of epithelial ovarian cancer (EOC), and many miRs are differentially expressed in EOC.[8] It is also reported that miRs play important roles in initiating and developing female infertility-associated diseases.[9] Furthermore, miRNAs are regarded as a group of key regulatory elements after transcription in POF, a common medical condition accompanying intricate molecular mechanisms.[10] It has been demonstrated that miR-22-3p has an association with POF.[6] It is confirmed that miR-22-3p can act as an indicator of resistance to platinum-based chemotherapy, thus benefiting chemotherapeutic efficiency improvement and personalized treatment optimization in high-grade serous ovarian cancer.[11]

Chemokine-like receptor 1 (CMKLR1) refers to a G protein-coupled receptor involved in multiple forms of human arthritis and macrophage-mediated inflammation.[12] Chemerin, abundantly expressed in barrier tissues, has been reported to modulate tissue inflammation via mediating CMKLR1, its functional receptor.[13] Chemerin is one multifunctional adipokine possessing established functions in adipogenesis, inflammation, and glucose homeostasis.[14] In addition, chemerin serves as a cytokine that captures much attention in the reproductive process, and the chemerin/CMKLR1 axis might play a critical role in maintaining early pregnancy probably by modulating ERK1/2 phosphorylation.[15] A previous study has indicated that the chemerin/CMKLR1 axis could be associated with steroidogenesis alterations in polycystic ovarian syndrome human-luteinized granulosa cells.[16] In addition, the chemerin-CMKLR1 axis regulates placental progression and spiral artery remodeling in early pregnancy.[17] However, the improving effects of the miR-22-3p/CMKLR1 axis in POF have been rarely investigated. Therefore, we designed this study to validate the role of the miR-22-3p/CMKLR1 axis in the symptoms of POF and our hypothesis was that the miR-22-3p/CMKLR1 axis could improve the symptoms of POF.


  Materials and Methods Top


Ethics statement

All animal experiments were conducted in strict accordance with the guidelines for the care and utilization of laboratory animals. The study was ratified by Central People's Hospital of Yichang (approval number: 20210107). All measures were taken for minimizing animal suffering.

Animal model establishment and treatment

A total of 48 female C57BL/6 mice, aged 10 weeks, were randomly grouped into 8 groups: the Control group, the POF group, the miR-negative control (NC) group, the miR-22-3p group, the short hairpin RNA (sh)-NC group, the sh-CMKLR1 group, the miR-22-3p + overexpression (oe)-NC group, and the miR-22-3p + oe-CMKLR1 group, 6 mice in each group. All mice were kept at a constant temperature of 26 ± 2°C, with a 12/12 h (light/dark) photoperiod, and supplied with water and feed on demand. In addition to the Control group, the other 7 groups were set up according to the following protocol to establish POF animal models.[18] The mice were intraperitoneally injected with cyclophosphamide (CTX) at a loading dose of 50 mg/kg for 15 consecutive days. After completing the last CTX injection, mice in the POF group were then injected with 5 μL of saline into ovaries on both sides, while the other groups, based on the grouping, were correspondingly interjected with 5 μL of lentiviral vectors containing miR-22-3p, sh-CMKLR1, oe-CMKLR1, and their NC lentiviral vectors into the ovaries on both sides.[19],[20] All the above-mentioned recombinant lentiviral vectors were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). pLenti-miR-22-3p, pLenti-sh-CMKLR1, and pLenti-oe-CMKLR1 were cotransfected into 293T cells with the helper vectors psPAX2 and pMD2 using Lipofectamine 3000 reagent (Invitrogen, USA), resulting in the production of the corresponding lentiviral particles. After 48 h of incubation, the virus-containing supernatant was collected, filtered through 0.45 μm cellulose acetate filters, and stored for experiments. Next, the titer of the recombinant lentivirus was 1 × 108 TU/mL. On the 45th d after completion of all injections, the specimens of the tail vein blood were collected from each mouse, stratified at room temperature, and centrifuged at 2800 rpm/min (10 cm radius), and the supernatant was isolated and preserved at -80°C for further use. After blood collection, the mice were dislocated by the neck and euthanatized, and the ovarian tissues of both sides were taken by dissection. The right side was fixed in 4% paraformaldehyde and the left side was stored at -80°C for future use.

