Expression of Long Noncoding RNA HOTAIR and Its Influence on the PTEN/PI3K/AKT Pathway and Inflammation in Patients With Osteoarthritis

Expression of Long Noncoding RNA HOTAIR and Its Influence on the PTEN/PI3K/AKT Pathway and Inflammation in Patients With Osteoarthritis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Expression of Long Noncoding RNA HOTAIR and Its Influence on the PTEN/PI3K/AKT Pathway and Inflammation in Patients With Osteoarthritis Xiaolu Chen, Jian Liu, Yanqiu Sun, Jianting Wen, Qin Zhou, Xiang Ding, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1156754/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jan, 2023 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted 5 You are reading this latest preprint version Abstract Objective: Our research was designed to investigate the correlations among expression of PTEN/PI3K/AKT pathway, clinical-related indicators, and long noncoding RNA HOX transcript antisense RNA (lncRNA HOTAIR) in osteoarthritis (OA). Methods: The expression of immune-inflammatory indicators was detected in OA patients and normal people, and peripheral blood mononuclear cells (PBMCs) were extracted to induce human chondrocytes (CHs). Reverse transcription-quantitative polymerase chain reaction was performed to measure lncRNA HOTAIR expression. The levels of inflammatory cytokines and adiponectin were detected using enzyme-linked immunosorbent assay. Cell Counting Kit-8 was used to assess the viability of CHs. Western blot analysis was utilized to evaluate related protein expression. Results: LncRNA HOTAIR showed high expression in PBMCs of OA patients with the sensitivity and specificity of receiver-operating characteristics (ROC) curve according to the area under the ROC curve, indicating that lncRNA HOTAIR might act as a biomarker of OA. Moreover, lncRNA HOTAIR was positively correlated with total cholesterol, high sensitivity C-reactive protein, immunoglobulin G, tumor necrosis factor-α (TNF-α), and visual analog scale score, which were found to be independent risk factors for lncRNA HOTAIR expression. Besides, overexpression of lncRNA HOTAIR diminished cell viability and IL-10 expression but augmented TNF-α expression in OA-CHs stimulated by OA-PBMCs. Meanwhile, lncRNA HOTAIR overexpression elevated the levels of PI3K, AKT proteins and reduced PTEN protein expression in OA-CHs. Conclusions: Conclusively, lncRNA HOTAIR was upregulated in PBMCs of OA patients, which facilitated the inflammatory response of OA by orchestrating inflammatory cytokines and the PTEN/PI3K/AKT pathway. Orthopedic Surgery Osteoarthritis Long noncoding RNA HOTAIR Inflammation Lipid metabolism Human chondrocytes PTEN/PI3K/AKT pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Osteoarthritis (OA) is a kind of degenerative disease featured by "low level" cartilage and synovial inflammation [ 1 , 2 ] , which is based on joint degeneration or aging and leads to the disappearance of joint structure and gradual aggravation of cartilage. It is a chronic arthritic disease with systemic pain in large joints and swelling or dysfunction of elbow and knee joints as the main clinical manifestations. It has been reported that there exist several risk factors for OA, including age, joint trauma, obesity, and genetic susceptibility [ 3 ] . To a certain extent, obesity is related to inflammation, namely, hyperlipidemia that is a common metabolic change and is characterized by an abnormal increase in serum total cholesterol (TC), triglycerides, and low-density lipoprotein cholesterol, as well as an abnormal decrease in high-density lipoprotein cholesterol and apolipoprotein A1, all of which contribute to the inflammatory response and OA deterioration. In addition, body fat, insulin resistance index, and body mass index are negatively related to adiponectin (APN) [ 4 , 5 ] . APN, a recently discovered cytokine released by adipose tissues, is a regulator for the repression of the inflammatory response in OA [ 6 ] . Through an animal experiment [ 7 ] , long noncoding RNAs (lncRNAs) are involved in the inflammation, lipid metabolism, and other processes of OA, which has become a research hotspot [ 8 ] . A prior research [ 9 ] has observed that lncRNA HOTAIR expression in OA-chondrocytes (CHs) was apparently higher than that in normal CHs and that the proliferation capacity was significantly higher in OA-CHs transfected with small interfering RNA (si)-lncRNA HOX transcript antisense RNA (HOTAIR) than in those transfected with si-negative control (NC). The tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten (PTEN) can dephosphorylate and inactivate phosphatidylinositol-3,4,5-trisphosphate (PIP3), an important molecule in the phosphoinositide 3-kinase (PI3K)/AKT pathway, to downregulate the PI3K/AKT pathway. Therefore, PTEN and the PI3K/AKT pathway together are called the PTEN/PI3K/AKT pathway, a classic inflammatory pathway. A recent study reported that PTEN can downregulate the expression of fatty acid synthase by inhibiting the PTEN/PI3K/AKT pathway [ 10 ] . Interleukin (IL)-10 and tumor necrosis factor-α (TNF-α) are critical pro-inflammatory factors and anti-inflammatory factors of OA [ 11 ] , respectively. Of note, the PTEN/PI3K/AKT pathway can increase TNF-α expression and decrease IL-10 expression [ 12 ] . Furthermore, it has been noted in a previous research that the content of TNF-α is obviously reduced and the content of IL-10 is remarkably enhanced after silencing lncRNA HOTAIR [ 13 ] . However, although lncRNA HOTAIR and the PTEN/PI3K/AKT pathway participate in the inflammation of OA, there has hitherto been little report on whether the former can assume a role in immune regulation by affecting the latter in the inflammatory response of OA. Therefore, this study was conducted to focus on observing lncRNA HOTAIR expression in OA patients and analyzing its correlation with clinical indicators and visual analog scale (VAS) scores. Finally, lncRNA HOTAIR was altered in cell experiments to assess the changes in the expression of the PTEN/PI3K/AKT pathway, inflammatory factors, and lipid metabolism indexes. 2 Materials And Methods 2.1 Subjects and samples OA patients and healthy control (HC) subjects matched on sex and age were enrolled from the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine between November 2020 and May 2021. Patients were excluded from the present research if they did not meet the following criteria: not conforming to the 2019 Guidelines for the diagnosis of OA [ 14 ] , severe mental illness, significantly impaired liver or renal function, the administration of immunosuppressive drugs, or pregnancy. The research was conducted following the relevant provisions of the Declaration of Helsinki [ 15 ] and was approved by the Ethical Committee of Scientific Research of Anhui University of Traditional Chinese Medicine's First Affiliated Hospital [2014AH-06(J)]. 2.2 Co-culture of peripheral blood mononuclear cells (PBMCs) with CHs from OA patients and their transfection Totally, 5 mL venous blood was collected from normal healthy subjects and OA patients, and diluted with an equal volume of normal saline. The same amount of lymphocyte separation solution was slowly added into the prepared samples and centrifuged at 2000 r/min for 20 min. The white floc in the centrifuge tube was transferred to the clean centrifuge tube. The same amount of normal saline was added into the tube and mixed, followed by 8-min centrifugation 800 r/min and two washes. After blowing, the samples were transferred into a culture flask and incubated for later use. The CHs were purchased from Saibaikang Biotechnology Co., Ltd. (Shanghai, China). PBMCs were extracted and seeded into the Transwell chamber, followed by the addition of 75 µL Dulbecco’s Modified Eagle Medium (DMEM). CHs were digested and centrifuged. Then, the medium was discarded, and CHs were washed twice with phosphate buffer saline, resuspended with medium, and seeded into the apical chamber, followed by the supplementation of 100 µL DMEM. Following cell adherence, the cells were activated with TNF-α (10 ng/mL), and OA-PBMCs and OA-CHs were treated at the ratio of 0.5:1. Co-culture was conducted in Roswell Park Memorial Institute-1640 media containing penicillin (the final concentration of 100 U/mL) and streptomycin (the final concentration of 0.1 mg/mL) with 5% CO 2 at 37°C until the CH confluence reached 70%-90%. OA-CHs were transfected with PCDNA3.1-lncRNA HOTAIR plasmids, si-lncRNA HOTAIR plasmids, and their corresponding NC plasmids as per the manuals of Lipofectamine 2000, followed by 48-h incubation. All plasmids were purchased from Shanghai GenePharma Company (Shanghai, China). 2.3 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) Total OA-CH RNA was obtained using Trizol, followed by reverse transcription reaction and amplification reaction. Agarose-gel electrophoresis was implemented for semi-quantitative analysis of PCR products using the Gelpro32 gel image analysis software. Relative quantitative analysis was performed using 2 −ΔΔCt with β-actin as an internal reference. All used primers were as follows: lncRNA HOTAIR: the forward primer: 5'-CCATAGCCGATTAGCTGTCA' and the reverse primer: 5'-AATGCCGAACTGGAGGTG-3'; β-actin: the forward primer: 5'-GGCAAATGTCAGAGGGTTCT-3' and the reverse primer: 5'-TTCTTAAATTGGGCTGGGTC-3'. 