Study on the value of Inhibin B in the diagnosis of nasopharyngeal carcinoma and its correlation with traditional Chinese medicine syndromes: An observational study.

To investigate the expression of Inhibin B between various clinical stages, Chinese medicine dialectic typing, and in nasopharyngeal carcinoma (NPC) tissues and serum, and to evaluate the potential of Inhibin B as a new biomarker for NPC. Paraffin specimens of pathologically confirmed NPC tissues and paracancerous tissues were retrospectively collected, and the expression of Inhibin α (INHA) and Inhibin ßB (INHBB) was detected by SP method, and their relationship with clinicopathological indexes was analyzed; in addition, patients with NPC who had received radiotherapy were included as the study subjects, and Epstein-Barr virus DNA (EBV-DNA), INHA, and INHBB in patients were detected by using the fluorescence quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and chemiluminescent immuno-sandwiching method, respectively. EBV-DNA, EBV-viral capsid antigen-immunoglobulin A (VCA IgA), INHA, and INHBB were detected in the patients, respectively, and their relationships with traditional Chinese medicine (TCM) patterns were also analyzed. The expression of INHA and INHBB in NPC tissues was lower than that in paracancerous tissues, and the expression of INHA in NPC patients was correlated with lymphatic metastasis, clinical staging, and TCM staging; the levels of EBV-DNA and VCA IgA were higher than that of healthy populations in NPC patients and were higher than that of patients with stage III + IV than that of patients with stage I + II, and the levels of INHA and INHBB were lower than those of healthy populations and were lower than those of patients with stage III + IV than that of patients with stage I + II. The levels of INHA and INHBB in nasopharyngeal cancer patients were lower than those in healthy people, and the levels in stage III + IV patients were lower than those in stage I + II patients. The levels of EBV-DNA and VCA IgA in nasopharyngeal cancer patients were correlated with the Chinese medicine patterns, and had different patterns. The expression of Inhibin B may be related to the progression of NPC, and it has certain typing significance for different TCM syndromes of NPC, which is helpful for TCM typing diagnosis.

Lactoferrin mediates epithelial-mesenchymal transformation by regulating the PI3K/AKT/mTOR pathway to inhibit nasopharyngeal carcinoma metastasis.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor of the head and neck. Epithelial-mesenchymal transition (EMT) is a major player in regulating NPC transfer. There is increasing evidence that lactotransferrin (LTF) is an important regulator of EMT conversion. However, the potential role and mechanisms of LTF in regulating NPC cell EMT remain unclear. In this study, quantitative real-time PCR (qRT‒PCR) and Western blotting were applied to measure the expression of LTF in NPC cells. Subsequently, the influences of LTF on the proliferation, migration and invasion of NPC cells were verified by functional acquisition experiments. Finally, Western blotting was used to analyze the effects of EMT-related proteins and phosphoinositol 3-kinase (PI3K)/Akt/mammalian rapamycin target (mTOR) signaling pathways. The data of this study indicate that LTF was underexpressed in human NPC cells, and upregulation of LTF could restrain NPC cell proliferation, invasion, migration and EMT transformation. Moreover, the effects of LTF on NPC cell metastasis and EMT are partly determined by the PI3K/AKT/mTOR pathway. This study suggests that LTF is a potential biomarker of NPC and that LTF-mediated EMT progression plays a tumor-suppressive role in the progression of NPC metastasis.

Low expression of INHB co-receptor TGFBR3 in connection with metastasis and immune infiltration in lung adenocarcinoma.