Detection of sex hormone levels

The levels of estradiol (E2), anti-Mullerian hormone (AMH), and follicle-stimulating hormone (FSH) were tested using ELISA kits (Mybiosource, USA) according to the instructions. Briefly, 50 μL of mouse serum was supplemented to each well and cultured at 37°C for 60 min. After the reaction, the wells were washed 3 times with washing buffer, and then enzyme-labeled antibodies were supplemented to each well and cultured at 37°C for 60 min. Finally, the stop buffer was supplemented and the optical density (OD) was assessed at 450 nm.[21]

Ovarian weight and follicle counts

The ovaries were harvested and the fatty tissue was removed. Then the blood was aspirated from the surface of the ovaries using filter paper, and the ovaries were weighed. Next, the ovarian tissues were fixed in 4% paraformaldehyde for 24 h and dehydrated. Following, these tissues were embedded in paraffin, sliced into 5-μm-thick sections, and put on numbered slides. For observing the morphological changes in mice's ovarian tissues, the prepared tissue sections were stained using hematoxylin and eosin (HE) (Servicebio, Wuhan, China), and these stained sections were observed under a microscope to determine follicle counts.[22] The primordial follicle only contained a single layer of spindle-shaped granulosa cells; the primary follicle had a single layer of granulosa cells with at least three columnar granulosa cells; the secondary follicle had at least two layers of granulosa cells, and the sinus follicle had at least two layers of granulosa cells with the follicular lumen.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining

TUNEL assay was conducted to detect apoptosis according to the manufacturer's instructions. Paraffin sections were dewaxed for 60 min at 60°C and gradually rehydrated with ethanol (100%, 90%, 80%, and 75%). The slides were rinsed with phosphate-buffered saline (PBS) (3 times for 5 min) and cultured with proteinase K for 25 min at 37°C. After drying, the slides were added with sufficient rupture solution, incubated at room temperature for 20 min, and rinsed again with PBS (3 times for 5 min). Then suitable microliters of terminal deoxynucleotidyl transferase (TdT) and deoxyuridine triphosphate (dUTP) enzyme reaction mixture (TUNEL Kit, Roche, Basel, Switzerland) were supplemented to cover the samples, and the entire setup was cultured for 2 h at 37°C in a dark humid atmosphere and rinsed 3 times for 5 min in PBS. Next, DAPI (Servicebio, Wuhan, China) was utilized to stain the nuclei for 10 min in the dark. Apoptotic cells in the ovaries were observed with a fluorescence microscope (ECLIPSE C1, Nikon, Japan).

Reverse transcription-quantitative polymerase chain reaction

Total RNA was isolated by TRIzol reagent (Invitrogen, USA). RNA was reverse-transcribed into cDNA strands following the instructions of the Mir-X miR First-Strand Synthesis kit (Takara, Dalian, China) or PrimeScript RT reagent kit (Takara). Next, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of gene expression was conducted utilizing cDNA and SYBR Premix Ex Taq II (Takara). RT-qPCR was conducted on a CFX96 system (Bio-Rad, Hercules, CA, USA), implementing U6 or β-actin as internal references. Relative gene expression was calculated utilizing the 2−ΔΔCt method. The primer sequences implemented in this research were displayed in [Table 1].
Table 1: Primer sequences for genes used in reverse transcription-quantitative polymerase chain reaction assay

Click here to view


Western blot

Total protein was isolated using RIPA lysate and protein concentration was tested by implementing the Bicinchoninic Acid assay (Beyotime, Shanghai, China). Protein samples were separated by 10%–12% SDS-PAGE, transferred to nitrocellulose membranes (Millipore, St. Louis, MO, USA), and incubated with primary antibodies. These primary antibodies were utilized: CMKLR1 (1:500, Abcam, Cambridge, MA, USA) and β-actin (1:1000, Cell Signaling Technology, Danvers, MA, USA). Bands were visualized with a chemiluminescence kit (Millipore) and then quantified by densitometric analysis (Bio-Rad, USA).[23]

Luciferase activity assay

To validate the targeting relationship between miR-22-3p and CMKLR1, the downstream regulatory site of miR-22-3p was predicted. The 3'-UTR sequence of the CMKLR1 gene (wild type, WT) was amplified, and the mutant sequence of the 3'-UTR binding site of miR-22-3p and CMKLR1 gene (mutant, Mut) was synthesized and cloned into pGL3-Control vector (Promega, Madison, WI, USA), respectively, and then cotransfected with miR-22-3p mimic and mimic NC, respectively, into mouse ovarian granulosa cells (Wuhan Procell Life Science and Technology Co., Ltd., Wuhan, China). To detect dual luciferase activity, a dual luciferase assay kit (Promega) was utilized.[24]

Statistics

SPSS 22.0 software (SPSS, Inc., IBM, Armonk, New York, NY, USA) and GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) were utilized for statistical analysis. All experimental data were expressed as mean ± standard deviation, and a t-test was applied for comparison between the two groups. P < 0.05 is an indicator of statistically significant differences.


