Study on serum miR-182 as a marker for diagnosis and prognosis of cervical cancer.

BACKGROUND: Cervical cancer (CC) is a common female malignancy, with a global incidence rate second only to breast cancer. OBJECTIVE: To propose a new idea for early treatment and auxiliary diagnosis of CC by exploring the diagnostic and prognostic implications of serum miR-182 in CC. METHODS: We enrolled 70 CC patients, 35 cervical intraepithelial neoplasia (CIN) patients and 35 healthy controls (HCs), who visited The First Affiliated Hospital of Hainan Medical College Hospital between January 2015 and April 2016. miR-182 expression was quantified by real-time quantitative PCR and compared among the three groups. The correlation of serum miR-182 expression with patients' clinical features was evaluated. The receiver operating characteristic curve (ROC) and the Kaplan-Meier method were used to evaluate the early diagnostic value and prognostic value of serum miR-182. Cox regression analysis was performed to determine serum miR-182 expression and its important role in predicting CC patients' prognosis. RESULTS: Serum miR-182 expression was determined to be 0.345 ± 0.094, 0.369 ± 0.076, and 0.586 ± 0.157 in CC patients, CIN patients, and HCs, respectively (P< 0.001). Serum miR-182 expression had an obvious association with lymph node metastasis and pathological differentiation (P< 0.05). The area under the ROC curve (AUC) of serum miR-182 was 0.709 (95% CI: 0.622-0.795), the critical value was 0.456, the sensitivity was 81.4%, and the specificity was 52.9%. CC patients were grouped as either the low- (miR-182 < 0.3) or high-level group (miR-182 ⩾ 0.03) based on serum miR-182 levels, and a Cox regression model of OS was established. Serum miR-182 expression was identified as a factor independently influencing CC patients' OS (P= 0.028); the death risk of the high-level group was 3.246 times that of the low-level group. CONCLUSION: Serum miR-182 expression is not only a biomarker for early diagnosis of CC, but also one of the independent factors influencing the survival and prognosis of CC patients.

Long noncoding RNA LINC00482 silencing sensitizes non-small cell lung cancer cells to cisplatin by downregulating CLASRP via E2F1.

Long noncoding RNA LINC00482 (LINC00482) is dysregulated in non-small cell lung cancer cells (NSCLC). Herein, this research examined the actions and specific mechanisms of LINC00482 in cisplatin (DDP) resistance in NSCLC. LINC00482 expression was assessed using RT-qPCR in clinical NSCLC tissues and cell lines. Knockdown and ectopic expression assays were conducted in A549 and HCC44 cells, followed by determination of cell proliferation with CCK-8 and clone formation assays, apoptosis with flow cytometry, and DDP sensitivity. The association between LINC00482, E2F1, and CLASRP was evaluated with dual-luciferase reporter, ChIP, and RIP assays. The role of LINC00482 in NSCLC was confirmed in nude mice. NSCLC tissues and cells had upregulated LINC00482 expression. LINC00482 was mainly localized in the cell nucleus, and LINC00482 recruited E2F1 to enhance CLASRP expression in NSCLC cells. LINC00482 knockdown enhanced the DDP sensitivity and apoptosis of NSCLC cells while reducing cell proliferation, which was negated by overexpressing CLASRP. LINC00482 knockdown restricted tumor growth and enhanced DDP sensitivity in NSCLC in vivo. LINC00482 silencing downregulated CLASRP through E2F1 to facilitate the sensitivity to DDP in NSCLC.

miR-182 promotes cervical cancer progression via activating the Wnt/ß-catenin axis.

Cervical cancer (CC) is among the leading causes of cancer-associated mortality in women worldwide; yet the molecular regulators involved in its progression are unclear. This study found that miR-182 was overexpressed in CC tissues when compared with adjacent normal tissues. Moreover, it found that miR-182 expression was significantly positively correlated with distant metastasis in patients with CC. Interestingly, in vitro experiments showed that overexpression and inhibition of miR-182 promoted and suppressed the growth of CC cells, respectively. The tumor-promoting effects of miR-182 on CC progression were achieved via the Wnt/ß-catenin axis and its downstream genes. Thus, this study revealed the potential of miR-182/ß-catenin as an effective new target for CC treatment.

Comprehensive Analysis of DMRT3 as a Potential Biomarker Associated with the Immune Infiltration in a Pan-Cancer Analysis and Validation in Lung Adenocarcinoma.