2.4 Enzyme-linked immunosorbent assay (ELISA) Following collection, the supernatant of OA-CHs was centrifuged at 2500 r/min for 20 min, and the precipitation was discarded. The supernatant was added to the ELISA plate (50 µL per well) and incubated at 37°C for 30 minutes after the plate was sealed with a sealing plate membrane. The levels of TNF-α, IL-10, APN, and adiponectin receptor 2 (ADIPOR2) were detected using the ELISA method strictly in the light of the kit instructions. 2.5 Cell Counting Kit-8 (CCK-8) assay Cell viability was measured with a CCK-8 assay kit (BIOSS, Beijing, China). At first, 3 × 10 4 OA-CHs were seeded into each well of 96-well plates and cultured until 70% and 90% confluence. Logarithmically growing cells were transfected by the above method. Three wells in each group were cultured for 0, 12, 24, 48, and 72 h, respectively. Next, 10 mL CCK-8 solution was supplemented into each well at each experimental site, followed by 1-4 h of cell incubation at 37°C. The viability of OA-CHs was determined by measuring the absorbance value of each well at 450 nm. 2.6 Western blot analysis Cells were lysed with Radio-Immunoprecipitation assay lysis solution and centrifuged at 12000 r/min for 10 min to harvest total proteins. A sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) gel preparation kit (Beyotime, Shanghai, China) was utilized. The 5 × SDS-PAGE protein loading buffer was added into cells at the ratio of 1:4 to obtain protein samples. The samples were heated for 10 min in a boiling water bath and supplemented into SDS-PAGE gel wells (5-10 µL per well). Filter papers and polyvinylidene fluoride membrane of the same size as the rubber strip were cut in advance (soaked in methanol for 2-3 min in advance) and immersed in the rotating film buffer for 5 min. Bubbles were removed at every step and the flow membrane was constant. After the protein was transferred into the membranes, the membranes were cooled to room temperature before the insertion of the membranes into the prepared Western washing solution and the removal of the membrane transfer solution by 5-min washing. The Western sealing solution (5% skim milk powder) was added, slowly shaken in the shaker, closed at room temperature for 2 h, and then washed with phosphate-buffered saline containing Tween 20 three times with 10 min for each time. The membrane underwent overnight probing with primary pig antibodies to phosphorylation (p)-AKT, PI3K, PTEN, AKT, P-PI3K, APN, ADIPOR1, and ADIPOR2 at 4°C. The horseradish peroxidase-tagged secondary antibody was diluted with secondary antibody diluent at the ratio of 1:20,000 and incubated with the membranes at room temperature for 1 h. The ratio of absorbance of target proteins to that of glyceraldehyde-3-phosphate dehydrogenase was calculated after electrogenerated chemiluminescence coloring, exposure in the darkroom, fixation, and photography. 2.7 Statistical analysis SPSS statistical software version 23.0 (IBM Corp. Armonk, NY, USA) was applied for statistical analysis, and GraphPad Prism software version 8.2 (GraphPad Software, La Jolla, CA, USA) was utilized to capture images. The significant difference between groups was compared with matching double tail Student t -test or Kruskal Wallis nonparametric tests. Classification variables were compared by the chi-square test. The Spearman correlation analysis was employed to evaluate the correlation of lncRNA HOTAIR with erythrocyte sedimentation rate (ESR), high sensitivity C-reactive protein (hs-CRP), immunoglobulin A (IgA), and other indicators. Data were expressed as mean ± standard deviation or median (quartile range). p < 0.05 was considered to be significantly different. 3 Results 3.1 Clinical characteristics of OA patients and HC subjects A total of 80 subjects were enrolled, including 30 HC subjects (10 males and 20 females, the mean age of 59.24 ± 11.94 years) in the control group and 50 OA patients (17 males and 33 females, the mean age of 65.54 ± 14.10 years) in the OA group. There were no statistically significant differences in age and gender distribution between the two groups but significant differences in ESR, TC, triglyceride, immunoglobulin G (IgG), complement C3 (Table 1 ). Table 1 Changes in clinical lipid metabolism indexes and immune-inflammatory indexes in OA patients Indexes OA (n = 50) HC (n = 30) p Age (years) 55.85 ± 10.49 a 56.48 ± 6.42 a 0.655 ESR (mm/h) 19.4 (11.2, 31) b 9.50 (5.66, 11.62) b < 0.01 TC (mmol/L) 5.25 ± 1.29 c 2.20 (1.53, 3.10) b < 0.01 TG (mmol/L) 1.00 (1.35, 1.73) b 0.50 (0.34, 1.03) b < 0.01 IgA (g/L) 2.48 ± 1.12 c 1.61 (1.20, 2.20) b < 0.01 C3 (g/L) 1.15 ± 0.22 c 1.05 (0.94, 1.09) b < 0.01 SDS score 53.09 ± 7.11 c NA - VAS score 5.72 ± 0.60 c - - SAS score 43.38 (40.62, 47.67) b - - Notes: a, Student’s t-test. b, Wilcoxon signed-rank test, median (25–75th percentile). c, mean ± standard error. NA, not applicable; NC, negative control; OA, osteoarthritis; ESR, erythrocyte sedimentation rate; TC, total cholesterol; TG, triglyceride; IgA, immunoglobulin A; C3, complement C3; SDS, Self-rating Depression Scale; SAS, Self-rating Anxiety Scale; VAS, visual analog scale; HC, healthy control. 3.2 LncRNA HOTAIR expression is upregulated in the PBMCs of OA patients This study intended to assess lncRNA HOTAIR expression in OA patients. LncRNA HOTAIR expression in the PBMCs of 50 OA patients and 30 HC subjects was detected using RT-qPCR. The findings revealed that lncRNA HOTAIR expression was increased in the PBMCs of OA patients (Figure 1 A). The diagnostic efficacy of lncRNA HOTAIR was evaluated using a receiver-operating characteristic (ROC) curve analysis, and the area under the ROC curve (AUC) was 0.8310 [95% confidence interval (CI): 0.74-0.92]. According to the Youden index, the ideal cut-off value for the separation of OA from HC subjects was 1.185 with a sensitivity of 60.00% and a specificity of 92.67% (Figure 1 B). 3.3 Differential expression of inflammatory cytokines and APN in OA with a positive correlation with lncRNA HOTAIR expression The levels of IL-10 and TNF-α in the OA group were dramatically lowered ( p < 0.01) and substantially increased ( p < 0.01), respectively, as compared to the normal group, showing that there were alterations in the levels of pro-inflammatory and anti-inflammatory factors in OA (Figure 2 A-B). Furthermore, the expression of APN and ADIPOR2 was considerably reduced in the OA group compared with the normal group ( p < 0.01; Figure 2 C-D). Then, to further explore the possible relationship between lncRNA HOTAIR and related research indicators, we conducted correlation analyses of lncRNA HOTAIR with TC, hs-CRP, IgG, TNF-α, and VAS respectively. The results displayed that lncRNA HOTAIR was a risk factor for TC, hs-CRP, IgG, TNF-α, and VAS [ 16 ] (Figure 2 E-I). These results further suggested the close relationship between lncRNA HOTAIR and the occurrence and development of OA. 3.4 Association rules analysis of lncRNA HOTAIR with laboratory indexes in OA patient Association rule analysis showed that the elevation of lncRNA HOTAIR was strongly correlated with the elevation of GSH, ESR, and VAS in OA patients. There was a strong correlation between the increase of lncRNA HOTAIR and the decrease of LDL-C, with a support degree greater than 20% and a confidence degree greater than 30%. The results are shown in Table 2 . Table 2 Association rules analyses between lncRNA HOTAIR and multiple laboratory indicators in OA patients Items (LHS ⇒ RHS) Support (%) Confidence (%) Lift p value {lncRNA HOTAIR↑}⇒{Hospitalization days↓} 63.636 76.087 1.02 < 0.01 {lncRNA HOTAIR↑}⇒{sex (female)} 56.364 67.391 1.05 < 0.01 {lncRNA HOTAIR↑}⇒{SDS↑} 63.636 76.087 1.03 < 0.01 {lncRNA HOTAIR↑}⇒{ESR↑} 61.818 73.913 1.03 < 0.01 {lncRNA HOTAIR↑}⇒{VAS↑} 67.273 80.435 1.01 < 0.01 {lncRNA HOTAIR↑}⇒{LDL-C↑} 27.273 32.609 1.02 < 0.01 Notes: For correlations, Aprior module analysis was used. The minimal level of support was set at 20%, while the minimum level of confidence was set at 30%. When the degree of the lift was set to greater than one, it was deemed important. 3.5 LncRNA HOTAIR overexpression enhanced the viability of OA-CHs LncRNA HOTAIR expression in the PCDNA3.1-lncRNA HOTAIR group was substantially higher ( p < 0.01) than that in the PCDNA3.1-NC group, whereas lncRNA HOTAIR expression in the si-lncRNA HOTAIR group was significantly lower than that in the si-NC group ( p < 0.01). The effect of lncRNA HOTAIR on the viability of OA-CHs was assessed by CCK-8. The results exhibited that the PCDNA3.1-lncRNA HOTAIR group had dramatically boosted cell viability when compared to the PCDNA3.1-NC group. In contrast to the si-NC group, cell viability of the si-lncRNA HOTAIR group was evidently diminished. Among the four groups, cell viability of the si-lncRNA HOTAIR group was the highest and that of the lncRNA HOTAIR group was the lowest. As shown in figure 3 . 3.6 LncRNA HOTAIR overexpression facilitated TNF-α expression but repressed IL-10 expression in OA-CHs The level of cytokines was evaluated by ELISA. As a result, versus the PCDNA3.1-NC group, TNF-α expression in the PCDNA3.1-lncRNA HOTAIR group was prominently augmented and IL-10 expression was remarkably decreased ( p < 0.01). Compared with the si-NC group, TNF-α expression was markedly lowered and IL-10 expression was noticeably elevated in the si-lncRNA HOTAIR group ( p < 0.01; Figure 4 A-B). 3.7 LncRNA HOTAIR manipulated the PTEN/PI3K/AKT pathway in OA-CHs In order to ascertain the impacts of lncRNA HOTAIR on the PTEN/PI3K/AKT pathway, Western blot analysis was adopted to identify the expression of PI3K, p-AKT, p-PI3K, AKT, APN, ADIPOR1, ADIPOR2, and PTEN. As discovered in Figure 5 , overexpression of lncRNA HOTAIR enhanced the expression of p-PI3K and p-AKT and reduced the expression of APN, ADIPOR1, and PTEN. On the contrary, lncRNA HOTAIR silencing contributed to the opposite trends. However, the overexpression or silence of lncRNA HOTAIR did not significantly change the expression of PI3K, AKT, and ADIPOR2. 