OBJECTIVE: Inhibin B (INHB) is one of the TGF-ß superfamily member, consisting of α (INHA) and ßB (INHBB) subunits. Studies have found that TGF-ß receptor 3 (TGFBR3) binds to a convex α subunit on the surface of INHB, and enhances the binding affinity of activin receptor type-2 (ACVR2A/B) to INHß subunit. This study tried to evaluate the roles of INHB subunits and its receptors (INHA, ACVR2A, ACVR2B, INHBB, TGFBR3) as prognostic biomarkers and therapeutic targets for the effective treatment of lung adenocarcinoma (LUAD). METHODS: We analyzed INHB subunits and its receptors' expression and the influence of LUAD from Oncomine, GEPIA, HCMDB, CancerSEA, TIMER databases and so on. Then, 41 cases of cancer tissue and 41 cases of adjacent epithelium were detected in LUAD patients by immunohistochemistry. RESULTS: INHA, ACVR2A, ACVR2B, INHBB were up-regulated while TGFBR3 was down-regulated in LUAD. INHA, ACVR2A and TGFBR3 were found to be strongly associated with high-grade malignancies and advanced TNM, only TGFBR3 expression was negatively correlated with LUAD metastasis probably mainly through cell adhesion molecules and the PI3K-Akt signaling pathway, univariate and multivariate analysis suggested that overall survival was lower in LUAD cases with low TGFBR3 levels. Further analysis revealed that low TGFBR3 expression was related to reduced infiltration of immune cells into the LUAD, promoting metastasis of LUAD cells. TGFBR3 expression negatively correlates with lymphatic metastasis and clinical stage in patients with LUAD. CONCLUSION: TGFBR3 could be a potential new metastatic biomarker for LUAD, with potential application as a prognostic marker and for immunotherapy of LUAD.

Alternative splicing associated with cancer stemness in kidney renal clear cell carcinoma.

BACKGROUD: Cancer stemness is associated with metastases in kidney renal clear cell carcinoma (KIRC) and negatively correlates with immune infiltrates. Recent stemness evaluation methods based on the absolute expression have been proposed to reveal the relationship between stemness and cancer. However, we found that existing methods do not perform well in assessing the stemness of KIRC patients, and they overlooked the impact of alternative splicing. Alternative splicing not only progresses during the differentiation of stem cells, but also changes during the acquisition of the stemness features of cancer stem cells. There is an urgent need for a new method to predict KIRC-specific stemness more accurately, so as to provide help in selecting treatment options. METHODS: The corresponding RNA-Seq data were obtained from the The Cancer Genome Atlas (TCGA) data portal. We also downloaded stem cell RNA sequence data from the Progenitor Cell Biology Consortium (PCBC) Synapse Portal. Independent validation sets with large sample size and common clinic pathological characteristics were obtained from the Gene Expression Omnibus (GEO) database. we constructed a KIRC-specific stemness prediction model using an algorithm called one-class logistic regression based on the expression and alternative splicing data to predict stemness indices of KIRC patients, and the model was externally validated. We identify stemness-associated alternative splicing events (SASEs) by analyzing different alternative splicing event between high- and low- stemness groups. Univariate Cox and multivariable logistic regression analysisw as carried out to detect the prognosis-related SASEs respectively. The area under curve (AUC) of receiver operating characteristic (ROC) was performed to evaluate the predictive values of our model. RESULTS: Here, we constructed a KIRC-specific stemness prediction model with an AUC of 0.968,and to provide a user-friendly interface of our model for KIRC stemness analysis, we have developed KIRC Stemness Calculator and Visualization (KSCV), hosted on the Shiny server, can most easily be accessed via web browser and the url https://jiang-lab.shinyapps.io/kscv/ . When applied to 605 KIRC patients, our stemness indices had a higher correlation with the gender, smoking history and metastasis of the patients than the previous stemness indices, and revealed intratumor heterogeneity at the stemness level. We identified 77 novel SASEs by dividing patients into high- and low- stemness groups with significantly different outcome and they had significant correlations with expression of 17 experimentally validated splicing factors. Both univariate and multivariate survival analysis demonstrated that SASEs closely correlated with the overall survival of patients. CONCLUSIONS: Basing on the stemness indices, we found that not only immune infiltration but also alternative splicing events showed significant different at the stemness level. More importantly, we highlight the critical role of these differential alternative splicing events in poor prognosis, and we believe in the potential for their further translation into targets for immunotherapy.

SPAG9/MKK3/p38 axis is a novel therapeutic target for liver cancer.

