miR-422a is an independent prognostic factor and functions as a potential tumor suppressor in colorectal cancer.

AIM: To determine the expression of miR-422a in colorectal cancer (CRC) tissues and to further explore the prognostic value and function of miR-422a in CRC carcinogenesis. METHODS: miR-422a expression was analyzed in 102 CRC tissues and paired normal mucosa adjacent to carcinoma by quantitative real-time PCR. The relationship of miR-422a expression with clinicopathological parameters was also analyzed. Kaplan-Meier analysis and Cox multivariate analysis were performed to estimate the potential role of miR-422a. Cell proliferation, migration, and invasion were used for in vitro functional analysis of miR-422a. RESULTS: The levels of miR-422a were dramatically reduced in CRC tissues compared with normal mucosa (P < 0.05), and significantly correlated with local invasion (P = 0.004) and lymph node metastasis (P < 0.001). Kaplan-Meier survival and Cox regression multivariate analyses revealed that miR-422a expression (HR = 0.568, P = 0.015) and clinical TNM stage (HR = 2.942, P = 0.003) were independent prognostic factors for overall survival in CRC patients. Furthermore, in vitro experiments showed that overexpression of miR-422a inhibited the proliferation, migration, and invasion of SW480 and HT-29 cells. CONCLUSION: Down-regulation of miR-422a may serve as an independent prognosis factor in CRC. MiR-422a functions as a tumor suppressor and regulates progression of CRC.

Overexpression of miR-223 correlates with tumor metastasis and poor prognosis in patients with colorectal cancer.

The purpose of the study was to investigate microRNA-223 (miR-223) expression in colorectal cancer (CRC) and its relationship with tumorigenesis and disease prognosis. Quantitative real-time PCR was used to measure levels of miR-223 in tumor samples and adjacent non-cancerous tissues from 62 patients undergoing radical resection for the treatment of CRC. The associations between miR-223 expression and patient age, sex, as well as clinicopathologic parameters, such as tumor size, differentiation, location, invasion depth, metastasis, tumor-node-metastasis (TNM) stage, and overall patient survival, were analyzed by Mann-Whitney U and Kruskal-Wallis tests. Kaplan-Meier method and Cox proportional hazards regression analyses were performed to estimate the prognostic factors for patient survival prediction. The expression of miR-223 was significantly upregulated in CRC tissues compared with adjacent non-cancerous tissues (P < 0.05). This overexpression was associated with TNM stage and lymph node and distant metastases, (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with high miR-223 expression had a poorer overall survival (OS) than those with low miR-223 expression (P = 0.002). Univariate analysis revealed a statistically significant correlation between OS and miR-223 level, histology grade, metastasis and TNM stage (P < 0.001). Furthermore, miR-223 levels and histology grade were independently associated with OS (HR 0.204, 95 % CI 0.101-0.415, P < 0.05 and HR 2.252, 95 % CI 1.429-3.546, P < 0.05, respectively). The overexpression of miR-223 may play an important role in the progression of CRC and can be used as an independent factor to determine CRC prognosis.

Decreased expression of miR-133a correlates with poor prognosis in colorectal cancer patients.

AIM: To investigate microRNA-133a (miR-133a) expression in colorectal cancer (CRC) and its relationship with tumorigenesis and disease prognosis. METHODS: Quantitative real-time polymerase chain reaction was used to measure levels of miR-133a in tumor samples and adjacent non-cancerous tissues from 169 patients undergoing radical resection for CRC. The associations between miR-133a expression and patient age, sex, as well as clinicopathologic parameters, such as tumor size, differentiation, location, invasion depth, metastasis, tumor-node-metastasis (TNM) stage and overall patient survival, were analyzed by Mann-Whitney U and Kruskal-Wallis tests. The Kaplan-Meier method and Cox proportional hazards regression analyses were performed to estimate the prognostic factors for patient survival prediction. RESULTS: The expression of miR-133a was significantly downregulated in CRC tissues compared with adjacent non-cancerous tissues (P < 0.05). This reduction was associated with the depth of the local invasion, poor differentiation, lymph node metastasis and advanced disease (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with low miR-133a expression had poorer overall survival (OS) than those with high miR-133a expression (P < 0.001). Univariate analysis revealed statistically significant correlations between OS and miR-133a level, tumor local invasion, lymph node metastasis and TNM stage (P < 0.001). Furthermore, miR-133a levels and TNM stage were independently associated with OS (HR = 0.590, 95%CI: 0.350-0.995, P < 0.05; and HR = 6.111, 95%CI: 1.029-36.278, P < 0.05, respectively). CONCLUSION: The downregulation of miR-133a may play an important role in the progression of CRC and can be used as an independent factor to determine CRC prognosis.