4 Discussion As one of the most frequent types of arthritis, OA is a chronic condition that causes cartilage loss, pain, and stiffness [ 17 , 18 ] . OA is particularly common among the elderly and obese, which is associated with a significant risk of joint injuries [ 19 ] . Joint space narrowing is a symptom of OA, which indicates cartilage loss. The function of inflammation has just recently been introduced in the pathophysiology of OA. Several studies [ 20 , 21 , 22 ] completed in the last ten years have documented a correlation between synovial inflammation and pain on one hand and radiographic progression on the other hand, demonstrating the critical role of inflammation in OA by resulting in the changes in the levels of inflammatory factors such as IL-10 and TNF-α. TNF-α is an essential pro-inflammatory factor involved in OA, which can activate osteoclasts of endothelial cells, leading to continuous inflammatory responses occurring in the gradual destruction of cartilage. IL-10 level was inhibited in the progression of OA. Existing studies [ 23 , 24 ] have shown that lipid metabolism imbalance plays an important role in the occurrence and development of OA, which is a risk factor for the occurrence of OA and hyperlipidemia and is closely related to the immune-inflammatory response of OA patients. APN, an indicator related to lipid metabolism, induces inflammatory responses [ 25 ] . Kabalyk et al. [ 26 ] confirmed that hyperlipidemia directly acts on articular tissues and causes cellular stress, manifested as changes in morphological and functional characteristics of CHs. Processes, including cell death, pathological mineralization of articular cartilages, and enhanced pathological angiogenesis can be observed in hyperlipidemia. Chen et al. constructed a lipopolysaccharide-induced human articular CH C28/I2 cell model and found that the expression and quantity of inflammatory cytokines (including IL-1β, IL-6, IL-8, and TNF-α) augmented in this cell model compared with the corresponding control group [ 27 , 28 ] . LncRNAs mediate critical mechanisms in the pathogenesis of OA, and these regulatory effects of lncRNA HOTAIR have also begun to attract extensive attention [ 29 , 30 ] , which is conducive to the development and progression of OA [ 31 ] . LncRNA HOTAIR, for example, is upregulated in OA, thus participating in IL-1β-induced matrix metalloproteinase overexpression and promoting inflammatory responses in CHs [ 32 ] . A prior research has elucidated the PI3K/AKT pathway assumes a key role in the development of inflammation in OA [ 33 ] . Moreover, another research has unraveled that the PI3K/AKT pathway exerts a certain effect on lipid metabolism [ 34 ] . This research provided evidence of substantially high expression of lncRNA HOTAIR in PBMCs from OA patients and OA-CHs induced by PBMCs of OA patients with the AUC of 0.8310, the optimal truncation value of 1.185, the sensitivity of 60.00%, and the specificity of 92.67%, illustrating that lncRNA HOTAIR had the high diagnostic value. Our results also exhibited that IL-10, APN, and ADIPOR2 levels were diminished and TNF-α level was augmented in OA patients. It has been documented that APN can control inflammation, thus functioning as an additional potential therapeutic target [ 35 ] . All in all, changes in levels of both cytokines and APN and its receptors can be indicated as the deregulation of pro-inflammatory and anti-inflammatory factors. In addition, clinical trials depicted that the level of lncRNA HOTAIR shared a positive correlation with TNF-α, clinical immune-inflammatory indicators, lipid metabolism index, and patient perception score scales, like TC, hs-CRP, IgG, and VAS. It has been reported that the imbalance of inflammatory factors is associated with lncRNA HOTAIR. In order to further dissect out the effects of lncRNA HOTAIR on the proliferation, inflammation, and lipid metabolism of OA-CHs, OA-PBMCs were utilized to stimulate OA-CHs as a cell model to conduct in vitro cell experiments. Subsequently, the impacts of lncRNA HOTAIR on the function of OA-CHs were assessed using CCK-8 assay. The results showed that the viability of OA-CHs was conspicuously decreased after overexpressing lncRNA HOTAIR. On the contrary, cell viability was enhanced following the transfection of lncRNA HOTAIR overexpression plasmids. Under the condition of lncRNA HOTAIR overexpression and silencing, inflammatory cytokines were further measured by the ELISA method. The results displayed that overexpression of lncRNA HOTAIR significantly enhanced TNF-α level and reduced IL-10 level. In contrast, the deletion of lncRNA HOTAIR triggered an opposite tendency to inflammation. These results suggested that si-lncRNA HOTAIR might be implicated in the occurrence and development of OA through orchestrating the levels of inflammatory factors in patients. Studies [ 36 , 37 , 38 , 39 ] have unveiled that lncRNA HOTAIR knockout can restrain cell viability through the PI3K/AKT pathway, reduce the cellular inflammatory response, inhibit drug resistance, and improve the quality of life of patients. Therefore, we next explored how lncRNA HOTAIR was involved in the progression of OA by manipulating the PTEN/PI3K/AKT pathway. The current study noted that the potential correlation of lncRNA HOTAIR with the level of the PTEN/PI3K/AKT pathway in OA-CHs through cell experiments. Western blot analysis was implemented to determine the influence of lncRNA HOTAIR on the pathway-related proteins and APN and its receptors proteins, manifesting that overexpression of lncRNA HOTAIR elevated the expression of p-PI3K and p-AKT but lowered the expression of PTEN, APN, and ADIPOR1 proteins. The cause of the insignificant effect on ADIPOR2 expression may be that ADIPOR1 is expressed abundantly in cartilages, bones, and synovial tissues, whereas ADIPOR R2 is rarely detected [ 40 ] . In summary, our obtained findings uncovered that lncRNA HOTAIR expression was high in the PBMCs of OA patients. Furthermore, its abnormal expression could modulate the viability of OA-CHs, which participated in the inflammatory response of OA by influencing the levels of inflammatory cytokines, APN, and the PTEN/PI3K/AKT pathway in OA-CHs, thus afflicting the occurrence and development of OA. Therefore, lncRNA HOTAIR may be utilized as a biomarker for OA and our research provides new insights into the pathogenesis of OA. The specific mechanism of action needs to be further explored. Declarations Acknowledges Not applicable. Funding This work was supported by Grants from Ministry of Science and Technology National Key Research and Development Program Chinese Medicine Modernization Research Key Project (2018YFC1705204); National Nature Fund Program (81973655); National Natural Science Foundation of China (82074373); Open Foundation of Key Laboratory of Xin'an Medical Ministry of Anhui University of Traditional Chinese Medicine (No.2020xayx10); The Key Research and Development Program Foreign Science and Technology Cooperation Project of Anhui (201904b11020011); Anhui Provincial Quality Engineering Teaching and Research Project (2018jyxm1068); Anhui Famous Traditional Chinese Medicine Liu Jian Studio Construction Project (Traditional Chinese Medicine Development Secret [2018] No. 11); National Key Innovative Talents Training Project of Traditional Chinese Medicine (National Education Letter of Traditional Chinese Medicine [2019] No.128); Key Research and Development Plan Project of Anhui Province (201904a07020004); Anhui Provincial Laboratory of Applied Basis and Development of Internal Medicine of Modern Traditional Chinese Medicine (2016080503B041); 12th batch of "115" Innovation team of Anhui Province (Anhui Talent Office [2019] No. 1). Contributions CXL and LJ contributed to the study design. CXL contributed to data analysis, wrote the first draft, and revised the manuscript. SYQ contributed to the specimen and data collection. ZXH, ZQ, and DX supervised the project and contributed to the manuscript revision. All authors reviewed and accepted the content of the final manuscript. Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. References Tu J, Huang W, Zhang W, et al. The emerging role of lncRNAs in chondrocytes from osteoarthritis patients. Biomed Pharmacother. 2020 Nov;131:110642. doi: 10.1016/j.biopha.2020.110642. Epub 2020 Sep 11. PMID: 32927251. X.D. Wang, J.N. Zhang, Y.H. Gan, et al. Current understanding of pathogenesis and treatment of TMJ osteoarthritis,J.Dent.Res.94(2015)666–673, https://doi.org/10.1177/0022034515574770. Schram B, Orr R, Pope R, et al. 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Linear regression analysis: part 14 of a series on evaluation of scientific publications. Dtsch Arztebl Int . 2010;107(44):776-782. doi:10.3238/arztebl.2010.0776 Martel-Pelletier J, Barr A J, Cicuttini F M, et al. Osteoarthritis. Nat. Rev. Dis. Primers 2016, 2, 16072. Di Paola R, Fusco R, Impellizzeri D, et al. Adelmidrol, in combination with hyaluronic acid, displays increased anti-inflammatory and analgesic effects against monosodium iodoacetateinduced osteoarthritis in rats. Arthritis Res. Ther. 2016, 18, 291. Sacitharan, P.K. Ageing and osteoarthritis. Subcell. Biochem. 