Sperm­associated antigen 9 (SPAG9) is a biomarker and potential therapeutic target for several cancers; however, its involvement in liver cancer progression is not clear. The aim of the present study was to determine whether SPAG9 regulates proliferation of liver cancer. Immunohistochemistry and cell immunofluorescence were used to confirm the expression and the localization of SPAG9 in human liver cancer tissues and the liver cancer­derived HepG2 cells. A small interfering RNA (siRNA) designed to target SPAG9 was transiently transfected into HepG2 cells using Lipofectamine™ 2000, and proliferation, apoptosis and cell cycle progression were analyzed using CCK­8 assay and flow cytometry; western blotting was used to detect the expression of SPAG9, JNK, p38, MKK3 and MKK6, and co­immunoprecipitation was used to assess the interaction between SPAG9 and JNK. SPAG9 was overexpressed in 16 out of 20 (80%) patients with liver cancer. The protein was localized in both the cytoplasm and nucleus of liver cancer cells obtained from patients and in HepG2 cells. Depletion of SPAG9 inhibited the proliferation of HepG2 cells, promoted apoptosis and arrested the cell cycle at the S phase. Moreover, cells deficient in SPAG9 had decreased expression of JNK, p38 and MKK3 compared to HepG2 cells not treated with an siRNA targeting SPAG9. In the present study, SPAG9 was revealed to regulate cell proliferation, apoptosis and cell cycle progression in liver cancer cells through the SPAG9/MKK3/p38 axis. This axis is a novel therapeutic target for liver cancer.

A splice site mutation causing exon 6 skipping in SLC20A2 gene in a primary familial brain calcification family.

BACKGROUND: Primary familial brain calcification (PFBC) is a rare degenerative disease characterized by symmetrical bilateral calcinosis in the basal ganglia and other brain regions. It has an autosomal dominant inheritance pattern in most cases and exhibits genetic heterogeneity. Previous studies reported that SLC20A2, PDGFRB, PDGFB, XPR1 and MYORG are associated with PFBC, with SLC20A2 the main culprit. However, other mutations may also cause PFBC. Here, we performed a study to reveal the contributing mutations that gave rise to PFBC in a Chinese PFBC family. METHODS: We recruited a PFBC family consisting of eight patients and eight healthy family members across three generations. Whole-exome sequencing, Sanger sequencing and RT-PCR were used to detect the genetic mutations. RESULTS: Whole-exome sequencing revealed that c.730 + 1G > A of SLC20A2 was the candidate pathogenic mutation for the proband in this family. Genomic DNA PCR amplification and Sanger sequencing confirmed that all the patients from the family carried this mutation, while the healthy subjects in the family did not. Complementary DNA (cDNA) PCR amplification and Sanger sequencing confirmed that the patients had a mutation that caused exon 6 skipping in SLC20A2. CONCLUSION: We identified a SLC20A2 splicing variant (c.730 + 1G > A) in a PFBC family. This mutation led to an alternative splicing event that skipped exon 6 in SLC20A2.

Inhibin B suppresses anoikis resistance and migration through the transforming growth factor-ß signaling pathway in nasopharyngeal carcinoma.

Inhibin B (INHBB), a heterodimer of a common α-subunit and a ßB-subunit, is a glycoprotein belonging to the transforming growth factor-ß (TGF-ß) family. In this study, we observed INHBB expression was reduced in nasopharyngeal carcinoma (NPC) tissues compared to non-tumor nasopharyngeal epithelium tissues, and INHBB was associated with lymph node metastasis, stage of disease, and clinical progress. Positive expression of INHBB in NPC predicted a better prognosis (overall survival, P = 0.038). However, the molecular mechanisms of INHBB have not been addressed in NPC. We induced anoikis-resistant cells in NPC cell lines under anchorage-independent conditions, then found epithelial-mesenchymal transition markers changed, cell apoptosis decreased, cell cycle was modified, and invasion strengthened in anoikis-resistant NPC cells. These anoikis-resistant NPC cells showed decreased expression of INHBB compared with adhesion cells. Furthermore, INHBB was found to influence the above-mentioned changes. In the anoikis-resistant NPC cells with INHBB overexpression, apoptotic cells increased, S phase cells weakened, vimentin, matrix metallopeptidase-9, and vascular endothelial growth factor A expression were downregulated, and E-cadherin expression was upregulated, and vice versa in knockdown of INHBB (INHBB shRNA) anoikis-resistant NPC cells. Diminished INHBB expression could activate the TGF-ß pathway to phosphorylate Smad2/3 and form complexes in the nucleus, which resulted in the above changes. Thus, our results revealed for the first time that INHBB could suppress anoikis resistance and migration of NPC cells by the TGF-ß signaling pathway, decrease p53 overexpression, and could serve as a potential biomarker for NPC metastasis and prognosis as well as a therapeutic application.

Curcumin inhibits liver cancer by inhibiting DAMP molecule HSP70 and TLR4 signaling.