Doublesex and Mab-3 related Transcription Factor 3 (DMRT3) is associated with the prognosis of some tumors. It is possible to explore the role of DMRT3 in the cancer process using bioinformatic approaches and experimental validation. We comprehensively explored the clinical and immunological characteristics of DMRT3. The DMRT3 expression is abnormal in human cancers and correlates with clinical staging. A high DMRT3 expression is significantly associated with poor overall survival (OS) in KIRC, KIRP, LUAD, and UCEC. Amplification was the greatest frequency of the DMRT3 alterations in pan-cancer. The OS was significantly lower in the DMRT3 altered group than in the DMRT3 unaltered group (P = 0.0276). The DMRT3 expression was significantly associated with MSI in three cancer types and TMB in six cancer types. The DMRT3 expression was significantly correlated with the level of the immune cell infiltration and the immune checkpoint genes. The DMRT3 was involved in some pathways in pan-cancer. DMRT3 may play a role in chemotherapy and may be associated with chemoresistance. A ceRNA network of KCNQ1OT1/miR-335-5p/DMRT3 was constructed in LUAD. DMRT3 was significantly upregulated in the LUAD cell lines. DMRT3 was aberrantly expressed in pan-cancer and may promote tumorigenesis and progression via different mechanisms. DMRT3 can be used as a therapeutic target to treat cancer in humans.

Clinical and pathological characteristics of 11 NSCLC patients with c-MET exon 14 skipping.

Background: The aim of the present study was to summarize the clinical and pathological characteristics of 11 non-small cell lung cancer (NSCLC) patients with mesenchymal-epithelial transition factor exon 14 skipping (METex14). Methods: From 2018 to 2021, medical records of 763 NSCLC patients were reviewed and 11 patients carrying METex14 were identified from the Affiliated Hospital of Guangdong Medical University. Their clinical data were subsequently examined for pathological and related clinical information including symptom and diagnosis, imaging and follow-up. Results: The METex14 cohort includes 9 males and 2 females and the age range was 69-85 years, with a median age of 77 years. Of the patients one is diagnosed with stage IVB lung adenosquamous carcinoma, 7 with lung adenocarcinoma (1 with stage IIIA and 6 with stage IV), and 3 with stage IV lung sarcomatoid carcinoma. 3 reached stable disease until the end of follow-up and 4 died within a year due to multiple metastases. In 4 cases, the patients received selective MET inhibitor treatment all lived longer than 7 months. There were 4 heterozygous point mutations and 1 deletion of the MET gene in this cohort, as follows: c.G3028T (p.D1010Y); c.G3028A (p.D1010N); c.G3005C (p.V1002A); c.3022C>G and MET c.3021_3028+20del (E14). Conclusions: According to the data that we collected, the incidence of NSCLC carrying METex14 is low and male outnumber female in our sample pool. Selective target therapy had better prognosis than multitargeted tyrosine kinase inhibitor (TKI) such as crizotinib or standard therapy.

The Value of Serum Exosomal miR-184 in the Diagnosis of NSCLC.

Lung cancer has the highest morbidity rate (11.6%) and mortality rate (18.4%) among all current tumors. The morbidity rate in China accounts for approximately one-third, and it is still rising. Nonsmall cell lung cancer is the most common type of lung cancer, accounting for 80%-85% of all lung cancers, and approximately 57% of patients with advanced nonsmall cell lung cancer have distant metastases at the time of diagnosis. To explore the expression changes in microRNA-184 (miR-184) and its clinical value in serum exosomes of patients with nonsmall cell lung cancer (NSCLC). This study adopted a case-control study method, selecting 88 patients (NSCLC group) from June 2015 to June 2017 in our hospital who are confirmed to have NSCLC by fiber-optic bronchoscopy, and 90 patients who are confirmed to have benign lung diseases by pathological examination during the same period (control group). Fluorescence quantitative PCR technology is used to detect the levels of miR-184 in serum exosomes of the two groups, and the differences in the levels of miR-184 in serum exosomes of NSCLC patients with different pathological characteristics are analyzed. According to the results of the 3-year follow-up, the miR-184 levels in serum exosomes of NSCLC patients are grouped and compared. The expression level of miR-184 in serum exosomes in the NSCLC group is significantly higher than that in the control group, and the difference between the two groups is statistically significant (p < 0.05). The ROC curve is drawn with the expression level of miR-184 in serum exosomes of the two groups of patients. The results showed that the area under the ROC curve for the differential diagnosis of NSCLC and benign lung tumors with the expression level of miR-184 in serum exosomes is 0.927, and the sensitivity is 87.61%, while the specificity is 84.02%. The expression levels of miR-184 in serum exosomes of NSCLC patients with different pathological characteristics, in different TNM stages [(I+II) vs. (III+IV)], lymph node metastasis (yes vs. no), and different degrees of differentiation [(High + Medium) vs. Poorly differentiated] are compared and showed statistical significance (p < 0.05). In 88 NSCLC patients, after 3 years of follow-up, 33 survived, and 55 died, with a survival rate of 37.50%. The expression of miR-184 in serum exosomes of the 33 surviving patients is significantly lower than that of the nonsurviving group (p < 0.05). The expression level of miR-184 in serum exosomes of NSCLC patients is significantly increased, which has a certain value for the differential diagnosis of the nature of benign and malignant lung diseases and is closely related to the prognosis of patients.