  Results Top


Successful establishment of premature ovarian failure mouse models

The main causes of POF include genetic, immunological, medical (chemotherapy and radiotherapy), and other idiopathic factors. With the development and application of chemotherapeutic drugs, the incidence of chemotherapy-induced POF is increasing, and among the various chemotherapeutic drugs, CTX possesses strong reproductive toxicity.[18],[25] Therefore, we implemented the intraperitoneal injection of the CTX method to induce POF models. The main manifestations of POF were apoptosis of ovarian granulosa cells, shortening of ovarian atresia, disturbance of sex hormone secretion, and structural and functional damage to ovarian tissues. Therefore, we conducted ELISA to measure the levels of serum sex hormones (E2, FSH, and AMH) in mice, and HE staining was performed to observe the changes in histopathological characteristics and the number of follicles at each stage (primordial follicles, primary follicles, secondary follicles, and atresia follicles) in mice's ovaries. Apoptosis in ovarian tissues was assessed by TUNEL staining. The results revealed that E2 and AMH levels were lower [Figure 1]a and [Figure 1]b and FSH was higher in POF mice compared with those in normal mice [Figure 1]c. The weight of ovaries in POF mice was reduced in comparison to normal mice [Figure 1]d. It was found by HE staining that, follicles of different developmental stages were visible in the ovaries of normal mice under the microscope, with more primordial follicles and normal follicle morphology, while the number of primordial follicles, primary follicles, and secondary follicles in the ovaries of POF mice was reduced and the number of atresia follicles was elevated [Figure 1]e and [Figure 1]f.
Figure 1: Successful establishment of POF mouse models. (a-c) The levels of E2, AMH, and FSH in normal and POF mice were determined by ELISA. (d) The ovarian weight of normal and POF mice was weighed. (e) HE staining results of ovarian tissues in normal and POF mice (arrows indicate developing follicles). (f) The number of follicles at each stage in normal and POF mice was observed under a microscope. (g and h) The expression levels of apoptotic genes Bax and Bcl2 and their ratios in normal and POF mice were measured by RT-qPCR. (i and j) Ovarian cell apoptosis rate in normal and POF mice was assessed by TUNEL staining (arrows indicate apoptotic ovarian cells); n = 6. *versus P < 0.05. miR-22-3p: MicroRNA-22-3p, CMKLR1: Chemokine-like receptor 1, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, POF: Premature ovarian failure.

Click here to view


RT-qPCR findings displayed that Bax expression was raised and Bcl2 expression was reduced [Figure 1]g, and the Bax/Bcl2 ratio was raised [Figure 1]h in POF mice in comparison to normal mice. Cell apoptosis was tested by TUNEL staining, and it was found that the apoptosis rate in POF mice was elevated versus normal mice [Figure 1]i and [Figure 1]j. The above results demonstrated that POF model mouse models were successfully established.

miR-22-3p overexpression ameliorates the symptoms of premature ovarian failure in mice

A previous study has reported that miR-22-3p expression is down-regulated in the plasma of POF patients.[26] To further probe the role of miR-22-3p in POF mice, we performed RT-qPCR to detect miR-22-3p expression in the ovaries of POF mice and found that the POF mice had reduced miR-22-3p expression in the ovaries versus normal mice [Figure 2]a.
Figure 2: miR-22-3p overexpression ameliorates the symptoms pf POF in mice. (a) miR-22-3p expression in the ovaries of normal and POF mice was tested by RT-qPCR. (b) The successful injection of lentiviral vectors containing miR-22-3p in POF mice was determined by RT-qPCR. (c-e) The levels of E2, AMH, and FSH in POF mice after overexpressing miR-22-3p were measured by ELISA. (f) The ovarian weight of POF mice after overexpressing miR-22-3p. (g) HE staining results of ovarian tissues of in POF mice after overexpressing miR-22-3p (arrows indicate developing follicles). (h) The number of follicles at each stage in POF mice after overexpressing miR-22-3p was observed by a microscope. (i and j) The expression of apoptotic genes Bax and Bcl2 and their ratios in POF mice after overexpressing miR-22-3p were measured by RT-qPCR. (k and l) The level of apoptosis in ovarian cells of in POF mice after overexpressing miR-22-3p was tested by TUNEL staining (arrows indicate apoptotic ovarian cells); n = 6. *versus P < 0.05. miR-22-3p: MicroRNA-22-3p, CMKLR1: Chemokine-like receptor 1, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, POF: Premature ovarian failure.