2019, 91, 123–159. Yusuf E, Kortekaas MC, Watt I, et al. Do knee abnormalities visualised on MRI explain knee pain in knee osteoarthritis? Asystematic review. Annals of the Rheumatic Diseases 2011;70:60e7. Berenbaum F. Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!). Osteoarthritis and Cartilage 2013;21:16e21. Brouwers H, von Hegedus J, Toes R, etal. 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Arthritis Research & Therapy,2011,13(6): Kabalyk MA, Kovalenko TS, Nevzorova VA, et al. [Effect of arterial hypertension and hyperlipidemia on remodeling of articular cartilage and the development of osteoarthritis (experimental study).]. Adv Gerontol.2019;32(4):550-557. Russian. PMID: 31800183. Chen H, Qi J, Bi Q, et al. Expression profile of long noncoding RNA (HOTAIR) and its predicted target miR-17-3p in LPS-induced inflammatory injury in human articular chondrocyte C28/I2 cells. Int J Clin Exp Pathol. 2017 Sep 1;10(9):9146-9157. PMID: 31966787; PMCID: PMC6966013. Luo X, Wang J, Wei X, et al. Knockdown of lncRNA MFI2-AS1 inhibits lipopolysaccharide-induced osteoarthritis progression by miR-130a-3p/TCF4. Life Sci . 2020;240:117019. doi:10.1016/j.lfs.2019.117019 Chen Yingchun, Zhang Lin, Li Ernan, et al. Long-chain non-coding RNA HOTAIR promotes the progression of osteoarthritis via sponging miR-20b/PTEN axis. [J]. Life Sci, 2020, 253: 117685. doi:10.1016/j.lfs.2020.117685. Yang Yang, Xing Dan, Wang Yawei, et al. A long non-coding RNA, HOTAIR, promotes cartilage degradation in osteoarthritis by inhibiting WIF-1 expression and activating Wnt pathway.[J].BMC Mol Cell Biol, 2020, 21: 53. doi:10.1186/s12860-020-00299-6 Marques-Rocha, J.L, Samblas, et al. (2015) Noncoding RNAs, cytokines, and inflammation-relateddiseases.FASEBJ.29,3595–3611,https://doi.org/10.1096/fj.14-260323. Jiang M, Liu J, Luo T, et al. LncRNA PACER is down-regulated in osteoarthritis and regulates chondrocyte apoptosis and lncRNA HOTAIR expression. Biosci Rep . 2019;39(6):BSR20190404. Published 2019 Jun 7. doi:10.1042/BSR20190404. Liu N, Fu D, Yang J, et al. Asiatic acid attenuates hypertrophic and fibrotic differentiation of articular chondrocytes via AMPK/PI3K/AKT signaling pathway. Arthritis Res Ther . 2020;22(1):112. Published 2020 May 12. doi:10.1186/s13075-020-02193-0. Li L, Liu F, Huang W, et al. Ricolinostat (ACY-1215) inhibits VEGF expression via PI3K/AKT pathway and promotes apoptosis in osteoarthritic osteoblasts. Biomed Pharmacother . 2019;118:109357. doi:10.1016/j.biopha.2019.109357. Yeon-Ah Lee, Hyun Mi Choi, Sang-Hoon Lee,et al. Synergy between adiponectin and interleukin-1β on the expression of interleukin-6, interleukin-8, and cyclooxygenase-2 in fibroblast-like synoviocytes[J]. Experimental & Molecular Medicine,2012,44(7) Li Z, Qian J, Li J, et al. Knockdown of lncRNA-HOTAIR downregulates the drug-resistance of breast cancer cells to doxorubicin via the PI3K/AKT/mTOR signaling pathway. Exp Ther Med . 2019;18(1):435-442. doi:10.3892/etm.2019.7629 Qi K, Zhong J. LncRNA HOTAIR improves diabetic cardiomyopathy by increasing viability of cardiomyocytes through activation of the PI3K/Akt pathway. Exp Ther Med . 2018;16(6):4817-4823. doi:10.3892/etm.2018.6755 Zhang XH, Hu P, Xie YQ, et al. Long Noncoding RNA HOTAIR Promotes Endometrial Carcinoma Cell Proliferation by Binding to PTEN via the Activating Phosphatidylinositol 3-Kinase/Akt Signaling Pathway. Mol Cell Biol . 2019;39(23):e00251-19. Published 2019 Nov 12. doi:10.1128/MCB.00251-19 Liu G, Zhang W. Long non-coding RNA HOTAIR promotes UVB-induced apoptosis and inflammatory injury by up-regulation of PKR in keratinocytes [retracted in: Braz J Med Biol Res. 2021 Jun 14;54(6):e6896retraction]. Braz J Med Biol Res . 2018;51(8):e6896. Published 2018 Jun 11. doi:10.1590/1414-431X20186896. Chen Tsu-Hsin, Chen Linda, Hsieh Ming-Shium, et al. Evidence for a protective role for adiponectin in osteoarthritis. [J]. Biochim Biophys Acta, 2006, 1762: 711-8. Cite Share Download PDF Status: Published Journal Publication published 12 Jan, 2023 Read the published version in Journal of Orthopaedic Surgery and Research → Version 1 posted Editorial decision: Major revision 30 Oct, 2022 Reviewers agreed at journal 07 Oct, 2022 Reviewers invited by journal 12 Dec, 2021 Editor assigned by journal 09 Dec, 2021 First submitted to journal 09 Dec, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1156754","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":69531476,"identity":"76b20fe5-3e2b-4977-bdc7-14e00dc6c204","order_by":0,"name":"Xiaolu Chen","email":"","orcid":"https://orcid.org/0000-0001-6064-6362","institution":"Anhui University of Traditional Chinese Medicine - Meishan Road Campus: Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaolu","middleName":"","lastName":"Chen","suffix":""},{"id":69531477,"identity":"db52b468-58e4-4ce1-8000-2b22074ac958","order_by":1,"name":"Jian Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYBACPmYGBoMEIMOAgYHxQUKFDWEtbEhamA0enEkjQguMAdTCJvmw7RARWth5DAoe1NyRN+c/Y1aRwHaAgb+9O4GAw3gMDBKOPTPc2XDG7EYCzx0GiTNnNxChhe0w44aDPUAtEs8YDCRyidHy77D9hsM8ZgUJBoeJ1JLYdjhxwzEeM4aEBKK0sBUYJPYdTt5whq1YIuFAGg9Bv/DzH95m+OPbYdsN5w9v/Pjzn40cf3svfi0giwwgNAeY5iGkHASYH0Bo9gfEqB4Fo2AUjIIRCACfiEc9wQsS0QAAAABJRU5ErkJggg==","orcid":"","institution":"Anhui University of Traditional Chinese Medicine - Meishan Road Campus: Anhui University of Traditional Chinese Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Liu","suffix":""},{"id":69531478,"identity":"e1d98a25-d7fd-4903-ae97-c42bf0fb721d","order_by":2,"name":"Yanqiu Sun","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine - Meishan Road Campus: Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanqiu","middleName":"","lastName":"Sun","suffix":""},{"id":69531479,"identity":"c2be155e-2e48-46c7-a186-f1c28b2f2961","order_by":3,"name":"Jianting Wen","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine - Meishan Road Campus: Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jianting","middleName":"","lastName":"Wen","suffix":""},{"id":69531480,"identity":"84527f64-4fe1-415d-bfe5-295d4cef5af2","order_by":4,"name":"Qin Zhou","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine - Meishan Road Campus: Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Zhou","suffix":""},{"id":69531481,"identity":"a2c52513-57fa-4f22-9ea8-0b4e50750b21","order_by":5,"name":"Xiang Ding","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine - Meishan Road Campus: Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Ding","suffix":""},{"id":69531482,"identity":"ebcf1ca2-6657-40af-a0e4-2fc7c7fdf06f","order_by":6,"name":"Xianheng Zhang","email":"","orcid":"","institution":"Anhui University of Traditional Chinese Medicine - Meishan Road Campus: Anhui University of Traditional Chinese Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xianheng","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2021-12-09 16:31:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1156754/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1156754/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13018-023-03505-1","type":"published","date":"2023-01-12T18:19:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":16870078,"identity":"db590314-f085-4808-86a5-160e6222ea9a","added_by":"auto","created_at":"2021-12-30 15:05:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":703596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLncRNA HOTAIR expression is high in the PBMCs of OA patients.\u003c/strong\u003e (A) LncRNA HOTAIR expression was considerably higher in OA patients than in HC subjects. (B) Comparison of ROC curves of lncRNA HOTAIR expression between PBMCs in OA patients and HC patients. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1156754/v1/0eef4d6925b7ebd14e3f6bd6.jpg"},{"id":16870079,"identity":"f88ecd56-30c8-4b59-b918-4a3a28f00bd7","added_by":"auto","created_at":"2021-12-30 15:05:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1646606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential expression of inflammatory cytokines in OA. \u003c/strong\u003e(A-D)\u003cstrong\u003e \u003c/strong\u003eThe levels of TNF-α (A), IL-10 (B), APN (C), and ADIPOR2 (D) in serum of OA patients and normal subjects. \u003csup\u003e*** \u003c/sup\u003e\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001. (F-I) Correlation analyses of lncRNA HOTAIR with TC (E), hs-CRP (F), IgG (G), TNF-α (H), and VAS (I) in the PBMCs of OA patients.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1156754/v1/23aee0e7be20deb3954aef0d.jpg"},{"id":16869941,"identity":"085afc61-80e7-4ad9-bc1c-93142dd8b69b","added_by":"auto","created_at":"2021-12-30 15:02:58","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":613140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLncRNA HOTAIR upregulation facilitates the viability of OA-CHs.\u003c/strong\u003e (A) The expression of lncRNA HOTAIR in OA-CHs measured by RT-qPCR. (B) The effect of lncRNA HOTAIR on the viability of OA-CHs tested by CCK-8. All tests were repeated three times and results were expressed as mean ± standard deviation. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1156754/v1/b7dad84ad04bc33941c6549f.jpg"},{"id":16869938,"identity":"577937c5-6801-4c8c-b323-82c4f29576e4","added_by":"auto","created_at":"2021-12-30 15:02:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":762755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLncRNA HOTAIR upregulation causes TNF-α upregulation and IL-10 downregulation in OA-CHs.\u003c/strong\u003e (A-B) The levels of IL-10 (A) and TNF-α (B) in OA-CHs determined by ELISA. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1156754/v1/105a87e921279b0fc2e47350.jpg"},{"id":16869939,"identity":"906a8cd3-deb7-4e83-8279-86f07fadedd3","added_by":"auto","created_at":"2021-12-30 15:02:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":173437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of lncRNA HOTAIR on the PTEN/PI3K/AKT pathway in OA-CHs. \u003c/strong\u003e(A) The representative Western blots of PI3K, p-AKT, p-PI3K, AKT, APN, ADIPOR1, ADIPOR2, and PTEN in OA-CHs. (B) The quantitative analysis of the expression of PI3K, p-AKT, p-PI3K, AKT, and PTEN in OA-CHs. (C) The quantitative analysis of the expression of APN, ADIPOR1, and ADIPOR2 in OA-CHs. All experiments were repeated three times and data were expressed as mean ± standard deviation. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1156754/v1/95c8e8c39bb561355faf7e66.jpg"},{"id":44716791,"identity":"bbb47272-8209-4dd4-9d0e-0be42454b46b","added_by":"auto","created_at":"2023-10-16 18:30:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":901115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1156754/v1/8dd05988-8857-4906-990b-0f365bdb9965.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eExpression of Long Noncoding RNA HOTAIR and Its Influence on the PTEN/PI3K/AKT Pathway and Inflammation in Patients With Osteoarthritis\u003c/p\u003e","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a kind of degenerative disease featured by \"low level\" cartilage and synovial inflammation \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, which is based on joint degeneration or aging and leads to the disappearance of joint structure and gradual aggravation of cartilage. It is a chronic arthritic disease with systemic pain in large joints and swelling or dysfunction of elbow and knee joints as the main clinical manifestations. It has been reported that there exist several risk factors for OA, including age, joint trauma, obesity, and genetic susceptibility \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. To a certain extent, obesity is related to inflammation, namely, hyperlipidemia that is a common metabolic change and is characterized by an abnormal increase in serum total cholesterol (TC), triglycerides, and low-density lipoprotein cholesterol, as well as an abnormal decrease in high-density lipoprotein cholesterol and apolipoprotein A1, all of which contribute to the inflammatory response and OA deterioration. In addition, body fat, insulin resistance index, and body mass index are negatively related to adiponectin (APN) \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. APN, a recently discovered cytokine released by adipose tissues, is a regulator for the repression of the inflammatory response in OA \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Through an animal experiment \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e, long noncoding RNAs (lncRNAs) are involved in the inflammation, lipid metabolism, and other processes of OA, which has become a research hotspot \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA prior research \u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e has observed that lncRNA HOTAIR expression in OA-chondrocytes (CHs) was apparently higher than that in normal CHs and that the proliferation capacity was significantly higher in OA-CHs transfected with small interfering RNA (si)-lncRNA HOX transcript antisense RNA (HOTAIR) than in those transfected with si-negative control (NC). The tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten (PTEN) can dephosphorylate and inactivate phosphatidylinositol-3,4,5-trisphosphate (PIP3), an important molecule in the phosphoinositide 3-kinase (PI3K)/AKT pathway, to downregulate the PI3K/AKT pathway. Therefore, PTEN and the PI3K/AKT pathway together are called the PTEN/PI3K/AKT pathway, a classic inflammatory pathway. A recent study reported that PTEN can downregulate the expression of fatty acid synthase by inhibiting the PTEN/PI3K/AKT pathway \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Interleukin (IL)-10 and tumor necrosis factor-α (TNF-α) are critical pro-inflammatory factors and anti-inflammatory factors of OA \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, respectively. Of note, the PTEN/PI3K/AKT pathway can increase TNF-α expression and decrease IL-10 expression \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Furthermore, it has been noted in a previous research that the content of TNF-α is obviously reduced and the content of IL-10 is remarkably enhanced after silencing lncRNA HOTAIR \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, although lncRNA HOTAIR and the PTEN/PI3K/AKT pathway participate in the inflammation of OA, there has hitherto been little report on whether the former can assume a role in immune regulation by affecting the latter in the inflammatory response of OA.\u003c/p\u003e \u003cp\u003eTherefore, this study was conducted to focus on observing lncRNA HOTAIR expression in OA patients and analyzing its correlation with clinical indicators and visual analog scale (VAS) scores. Finally, lncRNA HOTAIR was altered in cell experiments to assess the changes in the expression of the PTEN/PI3K/AKT pathway, inflammatory factors, and lipid metabolism indexes.\u003c/p\u003e"},{"header":"2 Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Subjects and samples\u003c/h2\u003e \u003cp\u003eOA patients and healthy control (HC) subjects matched on sex and age were enrolled from the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine between November 2020 and May 2021. Patients were excluded from the present research if they did not meet the following criteria: not conforming to the 2019 Guidelines for the diagnosis of OA \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e, severe mental illness, significantly impaired liver or renal function, the administration of immunosuppressive drugs, or pregnancy. The research was conducted following the relevant provisions of the \u003cem\u003eDeclaration of Helsinki\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e and was approved by the Ethical Committee of Scientific Research of Anhui University of Traditional Chinese Medicine's First Affiliated Hospital [2014AH-06(J)].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Co-culture of peripheral blood mononuclear cells (PBMCs) with CHs from OA patients and their transfection\u003c/h2\u003e \u003cp\u003eTotally, 5 mL venous blood was collected from normal healthy subjects and OA patients, and diluted with an equal volume of normal saline. The same amount of lymphocyte separation solution was slowly added into the prepared samples and centrifuged at 2000 r/min for 20 min. The white floc in the centrifuge tube was transferred to the clean centrifuge tube. The same amount of normal saline was added into the tube and mixed, followed by 8-min centrifugation 800 r/min and two washes. After blowing, the samples were transferred into a culture flask and incubated for later use.\u003c/p\u003e \u003cp\u003eThe CHs were purchased from Saibaikang Biotechnology Co., Ltd. (Shanghai, China). PBMCs were extracted and seeded into the Transwell chamber, followed by the addition of 75 \u0026micro;L Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM). CHs were digested and centrifuged. Then, the medium was discarded, and CHs were washed twice with phosphate buffer saline, resuspended with medium, and seeded into the apical chamber, followed by the supplementation of 100 \u0026micro;L DMEM. Following cell adherence, the cells were activated with TNF-α (10 ng/mL), and OA-PBMCs and OA-CHs were treated at the ratio of 0.5:1. Co-culture was conducted in Roswell Park Memorial Institute-1640 media containing penicillin (the final concentration of 100 U/mL) and streptomycin (the final concentration of 0.1 mg/mL) with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C until the CH confluence reached 70%-90%. OA-CHs were transfected with PCDNA3.1-lncRNA HOTAIR plasmids, si-lncRNA HOTAIR plasmids, and their corresponding NC plasmids as per the manuals of Lipofectamine 2000, followed by 48-h incubation. All plasmids were purchased from Shanghai GenePharma Company (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal OA-CH RNA was obtained using Trizol, followed by reverse transcription reaction and amplification reaction. Agarose-gel electrophoresis was implemented for semi-quantitative analysis of PCR products using the Gelpro32 gel image analysis software. Relative quantitative analysis was performed using 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e with β-actin as an internal reference. All used primers were as follows: lncRNA HOTAIR: the forward primer: 5'-CCATAGCCGATTAGCTGTCA' and the reverse primer: 5'-AATGCCGAACTGGAGGTG-3'; β-actin: the forward primer: 5'-GGCAAATGTCAGAGGGTTCT-3' and the reverse primer: 5'-TTCTTAAATTGGGCTGGGTC-3'.