Serum exosomal miR-16-5p functions as a tumor inhibitor and a new biomarker for PD-L1 inhibitor-dependent immunotherapy in lung adenocarcinoma by regulating PD-L1 expression.

OBJECTIVES: We aimed at investigating whether serum exosomal miR-16-5p could be utilized as an immunotherapy biomarker in lung adenocarcinoma (LUAD) patients administered by programmed cell death ligand-1 (PD-L1) inhibitors, and to evaluate its functions in LUAD progression. METHODS: Sixty LUAD sufferers and 20 healthy controls (HCs) were covered in this work. We applied both IHC and WB to examine PD-L1 level in clinical tissue samples and utilized WB to quantify PD-L1 expression in LUAD cells and LUAD xenograft tissues, respectively. Transmission electron microscopy (TEM), WB, and nanoparticle tracking analysis (NTA) were executed to confirm the exosomes isolated from serum specimens and cell culture media. To quantify of exosomal miR-16-5p level from serum and culture medium of cultured cell, qRT-PCR experiment was utilized. The connection between tissue PD-L1 level and serum exosomal miR-16-5p expression in PD-L1-positive sufferers administered by PD-L1 inhibitors was verified using Spearman correlation coefficient analysis. In addition, the overall survival (OS) and progression-free survival (PFS) rates among PD-L1 inhibitor managed sufferers were acquired through a follow-up visit. Finally, we used a group of assays, including 5-bromo-2'-dexoyuridine (BrdU) and colony formation test, wound healing experiment, flow cytometry, and nude mice xenograft experiment, to explore the functions of circulating exosomal miR-16-5p on LUAD cell proliferation, apoptosis, and migration, as well as tumor development, respectively. RESULTS: PD-L1 expression was positively related to T stage (tumor size stage), and PD-L1 inhibitor treatment reduced the PD-L1 expression and mitigated T stage in PD-L1-positive LUAD sufferers. For PD-L1-positive LUAD sufferers, elevated PD-L1 expression or reduced serum exosomal miR-16-5p level were linked to longer PFS and OS upon PD-L1 inhibitor treatment. The number of exosomes in patient's serum was more than that in the serum of healthy individuals, and PD-L1 inhibitor treatment decreased the number of serum-derived exosomes in PD-L1-positive LUAD sufferers. Exosome-derived miR-16-5p was downregulated in patient's serum and cell culture medium, and this was negatively linked to tumor stage and PD-L1 expression. Meanwhile, PD-L1 inhibitor treatment could increase the serum exosomal miR-16-5p expression, and the expression change of serum exosomal miR-16-5p was diametrically related to PD-L1 after the treatment. Moreover, the overexpression of PD-L1 accelerated tumor growth and decreased the exosomal miR-16-5p content in cell culture media, while exosomal miR-16-5p overexpression in cell culture media inhibited tumor development by decreasing the PD-L1 expression. Exosomal miR-16-5p overexpression in cell culture media also depressed LUAD cell proliferation and migration, and stimulated cell apoptosis, especially in the cells which cultured in the mediums with PD-L1 inhibitor in vitro. CONCLUSIONS: Serum exosomal miR-16-5p may be a latent tumor inhibitor and a new biomarker for PD-L1 inhibitor-dependent immunotherapy in LUAD by regulating the PD-L1 expression.

Activation of the Hedgehog pathway mediates resistance to epidermal growth factor receptor inhibitors in non-small cell lung cancer.