Click here to view


To probe the effects of miR-22-3p up-regulation on the ovaries of POF mice, lentivirus containing miR-22-3p was injected into the ovaries of both sides of POF mice, and RT-qPCR demonstrated that miR-22-3p was significantly elevated in the ovarian tissues of POF mice [Figure 2]b. Subsequently, we measured serum sex hormone (E2, FSH, and AMH) levels in POF mice by ELISA, and the findings suggested that the contents of sex hormones E2 and AMH were raised [Figure 2]c and [Figure 2]d and FSH levels were decreased [Figure 2]e in POF mice after miR-22-3p overexpression. Moreover, the ovaries were weighed and HE staining was utilized to observe the pathological changes in the ovaries. The ovarian mass of POF mice was larger after overexpressing miR-22-3p [Figure 2]f, and HE staining unraveled that after miR-22-3p overexpression, the number of primordial follicles, primary follicles, and secondary follicles was elevated, and the number of atresia follicles in the ovaries of POF mice was reduced [Figure 2]g and [Figure 2]h.

Afterward, RT-qPCR and TUNEL staining were implemented to evaluate the apoptosis of ovarian cells in each group of mice. RT-qPCR found a reduction in Bax expression and an increase in Bcl2 expression in POF mice after overexpressing miR-22-3p [Figure 2]i, and there was an obvious decrease in the Bax/Bcl2 ratio [Figure 2]j. Apoptosis was measured by TUNEL staining, and it was found that there were fewer TUNEL apoptosis-positive cells in POF mice after overexpressing miR-22-3p [Figure 2]k and [Figure 2]l. The above results unraveled that miR-22-3p overexpression could improve the symptoms of POF in mice.

Chemokine-like receptor 1 is a target gene of miR-22-3p

To explore the mechanism by which miR-22-3p affects the symptoms of POF, we predicted potential target genes of miR-22-3p using the bioinformatics website (https://starbase.sysu.edu.cn/). It is predicted by the bioinformatics website that miR-22-3p could bind to CMKLR1 [Figure 3]a. To verify this prediction, a dual luciferase reporter gene assay was performed, which demonstrated that overexpression of miR-22-3p decreased luciferase activity in the CMKLR1-WT group, but had no significant impact on the CMKLR1-Mut group [Figure 3]b, indicating that miR-22-3p could bind to CMKLR1. CMKLR1 expression after overexpressing miR-22-3p was tested by RT-qPCR and western blot, and the experimental results demonstrated that CMKLR1 expression was reduced in POF mice after overexpressing miR-22-3p [Figure 3]c and [Figure 3]d, which indicated that miR-22-3p negatively modulated CMKLR1 expression. In summary, miR-22-3p negatively regulated CMKLR1 expression.
Figure 3: CMKLR1 is a target gene of miR-22-3p. (a) Bioinformatics website predicted that there were binding sites between miR-22-3p and CMKLR1. (b) The targeting relationship between miR-22-3p and CMKLR1 was verified by dual luciferase reporter gene assay. (c and d) CMKLR1 expression in POF mice after overexpressing miR-22-3p was tested by RT-qPCR and western blot; b: n = 3, c and d: n = 6. *versus P < 0.05. miR-22-3p: MicroRNA-22-3p, CMKLR1: Chemokine-like receptor 1, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, POF: Premature ovarian failure.

Click here to view


Silencing chemokine-like receptor 1 improves the symptoms of premature ovarian failure in mice

To explore the influence of the CMKLR1 gene on POF mice, the changes in CMKLR1 in POF mice were tested by RT-qPCR and western blot, and the expression of CMKLR1 was elevated in POF mice [Figure 4]a and [Figure 4]b.
Figure 4: Silencing CMKLR1 improves the symptoms of POF in mice. (a and b) CMKLR1 expression in the ovaries of normal and POF mice was assessed by RT-qPCR and western blot. (c and d) Lentiviral vectors containing sh-CMKLR1 injection success was assessed by RT-qPCR and western blot. (e-g) E2, AMH, and FSH levels in POF mice after silencing CMKLR1 were measured by ELISA. (h) Ovarian weight in POF mice after silencing CMKLR1 was weighed. (i) H and E staining results of ovarian tissues in POF mice after silencing CMKLR1 (arrows indicate developing follicles). (j) The number of follicles at each stage in POF mice after silencing CMKLR1 was observed under a microscope. (k and l) The expression of apoptotic genes Bax and Bcl2 and their ratios in POF mice after silencing CMKLR1 were tested by RT-qPCR. (m and n) The level of apoptosis in ovarian cells of POF mice after silencing CMKLR1 was determined by TUNEL staining (arrows indicate apoptotic ovarian cells); n = 6. *versus P < 0.05. miR-22-3p: MicroRNA-22-3p, CMKLR1: Chemokine-like receptor 1, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, POF: Premature ovarian failure.