Krüppel-like factor 8 overexpression is correlated with angiogenesis and poor prognosis in gastric cancer.

AIM: To investigate Krüppel-like factor 8 (KLF8) expression in gastric cancer and its relationship with angiogenesis and prognosis of gastric cancer. METHODS: One hundred and fifty-four patients with gastric cancer who underwent successful curative resection were retrospectively enrolled in the study. Fifty tumor-adjacent healthy gastric tissues (≥ 5 cm from the tumor margin) obtained during the original resection were randomly selected for comparative analysis. In situ expression of KLF8 and CD34 proteins were examined by immunohistochemistry. The intratumoral microvessel density (MVD) was determined by manually counting the immunostained CD34-positive endothelial cells in three consecutive high-magnification fields (× 200). The relationship between differential KLF8 expression and MVD was assessed using Spearman's correlation coefficient test. χ² test was performed to evaluate the effects of differential KLF8 expression on clinicopathologic factors. Kaplan-Meier and multivariate Cox survival analyses were used to assess the prognostic value of differential KLF8 expression in gastric cancer. RESULTS: Significantly higher levels of KLF8 protein were detected in gastric cancer tissues than in the adjacent non-cancerous tissues (54.5% vs 34.0%, P < 0.05). KLF8 expression was associated with tumor size (P < 0.001), local invasion (P = 0.005), regional lymph node metastasis (P = 0.029), distant metastasis (P = 0.023), and tumor node metastasis (TNM) stage (P = 0.002), as well as the MVD (r = 0.392, P < 0.001). Patients with KLF8 positive expression had poorer overall survival (P < 0.001) and cancer-specific survival (P < 0.001) than those with negative expression. Multivariate analysis demonstrated that KLF8 expression independently affected both overall and cancer-specific survival of gastric cancer patients (P = 0.035 and 0.042, respectively). CONCLUSION: KLF8 is closely associated with gastric tumor progression, angiogenesis and poor prognosis, suggesting it may represent a novel prognostic biomarker and therapeutic target for gastric cancer.

RNA interference of human papillomavirus type 16 E7 increases HLA class I antigen expression in HaCaT-E7 cells.

BACKGROUND: High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer. The E7 protein of high-risk HPV disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting an ideal target for development of the immunotherapy in HPV-positive cervical cancers. We previously reported that HPV16 E7 could down-regulate cell-surface HLA class I antigen accompanying decreased expression of transporter associated with antigen processing 1 (TAP-1). The purpose of this study was to determine whether knockdown of HPV16 E7 could up-regulate surface HLA class I antigen expression in HPV16 E7 expressing HaCaT cells (HaCaT-E7). METHODS: An E7-specific small interfering RNA (siRNA) was transfected into the HaCaT-E7 cells, and the expression of HPV16 E7 was measured by real-time reverse transcriptase polymerase chain reaction and Western blot. With the use of flow cytometry analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 expression were detected. RESULTS: It was found that transfection of HPV16 E7-siRNA reduced HPV16 E7 expression as measured on messenger RNA and protein levels. The flow cytometry analysis showed that, compared with mock transfection, a statistically significant increase of approximately 75% in surface HLA class I levels was observed in HaCaT-E7 cells at 72 hours after transfection of E7 siRNA. Moreover, he knockdown of E7 in HaCaT-E7 cells could result in an increase of intracellular TAP-1 expression, which is essential for the expression of HLA class I at cell surface. CONCLUSIONS: Our study showed that the knockdown of HPV16 E7 could increase cell surface HLA class I antigen expression in HaCaT-E7 cells. In addition, for HPV-positive human cervical cancer, our observations indicate that the HPV E7 gene is a target of choice.

Down-regulation of HLA class I antigen in human papillomavirus type 16 E7 expressing HaCaT cells: correlate with TAP-1 expression.

OBJECTIVES: High-risk human papillomaviruses (HPVs) are the major causative agents of cervical cancer, and the E6 and E7 genes encode the major HPV oncoproteins. The E7 protein of high-risk HPV types disturbs cell cycle control and down-regulates components of the antigen presentation pathway, suggesting a role for E7 in tumor immune evasion. We previously reported that HPV-16 E7 expression and down-regulation of HLA class I was highly correlated in cervical lesions. This study was aimed to determine whether HPV-16 E7 oncoprotein could down-regulate surface HLA class I antigen in HPV-16 E7-transfected cells, and whether it had correlation with the expression of the transporter associated with antigen processing (TAP). METHODS: The HPV-16 E7 open reading frame was transfected into HaCaT cells. After G418 selection, resistant colonies were individually picked and expanded into clonal cell lines. Using the fluoresence-activated cell sorting analysis, the levels of cell surface HLA class I antigen and intracellular TAP-1 and TAP-2 expressions were detected. RESULTS: Compared with the empty vector control, a statistical significant decrease of approximately 50% in cell surface HLA class I expression was observed in HPV-16 E7 expressing HaCaT cells (P < 0.001). Moreover, the expression of HPV-16 E7 in HaCaT cells resulted in decreased expression of TAP-1 that was essential for HLA class I expression at the cell surface, a statistical significant decrease of approximately 40% compared with that with the empty vector control (P < 0.001). CONCLUSIONS: Our finding demonstrates that HPV-16 E7 down-regulates surface HLA class I antigen, which in part correlates with the decrease of TAP-1.