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eFollowing collection, the supernatant of OA-CHs was centrifuged at 2500 r/min for 20 min, and the precipitation was discarded. The supernatant was added to the ELISA plate (50 \u0026micro;L per well) and incubated at 37\u0026deg;C for 30 minutes after the plate was sealed with a sealing plate membrane. The levels of TNF-α, IL-10, APN, and adiponectin receptor 2 (ADIPOR2) were detected using the ELISA method strictly in the light of the kit instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell Counting Kit-8 (CCK-8) assay\u003c/h2\u003e \u003cp\u003eCell viability was measured with a CCK-8 assay kit (BIOSS, Beijing, China). At first, 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e OA-CHs were seeded into each well of 96-well plates and cultured until 70% and 90% confluence. Logarithmically growing cells were transfected by the above method. Three wells in each group were cultured for 0, 12, 24, 48, and 72 h, respectively. Next, 10 mL CCK-8 solution was supplemented into each well at each experimental site, followed by 1-4 h of cell incubation at 37\u0026deg;C. The viability of OA-CHs was determined by measuring the absorbance value of each well at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blot analysis\u003c/h2\u003e \u003cp\u003eCells were lysed with Radio-Immunoprecipitation assay lysis solution and centrifuged at 12000 r/min for 10 min to harvest total proteins. A sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) gel preparation kit (Beyotime, Shanghai, China) was utilized. The 5 \u0026times; SDS-PAGE protein loading buffer was added into cells at the ratio of 1:4 to obtain protein samples. The samples were heated for 10 min in a boiling water bath and supplemented into SDS-PAGE gel wells (5-10 \u0026micro;L per well). Filter papers and polyvinylidene fluoride membrane of the same size as the rubber strip were cut in advance (soaked in methanol for 2-3 min in advance) and immersed in the rotating film buffer for 5 min. Bubbles were removed at every step and the flow membrane was constant. After the protein was transferred into the membranes, the membranes were cooled to room temperature before the insertion of the membranes into the prepared Western washing solution and the removal of the membrane transfer solution by 5-min washing. The Western sealing solution (5% skim milk powder) was added, slowly shaken in the shaker, closed at room temperature for 2 h, and then washed with phosphate-buffered saline containing Tween 20 three times with 10 min for each time. The membrane underwent overnight probing with primary pig antibodies to phosphorylation (p)-AKT, PI3K, PTEN, AKT, P-PI3K, APN, ADIPOR1, and ADIPOR2 at 4\u0026deg;C. The horseradish peroxidase-tagged secondary antibody was diluted with secondary antibody diluent at the ratio of 1:20,000 and incubated with the membranes at room temperature for 1 h. The ratio of absorbance of target proteins to that of glyceraldehyde-3-phosphate dehydrogenase was calculated after electrogenerated chemiluminescence coloring, exposure in the darkroom, fixation, and photography.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eSPSS statistical software version 23.0 (IBM Corp. Armonk, NY, USA) was applied for statistical analysis, and GraphPad Prism software version 8.2 (GraphPad Software, La Jolla, CA, USA) was utilized to capture images. The significant difference between groups was compared with matching double tail Student \u003cem\u003et\u003c/em\u003e-test or Kruskal Wallis nonparametric tests. Classification variables were compared by the chi-square test. The Spearman correlation analysis was employed to evaluate the correlation of lncRNA HOTAIR with erythrocyte sedimentation rate (ESR), high sensitivity C-reactive protein (hs-CRP), immunoglobulin A (IgA), and other indicators. Data were expressed as mean \u0026plusmn; standard deviation or median (quartile range). \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered to be significantly different.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e3.1 Clinical characteristics of OA patients and HC subjects\u003c/h2\u003e\n\u003cp\u003eA total of 80 subjects were enrolled, including 30 HC subjects (10 males and 20 females, the mean age of 59.24 \u0026plusmn; 11.94 years) in the control group and 50 OA patients (17 males and 33 females, the mean age of 65.54 \u0026plusmn; 14.10 years) in the OA group. There were no statistically significant differences in age and gender distribution between the two groups but significant differences in ESR, TC, triglyceride, immunoglobulin G (IgG), complement C3 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eChanges in clinical lipid metabolism indexes and immune-inflammatory indexes in OA patients\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eIndexes\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eOA (n = 50)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eHC (n = 30)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge (years)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e55.85 \u0026plusmn; 10.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e56.48 \u0026plusmn; 6.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.655\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eESR (mm/h)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19.4 (11.2, 31)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.50 (5.66, 11.62)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTC (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.25 \u0026plusmn; 1.29\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.20 (1.53, 3.10)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTG (mmol/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.00 (1.35, 1.73)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.50 (0.34, 1.03)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIgA (g/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.48 \u0026plusmn; 1.12\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.61 (1.20, 2.20)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC3 (g/L)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.15 \u0026plusmn; 0.22\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1.05 (0.94, 1.09)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSDS score\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e53.09 \u0026plusmn; 7.11\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eVAS score\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.72 \u0026plusmn; 0.60\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSAS score\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e43.38 (40.62, 47.67)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"4\"\u003eNotes: a, Student\u0026rsquo;s t-test. b, Wilcoxon signed-rank test, median (25\u0026ndash;75th percentile). c, mean \u0026plusmn; standard error. NA, not applicable; NC, negative control; OA, osteoarthritis; ESR, erythrocyte sedimentation rate; TC, total cholesterol; TG, triglyceride; IgA, immunoglobulin A; C3, complement C3; SDS, Self-rating Depression Scale; SAS, Self-rating Anxiety Scale; VAS, visual analog scale; HC, healthy control.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e3.2 LncRNA HOTAIR expression is upregulated in the PBMCs of OA patients\u003c/h2\u003e\n\u003cp\u003eThis study intended to assess lncRNA HOTAIR expression in OA patients. LncRNA HOTAIR expression in the PBMCs of 50 OA patients and 30 HC subjects was detected using RT-qPCR. The findings revealed that lncRNA HOTAIR expression was increased in the PBMCs of OA patients (Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The diagnostic efficacy of lncRNA HOTAIR was evaluated using a receiver-operating characteristic (ROC) curve analysis, and the area under the ROC curve (AUC) was 0.8310 [95% confidence interval (CI): 0.74-0.92]. According to the Youden index, the ideal cut-off value for the separation of OA from HC subjects was 1.185 with a sensitivity of 60.00% and a specificity of 92.67% (Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003ch2\u003e3.3 Differential expression of inflammatory cytokines and APN in OA with a positive correlation with lncRNA HOTAIR expression\u003c/h2\u003e\n\u003cp\u003eThe levels of IL-10 and TNF-\u0026alpha; in the OA group were dramatically lowered (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) and substantially increased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01), respectively, as compared to the normal group, showing that there were alterations in the levels of pro-inflammatory and anti-inflammatory factors in OA (Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Furthermore, the expression of APN and ADIPOR2 was considerably reduced in the OA group compared with the normal group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). Then, to further explore the possible relationship between lncRNA HOTAIR and related research indicators, we conducted correlation analyses of lncRNA HOTAIR with TC, hs-CRP, IgG, TNF-\u0026alpha;, and VAS respectively. The results displayed that lncRNA HOTAIR was a risk factor for TC, hs-CRP, IgG, TNF-\u0026alpha;, and VAS \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e (Figure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE-I). These results further suggested the close relationship between lncRNA HOTAIR and the occurrence and development of OA.