Click here to view


To better understand the role of CMKLR1 in the symptoms of POF, lentivirus containing inhibited CMKLR1 was injected into the ovaries of both sides of POF mice and RT-qPCR demonstrated that CMKLR1 was notably reduced in the ovarian tissues of POF mice [Figure 4]c and [Figure 4]d. Next, we measured serum sex hormone (E2, FSH, and AMH) levels in POF mice by ELISA, and the findings displayed that the contents of sex hormones E2 and AMH were raised [Figure 4]e and [Figure 4]f and FSH levels were diminished in POF mice upon suppression of CMKLR1 [Figure 4]g. Besides, the ovaries were weighed and HE staining was utilized to observe the pathological changes in the ovaries. There was a larger ovarian mass of POF mice after silencing CMKLR1 [Figure 4]h, and HE staining indicated that the number of primordial follicles, primary follicles, secondary follicles, and sinus follicles in POF mice's ovaries was increased and the number of atresia follicles was decreased after silencing CMKLR1 [Figure 4]i and [Figure 4]j.

Furthermore, RT-qPCR and TUNEL staining were implemented to evaluate the apoptosis of ovarian cells in each group of mice. The findings of RT-qPCR revealed that Bax expression was reduced and Bcl2 expression was elevated [Figure 4]k, and the Bax/Bcl2 ratio was decreased [Figure 4]l in POF mice after silencing CMKLR1. TUNEL staining unveiled that the TUNEL apoptosis-positive cells were reduced in POF mice after silencing CMKLR1 [Figure 4]m and [Figure 4]n. Taken together, the above results suggested that silencing CMKLR1 could also effectively improve the symptoms of POF in mice.

Chemokine-like receptor 1 overexpression reverses the ameliorative effects of miR-22-3p expression on the symptoms of premature ovarian failure

To elucidate the impacts of the miR-22-3p/CMKLR1 axis in POF mice, the miR-22-3p + oe-NC group and the miR-22-3p + oe-CMKLR1 group were set up. The expression of CMKLR1 in the ovarian tissues of POF mice was tested by RT-qPCR and western blot. The results indicated that the expression of CMKLR1 was raised in the ovarian tissues of POF mice in the miR-22-3p + oe-CMKLR1 group in comparison with that in the miR-22-3p + oe-NC group [Figure 5]a and [Figure 5]b.
Figure 5: CMKLR1 overexpression reverses the ameliorative effects of miR-22-3p expression on the symptoms of POF. (a and b) CMKLR1 expression was measured after treatment of miR-22-3p and CMKLR1 overexpression by RT-qPCR and western blot. (c-e) E2, AMH, and FSH levels after treatment of miR-22-3p and CMKLR1 overexpression were tested by ELISA. (f) The ovarian weight after treatment of miR-22-3p and CMKLR1 overexpression was weighed. (g) HE staining results of ovarian tissues in each group of mice after treatment of miR-22-3p and CMKLR1 overexpression (arrows indicate developing follicles). (h) The number of follicles at each stage after treatment of miR-22-3p and CMKLR1 overexpression was observed under a microscope. (i and j) Apoptotic genes Bax and Bcl2 expression and their ratios after treatment of miR-22-3p and CMKLR1 overexpression were determined by RT-qPCR. (k and l) Apoptosis rate in ovarian cells of each group of mice after treatment of miR-22-3p and CMKLR1 overexpression was determined by TUNEL staining (arrows indicate apoptotic ovarian cells); n = 6. *versus P < 0.05. miR-22-3p: MicroRNA-22-3p, CMKLR1: Chemokine-like receptor 1, RT-qPCR: Reverse transcription-quantitative polymerase chain reaction, POF: Premature ovarian failure.

Click here to view


The levels of serum sex hormones (E2, FSH, and AMH) in POF mice were measured by ELISA. The ovaries were weighed and HE staining was implemented to observe the pathological changes in the ovaries. Subsequently, RT-qPCR and TUNEL staining were employed to determine the apoptosis of ovarian cells in each group of mice. The results demonstrated that compared with those in the miR-22-3p + oe-NC group, E2 and AMH levels were decreased [Figure 5]c and [Figure 5]d, FSH levels were increased [Figure 5]e, the ovarian weight was decreased [Figure 5]f, the number of primordial follicles, primary follicles, and secondary follicles was decreased; the number of atresia follicles was increased [Figure 5]g and [Figure 5]h, the apoptosis-related gene Bax expression was increased, while Bcl2 expression was decreased [Figure 5]i, and the Bax/Bcl2 ratio was increased [Figure 5]j, and apoptosis rate was increased [Figure 5]k and [Figure 5]l in the miR-22-3p + oe-CMKLR1 group. In summary, CMKLR1 overexpression could reverse the ameliorative effects of miR-22-3p expression on the symptoms of POF.