[Evaluation of genotyping of human papilomavirus by a new DNA liquid chip based on Luminex XMAP].

OBJECTIVE: To evaluate the diagnostic value of Luminex XMAP liquid chip for HPV detection. METHODS: Detect HPV DNA with the liquid chip and HC II in 264 cases, including 231 of cervical cancer and 33 of cervical scrapes. The accordance of two methods were compared and DNA sequencing was performed in conflicting samples and single (infection samples. RESULTS: There is an excellent agreement between the methods. The overall incidence of HPV was 82.95%, the most common genotypes were HPV 16, 52, 58, 18, 11, 31, 6, 39, 33, 56, 70. Among all the positive types, 117 were single type and 102 were multiple types, and 87.43% were high-risk HPV genotypes and 12.57% were low-risk genotypes. Based on the criteria of histology and pathology, the sensitivity, specificity, positive and negative-predictive value of HPV liquid chip and HC II for detecting all cases of CIN II, III and carcinomas were respectively 93.07%, 87.88%, 98.17%, 64.44% and 94.81%, 87.88%, 98.21%, 70.73%. CONCLUSION: The common types of HPV infection are 16, 52, 58, 18, 11, 6, 56 and 31. Multiplex HPV genotyping by Luminex XMAP liquid chip appears to be highly suitable for diagnostic screening and large-scale epidemiological studies.

[Study on the genotyping of human papillomavirus using a new DNA liquid chip in women of high-risk group of Shandong province].

OBJECTIVE: To evaluate the diagnostic applicability of human papillomavirus (HPV) liquid chip assay which is based on Luminex XMAP System, and perform a HPV epidemiologic study with the liquid chip in women of Shandong province. METHODS: To detect HPV genotypes on a 96-well plate with the liquid chip which can simultaneously detect and identify 26 common HPV genotypes in a total of 2925 cervical scrapes obtained from gynecological outpatients as well as to analyze the relationship between HPV types and different cervical diseases by studying the distribution of HPV genotypes and pathologic diagnosis. RESULTS: Among 639 cases who performed pathologic/cytological and histological diagnoses, 184 cases are in group of normal cytology, 266 cases in group of, 77 cases in group of cervical intra-epithelial neoplasia (CIN) I, 7 cases in group of CIN I - II, 46 cases in group of CIN I - II, 46 cases in group of CIN I - II and 13 cases in group of cervical cancer. The overall incidence of HPV in our samples is 36.0% (1054/2925) and 23 types of all 26 types on liquid chip are found. The most common genotypes found are HPV-16 (26.75%), HPV-52 (25.75%), HPV-58 (10.47%), HPV-18 (8.87%) and HPV-11 (6.94%). Among all the positive types, 87.32% are high-risk HPV and 13.68% are low-risk HPV genotypes. Both single and multiple types are easily identified, showing 66.22% ( n = 698) single type and 33.78% ( n = 356) multiple types. Of all the 1054 HPV-positive cases, 261 (24.8%) is occupied by women 21 to 25 years of age and progressively lower by older age groups, reaching 4.9% by women between 51 to 67 years old. The incidence of HPV in our samples is 23.37%, 33.08%, 54.54%, 57.14%, 82.61%, 91.30% and 100% for normal cytology, inflammation,CIN I ,CIN I - II, CIN II ,CIN III, and carcinomas specimens, respectively. Infections with more that one virus are common, accounted for 4.89%, 7.14%, 18.18%, 28.57%, 41.30%, 43.37% and 38.46% for normal cytology, inflammation, CIN I, CIN I - II, CIN II, CIN III, and carcinomas specimens, respectively. Based on the criteria of histology and pathology, the sensitivity, specificity, positive-predictive value and negative-predictive value of HPV liquid chip assay for detecting all cases of CIN II, III are 88.57%, 76.63%, 68.89% and 92.16% respectively. Conclusion The common types of HPV infection are 16, 52, 58, 18, 11, 6, 56 and 31. The HPV-positive rate increased along with the increase of grading on cervical lesions. There are more younger women among all the HPV-positive ones. Multiplex HPV genotyping by liquid chip appears to be highly suitable for diagnostic screening as well as the conduction of large-scale epidemiological studies.