Curcumin has been revealed to inhibit liver cancer, however, no studies have reported that the mechanism of curcumin's action on liver cancer is related to damage-associated molecular pattern (DAMP) molecules heat shock protein 70 (HSP70) and the toll-like receptor 4 (TLR4) signaling. This study aimed to investigate whether the activation of TLR4 signaling by HSP70 could be inhibited by curcumin, thus investigating the possible mechanism of curcumin in the inhibition of liver cancer. Western blotting was used to evaluate the expression of the HSP70 and TLR4 in HepG2 cells and ELISA was used to detect the concentration of HSP70 in cell culture medium. A thermal tolerance HepG2 (HepG2TT) cell model was established to simulate HSP70 accumulation in the microenvironment. A certain concentration of curcumin was co-cultured with HepG2 and HepG2TT cells to observe the changes of HSP70 and TLR4. Our results revealed that heat stress significantly increased the expression of extracellular HSP70 (eHSP70) and TLR4 (P<0.01), but significantly reduced the expression of intracellular HSP70 (P<0.01). Curcumin inhibited proliferation, invasion, and metastasis of HepG2 cells, caused cells to remain in the DNA S phase, promoted apoptosis, and significantly reduced intracellular HSP70, eHSP70 and TLR4 levels of HepG2TT cells. Following the removal of curcumin, eHSP70 increased again. In summary, our results demonstrated that the antitumor effect of curcumin was related to the inhibition HSP70-TLR4 signaling.

Sperm-associated antigen 9 is upregulated in hepatocellular carcinoma tissue and enhances QGY cell proliferation and invasion in vitro.

The incidence and mortality rates of hepatocellular carcinoma (HCC) are higher in China compared with in other countries. Further research is required in order to improve the diagnosis and treatment of HCC. Sperm-associated antigen 9 (SPAG9) protein has been revealed to serve an important function in cancer progression; however, the underlying mechanisms remain to be elucidated. The present study investigated the expression level of SPAG9 in HCC tissues using quantitative-polymerase chain reaction, immunohistochemistry and western blotting, and the results demonstrated that SPAG9 was overexpressed in HCC tissues compared with the adjacent non-cancerous tissues. To explore the potential mechanisms underlying SPAG9 in HCC, the effect of SPAG9 on cell proliferation, cell cycle, migration and invasion capacities were investigated in the QGY HCC cell line by RNA interference. It was revealed that inhibition of SPAG9 mRNA in QGY cells significantly inhibited the expression level of SPAG9 compared with the control. Depletion of SPAG9 expression decreased cell proliferation (P<0.01) and increased the percentage of cells in the G1/G2 cell cycle phase. The percentage of cells in the S phase was decreased, and cell migration and invasion capabilities in vitro were reduced (P<0.01). In summary, the results of the present study suggested that SPAG9 was upregulated in HCC and may serve an important function in cancer cell proliferation, differentiation and invasion. Whether SPAG9 is a potential diagnostic marker and therapeutic target of human HCC requires additional study.

Serum levels of anti-sperm-associated antigen 9 antibody are elevated in patients with hepatocellular carcinoma.

At present, there is a high incidence of viral hepatitis and high mortality rates due to hepatocellular carcinoma (HCC) in China. In the current study, the quantification of antibodies against the cancer-testis antigen sperm-associated antigen 9 (SPAG9), alone and combined with α-fetoprotein (AFP), were evaluated as biomarkers for the diagnosis of HCC. The levels of anti-SPAG9 antibody and AFP were quantified in serum samples from patients with HCC and hepatitis or cirrhosis, as well as healthy volunteers. The results revealed that the serum levels of anti-SPAG9 immunoglobulin G antibody in patients with HCC were significantly higher compared with those in patients with hepatitis/cirrhosis and healthy controls. Using receiver operator characteristic curves, the area under the curve (AUC, 0.870) of SPAG9 as a diagnostic marker of HCC was significant [P<0.001; 95% confidence interval (CI), 0.793-0.947], whereas the AUC of AFP was 0.832 (P<0.001; 95% CI, 0.736-0.928). Serum anti-SPAG9 antibody levels exhibited significant potential for the differential diagnosis of HCC, with an AUC value of 0.729, (P=0.008; 95% CI, 0.559-0.899). Similarly, serum AFP levels exhibited significant value for the differential diagnosis of HCC, with an AUC value of 0.842 (P<0.001; 95% CI, 0.732-0.953). When combined with quantification of AFP, the diagnostic sensitivity and specificity of anti-SPAG9 levels were increased. In summary, the results suggested that anti-SPAG9 antibody is a potential early diagnostic marker of HCC.

The expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in peripheral blood of patients with HCC and lung cancer.

There are different views of how the immune system participates in the reaction to cancer. Here, we evaluated expression of DAMP proteins HSP70 and cancer-testis antigen SPAG9 in patients with hepatocellular carcinoma (HCC) and lung cancer to explore tumor immunity. Our analysis showed that levels of HSP70 and SPAG9 antibody were significantly higher in the serum of lung cancer and HCC patients than in the serum of healthy subjects (P < 0.001), but there were no differences in levels of HSP70 antibody in patients and controls. Levels of serum SPAG9 antibody in newly diagnosed lung cancer patients were significantly higher than in treated lung cancer patients (P < 0.05), but there were no differences in levels of HSP70 or HSP70 antibody. Levels of serum HSP70 and SPAG9 antibody, but not HSP70 antibody, were also higher in hepatitis/cirrhosis patients than in healthy subjects (P = 0.005, P < 0.001). Levels of serum SPAG9 antibody were significantly higher in HCC patients than in hepatitis/cirrhosis patients, but there were no differences in HSP70 or HSP70 antibody levels. Finally, levels of serum HSP70 and SPAG9 antibody were significantly higher in HCC patients than in lung cancer patients (P < 0.05, P < 0.001). These results indicate that cancer-testis antigen SPAG9 induces a strong humoral immune response in cancer patients but HSP70 does not. These results show that SPAG9 has potential as a tumor-specific biomarker.

Cancer testis antigen SPAG9 is a promising marker for the diagnosis and treatment of lung cancer.

Cancer testis antigen sperm-associated antigen 9 (SPAG9) is highly expressed in many types of cancers. In the present study, to obtain a better understanding of the relevance of SPAG9 in cancer diagnosis and treatment, the expression of SPAG9 mRNA and protein in lung cancer specimens was evaluated by RT-PCR, western blotting and immunohistochemistry. ELISA was used to quantify the SPAG9 autoantibody in the peripheral blood of lung cancer patients. The results showed that the expression of SPAG9 mRNA and protein in the lung cancer tissues was significantly higher than that in the adjacent non-cancerous tissues (P<0.01). The level of the SPAG9 autoantibody in the serum of lung cancer patients was significantly higher than the level in the healthy controls (P<0.001), and the level of the SPAG9 autoantibody in the serum of untreated patients was significantly higher than that in treated patients (P=0.002). SPAG9 IgG antibody levels were significantly lower in treated adenocarcinoma and small cell lung cancer patients than these levels in the untreated patients (P=0.006, P=0.026, respectively), while no statistical difference was found between treated and untreated squamous cell carcinoma patients. Our results suggest that the SPAG9 antibody in serum is a promising marker for the diagnosis of lung cancer, and the level of the humoral immune response to this antigen appears to be related to the type of lung cancer.

Lactotransferrin could be a novel independent molecular prognosticator of nasopharyngeal carcinoma.

Lactotransferrin (LTF), also known as lactoferrin, is a key component of innate immune defense. We previously reported that LTF was downregulated in nasopharyngeal carcinoma (NPC) and could suppress NPC cell proliferation. However, the relevance of the relationship between LTF expression and NPC clinical outcome has not been reported. This study aims to assess the possible correlations between LTF expression and clinical parameters and its potential prognostic predictive ability in the outcomes of patients with NPC. Complementary DNA (cDNA) microarray, quantitative real-time PCR (qRT-PCR), and immunohistochemistry (IHC) results suggested that LTF expression was significantly downregulated in NPC tissues compared to non-NPC tissues. LTF was negatively correlated with lymph node metastasis (P = 0.042), T stage (P < 0.001), clinical tumor-node-metastasis (TNM) stage (P = 0.022), and EBV-encoded RNA 1 (EBER-1) expression (r = -.167, P = 0.016). A survival analysis of 108 patients with NPC revealed that positive expression of LTF could predict a good prognosis [disease-free survival (DFS): P = 0.043, overall survival (OS): P = 0.040]. Multivariable analysis revealed that LTF could independently predict prognosis (DFS: HR = 0.414, P = 0.003; OS: HR = 0.309, P = 0.005). These observations indicated that LTF is a potential prognostic factor of NPC.