  Discussion Top


POF can be considered as a series of symptoms of perimenopausal hot flashes, vaginal dryness, reduced menstruation, insomnia, night sweats, amenorrhea, and even infertility due to ovarian function decline, which is a prevalent and harsh disease in gynecology.[27] This study focused on the ameliorative effects of the miR-22-3p/CMKLR1 axis on the symptoms of POF, and we discovered that miR-22-3p ameliorated the symptoms of POF in mice by repressing CMKLR1 expression.

As previously reported, miRNAs take part in the post-transcription modulation of gene expression. Meanwhile, miRNA deregulation has an epigenetic component and abnormal miRNA expression is often involved in the form of EOC tumor, prognosis, histological grade, and the International Federation of Gynecology and Obstetrics stage.[28] It is reported that miR-22-3p is lowly expressed in POF and is efficient in distinguishing POF from control subjects, which may reflect the down-regulated ovarian reserve and act as a consequence of the POF pathologic process.[26] Besides, the pathogenic factors of POF include autoimmune factors,[29] and a previous study has suggested that miR-22-3p has the ability to modulate the function of several types of immune cells and may be implicated in autoimmune disease development.[30] In addition, overexpressed miR-22-3p inhibits cell activity and promotes cell apoptosis via modulating apoptosis-related genes.[31] Similar to the above references, we found in our research that the expression of miR-22-3p was reduced in POF mice, and overexpression of miR-22-3p could improve the symptoms of POF in mice.

Increasing evidence has indicated that miRs are associated with a diversity of biological signaling pathways and have a critical role in the development of several diseases, including not only oncological but also non-oncological diseases. In addition, these small non-coding RNAs can block translation, leading to lowly-expressed target genes.[32] Besides, miRNAs are capable of modulating gene expression via targeting the binding sites of mRNAs, thus influencing multiple biological processes.[33] To further explore the mechanism of miR-22-3p affecting the symptoms of POF, we predicted that miR-22-3p could bind to CMKLR1 through the bioinformatics website, and further assays revealed that there was a targeting relationship between miR-22-3p and CMKLR1, and miR-22-3p had a negative regulation with CMKLR1 expression. CMKLR1, as a receptor for chemerin, has a broad range of functions in both physiological and pathological activities, such as inflammation, innate and adaptive immunity, metabolism, as well as reproduction.[34] As previously demonstrated, CMKLR1 expression is elevated in the polycystic ovary syndrome model,[35] which is in accordance with our experimental results. In our study, we found that CMKLR1 was up-regulated in POF. Moreover, increasing evidence has displayed that CMKLR1 is correlated with the regulation of ovarian functions.[36] We conducted some assays to evaluate the role of CMKLR1 in POF, and found that silencing CMKLR1 could also effectively improve the symptoms of POF in mice. Furthermore, our paper also revealed that overexpression of CMKLR1 could reverse the ameliorative effects of miR-22-3p expression on the symptoms of POF. Similarly, a study has demonstrated that the regulatory impact of miR-7-5p on osteogenic differentiation of human mesenchymal stem cells was reversed by CMKLR1 overexpression.[37] This signifies that CMKLR1 overexpression can reverse the influences of miRNAs on different diseases. Nevertheless, the specific mechanism needs further validation.


  Conclusion Top


In conclusion, our research demonstrates that miR-22-3p could improve the symptoms of POF in mice by suppressing CMKLR1 expression. This study offers a foundation for the future and further investigation of the miR-22-3p/CMKLR1 axis in treating the symptoms of POF. Nevertheless, further evidence is still needed to explore the role of the miR-22-3p/CMKLR1 axis in POF. This study is based on animal trials without clinical data, which is the major limitation of our study. In addition, the regulatory mechanism of CMKLR1 on miR-22 should be validated to further confirm our findings.

Acknowledgment

We would like to thank Ebubechukwu Orji for English language editing.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Jankowska K. Premature ovarian failure. Prz Menopauzalny 2017;16:51-6.  Back to cited text no. 1
    
2.
Wang J, Liu W, Yu D, Yang Z, Li S, Sun X. Research progress on the treatment of premature ovarian failure using mesenchymal stem cells: A literature review. Front Cell Dev Biol 2021;9:749822.  Back to cited text no. 2
    
3.
Ghahremani-Nasab M, Ghanbari E, Jahanbani Y, Mehdizadeh A, Yousefi M. Premature ovarian failure and tissue engineering. J Cell Physiol 2020;235:4217-26.  Back to cited text no. 3
    
4.
Shareghi-Oskoue O, Aghebati-Maleki L, Yousefi M. Transplantation of human umbilical cord mesenchymal stem cells to treat premature ovarian failure. Stem Cell Res Ther 2021;12:454.  Back to cited text no. 4
    