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Association rules analysis of lncRNA HOTAIR with laboratory indexes in OA patient\u003c/h2\u003e\n\u003cp\u003eAssociation rule analysis showed that the elevation of lncRNA HOTAIR was strongly correlated with the elevation of GSH, ESR, and VAS in OA patients. There was a strong correlation between the increase of lncRNA HOTAIR and the decrease of LDL-C, with a support degree greater than 20% and a confidence degree greater than 30%. The results are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eAssociation rules analyses between lncRNA HOTAIR and multiple laboratory indicators in OA patients\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eItems (LHS \u0026rArr; RHS)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSupport (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eConfidence (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLift\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e{lncRNA HOTAIR\u0026uarr;}\u0026rArr;{Hospitalization days\u0026darr;}\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e63.636\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e76.087\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e{lncRNA HOTAIR\u0026uarr;}\u0026rArr;{sex (female)}\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e56.364\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e67.391\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e{lncRNA HOTAIR\u0026uarr;}\u0026rArr;{SDS\u0026uarr;}\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e63.636\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e76.087\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e{lncRNA HOTAIR\u0026uarr;}\u0026rArr;{ESR\u0026uarr;}\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e61.818\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e73.913\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e{lncRNA HOTAIR\u0026uarr;}\u0026rArr;{VAS\u0026uarr;}\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e67.273\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e80.435\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.01\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e{lncRNA HOTAIR\u0026uarr;}\u0026rArr;{LDL-C\u0026uarr;}\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e27.273\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32.609\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.02\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e\u0026lt; 0.01\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eNotes: For correlations, Aprior module analysis was used. The minimal level of support was set at 20%, while the minimum level of confidence was set at 30%. When the degree of the lift was set to greater than one, it was deemed important.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e3.5 LncRNA HOTAIR overexpression enhanced the viability of OA-CHs\u003c/h2\u003e\n\u003cp\u003eLncRNA HOTAIR expression in the PCDNA3.1-lncRNA HOTAIR group was substantially higher (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) than that in the PCDNA3.1-NC group, whereas lncRNA HOTAIR expression in the si-lncRNA HOTAIR group was significantly lower than that in the si-NC group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). The effect of lncRNA HOTAIR on the viability of OA-CHs was assessed by CCK-8. The results exhibited that the PCDNA3.1-lncRNA HOTAIR group had dramatically boosted cell viability when compared to the PCDNA3.1-NC group. In contrast to the si-NC group, cell viability of the si-lncRNA HOTAIR group was evidently diminished. Among the four groups, cell viability of the si-lncRNA HOTAIR group was the highest and that of the lncRNA HOTAIR group was the lowest. As shown in figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e3.6 LncRNA HOTAIR overexpression facilitated TNF-\u0026alpha; expression but repressed IL-10 expression in OA-CHs\u003c/h2\u003e\n\u003cp\u003eThe level of cytokines was evaluated by ELISA. As a result, versus the PCDNA3.1-NC group, TNF-\u0026alpha; expression in the PCDNA3.1-lncRNA HOTAIR group was prominently augmented and IL-10 expression was remarkably decreased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Compared with the si-NC group, TNF-\u0026alpha; expression was markedly lowered and IL-10 expression was noticeably elevated in the si-lncRNA HOTAIR group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-B).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7 LncRNA HOTAIR manipulated the PTEN/PI3K/AKT pathway in OA-CHs\u003c/h2\u003e\n\u003cp\u003eIn order to ascertain the impacts of lncRNA HOTAIR on the PTEN/PI3K/AKT pathway, Western blot analysis was adopted to identify the expression of PI3K, p-AKT, p-PI3K, AKT, APN, ADIPOR1, ADIPOR2, and PTEN. As discovered in Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, overexpression of lncRNA HOTAIR enhanced the expression of p-PI3K and p-AKT and reduced the expression of APN, ADIPOR1, and PTEN. On the contrary, lncRNA HOTAIR silencing contributed to the opposite trends. However, the overexpression or silence of lncRNA HOTAIR did not significantly change the expression of PI3K, AKT, and ADIPOR2.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eAs one of the most frequent types of arthritis, OA is a chronic condition that causes cartilage loss, pain, and stiffness \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. OA is particularly common among the elderly and obese, which is associated with a significant risk of joint injuries \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Joint space narrowing is a symptom of OA, which indicates cartilage loss. The function of inflammation has just recently been introduced in the pathophysiology of OA. Several studies \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e] completed in the last ten years have documented a correlation between synovial inflammation and pain on one hand and radiographic progression on the other hand, demonstrating the critical role of inflammation in OA by resulting in the changes in the levels of inflammatory factors such as IL-10 and TNF-\u0026alpha;. TNF-\u0026alpha; is an essential pro-inflammatory factor involved in OA, which can activate osteoclasts of endothelial cells, leading to continuous inflammatory responses occurring in the gradual destruction of cartilage. IL-10 level was inhibited in the progression of OA. Existing studies \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e have shown that lipid metabolism imbalance plays an important role in the occurrence and development of OA, which is a risk factor for the occurrence of OA and hyperlipidemia and is closely related to the immune-inflammatory response of OA patients. APN, an indicator related to lipid metabolism, induces inflammatory responses \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Kabalyk et al. \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e confirmed that hyperlipidemia directly acts on articular tissues and causes cellular stress, manifested as changes in morphological and functional characteristics of CHs. Processes, including cell death, pathological mineralization of articular cartilages, and enhanced pathological angiogenesis can be observed in hyperlipidemia. Chen et al. constructed a lipopolysaccharide-induced human articular CH C28/I2 cell model and found that the expression and quantity of inflammatory cytokines (including IL-1\u0026beta;, IL-6, IL-8, and TNF-\u0026alpha;) augmented in this cell model compared with the corresponding control group \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. LncRNAs mediate critical mechanisms in the pathogenesis of OA, and these regulatory effects of lncRNA HOTAIR have also begun to attract extensive attention \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, which is conducive to the development and progression of OA \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. LncRNA HOTAIR, for example, is upregulated in OA, thus participating in IL-1\u0026beta;-induced matrix metalloproteinase overexpression and promoting inflammatory responses in CHs \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. A prior research has elucidated the PI3K/AKT pathway assumes a key role in the development of inflammation in OA \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Moreover, another research has unraveled that the PI3K/AKT pathway exerts a certain effect on lipid metabolism \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis research provided evidence of substantially high expression of lncRNA HOTAIR in PBMCs from OA patients and OA-CHs induced by PBMCs of OA patients with the AUC of 0.8310, the optimal truncation value of 1.185, the sensitivity of 60.00%, and the specificity of 92.67%, illustrating that lncRNA HOTAIR had the high diagnostic value. Our results also exhibited that IL-10, APN, and ADIPOR2 levels were diminished and TNF-\u0026alpha; level was augmented in OA patients. It has been documented that APN can control inflammation, thus functioning as an additional potential therapeutic target \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. All in all, changes in levels of both cytokines and APN and its receptors can be indicated as the deregulation of pro-inflammatory and anti-inflammatory factors. In addition, clinical trials depicted that the level of lncRNA HOTAIR shared a positive correlation with TNF-\u0026alpha;, clinical immune-inflammatory indicators, lipid metabolism index, and patient perception score scales, like TC, hs-CRP, IgG, and VAS. It has been reported that the imbalance of inflammatory factors is associated with lncRNA HOTAIR. In order to further dissect out the effects of lncRNA HOTAIR on the proliferation, inflammation, and lipid metabolism of OA-CHs, OA-PBMCs were utilized to stimulate OA-CHs as a cell model to conduct in vitro cell experiments.\u003c/p\u003e\n\u003cp\u003eSubsequently, the impacts of lncRNA HOTAIR on the function of OA-CHs were assessed using CCK-8 assay. The results showed that the viability of OA-CHs was conspicuously decreased after overexpressing lncRNA HOTAIR. On the contrary, cell viability was enhanced following the transfection of lncRNA HOTAIR overexpression plasmids. Under the condition of lncRNA HOTAIR overexpression and silencing, inflammatory cytokines were further measured by the ELISA method. The results displayed that overexpression of lncRNA HOTAIR significantly enhanced TNF-\u0026alpha; level and reduced IL-10 level. In contrast, the deletion of lncRNA HOTAIR triggered an opposite tendency to inflammation. These results suggested that si-lncRNA HOTAIR might be implicated in the occurrence and development of OA through orchestrating the levels of inflammatory factors in patients.