The current study aimed to investigate the function of the Hedgehog pathway and its association with epithelial-mesenchymal transition (EMT) in epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC). Lung tumor tissue specimens from EGFR TKI-resistant patients, including those with brain metastases, had hyperactive Hedgehog signaling compared with those from TKI-sensitive patients. SHH stimulation promoted GLI1 activation as well as cell motility in parental PC9 cells while suppressing gefitinib-induced apoptosis in gefitinib-resistant cells. SHH also promoted EMT in parental PC9 cells via E-cadherin suppression and N-cadherin and vimentin upregulation. The knockdown of GLI1 exhibited the opposite effects. Besides, SHH induced, whereas GLI1 knockdown reversed gefitinib resistance in xenograft tumors. The Hedgehog pathway inhibitor GDC-0449 synergized with gefitinib to increase xenograft tumor sensitivity to chemotherapy and extend survival in tumor-bearing animals. These results suggest the Hedgehog pathway mediates EGFR TKI resistance and induces EMT in NSCLC, representing a potential therapeutic target to defeat TKI resistance.

Concomitant genetic alterations are associated with response to EGFR targeted therapy in patients with lung adenocarcinoma.

BACKGROUND: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are considered to be more effective than chemotherapy in the treatment of EGFR-mutant advanced non-small cell lung cancer (NSCLC). However, in addition to EGFR-sensitive mutations, the genetic factors that affect the prognosis of patients who receive TKI treatment are not yet clear. METHODS: The clinical data of 36 NSCLC patients with EGFR mutation who received TKI treatment were retrospectively analyzed. Liquid re-biopsy with next generation sequencing (NGS) analysis was performed to analyze genetic alterations and potential resistance mechanisms. RESULTS: All of the patients harbored actionable sensitive EGFR mutations by NGS, with the major types being 19del or 21L858R (52.78%, 19/36 and 55.56%, 20/36, respectively). The 3 most frequent accompanying somatic mutations were TP53 (12, 48.4%), KRAS (7, 19.44%) and PIK3CA (3, 8.33%). Concomitant mutations were present in 16 patients (44.44%). The occurrence of co-mutation was found to be significantly related to a history of smoking [87.5% (7 of 8) vs. 32.14% (9 of 28); Pearson chi-square, P=0.005]. Patients who received EGFR-TKIs treatment (P=0.0079) or third-generation EGFR-TKIs only (P=0.0468) had better progression-free survival (PFS). Concomitant mutations were significantly related to lower objective response rates (43.75% vs. 80.0%; P=0.024) and poorer PFS (P<0.001). Patients with concomitant genetic alterations had a worse response after receiving EGFR-TKIs treatment (P=0.0033). CONCLUSIONS: Our research underscores the importance of using multiple molecular profiles. Concomitant genetic alterations were significantly associated with response to EGFR targeted therapy in NSCLC. Therefore, research on multi-drug or sequential therapy to address the covariation that drives drug resistance is urgently needed.

[Study of isobutyrylshikonin inhibiting proliferation of colon carcinoma cells through PI3K/Akt/m-TOR pathway].

To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells in vitro and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G0/G1 or G2/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.

Notch3 functions as a regulator of cell self-renewal by interacting with the ß-catenin pathway in hepatocellular carcinoma.

The Notch signaling pathway plays a role in cell proliferation, differentiation. Emerging data have revealed aberrant Notch3 expression in hepatocellular carcinoma (HCC). However, whether Notch3 plays a role in tumorigenesis or tumor progression is unclear. In this study, we found that over 71.8% of the cases studied had high Notch3 expression levels (n = 32); Notch3 expression positively correlated with alpha-fetoprotein (AFP) levels (p = 0.0311) and negatively correlated with the differentiation grade (p = 0.042). We demonstrated that the patients with higher levels of Notch3 expression commonly had a poor prognosis. We discovered that Notch3 expression is inversely correlated with ß-catenin content but positively associated with the protein level of Nanog. In parallel, we found that Notch3 attenuation resulted in the upregulation of ß-catenin and the downregulation of Nanog in the hepatoma cell lines QGY7701 and HepG2. The downregulation of Notch3 enhanced the sensitivity to cisplatin in the QGY7701 and HepG2 cells and inhibited the ability of QGY7701 cells to form tumors. The Notch3-positive cells had higher levels of aldehyde dehydrogenase (ALDH) activity, and a tendency to differentiate into Notch3-negative cells. In conclusion, our study demonstrated that Notch3 plays a role in modulating the stemness of tumor cells via the inactivation of the Wnt/ß-catenin pathway.