5.
Laissue P. Aetiological coding sequence variants in non-syndromic premature ovarian failure: From genetic linkage analysis to next generation sequencing. Mol Cell Endocrinol 2015;411:243-57.  Back to cited text no. 5
    
6.
Guo Y, Sun J, Lai D. Role of microRNAs in premature ovarian insufficiency. Reprod Biol Endocrinol 2017;15:38.  Back to cited text no. 6
    
7.
Yang X, Zhou Y, Peng S, Wu L, Lin HY, Wang S, et al. Differentially expressed plasma microRNAs in premature ovarian failure patients and the potential regulatory function of mir-23a in granulosa cell apoptosis. Reproduction 2012;144:235-44.  Back to cited text no. 7
    
8.
Muralidhar GG, Barbolina MV. The miR-200 family: Versatile players in epithelial ovarian cancer. Int J Mol Sci 2015;16:16833-47.  Back to cited text no. 8
    
9.
Bahmyari S, Jamali Z, Khatami SH, Vakili O, Roozitalab M, Savardashtaki A, et al. microRNAs in female infertility: An overview. Cell Biochem Funct 2021;39:955-69.  Back to cited text no. 9
    
10.
Nouri N, Shareghi-Oskoue O, Aghebati-Maleki L, Danaii S, Ahmadian Heris J, Soltani-Zangbar MS, et al. Role of miRNAs interference on ovarian functions and premature ovarian failure. Cell Commun Signal 2022;20:198.  Back to cited text no. 10
    
11.
Qi X, Yu C, Wang Y, Lin Y, Shen B. Network vulnerability-based and knowledge-guided identification of microRNA biomarkers indicating platinum resistance in high-grade serous ovarian cancer. Clin Transl Med 2019;8:28.  Back to cited text no. 11
    
12.
Serafin DS, Allyn B, Sassano MF, Timoshchenko RG, Mattox D, Brozowski JM, et al. Chemerin-activated functions of CMKLR1 are regulated by G protein-coupled receptor kinase 6 (GRK6) and β-arrestin 2 in inflammatory macrophages. Mol Immunol 2019;106:12-21.  Back to cited text no. 12
    
13.
Lin Y, Cai Q, Luo Y, Li B, Chen Y, Yang X, et al. Epithelial chemerin-CMKLR1 signaling restricts microbiota-driven colonic neutrophilia and tumorigenesis by up-regulating lactoperoxidase. Proc Natl Acad Sci U S A 2022;119:e2205574119.  Back to cited text no. 13
    
14.
Treeck O, Buechler C, Ortmann O. Chemerin and cancer. Int J Mol Sci 2019;20:3750.  Back to cited text no. 14
    
15.
Yang X, Yao J, Wei Q, Ye J, Yin X, Quan X, et al. Role of chemerin/CMKLR1 in the maintenance of early pregnancy. Front Med 2018;12:525-32.  Back to cited text no. 15
    
16.
Estienne A, Mellouk N, Bongrani A, Plotton I, Langer I, Ramé C, et al. Involvement of chemerin and CMKLR1 in the progesterone decrease by PCOS granulosa cells. Reproduction 2021;162:427-36.  Back to cited text no. 16
    
17.
Zhang Q, Xiao Z, Lee CL, Duan YG, Fan X, Yeung WS, et al. The regulatory roles of chemerin-chemokine-like receptor 1 axis in placental development and vascular remodeling during early pregnancy. Front Cell Dev Biol 2022;10:883636.  Back to cited text no. 17
    
18.
Li J, Yu Q, Huang H, Deng W, Cao X, Adu-Frimpong M, et al. Human chorionic plate-derived mesenchymal stem cells transplantation restores ovarian function in a chemotherapy-induced mouse model of premature ovarian failure. Stem Cell Res Ther 2018;9:81.  Back to cited text no. 18
    
19.
Sun XF, Li YP, Pan B, Wang YF, Li J, Shen W. Molecular regulation of miR-378 on the development of mouse follicle and the maturation of oocyte in vivo. Cell Cycle 2018;17:2230-42.  Back to cited text no. 19
    
20.
Wang X, He Y, Liu M, Fu X. Lentivirus-mediated bcl-2 gene therapy improves function and structure of chemotherapy-damaged ovaries in wistar rats. Am J Reprod Immunol 2013;69:518-28.  Back to cited text no. 20
    
21.
Liu M, Qiu Y, Xue Z, Wu R, Li J, Niu X, et al. Small extracellular vesicles derived from embryonic stem cells restore ovarian function of premature ovarian failure through PI3K/AKT signaling pathway. Stem Cell Res Ther 2020;11:3.  Back to cited text no. 21
    