\u003c/p\u003e\n\u003cp\u003eStudies \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e have unveiled that lncRNA HOTAIR knockout can restrain cell viability through the PI3K/AKT pathway, reduce the cellular inflammatory response, inhibit drug resistance, and improve the quality of life of patients.\u003c/p\u003e\n\u003cp\u003eTherefore, we next explored how lncRNA HOTAIR was involved in the progression of OA by manipulating the PTEN/PI3K/AKT pathway. The current study noted that the potential correlation of lncRNA HOTAIR with the level of the PTEN/PI3K/AKT pathway in OA-CHs through cell experiments. Western blot analysis was implemented to determine the influence of lncRNA HOTAIR on the pathway-related proteins and APN and its receptors proteins, manifesting that overexpression of lncRNA HOTAIR elevated the expression of p-PI3K and p-AKT but lowered the expression of PTEN, APN, and ADIPOR1 proteins. The cause of the insignificant effect on ADIPOR2 expression may be that ADIPOR1 is expressed abundantly in cartilages, bones, and synovial tissues, whereas ADIPOR R2 is rarely detected \u003csup\u003e[\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn summary, our obtained findings uncovered that lncRNA HOTAIR expression was high in the PBMCs of OA patients. Furthermore, its abnormal expression could modulate the viability of OA-CHs, which participated in the inflammatory response of OA by influencing the levels of inflammatory cytokines, APN, and the PTEN/PI3K/AKT pathway in OA-CHs, thus afflicting the occurrence and development of OA. Therefore, lncRNA HOTAIR may be utilized as a biomarker for OA and our research provides new insights into the pathogenesis of OA. The specific mechanism of action needs to be further explored.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledges\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Grants from Ministry of Science and Technology National Key Research and Development Program Chinese Medicine Modernization Research Key Project (2018YFC1705204); National Nature Fund Program (81973655); National Natural Science Foundation of China (82074373); Open Foundation of Key Laboratory of Xin\u0026apos;an Medical Ministry of Anhui University of Traditional Chinese Medicine (No.2020xayx10); The Key Research and Development Program Foreign Science and Technology Cooperation Project of Anhui (201904b11020011); Anhui Provincial Quality Engineering Teaching and Research Project (2018jyxm1068); Anhui Famous Traditional Chinese Medicine Liu Jian Studio Construction Project (Traditional Chinese Medicine Development Secret [2018] No. 11); National Key Innovative Talents Training Project of Traditional Chinese Medicine (National Education Letter of Traditional Chinese Medicine [2019] No.128); Key Research and Development Plan Project of Anhui Province (201904a07020004); Anhui Provincial Laboratory of Applied Basis and Development of Internal Medicine of Modern Traditional Chinese Medicine (2016080503B041); 12th batch of \u0026quot;115\u0026quot; Innovation team of Anhui Province (Anhui Talent Office [2019] No. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCXL and LJ contributed to the study design. CXL contributed to data analysis, wrote the first draft, and revised the manuscript. SYQ contributed to the specimen and data collection. ZXH, ZQ, and DX supervised the project and contributed to the manuscript revision. All authors reviewed and accepted the content of the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTu J, Huang W, Zhang W, et al. The emerging role of lncRNAs in chondrocytes from osteoarthritis patients. Biomed Pharmacother. 2020 Nov;131:110642. doi: 10.1016/j.biopha.2020.110642. Epub 2020 Sep 11. PMID: 32927251.\u003c/li\u003e\n \u003cli\u003eX.D. Wang, J.N. Zhang, Y.H. Gan, et al. Current understanding of pathogenesis and treatment of TMJ osteoarthritis,J.Dent.Res.94(2015)666\u0026ndash;673,\u003ca href=\"https://doi.org/10.1177/0022034515574770.\"\u003ehttps://doi.org/10.1177/0022034515574770.\u003c/a\u003e\u003c/li\u003e\n \u003cli\u003eSchram B, Orr R, Pope R, et al. Risk factors for development of lowerlimb osteoarthritis in physically demanding occupations: A narrativeumbrella review. [J]. J Occup Health, 2019, 62 (1):e12103. DOI:10.1002/1348-9585.12103.\u003c/li\u003e\n \u003cli\u003eY. Arita, S. Kihara, N. Ouchi, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity, Biochem. Biophys. Res. Commun. 257(1999) 79-83.\u003c/li\u003e\n \u003cli\u003eM. Cnop, P. J. Havel, K.M. Utzschneider, et al. 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Biochim Biophys Acta, 2006, 1762: 711-8.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-orthopaedic-surgery-and-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"josr","sideBox":"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)","snPcode":"13018","submissionUrl":"https://submission.nature.com/new-submission/13018/3","title":"Journal of Orthopaedic Surgery and Research","twitterHandle":"@MSKmedBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Osteoarthritis, Long noncoding RNA HOTAIR, Inflammation, Lipid metabolism, Human chondrocytes, PTEN/PI3K/AKT pathway","lastPublishedDoi":"10.21203/rs.3.rs-1156754/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1156754/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e Our research was designed to investigate the correlations among expression of PTEN/PI3K/AKT pathway, clinical-related indicators, and long noncoding RNA HOX transcript antisense RNA (lncRNA HOTAIR) in osteoarthritis (OA). \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e The expression of immune-inflammatory indicators was detected in OA patients and normal people, and peripheral blood mononuclear cells (PBMCs) were extracted to induce human chondrocytes (CHs). Reverse transcription-quantitative polymerase chain reaction was performed to measure lncRNA HOTAIR expression. The levels of inflammatory cytokines and adiponectin were detected using enzyme-linked immunosorbent assay. Cell Counting Kit-8 was used to assess the viability of CHs. Western blot analysis was utilized to evaluate related protein expression.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eLncRNA HOTAIR showed high expression in PBMCs of OA patients with the sensitivity and specificity of receiver-operating characteristics (ROC) curve according to the area under the ROC curve, indicating that lncRNA HOTAIR might act as a biomarker of OA. Moreover, lncRNA HOTAIR was positively correlated with total cholesterol, high sensitivity C-reactive protein, immunoglobulin G, tumor necrosis factor-α (TNF-α), and visual analog scale score, which were found to be independent risk factors for lncRNA HOTAIR expression. Besides, overexpression of lncRNA HOTAIR diminished cell viability and IL-10 expression but augmented TNF-α expression in OA-CHs stimulated by OA-PBMCs. Meanwhile, lncRNA HOTAIR overexpression elevated the levels of PI3K, AKT proteins and reduced PTEN protein expression in OA-CHs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Conclusively, lncRNA HOTAIR was upregulated in PBMCs of OA patients, which facilitated the inflammatory response of OA by orchestrating inflammatory cytokines and the PTEN/PI3K/AKT pathway.\u003c/p\u003e","manuscriptTitle":"Expression of Long Noncoding RNA HOTAIR and Its Influence on the PTEN/PI3K/AKT Pathway and Inflammation in Patients With Osteoarthritis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-12-30 15:02:57","doi":"10.21203/rs.3.rs-1156754/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-10-30T04:37:41+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2022-10-08T00:59:39+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-12-12T06:59:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-12-10T04:27:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Orthopaedic Surgery and Research","date":"2021-12-09T11:31:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-orthopaedic-surgery-and-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"josr","sideBox":"Learn more about [Journal of Orthopaedic Surgery and Research](http://josr-online.biomedcentral.com)","snPcode":"13018","submissionUrl":"https://submission.nature.com/new-submission/13018/3","title":"Journal of Orthopaedic Surgery and Research","twitterHandle":"@MSKmedBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"84e373a7-bcf7-4284-9491-c8ee78a79d07","owner":[],"postedDate":"December 30th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":9434325,"name":"Orthopedic Surgery"}],"tags":[],"updatedAt":"2023-10-16T18:26:35+00:00","versionOfRecord":{"articleIdentity":"rs-1156754","link":"https://doi.org/10.1186/s13018-023-03505-1","journal":{"identity":"journal-of-orthopaedic-surgery-and-research","isVorOnly":false,"title":"Journal of Orthopaedic Surgery and Research"},"publishedOn":"2023-01-12 18:19:12","publishedOnDateReadable":"January 12th, 2023"},"versionCreatedAt":"2021-12-30 15:02:57","video":"","vorDoi":"10.1186/s13018-023-03505-1","vorDoiUrl":"https://doi.org/10.1186/s13018-023-03505-1","workflowStages":[]},"version":"v1","identity":"rs-1156754","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1156754","identity":"rs-1156754","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}