22.
Devine PJ, Sipes IG, Hoyer PB. Initiation of delayed ovotoxicity by in vitro and in vivo exposures of rat ovaries to 4-vinylcyclohexene diepoxide. Reprod Toxicol 2004;19:71-7.  Back to cited text no. 22
    
23.
Xie Y, Huang Y, Ling X, Qin H, Wang M, Luo B. Chemerin/CMKLR1 axis promotes inflammation and pyroptosis by activating NLRP3 inflammasome in diabetic cardiomyopathy rat. Front Physiol 2020;11:381.  Back to cited text no. 23
    
24.
Wang Y, Lin C. Exosomes miR-22-3p derived from mesenchymal stem cells suppress colorectal cancer cell proliferation and invasion by regulating RAP2B and PI3K/AKT pathway. J Oncol 2021;2021:3874478.  Back to cited text no. 24
    
25.
Yang M, Lin L, Sha C, Li T, Zhao D, Wei H, et al. Bone marrow mesenchymal stem cell-derived exosomal miR-144-5p improves rat ovarian function after chemotherapy-induced ovarian failure by targeting PTEN. Lab Invest 2020;100:342-52.  Back to cited text no. 25
    
26.
Dang Y, Zhao S, Qin Y, Han T, Li W, Chen ZJ. MicroRNA-22-3p is down-regulated in the plasma of Han Chinese patients with premature ovarian failure. Fertil Steril 2015;103:802-7.e1.  Back to cited text no. 26
    
27.
Niu J, Yu F, Luo X, Chen S. Human umbilical cord mesenchymal stem cells improve premature ovarian failure through cell apoptosis of miR-100-5p/NOX4/NLRP3. Biomed Res Int 2022;2022:3862122.  Back to cited text no. 27
    
28.
Kumar V, Gupta S, Varma K, Sachan M. MicroRNA as biomarker in ovarian cancer management: Advantages and challenges. DNA Cell Biol 2020;39:2103-24.  Back to cited text no. 28
    
29.
Chen H, Liu C, Zhu S, Li S, Zhang Q, Song L, et al. The therapeutic effect of stem cells on chemotherapy-induced premature ovarian failure. Curr Mol Med 2021;21:376-84.  Back to cited text no. 29
    
30.
Wang B, Yao Q, Xu D, Zhang JA. MicroRNA-22-3p as a novel regulator and therapeutic target for autoimmune diseases. Int Rev Immunol 2017;36:176-81.  Back to cited text no. 30
    
31.
Lu W, Liu X, Zhao L, Yan S, Song Q, Zou C, et al. MiR-22-3p in exosomes increases the risk of heart failure after down-regulation of FURIN. Chem Biol Drug Des 2023;101:550-67.  Back to cited text no. 31
    
32.
Deng B, Tang X, Wang Y. Role of microRNA-129 in cancer and non-cancerous diseases (Review). Exp Ther Med 2021;22:918.  Back to cited text no. 32
    
33.
Xia S, Wang X, Wu Y, Zhou T, Tian H, Liu Z, et al. miR-22 suppresses EMT by mediating metabolic reprogramming in colorectal cancer through targeting MYC-associated factor X. Dis Markers 2022;2022:7843565.  Back to cited text no. 33
    
34.
Huang C, Wang M, Ren L, Xiang L, Chen J, Li M, et al. CMKLR1 deficiency influences glucose tolerance and thermogenesis in mice on high fat diet. Biochem Biophys Res Commun 2016;473:435-41.  Back to cited text no. 34
    
35.
Kian M, Hosseini E, Abdizadeh T, Langaee T, Khajouei A, Ghasemi S. Molecular docking and mouse modeling suggest CMKLR1 and INSR as targets for improving PCOS phenotypes by minocycline. EXCLI J 2022;21:400-14.  Back to cited text no. 35
    
36.
Mellouk N, Ramé C, Diot M, Briant E, Touzé JL, Guillaume D, et al. Possible involvement of the RARRES2/CMKLR1-system in metabolic and reproductive parameters in Holstein dairy cows. Reprod Biol Endocrinol 2019;17:25.  Back to cited text no. 36
    
37.
Chen B, Meng J, Zeng YT, Du YX, Zhang J, Si YM, et al. MicroRNA-7-5p regulates osteogenic differentiation of hMSCs via targeting CMKLR1. Eur Rev Med Pharmacol Sci 2018;22:7826-31.  Back to cited text no. 37
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures
Article Tables

 Article Access Statistics
    Viewed1259    
    Printed47    
    Emailed0    
    PDF Downloaded71    
    Comments [Add]    

Recommend this journal